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Characterization of hemopoietic stem cells in chronic myeloid leukemia (CML) Udomsakdi, Chirayu 1992

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CHARACTERIZATION OF HEMOPOIETIC STEM CELLS IN CHRONIC MYELOID LEUKEMIA (CML)  by  CHIRAYU UDOMSAKDI MD (First Class Honor), Mahidol University, Thailand, 1988  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  in  THE FACULTY OF GRADUATE STUDIES (Department of Pathology)  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA September 1992 © Chirayu Udomsakdi, 1992  In  presenting  degree freely  at  this  the  thesis  in  partial  University  of  British  available for  copying  of  department publication  this or of  reference  thesis by  this  for  his thesis  scholarly  or for  her  The University of British C o l u m b i a Vancouver, Canada  (2/88)  1 further  purposes  the  requirements  gain  that  agree that  may  representatives.  financial  Department  of  Columbia, I agree  and study.  permission.  DE-6  fulfilment  It  shall not  be  the  advanced  Library shall make  by  understood be  an  permission for  granted  is  for  the that  allow/ed without  it  extensive  head  of  my  copying  or  my  written  ABSTRACT  Much evidence indicates that the target of neoplastic transformation In chronic myeloid leukemia (CML) is a pluripotent hemopoietic stem cell, from which differentiated blood cells of the myeloid and lymphoid lineages Eire normally derived throughout adult life. CML is one of the best defined hematologic malignancies, being characterized by a consistent chromosomal and molecular abnormality, the Philadelphia (Ph^) chromosome and the BCR/ABL fusion gene, respectively. In most patients with CML, expansion of the leukemic clone is evident both at the level of mature blood cells and of their immediate precursors. Relatively little is known about the number or characteristics of the most primitive cells responsible for maintaining the leukemic clone. This has been due to a lack of suitable assays for these cells. In this thesis, I describe the development of a quantitative assay for primitive CML hemopoietic cells based on the long-term culture-initiating cell (LTC-IC) assay recently established for very primitive hemopoietic cells in normal human bone marrow. Blood samples from CML patients with high WBC counts were used as an highly enriched source of Ph^-positive leukemic progenitors. Clonogenic cell output after 5 weeks in LTC was shown to be a linear function of the number of Input blood cells, enabling this endpoint to be used as a quantitative, albeit relative, measure of CML LTC-IC. The application of limiting dilution methods allowed derivation of absolute CML LTC-IC frequencies. LTC-IC in CML blood were found to be markedly increased in proportion both to the WBC count and other clonogenic progenitors. This is in contrast to the situation in CML marrow where in the specimens examined, the frequency of leukemic LTC-IC was found to be decreased, on average >20 fold relative to normeil LTC-IC in normal marrow.  Characterization of LTC-IC and clonogenic cells in CML blood and their comparison to these cells in normal marrow and blood showed some similarities and differences. Most (but not all) CML clonogenic cells were similar to clonogenic cells in normal marrow, but different from clonogenic cells in normal blood in terms of thefr apparent activation state, as measured by Rh-  123 staining, HLA-DR expression, forward light scatter, and sensitivity to 4hydroperoxycyclophosphamide. Most but not all CML LTC-IC were also found to express an activated phenotype, and thus differed from the LTC-IC in normal marrow and blood which exhibit a phenotype expected of quiescent cells.  The proliferative/self-maintenance and differentiative capacities of CML LTC-IC were also studied. CML LTC-IC were not different from normal LTC-IC in terms of either the total number or different types of clonogenic cells they produced after 5 weeks. However, CML LTC-IC were defective In their self-maintenance. These results, together with the decreased quantity of these cells in CML marrow, explains why leukemic progenitor output rapidly declines in LTC initiated with CML marrow.  In summeiry, my thesis demonstrates that primitive CML hemopoietic cells can be detected, quantltated, and phenotypically and functionally characterized using the LTC-IC assay recently developed for normsd marrow. The data obtained provide an explanation for the rapid and selective decline of CML progenitors observed in LTC initiated with CML marrow, but not CML blood. The phenotypic differences of leukemic and normal LTC-IC should provide Important Information for the design of new treatment strategies and for further studies into the pathogenetic mechanisms leading to the development of the neoplastic clone in CML.  TABLE OF CONTENTS ABSTRACT TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS ACKNOWLEDGEMENTS  CHAPTER I  11 Iv vU vlll x xl  INTRODUCTION  (1) Normal Hemopoiesis 1.1 Ontogeny and organization of the hemopoietic system 1.2 Hierarchy of hemopoietic cell differentiation 1.2.1. Primitive hemopoietic stem cells 1.2.1.1 In vivo repopulation assays 1.2.1.2 The Ogawa Blast-colony assay 1.2.1.3 The HPP-CFC assay 1.2.1.4 Stromïd-dependent Blast cell colonies 1.2.1.5 LTC-IC assay 1.3 Isolation of primitive hemopoietic stem cells 1.3.1 CD34 1.3.2 HLA-DR 1.3.3 Rhodamine-123 1.3.4 Light scattering properties 1.3.5 4-hydroperoxycyclophosphamide (4-HC) sensitivities 1.4 Regulation of normal hemopoietic stem cells 1.4.1 Regulation of cell viabiUty 1.4.2 Regulation of population turnover 1.4.3 Regulation of seLF-renewal and differentiation  1 5 6 8 9 10 11 12 17 17 18 18 19 19 20 22 22 24  (2) Chronic Myeloid Leukemia (CML) 2.1 CML as a clinically defined entity 2.1.1 Diagnosis and classification of disease stages 2.1.2 Therapy of CML 2.2 Cytogenetic and molecular correlates 2.2.1 The Philadelphia (Phh chromosome 2.2.2 The BCR-ABL fusion gene 2.2.3 Is the BCR-ABL gene the initiating event In CML ? 2.3 Cellular studies 2.3.1 The persistence of normal stem cells in CML 2.3.2 Regulation of hemopoiesis in CML 2.3.2. l a CeU division 2.3.2. l b Mechanisms of altered growth behaviour 2.3.2.2 Cell adhesion (3) Thesis objectives References  26 29 29 31 31 35 38 39 41 42 43 44 46 47 50  CHAPTER n  MATERIALS AND METHODS  1) Preparation of cells 1.1 Normal marrow 1.2 Normal blood 1.3 CML blood 2) Staining 3) Flow C3rtometiy 4) Functional Assays 4.1 Short-term colony assays 4.2 LTC-IC assays 5) Limiting dilution analysis 6) Maintenance studies 7) 4-HC studies References CHAPTER m  SEPARATION OP PRIMITIVE HEMOPOIETIC STEM CELLS IN NORMAL HUMAN MARROW BY RHODAMINE-I23  1) Introduction 2) Results 2.1 Dose-response of Rh-123 2.2 Uptake ofRh-123 by clonogenic cells and LTC-IC 2.3 Double-staining of human marrow with CD34 and Rh-123 2.4 Frequency and enrichment of LTC-IC by CD34 and Rh-123 3) Discussion 4) References CHAPTER IV  94 95 95 97 101 102 103 105  CHARACTERIZATION OF PRIMITIVE HEMOPOIETIC CELLS IN NORMAL HUMAN BLOOD  1) Introduction 2) Results 2.1 Quantitation of LTC-IC in the normal blood circulation 2.2 Phenotype of circulating LTC-IC 2.3 4-HC sensitivities of circulating progenitors 2.4 Differentiative potential of circulating LTC-IC 3) Discussion 4) References CHAPTER V  79 79 80 81 83 84 87 87 88 89 89 91 92  107 108 108 112 122 124 126 128  RAPID DECLINE OF CHRONIC MYELOID LEUKEMIC CELLS IN LONG-TERM CULTURE IS DUE TO A DEFECT AT THE LEUKEMIC STEM CELL LEVEL  1) Introduction 2) Results 2.1 Validation of an in vitro quantitative assay for CML LTC-IC 2.2 Quantitation of CML LTC-IC and clonogenic cells in the circulation of CML patients 2.3 Proliferative capabilities of CML versus normal LTC-IC 2.4 Self-maintenance in vitro of CML versus normal LTC-IC 3) Discussion 4) References  131 132 132 135 137 140 142 145  vl CHAPTER VI  PHENOTTPIC HETEROGENEITY OF PRIMITIVE LEUKEMIC HEMOPOIETIC STEM CELLS IN PATIENTS WITH CML  1) Introduction 2) Results 2.1 Phenotype of CMLLTC-IC 2.2 Sensitivities of CML and normal LTC-IC to 4-HC 2.4 Differentiative potential of CML LTC-IC 3) Discussion 4) References CHAPTER V n SUMMARY AND FUTURE STUDIES  148 149 149 160 162 163 166 168  vil LIST OF TABLES page Table 1.  Criteria for Identifying Committed Progenitors in Vitro  6  Table 2.  Properties of Primitive Hemopoietic Stem CeUs  16  Table 3.  Criteria for Classifying the Three Phases of CML  29  Table 4.  Landmarks In the Treatment of CML  31  Tables.  Characteristics of the CML Patients Studied  82  Table 6.  Frequency and Enrichment of Normal Human Marrow Progenitors By Multi-parameter Sorting  102  Table 7.  Quantitation of LTC-IC and Clonogenic Cells In Normal Blood  111  Table 8.  Frequency and Enrichment of Normal Circulating Progenitors By Multi-parameter sorting  119  Table 9.  DIfferentiative Potential of Normal Marrow and Blood LTC-IC  125  Table 10.  Proliferative Potential of Normal and Leukemic LTC-IC  137  Table 11.  Quantitation of LTC-IC In CML and Normal Marrow  139  Table 12.  Frequency of Primitive Progenitors In the CML Patients Studied  151  Table 13.  Frequency, Enrichment and Recovery of Primitive CML Progenitors By Multi-parameter Sorting  158  Table 14.  Concentration of Various Phenotypicedly Defined Subpopulations of CML Blood and Comparison to Normal Values  159  Table 15.  DIfferentiative Potential of CML LTC-IC  162  Table 16.  Properties of Normal and CML LTC-IC  172  Table 17.  Properties of Normal and CML Clonogenic Cells  172  vlll LIST OF FIGURES page FIGURE 1.  LTC-IC Assay  14  FIGURE 2.  Model of Hemopoiesis In LTC  15  FIGURES.  Options for Hemopoietic Stem Cells  21  FIGURE 4.  Incidence of CML in Canada in 1983  27  FIGURE 5.  Cytogenetic and Molecular Abnormalities in CML  33  FIGURE 6.  Principles of FACS  85  FIGURE 7.  Maintenance Studies  90  FIGURE 8.  Survival of clonogenic cells and LTC-IC after exposure to Rh-123 at variable doses.  96  FIGURE 9.  Contour Plots of FSC versus SSC and Log Rh-123 Fluorescence Intensity of Low Density Normal Marrow Cells.  98  FIGURE 10. Expression of Rh-123 on Marrow Clonogenic cells and LTC-IC.  99  FIGURE 11. Distribution of BFU-E and CFU-GM in the Rh-123^^^ and Rh-123^'^êht Fractions from Normal Marrow.  99  FIGURE 12. FACS Contour Plots of Anti-CD34 and Rh-123 Stained Normal Human Marrow Cells.  101  FIGURE 13. Input-output Relationship in LTC of Normal T cell-depleted Blood.  109  FIGURE 14. Limiting Dilution Analysis of Normal Blood LTC-IC.  110  FIGURE 15. Bivariate Contour Histograms of Light density T cell-depleted Normal Blood Cells Stained With Anti-CD34 and Anti-HLA-DR  113  FIGURE 16. Light Scatter Profiles of Normal T ceU-Depleted Blood.  115  FIGURE 17. A Representative Histogram of CD34+, Low Density T-cell depleted Normal Blood Cells Double-stained with Anti-HLA-DR.  117  FIGURE 18. A Representative Histogram of CD34+. Low density T cell-depleted Normal Blood Cells Double-stained with Rh-123.  121  FIGURE 19. Sensitivities of Marrow and Blood Clonogenic cells and LTC-IC to 4-HC.  123  FIGURE 20.  Input-output Relationship in Leukemic LTC.  134  FIGURE 21.  Limiting Dilution Analysis of CML LTC-IC.  136  Ix page FIGURE 22.  LTC-IC and Clonogenic Cell Concentration in the Peripheral Blood of 26 CML Patients and 23 Normal Individuals.  138  FIGURE 23.  Differential Kinetics of CML versus Normal LTC-IC  141  FIGURE 24.  Light Scatter Characteristics of Clonogenic Cells and LTC-IC of CML blood.  152  FIGURE 25.  Bivariate Contour Plots of a Single Representative Sample of Normal and CML Light Density Blood Cells.  154  FIGURE 26.  HLA-DR Expression of CML Clonogenic Cells and LTC-IC  155  FIGURE 27.  Rh-123 Uptake of CML Clonogenic Cells and LTC-IC  156  FIGURE 28.  Survival of CML Clonogenic Cells and LTC-IC After 4-HC Treatment  161  FIGURE 29.  Proposed Phenotypic Organization of Normal and CML Primitive Cells  173  LIST OF ABBREVIATIONS ABL abelson BCR breakpolnt-cluster-region BFU-E burst-formlng-unlt-erythrold BL-CFC blast-colony-forming cell BMT bone marrow transplemtatlon CFU-E colony-formlng-unlt-erj^throld CFU-GM colony-formlng-unlt-granuloc5d;e/macrophage CFU-GEMM colony-formlng-unltgranulocyte-ers^hrold-macrophage-megakaiyocyte CFU-MK colony-formlng-unlt-megakaryoc3^e CFU-S colony-formlng-unit-spleen CML chronic myeloid leukemia CRU competitive repopulating unit CSF colony-stimulating factors CSF-1 colony-stimulating factor-1 ECM extracellular matrix FACS fluorescence-activated cell sorter FCS fetal calf serum FH ficoll-hypaque FITC fluorescein isothiocyanate FSC forward light scatter characteristics FU fluorouracil G6PD glucose 6-phosphate dehydrogenase G-CSF granulocj^e colony-stimulating factor GM-CSF granulocyte-macrophage colony-stimulating factor Gy gray HPP-CFC h i ^ prollferative-potential colony-forming cell HPRT hypoxanthlne phosphoribosyltransferase IFN interferon ILinterleukin LFA-3 leucocjrte-function-associated-S LAK lymphokine-activater killer cell LTC long-term culture LTC-IC long-term culture-initiating cell MDR multidrug resistance MHC major histocompatibility complex MIP-1 a macrophage inflammatory protein-1 -alpha NK natural killer ceUs nm nanometers PE phycoerythrin PGK phosphoglycerate kinase Ph^ Philadelphia-chromosome PI phosphatidyllnositol Rh-123 rhodamlne-123 SEM standard error of the mean SH SRC homology TC tetramerlc complexes TGF-p transforming growth factor-beta U units VLA very late antigen WBC white blood cell  ACKNOWLEDGEMENT I would like to gratefulty acknowledge the assistance, support and instruction provided by many people during the course of my study and my stay In Canada. I have been priviledged to have Dr. Connie Eaves as my PhD supervisor, who in spite of her demanding schedule, always generously shared her time, support and intelligent advice, and also for critical review of the thesis. I am particularly grateful to Dr. Allen Eaves for giving me the opportunity to come to Canada to study at the Teny Fox Laboratory and for his administrative support necessary for the successful conduct of this research. I would also like to thank Dr. K. Dorovini-Zls, Chairman of my supervisoiy committee. Dr. Keith Humphries, Dr. M. Hayden and Dr. J . O'Kuslqr, members of the committee and Dr. Dagmar Kalousek and Dr. Dana Devine, the external examiners for my PhD departmental defense for their critical advice and review of the thesis. Discussions with Dr. Peter Lansdorp, Dr. Heather Sutherland and Dr. Christian Schmitt were highly valued. I am indebted to Dianne Reld for patiently teaching me how to use laboratory equipments and for excellent technical assistance with long-term culture experiments. I would also like to thank Jessyca Maltman, Gayle Thombury for expert technical assistance and staffs of the Stem Cell Assay Service, particularly Karen Lamble and Glovanna Cameron for processing patient marrow and blood samples and preparing feeder cells. I also thank Don Henkelman for statistical advice. I am very grateful to the National Cancer Institute of Canada (NCI-C), the Cancer Research Society, and the University of British Columbia for financial support. Finally, I would like to especially thank my mother. Dr. Yupha Udomsakdl, and my sisters (Nol and Noo) for thelr love and encouragement that were vital to the completion of this thesis.  CHAPTER  I  INTRODUCTION  1. NORMAL HEMOPOIESIS  1.1  ONTOGENY AND ORGANIZATION OF THE HEMOPOIETIC SYSTEM Most types of mature blood cells are short-lived and therefore must be replaced  continuously throughout adult life to maintain their numbers at constant levels in the blood and tissues. Blood cells originate from a rare population of totlpotential hemopoietic stem cells capable of self-renewal of thefr very extensive proliferation and differentiation potentialities. ^ The principal site of blood cell formation (hemopoiesis) In normal adult is the bone marrow, where approximately 2.5x10^ red blood cells, 2.5x10^ platelets, and 1x10^ granulocjrtes are normally produced per kg body weight every day.^ The bone marrow is also capable of increasing blood cell production upon demand (e.g., increasing the output of gremuloc3^es and red blood cells during Infection and hemorrhage, respectively).^*^ The hemopoietic system is thus a typical cell renewal system In which end cell production from more primitive precursors Is regulated by a complex series of mechanisms that balance cell proliferation, differentiation and survival.  In the developing embryo, the yolk sac is the first site of recognizable hemopoiesis.^'^ The liver then becomes the major hemopoietic organ towards the end of the first trimester and continues in this role through most of the second trimester; however the spleen also has a minor hemopoietic role In this middle period of fetal development. In the last trimester, hemopoiesis in the liver and spleen declines. At the same time, the bone marrow becomes the almost exclusive site of hemopoiesis and this remains so throughout subsequent development and adulthood. At birth, all bones that contain marrow have red (hemopoletically active) marrow. With age.  hemopoiesis undergoes regression in a centripetal direction so that, in adults, the more peripherally located marrow consists entirely of adipose tissues. Yellow marrow, however, can also transform into red marrow upon demand.^  The extravascular spaces between the marrow vascular sinuses are the site where hemopoiesis in humans takes place. The sinus wall forms a barrier between the hemopoietic compartment and the circulation: the so-called marrow-blood barrier.® This barrier is composed of a luminal layer of endothelial cells that form a complete inner lining and an abluminal coat of cells described as adventitial reticular cells that form an incomplete outer coat. The endothelial cells overlap extensively, but unlike extramedullaiy vessels, the overlapping areas lack tight Junctions, and therefore are thought to be able to slide over one another. Marrow endothellEd cells are also actively endocytlc and form the control system for particles and molecules entering eind leaving the hemopoietic spaces. The adventitial reticular cells are neither phagocytic nor capable of developing into hemopoietic cells. They synthesize reticular fibers, that Eilong with their cjrtoplasmic processes, provide the physical support of hemopoietic cells. ^ The reduction in adventitial cell covering on the abluminal surface of the sinus may facilitate penetration of the endothelial cells by mature cells.  A network of fibroblasts, macrophages and adipocytes are also components of the marrow "stromal" microenvironment.^'^ ^ It has been suggested that these cells may affect the development of blood cells through direct surface-to-surface interactions with their precursors. This is based on observed associations between granulocytes and adipocjrtes and fibroblasts, between erythroblasts and macrophages, and between l5miphoc)d:es and  fibroblasts.  The  association of multiple erythroblasts with a central macrophage-like cell is known as an "erythroblastic island". The function of these macrophages is thought to be to ingest extruded red cell and megakaryocyte nuclei, and also to serve as a localized source of growth factors. Some macrophages also have a parasinal position and therefore presumably are active in  phagocytosis of defective and senescent blood cells. Mature granulocytes have been identified to exist in close association with large adventitial reticular cells. Megakaryoc3rtes are typically located on the abluminal surface of the sinus endothelium. They extend projections into the lumen and are in contact with blood circulation. Early B-lineage l5miphoc5rtes develop from a plurlpotent hemopoietic stem cell In bone marrow.  However, areas of focal proliferation  comparable to the follicles in the avian bursa of Fabriclus are not readily apparent in humans. T lymphocytes are also believed to have a common precursor with B lymphocytes, however, their normal development appears restricted to the th3mius.  To enter the circulation, developing blood cells have to exert pressure on the endothelial membrane and form a migration pore through the c3d;oplasm of the endothelial cells (I.e., their migration is transcellular rather than intercellular).  Several factors are known to Influence  the process of marrow cell egress. ^® These include the C3e component of plasma complement, glucocorticoids and androgenic steroids. All of these molecules are believed to be capable of accelerating granulocyte release, cilthough their physiologic role is not clceir. Erythropoietin also has reticulocytlc cell-releasing activity, probably by reducing the adventltieil cell cover of the sinus wall and facilitating the access of the reticulocyte to the endothelial cell.  Primitive hemopoietic cells are also known to circulate in normal adults. ^^'^^ Infusion of peripheral blood mononucleeir cells into lethally Irradiated recipients (both animals and man) has been known for many years to result in successful engraftment and hemopoietic reconstitution with donor cells.-^^'^^ However, long-term survival in such situations has been variable and may reflect differences in the numbers of different types of primitive hemopoietic cells infused in different situations. Mechanisms and factors that are specifically involved in the release and homing of primitive hemopoietic cells to bone marrow have not yet been delineated. However, this is now £m eirea of Intensive investigation. It seems likely that the coordinate expression of various integrins^^ and other adhesion molecules^^ on primitive hemopoietic cells  will be found to play an Important part in these processes as In other homing mechanisms.2^"^ ^  Another putative role of stromal cells in creating a microenvironment suitable for the maintenance of hemopoiesis is to secrete certain extracellular matrix (ECM) proteins to provide anchorage sites for colonization of hemopoietic cells and/or fixation of growth factors. The ECM is a highly organized structure of macromolecules (e.g., laminin, fibronectin, glycosaminoglycans and collagen) that aire capable of influencing ceU proliferation and differentiation in vivo and in vitro.^^"^'* Devitalized extracts of bone ECM implanted into soft tissues are capable of inducing a weU-deflned developmented sequence that results in the formation of ectopic bone followed by vasculcirization eind the development of a functioned bone marrow from circulating stem cells.^^ There is also evidence that at least some hemopoietic growth factors are concentrated in a biologically active form by binding to certain E C M components. For example, IL-3 and GM-CSF have been shown to bind to heparan sulfate and glycosamlnoglycans."^^-^^ WUliams et al recently showed that murine CFU-S day 12 primitive hemopoietic ceUs express the a4 subunit of the VLA-4 integrln receptor, which is known to be a receptor for the CS-1 peptides of human fibronectin. This suggests that the adhesion of primitive stem ceUs to stromal ceU ECM is partly mediated by an Interaction between VLA-4 andfibronectin.^®Specific binding of granulocytic cells to a 60-Kd hemonectin protein has also been demonstrated."^^-'*^^ Unlike marrow neutrophfls, circulating neutrophfls faUed to bind to purified hemonectin in vitro. Some growth factors are also present locsilly in bone marrow stroma in an Inactive (latent) form, but can be released in an active form by a proteolytic process. Transforming growth factor-beta (TGF-P) is one example where such activation is seen (Review in Keller et al."*^^). Recently, a potent growth factor for primitive hemopoietic ceUs was cloned and found to exist in both transmembrane and soluble forms.'*2 This factor is the product of the Steel gene (hence the term "Steel factor"). It Is produced by many ceU types, including stromed fibroblasts In adult marrow and fetal Uver.'*^^'^^ Deletion of the tréinsmembreine form of Steel factor occurs in geneticedly defective Sl/Sl mice and  this results In hemopoietic failure, suggesting an Important role of the stromal mlcroenvlronment In the regulation of hemopoiesis.'^^  1.2  HIERARCHY OF HEMOPOIETIC CELL DIFFERENTIATION The hemopoietic system Is thought to represent a continuum of cells of varying self-  renewal, proliferative and dlfferentiative capacities.  Conceptually, It has been useful to  consider Its organization In terms of three levels of cellular development. The most primitive of these comprises cells that share extensive proliferative and dlfferentiative potential, but vary in the extent to which they maintain their pluripotentiallty. These cells give rise to an Intermediate category of committed progenitor cells. These have less proliferative capacity and are not thought to be self-malntalnlng In vivo. Hence they must be derived by the continuing differentiation of primitive hemopoietic cells. Committed progenitor cells can be detected in short-term In vitro colony assays and are distinguished by their ability to produce different numbers and arrangements of mature cells of single lineages In semi-solid media.'^^ The nomenclature currentiy used for defining different types of committed progenitors, the kinds of colonies they produce and the criteria used for their Identification Is summarized in Table 1. Proliferation and differentiation of committed progenitor cells leads to the formation of precursor cells which represent the first stage when hemopoietic cells can be morphologically identifiable as belonging to a specific lineage. However, most morphologically recognizable precursors carmot be detected In colony assays due to their very limited proliferative capacity prior to terminal differentiation into mature blood cells. The process of blood cell development is thus a complex, multi-step process associated with enormous capacity for cell amplification.  TABLE 1 Criteria For Identifying Committed Progenitors In Vitro  TYPES OF COLONIES  SCORING CRITERIA  Erythroid Colonies CFU-E  No.of Hemoglobinized Erythroblast Clusters 1-2 clusters at day 10-12 (>8 ersdhroblasts per cluster) 3-8 clusters at day 18-20 9-16 clusters at day 18-20 > 16 clusters at day 18-20  Mature BFU-E Intermediate BFU-E Primitive BFU-E Granulocytic Colonies  1.2.1  Mature CFU-GM Primitive CFU-GM  Numbers of Granulocytes and/or Macrophages progeny <500 cells >500-1000 cells  Megakaryocytic Colonies Mature CFU-MK Primitive BFU-MK  No. of Clusters of Megakaryocytes unifocal cluster at day 12 (> 2 megakaryocytes) multiple, larger clusters at day 21  Primitive Hemopoietic CeMs Present models of hemopoiesis assume the persistence into adulthood of a population of  very primitive "totipotent" stem cells that serve as the origin of all of the mature lymphoid and myeloid cells produced throughout Ufe.^^'^'* The first definitive evidence for the existence of such cells came from mouse bone marrow trgmsplantatlon experiments using three types of markers for tracking repopulation of recipients with donor-derived cells: radiation-induced chromosome markers; differing hemoglobin, isozjmie, and immunoglobulin genotypes 52,55-61 and unique retroviral vector Integration sites.®^"®^ clonal reconstitution of myeloid and lymphoid tissues In the recipient animals with donor-derived cells was demonstrated In some of these experiments.  Early studies In mice also Indicated that normal adult marrow contained cells (called CFU-S) capable of forming macroscopic, multillneage colonies In the spleens of transplanted, myeloablated or genetically compromised (W/W^) reclpients.^^'^^'®^ Further examination of  these spleen colonies showed that they, themselves, could contain varying numbers of CFU-S, I.e., cells able to generate new spleen colonies upon Injection into secondary irradiated hosts.^ CFU-S therefore seemed to possess the property considered to be an essential hedlmark of a hemopoietic stem cell. However, recent observations have demonstrated that CFU-S are heterogeneous and there probably exists a hierarchy amongst cells thus deflned.^^-^^ The extent to which cells responsible for long-term multilineage hemopoietic reconstitution in vivo have the potentied to be detected as CFU-S remains controversial.  Since comparable in vivo assays suitable for detecting human hemopoietic stem cells are not possible, the existence of primitive cells with myeloid and lymphoid reconstituting potential in normal adult human marrow has been more difficult to establish. Initial evidence for the existence of such cells came from analyses of glucose-6-phosphate dehydrogenase (G6PD) isoenz3mie expression in hemopoietic cells from heterozygous women with myeloproliferative and myelodysplastic disorders. Clonal Involvement of myeloid and Ijnnphoid cells and their precursors demonstrated in such cases suggested that the initial neoplastic transformation event had occurred in a cell that still had all of these potentialities.^^ The demonstration of a consistent chromosomal abnormality, the Philadelphia chromosome (Ph^), in multiple cell types as well as B-l3miphoid cells in patients with chronic myeloid leukemia (CML) has also lent support to the hypothesis. ^^'^^ Recent studies of circulating blood cells In patients transplanted with marrow from normal female donors heterozygous for certain methylationsensitive restriction fragment length polymorphisms (RFLP) in the X-chromosome-Unked phosphoglycerate kinase (PGK) or hs^poxanthine phosphoribosyltransferase (HPRT) loci have provided evidence for clonal granulocytes and T cells originating from a common, donor-derived normal precursor, thus indicating the presence of transplantable lymphomyeloid stem cells In normal adult human marrow.^'*  Despite major Impediments to the characterization and direct identification of hemopoietic stem cells due to their very low frequency In hemopoietic tissues and their lack of vinlque morphological features to distinguish them from other l)miphoc5d;e-lIke cells, significant progress in these areas has been made during the past thirty years. The following sections will briefly summarize the developments that have occurred leading to the very recent establishment of quantitative assays that appear to be specific for totipotent hemopoietic stem cells.  1.2.1.1  In Vivo Repopulation Assays  TlU and McCulloch were the first to develop a quantitative in vivo assay for hemopoietic progenitor cells.^^ This assay is based on the ability of certain hemopoietic cells present In adult mouse bone marrow to form macroscoplccdly visible colonies on the surface of the spleen in lethaUy Irradiated recipients. The precursors of these colonies are called CFU-S (colonyforming-unlt spleen). Each CFU-S has the potential to form a colony containing 10^-10*^ ceUs within 8 to 14 days, and the cells within each colony Include one or more of the following types: erythrocytes, gremulocytes, monocjrtes cind megakaryocytes as well as progeny CFU-S.^^-^^'^® CFU-S were InitiaUy regarded as the most primitive type of hemopoietic stem ceU.'*^ However, subsequent studies showed that cells defined as CFU-S are phenotypicaUy as weU as functionally heterogeneous. ^^-^^'^^ Moreover, measurements of CFU-S do not necessarily correlate with life-sparing and long-term repopulating ability.^^'^^'^^ Thus accumulating evidence over the last 10 years indicates that aU CFU-S are not pluripotent, self-renewing stem ceUs and that a distinction must be made between CFU-S giving rise to colonies that appear on the spleen earlier than 11 days after the cell transfer (CFU-S day 8-11), compared to CFU-S giving rise to colonies that appear at later times (e.g., CFU-S day 12-16).®® Even many CFU-S day 12 may not represent long-term repopulating stem cells. At most about 10% of CFU-S day 12 survive in mice treated with 150 mg/kg 5-fluorouracfl (5-FU) treatment, although this is not thought to have any effect on cells with long-term repopulating potential.^^'®'^'®^ Recently, both in vivo and in vitro assays for precursors of CFU-S have been developed.^^•®^'®® These  cells have high marrow-repopvilatlng ability (MRA)'^^ g^^d radioprotective ability (RPA)'^^ and also the ability to generate CFU-S day 12 in vivo (and In vitro on marrow-derived stromal layers) despite depletion of CFU-S day 12 in the original samples.®^ Isolated subsets of cells with longterm repopulatlng ability were also found and did not contain CFU-S. These studies have been interpreted as indicative of a hierarchical structure within the CFU-S population. Such assays allow relative measurements of these cells to be made but do not allow absolute numbers to be derived.  A practical quantitative assay for long-term repopulatlng cells with potential for reconstituting all hemopoietic tissues and that allows derivation of absolute number of cells has been described by Szllvassy et al.^^ In this assay, limiting number of cells derived from genetically distinguishable (male) donors are Injected into Irradiated (female) recipients together with a myeloprotective gréift of (female) bone marrow cells that, however, are signiflcantiy compromised in their long-term reconstituting potential. This is achieved by subjecting normal marrow to two serial rounds of transplantation and regeneration. The proportion of recipients showing detectable (>5%) reconstitution of their tissues with test (male) cells Is then used to calculate the frequency of repopulatlng precursors (termed CRU for competitive repopulating units) In the original male cell suspension assayed using Poisson statistics.^^-^^ Analysis of thymic eind marrow reconstitution by retrovireilly marked CRU has provided evidence that CRU are capable of reconstituting both Ijnnphold and myeloid tissues long-term.  Recentiy, new  approaches for engrafting human cells Into Immune-deficient mice have been described, providing the foundation for the potential development of a corresponding in vivo assay to enable characterization of the normal developmental program of human hemopoietic stem cells along both myeloid sind lymphoid llneages.^^'^^  1.2.1.2  The Ogawa Blast Colony Assay  In vitro colony assays for veiy primitive subpopulations of plurlpotent clonogenic progenitors (CFU-GEMM)®^ have also been recently developed by several groups using different combinations of growth factors and distinct read-outs. Ogawa et al. described a class of primitive hemopoietic cells that produce colonies containing large numbers of daughter cells detectable as clonogenic progenitors when replated Into secondary colony assays. Such colonies are distinguished from CFU-GEMM-derived colonies In that they consist exclusively of blasts at the time they are recognized. The cell from which they derive was therefore called a CPUBlast.^^'^^ These progenitors are thought to represent a primitive subset of C F U - G E M M since most take a longer time to initiate colony formation under conditions when many C F U - G E M M colonies have matured and may not yet show cmy signs of terminal differentiation. It appears that CFU-Blast can remain dormant In vitro In GQ for at least two weeks before proliferation Is initiated, although this dormancy period can be modulated by growth factor stimulation. lOO^lOS CFU-Blast have been demonstrated in a variety of murine and human hemopoietic tissues including marrow, spleen and cord blood. 106-108 CFU-Blast are not actively cycling In vivo and are thus relatively resistant to 5-FU.  j^ie relationship of CFU-  Blast to other cells In the hemopoietic hierarchy is not yet clear, since few comparisons with highly purified populations have as yet been performed. As expected, some CFU-Blast (like some C F U - G E M M ) can give rise to daughter CFU-S and CFU-Blast at low frequency.  In man  CFU-Blast are found amongst the CD34"'", HLA-DR', CD38''", CD33" cells in normal human marrow. ^  1.2.1.3  The HPP-CFC Assay  In 1979, Bradley and Hodgson described the formation of extremely Isirge colonies of granxolocytes and macrophages by murine bone marrow cells stimulated with certain combinations of factor-containing media, and called the progenitor of such colonies high proliferative potential colony-forming cells (HPP-CFC). ^ ^ ^ HPP-CFC also have the following properties suggesting that they are primitive cells: 1) relative resistance to 5-FU; 2) correlation of  their numbers with cells capable of repopulating the bone marrow of lethally Irradiated mice; and in some cases, 3) ability of some H P P - C F C to also generate cells of the megakaryocyte and erythroid lineages. 1 1 2 - 1 1 4 H P P - C F C have been described in both mouse and human bone marrow. 1 1 L 1 1 5 Three subpopulations of H P P - C F C have been defined and characterized, suggesting a hierarchical structure within the H P P - C F C compartment. 1 1 ^ Less is known about the relationship of H P P - C F C to other candidate hemopoietic stem cells identified by other assays. Ogawa et al reported that at least some CFU-blast give rise to multiple H P P - C F C and perhaps the most primitive H P P - C F C may in fact be the same as the CFU-blast (reviewed in McNiece et al. 11®). H P P - C F C have been shown to be present In murine LTC, where their maintenance (production) may be dependent on the production of growth factors produced by stromal cells. H ' '  1.2.1.4  Stromal-dependent Blast Cell Colonies  In 1985, Gordon et al. identified a population of human hemopoietic cells characterized by an ability to specifically adhere to preformed marrow cell adherent layers within 2 hours and then to generate colonies on the surface of such stroma in the absence of added growth factors. 11® Meiny of colonies produced consisted of blasts, hence the term BL-CFC for their precursors. 1 1 ^ The frequency of BL-CFC was found to be about 1/10^ unfractionated normal human bone marrow cells. Some BL-CFC give rise to CFU-GM, BFU-E and CFU-GEMM as well as BL-CFC, as demonstrated by replatlng experiments. 1^0 However, as for HPP-CFC, a hierarchy appears to exist in the BL-CFC compsirtment. Bol et al. showed that colonies derived from BL-CFC that are detected on day 5, day 14 and day 2 1 are derived from different types of BL-CFC, and that these can be separated on the basis of their forward light scattering properties. 1^1 Day 2 1 BL-CFC are believed to represent a much earlier cell type than day 5-BLCFC. The relationship of BL-CFC to progenitors detected in other assays remains to be determined. Some recent studies, however, indicate that day 5 BL-CFC are more mature than  either the Ogawa CFU-Blast or UTC-IC since day 5 BL-CFC are 0033+ and HLA-DR"*" in contrast to the latter two, which are CD33' and HIA-DR' as shown by sorting experiments.  1.2.1.5  ^"^^  LTC-IC Assay  In 1977, Dexter et al. described a liquid culture system in which production of CFU-S, CFU-GM and their mature progeny could be maintained in vitro from an initial inoculum of mouse bone marrow cells for several months. 1^'* Subsequently this system was shown to be able to sustain long-term hemopoiesis from marrow cells from other species, including man. In this long-term culture (LTC) system the cells are distributed between two fractions: the adherent layer, which contains several types of stromal cells, including endothelial cells, fibroblasts, adipocytes and macrophages as well as the majority of the more primitive hemopoietic cells; and the nonadherent fraction, which consists mostly of mature granulocytes, macrophages and their immediate, morphologically recognizable precursors.  The  Importance of stroma cells in LTC was ffrst suggested by the duplication and cure of genetically defective hemopoiesis in vitro.  Preferential localization of primitive hemopoietic cells In the  adherent layer of LTC provided evidence that close contact with stromal cells may be Important for the maintenance and proliferation of hemopoietic stem cells. 128,129 Subsequent studies showed that the adherent layer produces both positive and negative signals that regulate the turnover of primitive hemopoietic cells.  Separation of early cells responsible for  initiating long-term hemopoiesis in this system from the stromal cells also revealed an absolute dependence of the former on the latter. ^"^^  Recentiy, Fraser et al.^^-l"^^ have shown that Ijmiphomyelold repopulatlng cells (CRU) In mice also persist and undergo self-renewal divisions in murine LTC, suggesting the possibility that the LTC system might detect an analogous, primitive reconstituting cell In human bone marrow. Sutherland et al. l ^ ' * pursued this approach concurrentiy. The assay as shown schematically In Figure 2, measures the output of progenitors detected in standéird in vitro  colony assays (I.e., CFU-GM, BFU-E and CFU-GEMM) at the end of 5 weeks to provide a relative measure of the number of so-called LTC-initiating cells (LTC-IC) present in the input suspension. These are edways seeded onto a pre-established irradiated normal marrow LTC adherent layer to ensure "optimal" stimulation of the input LTC-IC. Under these conditions, clonogenic cell output Is linearly related to marrow cell output down to limiting numbers of cells, so that at limiting dilution Poisson statistics can be used to derive absolute LTC-IC frequencies.  A unique feature of the LTC-IC assay over standard clonogenic assays is that it  detects and maintains a hemopoietic cell which is distinct from, and more primitive than, the majority if not all, cells detected In standard clonogenic cell assays. Thus initiation of cultures with highly purified populations of clonogenic cells seeded onto the same Irradiated feeder layers used to detect LTC-IC leads to a rapid decline in clonogenic cells.  This is the basis of the  model of hemopoiesis in the LTC system shown in Figure 2. Murine studies have also suggested that the cell responsible for the production of hemopoietic cells seen after 4 weeks (Identified as "cobblestone" areas in the cultures®^) share several characteristics with In vivo reconstituting cells,  some of which are also features of human LTC-IC (Table 2).  The frequency of LTC-IC in human marrow is 1 per lO'^ cells which is about the same as the frequency of CFU-GEMM, CFU-Blast. BL-CFC and HPP-CFC.  By Umltlng dilution  analysis. It was shown that LTC-IC have an average clonogenic ceU output at the 5-week time point of 4 clonogenic cells (CFU-GM plus BFU-E and occasionally CFU-GEMM) per LTC-IC and that under the conditions preveilling In these cultures, at least a quarter of LTC-IC can be shown to exhibit plurlpotentlal capacity,  ASSAY FOR LTC INITIATING CELLS  BM Cells  Dishes with ^ Pre-established \jj^l:A LTC Adh Layers , (+15 Gy) 0  Total Clonogenic Cells Per LTC (Adh + NA Cells)  33°C weekly 1/2 med. change  f  weeks in culture  FIGURE 1 LTC-IC ASSAY Low density marrow cells were suspended in 2.5 mL of LTC media and seeded on top of pre-established irradiated (15 Gy) normal marrow adherent layer. Cultures were maintained at 37°C for 3 to 4 days then switched to 33°C in 5% CO2. Weekty half-media changes were performed, as described in Chapter II. After 5 weeks, all nonadherent cells were removed and adherent cells suspended by trypsinlzation. Nonadherent and adherent cells were then plated separately in methylcellulose for assessment of tot£d clonogenic cell content of the LTC.  S  \  10^ 2L O  Total Clonogenic Progenitors  I  10'  Number of Clonogenic Progenitors per LTC-IC  4)  2 10^  0) E 3  10  0  1  2  3  4  5  6  7  -I 8  Weeks in Culture FIGURE 2 . Model of Hemopoiesis in LTC (From Dr. Heather J . Sutherland. PhD thesis, UBC, 1991) The total number of clonogenic cells present in LTC (represented by the dotted line) was postulated to be the result of the behavior of two separable types of cells. Directly clonogenic cells (dashed line) contribute to the majority of the clonogenic cells in these cultures during the initial 4 weeks of the culture. LTC-IC (solid line) are present in lower numbers but proliferate to produce clonogenic cells which are present in the cultures at >4 weeks after initiation. Measurement of the total number of LTC-IC at initiation (using limiting dilution analysis) and the total number of clonogenic cells in the cultures, allows calculation of the average number of clonogenic cells produced per LTC-IC.  A summary of the properties of primitive murine and human hemopoietic cells detected by the assays just described is given in Table 2 below.  TABLE 2 PROPERTIES OP PRIMITIVE HEMOPOIETIC STEM CELLS  Murine  Assays  5-FU-resistant Light buoyeint density High class 1 MHC Thy-llow  CRU. MRA, HPP-CFC 137CFU-S 13® CRU, CFU-S, RPA139'140 CRU, HPP-CFC CFU-S, CFU-Blastl37.139,141,142 RPA, CFU-S 140.142,143 RPA,CFU-Sl40.142.143 MRA, LTC-IC77.144 CFU-S, HPP-CFC 109.138 CFU-S. RPA, MRA, LTC-IC l^S-l^^'l^^S CFU-S® 1-13® CFU-S, MRA142,146  SCA-1-positive Lineage marker-negative Low Rh-123 uptake Low Hoechst 33342 dye uptake Low FSC Slow sedimentation rate c-kit Human  Assays  4-HC resistant Light density No or low HLA-DR CD34-posltlve  LTC-IC, BI^CFC, HPP-CFCll6.147,148 LTC-IC 134 LTC-IC. CFU-Blastl34.149-151 LTC-IC. CFU-Blast BL-CFC. HPP-CFCl34.150.152-155 LTC-IC 156 LTC-IC. HPP-CFC9'*'123.157 LTC-IC 15® LTC-IC. CFU-Blast. day 21 BI^CFCl21.134.157 CFU-Blast, LTC-IC123.134 CFU-Blast  CD45RO Isoform-posltive Lineage marker-negative LOWCD71 Low FSC and SSC Low CD33 Low CD38  1.3  ISOLATION OF PRIMITIVE HEMOPOIETIC STEM CELLS The recognized importance of hemopoietic stem cells for the reconstitution of  hemopoiesis after bone mcirrow transplantation and the need for pure populations to define early differentiation events in molecular terms accelerated research aimed at hemopoietic stem cell isolation more than 20 years ago. Considerable enrichment of various types of human progenitor cells in bone meirrow can now be achieved by positive or negative selection using monoclonal antibodies to cell surface molecules, or on the basis of other physical or biological properties including cell size, density, internal structure or sensitivity to various chemotherapeutlc agents. ^  A number of these, which have been particularly useful for  distinguishing subpopulations of primitive cells, are discussed in more details below.  1.3.1  CD34 The CD34 antigen is a 110 Kd phosphoglycoprotein that was originally purified from  leukemic cells.  -Yhis cell membrane component has an unusually restricted expression on  normal human hemopoietic progenitor cells but is also expressed on endothelial cells and their precursors.  It has been demonstrated on all multlpotent cells (CFU-Blast, BL-CFC, CFU-  GEMM, LTC-IC and HPP-CFC), and most bipotent and unlpotent colony-forming cells thus far examined, as well as on early B-lymphold precursors. 152,155,162-164 Recent transplantation studies in primates and humans Indicate that the cells capable of reconstituting lymphohemopolesls in myeloablated recipients and in SCID mice are CD34'''.  1^^ It Is  generally agreed that expression of CD34 is highest on the earliest cells and Is progressively lost as these cells mature. 159,167,168 L T C - I C appear to express a higher level of CD34 than their clonogenic progeny , l"^"* whereas BL-CFC day 5 fall In the low to medium CD34'*" range. 1^2 The human gene for CD34 Is located on chromosome 1.1®^ The CD34 cDNA sequence has been determined, predicting an approximately 40 Kd transmembrane pol)T)eptide. 1^^  j-jryc  function of  the CD34 molecule remains elusive, although Its structure has suggested a role In cell-cell adhesion Interactions .171,172  1.3.2  HLA-DR Human HLA-DR antigens are a group of histocompatibility antigens that closely resemble  the murine I-E antigen system. In mice, I-E antigens are not expressed on CFU-S, but are present on most CFU-GM and BFU-E.  humans, whether there is expression of HLA-DR  on primitive hemopoietic stem cells is still controversial, l'^^-151,153,174 ^^g^ clonogenic cells. Including CFU-GM, BFU-E, CFU-MK, CFU-GEMM and day 5 BL-CFC express detectable levels of HLA-DR. LTC-IC, CFU-Blast and HPP-CFC have been reported to express no or low levels of HLA-DR. 134,150  1.3.3  Rhodamine-123 Rhodamine-123 (Rh-123) is a cationic fluorescent dye that has relatively high aflflnity for  mitochondrial membranes and appears to be retained much better In cycling cells than in quiescent cells. 175-178 Recently this differential retention of Rh-123 In hemopoietic cells has been shown to be related to the level of expression of P-glycoproteln, the product of the multidrug resistance gene (MDR). 179 Studies in mice have demonstrated that Rh-123 can be used to separate subpopulations of CFU-S. 1®0 The Rh-123^"^ fraction is virtually depleted of CFU-S day 8 and CFU-S day 12 but contains the majority of cells that generate CFU-S day 12 (i.e., MRA) and cells that protect otherwise lethally-irradiated recipients. Conversely, Rh-123^^^611* cells are enriched for CFU-S day 8 and CFU-S day 12 but depleted in MRA, RPA, and ceUs with an ability to generate new cells that can rescue recipients from radiation-inflicted death.77.144,181 Recent research has also shown that the more mature HPP-CFC can be partiaUy resolved from the primitive HPP-CFC on the basis of thefr Rh-123 uptake. 1^4 Prior to the studies reported in Chapter III, the ability of human ceUs to take up or exclude Rh-123 had not been investigated, nor were the mechanisms for the selective staining of mitochondria wellestablished.  1.3.4  Light Scattering Properties Studies of the forward light scatter (FSC) and side scatter (SSC) characteristics of human  marrow or blood cells indicated that these parameters would be useful to distinguish major subpopulations.  ^'^5,182,183 since every cell gives a light scatter signal, whereas many  cells may give no fluorescence signed after labelling with fluorescent antibody molecules, the detection of light scatter can be used to trigger the FACS to specify whether ceUs should be sorted or not. This can be accomplished simultaneously with the determination of the amoimt of fluorescence from the bound conjugated antibodies or from supravital dyes.  CD34"'' ceUs  in marrow are found to have a broad range of FSC properties, but are of low SSC. Primitive hemopoietic cells including human LTC-IC and day 21 BL-CFC have been reported to exhibit low to medium FSC and low SSC.  The majority of clonogenic cells in human marrow, however,  are found to have medium to high FSC.  A number of other monoclonal antibodies have been successfully used In combination with anti-CD34 antibodies to enrich for primitive cells. Robertson et al. reported that CD33, another myeloid differentiation antigen is expressed at very low or neglegible level on transplantable cells.  LTC-IC and CFU-Blast are also CD34+CD33', whfle virtually aU CFU-  GM and day 5 BL-CFC are CD33"'". 1^3,186 Recentiy, a monoclonal antibody against the receptor for Steel factor (c-kit), has been used to stain normal marrow cells.  1^^, 188  ^.Q  80% of 0034"*" cells express c-kit, including most committed progenitors. However, the expression of c-klt on more primitive human hemopoietic cells has not yet been reported.  1.3.5  4-hydroperoxycyclophosphamide (4-HC) Sensitivity 4-HC Is a derivative of cyclophosphamide that exhibits In vitro chemical and biological  properties simflar to those of mlcrosomaUy activated cyclophosphamide, l^®-  j^^ ^^s shown  that 4-HC can be used in autologous bone marrow trernsplantations to eliminate leukemic cells  without affecting hemopoietic stem cell reconstituting ability. 191-193 in vitro studies also demonstrated that 4-HC has a differential effect on subpopulations of hemopoietic cells; the more mature progenitors, including BFU-E, CFU-GM, and CFU-GEMM being more sensitive to 4-HC than LTC-IC and BL-CFC. 147,148,194,195  1.4  REGULATION OF NORMAL HEMOPOIETIC STEM CELLS  The available data favor both the existence of a continuum of hemopoietic stem cells, changing gradually In self-renewal and differentiation potential, and the existence of a concatenated series of hemopoietic stem cells with distinct properties. Various studies have provided support for the notion that the proportion of cycling cells Increases with maturation In the hemopoietic stem cell/progenitor compartments and that the potential for self-renewal may concomitantly decrease. 196  Hemopoietic stem cells Eire thought to have the following key decisions or options (Figure 3). These are 1) to remain alive or die, 2) to move from a quiescence (GQ) phase into an active cycling state or vice versa, 3) to maintain or become restricted with respect to proliferative capacity, 4) to maintain or become further restricted in differentiative potential.  o  Self - renewal  Determination  Functional End Cells  FIGURE 3 Options for Hemopoietic Stem CeUs  1.4.1  Regulation of CeU Viability Cell death plays a critical role in normal tissue growth and development.  Steady-state  levels of mature hemopoietic cells can be actively regulated by the availability of growth factors that are essential for their survival. The removal of growth factors from dependent cells does not always result simply in the arrest of their groAvth, but can in some cases lead to the rapid initiation of a cellular program of self-destruction or apoptosls.  Apoptosls is an active  process characterized by initiation of gene expression and protein synthesis, and by chromatin condensation and DNA fragmentation. Many anti-cancer agents are believed to operate through the activation of an apoptotlc process.  Recently, a novel proto-oncogene, BCL-2 discovered  at the chromosomal breakpoint of the t{14; 18) found in human follicular lymphoma has been demonstrated to have a unique function in blocking apoptotlc cell death In hemopoietic ceU lines following growth factor deprivation, implicating the product of BCI^2 as a component of the molecular processes that decide whether a cell lives or dies.^00,201 B C L - 2 IS edso expressed In CD34"''CD33"HLA-DR' hemopoietic cells In normal marrow where It presumably contributes to their long life span and resistance to external Insults.^^^  1.4.2  Regulation of Population Timiover In normal, steady state human hemopoiesis, It Is generally believed that the great  majority of the hemopoietic stem cells are dormant, l.e. In a non-cycling, GQ state and that they serve largely as a reserve from which the system may be replenished If Injured or depleted.^03-205 jj^g concept came from a number of studies including measurements of the ^H-thymldine suicide of CFU-S, CFU-GEMM, BFU-E and CFU-GM, analysis of the sensitivity of CFU-S to high dose 5-FU, and serial observations (mapping studies) of the formation of multipotential CFU-Blast-derlved colonies in culture.69'100'206.207 -pj^g transition of hemopoietic cells from a non-cycling to cycling state is thought to be reversible and to alter In vivo in accordance with physiologic requirements for hemopoiesis. 101.204 Such a model predicts that at any given time only a small number of stem cells will be contributing new cells  Into the entire lymphohemopoletic system.20®'209 j h i g situation can be readily demonstrated after transplantation of cells bearing markers or unique retroviral Integration fragments where the sequential activation of different stem cell clones may be seen.62.65,74,210-212 some studies have also shown that more clones are seen ezirller after bone marrow transplantation than are seen later.® 1-213,214  A number of humoral factors appear to be capable In regulating hemopoietic stem cell proliferation. For example. It was suggested that 11^3 and G M - C S F do not trigger quiescent stem cells into a cycling state but may be able to support their continued proliferation once they leave Gg. 1^0 o n the other hand, IL-1, IL-6 and G-CSF appear as synergistic factors for IL-3dependent proliferation of CFU-Blast since their addition to cultures of CFU-Blast can significantly shorten the average length of the GQ period, perhaps by inducing/increasing expression of receptors for molecules such as IL-3.102,215 inhibition of proliferation has cdso been described and several inhibitors of primitive hemopoietic cell cycling have been identlfled.216 For example, transforming growth factor-p (TGF-P) has been shown to have a reversible and specific inhibitory effect on the cycling of primitive hemopoietic cells In the adherent layer of LTC. 1® 1 The growth of all HPP-CFC colonies is also Inhibited by TGF-p in a dose-dependent manner.217 Recently, it has been shown that the inhibitory effect of TGF-p is linked to its ability to prevent Inactlvatlon of the product of the retinoblastoma susceptibility gene, RB, an anti-oncogene. RB acts by binding and sequestering treinscriptlon factors needed for the expression of genes required for Gj/S progression.21®'219 Addition of exogenous TGF-p to the cultures completely inhibits colony formation by CD34''" marrow cells, and this inhibition is partially reversed by the addition of antlsense RB oligonucleotides.220 inactivation of RB protein through phosphorylation may be a key step in allowing cells to cross G j / S botmdary. TGF-P may play an Important role by arresting cells with RB in the underphosphorylated form. p53, another tumor suppressor gene, causes growth arrest of cells in Gj,221 suggesting that multiple regulatory molecules may act together to control normal ceU proliferation. 222 'phg stem  cell Inhibitor, macrophage Inflgmimatory protein-la (MlP-la) Is another recently described Inhibitor with similar effects and specificity for primitive hemopoietic cells as TGF-p.223,224 Increased production of, or sensitivity to, growth factors normally required for progenitor cell stimulation would be expected to lead to an excess proliferation of these cells and hence an abnormal expansion of the whole hemopoietic system. Similarly, reduced production of, or decreased sensitivity or unresponsiveness to, inhibitors would be expected to jTleld similar results. An appropriate balance between the stimulators and Inhibitors to which primitive hemopoietic cells are exposed could thus be of major Importance to the control of normal hemopoietic cell production.225  1.4.3  SELF-RENEWAL AND DIFFERENTIATION Self-renewal and differentiation are considered to be closely linked and opposing  processes.2^6 Self-renewal, by definition. Is a process by which cell division leads to the production of two daughter cells, at least one of which retains the phenotype and differentiation potentialities of the parent cell. However, some of the daughter cells generated by divisions of stem cells must differentiate to supply cells Into the mature cell pool. On the other hand. If all daughter cells differentiated, there would be no more stem cells and after a certain period further production of mature cells would cease. In general, self-renewal of primitive hemopoietic cells has been demonstrated by the ability of single cells present In primary colonies to give rise to secondary colonies of slmfiar characteristics (size and/or multillneage composition).^^^'228 Analysis of Individual colonies has shown that there exists marked heterogeneity In the selfrenewed behavior of plurlpotent highly proliferative cells even when these are stimulated to divide under apparentiy Identical condltions.^^'-^^^'^^O  Till and colleagues proposed a model to describe this stem cell proliferative behavior based on probabilistic (stochastic) prlnclples.^^^ In this model, self-renewal and differentiation are considered to be mutually exclusive. Stem cells are thus assumed to have an Intrinslceilly  determined probability p of self-renewal, and 1-p of differentiation (with an accompanying loss of self-renewal capacity). According to this model the final choice to self-renew or differentiate at the single cell level is determined by a mechanism that involves a random component. If it is assumed that conditions extrinsic to the cell can be Identical, then this random component would appear to be intrinsic to the stem cell, itself. Such a model, however, does not preclude the possibility that the value of p may also be regulated by humoral factors extrinsic to the cell. 231 An alternative model was proposed by Curry, Trentin and Wolf.232 This model, emphasized the role of deterministic factors in the hemopoietic inductive microenvironment (HIM), in contrast to the model of Till et al. which hypothesized a random component (hemopoiesis engendered at random, HER). The HIM model proposed the existence of instructive extrinsic factors either of a humored or fixed microenvironmental nature that determined the specific pathway along which a stem cell would be forced to differentiate. A prediction of the HIM model is that stimulation of stem cells to differentiate down one pathway will, in a competitive fashion, decrease stem cell differentiation down alternate pathways. As is often the case, these models are not mutually exclusive and await Investigation at the molecular level. However, the observation that c-FMS (CSF-l receptor)-transfected pre-B cell lines can "llneage-swltch" to become functional macrophages upon transfer into CSF-1 medium that preferentially supports the growth of myeloid cells, suggests that the C S F - l receptor may be capable of transducing a deterministic signal imder certain circumstances.^33  2. CHRONIC MYELOID UBUKEMIA (CML)  2.1  CML AS A CLINICAIXY DEFINED ENTITY  2.1.1  Diagnosis and Classification of Disease Stages CML is a myeloproliferative disorder characterized by an overproduction of granuloc5rtes  and monocytes. Other myeloproliferative diseases (MPD) Include polycythemia vera (PV), essential thromboc5i:osis (ET), and agnogenic myeloid metaplasia (AMM). The latter Is also known as myelofibrosis with myeloid metaplasia, or myelofibrosis with extramedullaiy hemopoiesis. All four diseases are characterized by an overproduction of at least one type of mature blood cells: gremulocytes and monocytes in CML, red blood cells In PV, and platelets in ETT, although often overproduction of more than one lineage occurs. 234-236  CML is the first and best defined among the four MPD. It was first recognized in 1845 independently by Craigie 237 gnd Bennett 238  Scotland and Virchow in Germany.239 Both  reports describe the classical features of patients with splenic enlargement, severe anemia, and "suppuration of the blood". However, It was not until 1924 that the unique natural history of this disease was described.240 c M L accounts for about 20% of all adult leukemias, with an annual incidence of approximately 1 case per 100,000 people. This incidence appears to be constant worldwide. 241 The incidence of CML is strongly age-related (Figure 4). It develops almost exclusively in adults, the median age being about 60 years with a slight male predominance (male:female ratio 1.4:1).242  30  (0 c 0) (0 Q. o^  20 H  ioH  o T—  I—  V  1 o T-  CM 1 o CM  o> CO 1 o CO  1 o  1 o in  O)  O)  o  o  o CO A  FIGURE 4. Incidence of CML By Age Reported in Canada in 1983 (From Statistics Canada, Ottawa, cat 82-207)  age (yr)  Patients with CML frequently present with complaints relating to fatigue, weight loss, night sweats and abdominal fullness. The symptoms Eire vague, nonspecific, gradual in onset and generally lead to a physical examination that may reveal pallor and splenomegaly, the latter being present in about 90% of patients at diagnosls.243  ^ proportion of patients, the disease  Is discovered accidentally, during a routine blood examination by the presence of a diagnostic profusion of granulocytic elements. The total blood leukocyte covmt is often elevated at the time of diagnosis and is usually near 10^ ^/L. Granulocytes and thefr morphologically recognizable precursors at all stages of development are the dominant population in the blood and are generally normal In appearance. Some patients may have an Increase in the number of eosinophils or basophils in the blood. A third or more of patients also have a pronoimced thrombocytosis. Examination of the bone marrow usually demonstrates a marked hypercellularlty (2- to 5- fold) with an abnormal granulocytic to erythroid ratio of 10:1 to 50:1, in contrast to the normal ratio of 2:1 to 5:1. The eimount of leukocyte alkaline phosphatase present In mature granuloc3^es Is strikingly decreased, which distinguishes CML from other MPD or diseases associated with a leukemoid reaction.244,245 Qn average, after 3.5 years, the disease progresses from an indolent chronic phase to an acute phase (blast crisis). This is generally characterized by a worsening of laboratory and cUnlcal signs and the appearance of increasing numbers of blast cells in the bone marrow and blood (see Table 3). In some cases the onset of blast crisis is preceded by a recognizable transition phase referred to as an accelerated phase.246  TABLE 3 CRITERIA FOR CLASSIFYING THE THREE PHASES OF CML Chronic Phase No significant sjnnptoms (after treatment) None of the features of accelerated phase or blastlc phase Accelerated Phase WBC count difficult to control with conventional use of Busulfan or Hydroxyurea in terms of doses required or Shortening of intervals between courses Rapid doubling of WBC (<5 days) ^10% blasts in blood or marrow >20% blasts plus promyeloc5d;es in blood or marrow >20% basophils plus eosinophils in blood Anemia or thrombocytopenia unresponsive to Busulfan or Hydrojqrurea Persistent thromboc)^osis Additional chromosome changes Increasing splenomegaly Development of chloromas or myelofibrosis Blastlc Phase >30% blasts plus promyelocytes in the blood or marrow  Patients in blast crisis usually die from marrow failure within 1 to 5 months.^'*'' Thus, CML epitomizes the progressive and multistep evolution now believed to occur in most cancers but is less well-defined in other types of tumors. ^'^^^"^^^  2.1.2  Therapy of CML fTable 4) Despite the first description of CML almost 150 years ago, neither the course of the  disease nor survival of CML patients changed until recently when bone marrow transplants [BMT) were introduced.254,255  jj^^g beginning of the century, irradiation was first Introduced  as a treatment modality. It was given to the whole body, spleen, bones, tumor masses, or in the form of radioactive phosphorus. Irradiation basically improved quality of life by reducing the white blood cell counts and spleen/tumor masses. However, most patients died of the related complications of the disease or radlatlon.^^^  In 1953, busulfan, a sulfonic acid alkylating agent, was first used as an oral drug In the treatment of CML patients.  offered better disease control and longer survival than  Irradiation. The median survival ranged firom 30-42 months with the use of busulfan, as compared to 6-32 months with Irradiation. ^58  patients now die in the chronic phase,  unmasking the natural course of CML. On average, patients will transform to a blastic phase within 3-4 years despite busulfan treatment. Busulfan is believed to affect early progenitors as indicated by the time that elapses before the white blood cells begin to decline. It also has many serious side effects, which stimulated attempts to find alternative drugs. In 1960, hydrojqoirea was introduced. It is an inhibitor of DNA S5^thesis and is thus specific for cycling cells. ^59 Although hydroxyurea has fewer side effects, it still does not offer better overall survival times than busulfan.260  The only demonstrated curative treatment of CML to date Is Intensive therapy supported by transplantation of bone marrow either from identical twins (syngeneic) ^61 or HLA-identical siblings (allogeneic).^62-266  jf allogeneic transplantation is carried out in chronic phase,  disease-free survival can be achieved in 40-80% of the patients, in contrast to 15% in patients treated after blastic phase has occurred.247.267,268 However, this treatment is of limited applicability due to the fact that only 25% of CML patients have HLA-matched siblings. Other alternatives currentiy under investigation eire the use of unrelated, but HLA-matched donor 269 and the use of purged autologous marrow transplants. 2^0-272  Recentiy, Interferon (IFN) has also been used In the treatment of CML patients since it is known to have anti-proliferative and differentiation-inducing activity.273-275 Therapy with IFNa has been most extensively studied.276-279 Talpaz et al showed 70% of IFNa-treated CML patients to achieve complete hematologic remission with a decrease of Ph^-positive cells to a very low percentage In many cases.276 unllke the responses observed with Intensive chemotherapy or BMT, the IFN-a-lnduced cytogenetic responses do not require profound  myelosuppression. The mechanism by which IFN exerts Its selective effect on Ph^-positive cells Is, however, still not established.  TABLE 4 Landmarks in tlie treatment of CML 1902 1930 1952 1960 1979 1981 1983 1985 1986  Radium Therapy Splenic Irradiation Busulfan Hydroxyurea Syngeneic bone marrow transplantation Autologous transplants with peripheral blood Allogeneic BMT Avlth HLA-ldentlcal siblings* a-lnterferon Unrelated donor transplants Autologous BMT  * First curative treatment  2.2  CYTOGENETIC AND MOLECULAR CORRELATES  2.2.1  The Pfailadelpiiia Cliromosome (Pli^) (Figure 5) CML was the first human neoplasm to be associated with a consistent chromosomal  abnormality, the Philadelphia (Ph^) chromosome. This abnormal chromosome was first described In 1960 by Nowell and Hungerford as a minute chromosome present In the dividing cells of many CML patlents.^^^ i^ter. In 1973, when banding techniques were developed, this minute chromosome known to belong to the G-group of chromosomes, was shown to be different from the extra-chromosome present In patients with Down's S5nidrome.^^^'^®^ Since the latter had been assigned number 21, the Ph^ chromosome became number 22.^81 Further cytogenetic emalysls demonstrated that the Ph^ chromosome usually results from a reciprocal translocation of cjrtogenetlc material on chromosome 9 distal to band q34.1 and on chromosome 22 distal to band q l 1.21. This unique translocation Is thus designated t(9;22)(q34.1 l ; q l 1.21). It Is considered the hallmark of CML since over 90% of cases with CML have been shown to have this translocation In their cells  and It Is rsirely observed In any other type of disease. About  5% of cases have variant forms of the Ph^ chromosome involving at least one additional chromosome.285  such instances, the third chromosome Is the recipient of the deleted part of  22q-, with 9 being the recipient of the deleted part of the third chromosome.286 A masked Ph^ chromosome occurs when there is an exchange of cytogenetic material involving chromosome 22q without the formation of a typical Ph^ chromosome.287 Analysis of these rare variant translocations were instrumental in providing the first evidence that it was the translocation of a gene on chromosome 9q to a specific locus on chromosome 22q (rather than the opposite) that was important to the pathogenesis of CML.252,288  ABL m-8CR  BCR  M-8CR n u l l 1 2 3 45  •  CML • • • •  II • 1 2 3  FIGURE 5. Cytogenetic and Molecular AbnormaUties in CML  The evidence that CML results from the transformation of a single plurlpotent stem ceU whose progeny eventually replace normal hemopoiesis comes primarily from cjdiogenetic and biochemical studies. In females who are G6PD hétérozygotes, monoclonality of the leukemic cells has been shown by demonstration of only one of these enzymes In aU of the blood or marrow cells, even though other somatic tissues in the same individual. Including resldued normal hemopoietic cells, are polyclonal; i.e. contain equal numbers of cells expressing one or the other alloenzymes.^^^ In almost all cases of CML examined, the presence of the Ph^ chromosome and/or a monoclonal pattern of G6PD Isoenzyme e3q)ression (in heterozygous individuals) has been identified in neutrophils ,^90 monocytes and macrophages ,^91 erythrocytes,292.293 megakaryocytes ,294,295 eosinophlls,^2'296 basophfis ^97 QJ. committed progenitors of each of these lineages and some B-cells.^^'298 Evidence that the clone arises in a very primitive hemopoietic cell with lymphopoietic potential was actually suggested even earlier by the demonstration of transformed subclones of cells with pre-B cell characteristics in a proportion of patients with blast crisis.  Involvement of T cells In the neoplastic clone in  CML remains controversial. Peripheral blood T cells that are stimulated to proliferate by lectins have been reported to remain Ph-^-negative and polyclonal by G6PD analysis. ^^0 However, a few cases of CML in blast phase have been reported where blasts with a T cell phenotype have been found.^^1-303 jj^ addition a case of T cell leukemia In which a Ph^ chromosome appeared as a secondary event has been described,^04g^ggggyj^g ^j^g^^ ^j^g underlying genetic abnormedity is not incompatible with T cell development. Tlie Ph^ chromosome Is not present in bone marrow fibroblasts and other mesenchymal tissues.^05,306 Recent application of DNA-based strategies for examination of the methylation status of certain X-llnked genes in women with CML who are heterozygous for an Informative restriction fragment length polymorphism (RFLP) at these loci have confirmed the single cell origin of the leukemic cells in patients with CML.307,308  2.2.2  The BCR-ABL Fusion Gene In 1982, Helsterkamp and co-workers, by analysis of mouse-human somatic cell hybrids,  determined that c-ABL, the normal human homolog of the transforming gene (v-ABL) of the Abelson murine leukemia virus (A-MuLV)®^^ was located on chromosome 9 at band q34.®10 The translocation of this cellular proto-oncogene In patients with Ph^-positive CML was then demonstrated simultaneously by de Klein® ^ ^ and GroflFen et al.®!^  Molecular investigation of the site of the breakpoints on chromosome 22 demonstrated that these are confined to a small region of 5.8 Kb referred to as M-BCR for major breakpoint cluster region.®12,313 jj^g j-egion was subsequently shown to be part of a gene that spans ~90 kb and is now called BCR-1  as several other related genes have since been identified.®  The BCR-1 gene has been cloned and found to express a 4.5 and 6.7 Kb mRNA (Reviewed in Grofifen et al ®16) Two proteins of 130 and 160 kD are encoded by this gene and these are widely expressed in normal tissues. Recent studies indicate that the product of the BCR-1 gene has GTPase activity®  and Is also a serine-threonine kinase.® 1® In most patients with CML,  the breakpoint within the BCR-1 gene generally occurs either between exons 2 and 3 or between exons 3 and 4.®  ^11 exons of BCR-1 that are distal to the breakpoint are typically  translocated to chromosome 9, the proximal 5' exons remaining on chromosome 22. Tliis exchange of genetic material results in the Juxtaposition of 5' BCR-1 sequences and 3' c-ABL sequences (exons 2 to 11) in a head-to-tail fashion.® ^9,320 This occurs also in patients with variant Ph^ translocations and in most patients considered clinically to have CML but whose leukemic cells do not contain a cytogeneticsdly detectable Phl.®^!  Extensive characterization of the c-ABL gene has revealed this gene to be highly conserved throughout evolutlon,®^^ being present not only in mammals, but also In other vertebrates and Drosophila, suggesting a major role In basic cellular processes. The human cABL gene contains 12 exons,®2®'®24 nine of which show dispersed v-abl homologous sequences  within the gene.^^^ Upstream of the second exon are two additional exons (la and lb).^25 j^i^ alternative use of these first exons results in the transcription of two mRNA messages of 6 and 7 kb."^!^ The 7-kb transcript differs firom the 6-kb transcript in that the former begins with exon l b and omits a 200-kb distance to exon 2 whfie the latter consists of exon l a through 11. The translated products of these two mRNA species correspond to similar c-ABL encoded proteins in mice ,316 both of which have a mass of 145 kD and an intrinsic tyrosine kinase activity.^^6 The latter suggests a normal role in the phosphorylation of Important substrates within the cell possibly as part of a signal transduction mechanlsm.327,328  The fusion of 5' BCR-1 sequences on chromosome 22 with 3' c-ABL sequences from chromosome 9 creates a new chimeric gene that Is transcribed and translated In the leukemic cells. The BCR-ABL mRNA produced can be uniquely detected by its size of 8.5 Kb329  ^jig  new fusion protein It encodes (p210) 330-331 is similarly distinguished by Its size and possession of a higher tyrosine kinase activity than its normal counterpart, p l 4 5 c-abl 332,333 In mice, the two protein products of the c-ABL gene cire referred to as Type I and. Type IV.334 Type I is located on the irmer surface of the plasma membrane and in the cytoplasm. Type IV may also be found in the nucleus. In contrast, the transforming 160-Kd fusion protein (pi60 gag-ablj  A-MuLV is predominantly cytoplasmic or membrane-associated. The transforming  potential of the type W c-ABL-encoded protein can be activated by deletion of its small Ntermlnal regulatory region.335 since this results in its redistribution from nucleus to cytoplasm it has been concluded that the locêdization of c-ABL within the cell is Important to its transforming potential.336 Kipreos and Wang337 recently showed that c-ABL contained a high affinity DNA-binding domain. This activity was found to be abolished in the p210^^1^"^l' fusion protein, consistent with its cj^oplasmlc localization.338  The possible consequences of BCR-ABL gene expression have recently been Investigated both In vitro and in vivo in animal models. Unlike the transforming protein encoded by v-ABL,  p210^^^'-^L  not able to transform N1H/3T3 cells unless the myristylatlon site for  membrane attachment was provided by the additional Introduction of viral gag sequences. However, p 2 1 0 ^ ^ ^ - ^ ^ can also transform IL-3-dependent hemopoietic cell Unes to factor independence and tumorigenicity.^^O HéiriharEin et al. showed that infection of cells with p 2 1 0 ^ ^ ^ - ' ^ ^ also induced a low level of IL-3 expression, as had been previously shown for vABL.®41,342 Initial attempts to recreate the generation of CML experimentally Involved infecting long-term murine bone marrow cultures with a BCR-ABL retrovirus. However, these universally yielded transformed pre-B cell llnes®43 gven when culture conditions that favor myeloid cell growth and are non-permissive for normal pre-B cells were used.®44 Recently Daley et al,®45 Elefanty®46 and KelUher et £d®47 separately demonstrated similar results of the ability of the p2ld^^^'^^  to induce a CMI^llke disease in mice, when the target population was  presumably enriched in the appropriate hemopoietic cell type using 5-FU treatment. Transgenic mice carrying a BCR-ABL constructors have also been shown to develop a CML-Uke disease upon trcinsplEintation of their marrow into irradiated recipients.®49  To dissect the molecular mechanisms underlying the transforming activity of BCR-ABL, the essential biochemiced and functional properties of the product of this gene continue to be intensively investigated. The c-ABL proto-oncogene is now known to be a member of the nonreceptor class of SRC-like tyrosine kinases.^^O C-ABL shares homology with C-SRC not only in its possession of a tyrosine kinase domain but edso in two other regions 5' to the kinase domain. These two domains, SH2 (src homology region 2) and SH3 (src homology region 3) are also present in other signalling molecules.^^S The SH2 region is believed to play a major role in serving as a binding site for phosphorylated tyrosine residues, thereby mediating tramslent protein-protein Interactions with substrates of other tyrosine kinases, which may include some of the growth factor receptors or tyrosine kinases that interact with growth factor receptors. ®51 Interestingly, the first exon of BCR-1 has also recently been found to contain at least 2 SH2binding sites and p2ld^^^'^^  binds specifically to the SH2 domain of the c-ABL product,  suggesting a role of the BCR in the activation o{p2l6^^^'^^.^^^ Activation of c-ABL can also occur by deletion of sequences encoding the SH3 domain, which are also deleted In v-ABL.^O^ Mutation in the SH3 domain coupled with mjrlstylated membrane localization sequences is sufiiclent to activate tyrosine phosphorylation and transformation by c-ABL.335,353,354 However, BCR-ABL Includes an intact SH3 domain and the encoded protein does not have the myrlstylatlon consensus sequence.3^5,356 Qn the other hand, Muller et al. have proposed that the first exon of BCR may substitute for the effects of both the myrlstylatlon site and the effects of deleting the SH3 domain by specifically Interfering with the negative regulation of ABL normally mediated by interaction of the SH3 domain with an as yet tmdefined host factor. ^57 EMdence of a candidate inhibitory activity that binds noncovalently to pMB^'-^^^ to inhibit its autophosphorylatlon has recently been pubUshed.^^S Thus several factors may be Involved In, or contribute to the activation of an ABL-based transforming activlly.  2.2.3  Is the Creation of the BCR/ABL Fusion Gene the Initiating Event in CML? Although the BCR-ABL translocation Is a consistent phenomenon in 98% of CML  patlents,359 ^ jg g^jn uncertain as to whether the creation of this fusion gene is the primary event In the development of the neoplastic clone seen in CML patients. For example, it is possible that this chromosome abnormality could occur in a pre-formed (abnormally expanded) Ph^-negative clone. Even though most CML patients do not have a history of exposure to radiation, an increased incidence of CML has been found in survivors of the atomic bomb explosions in 1945 and in anlqrloslng spondylitis patients receiving radiation therapy. ^^4 other available evidence indicating that CML is an acquired disease comes from the demonstration that CML does not affect both members of identical twin pairs, nor do the offspring of mothers with CML subsequently develop disease. ^^4 j^. gggms likely that the "causes" of CML may be diverse even though aU lead to the same crucial type of mutation and the production of a BCR/ABL fusion protein. The evidence that has been cited in support of the idea that the acquisition of the Ph^ chromosome may not necessarily be the primary event has come from  studies of patients, who appeared to have the disease before Ph^-positive metaphases could be detected in their tissues 360-362 g^^d from studies revealing a skewed distribution of allotypes amongst Ph^-negative EBV-transformed pre-B cell lines established from G6PD heterozygous females with CML.249 However, definitive studies in all of these cases are lacking, so that alternative, less Interesting explanations based on differential sampling cannot yet be ruled out.  It should be noted that a number of additional non-random and random chromosomal abnormalities are also seen in -10-20% of CML patients diagnosed in chronic phase.363 fhe most common of these are duplication of the Ph-^ chromosome or presence of an isochromosome 17q, trisomy 8 or trisomy 19. The isochromosome 17 is highly predictive of £in Impending transformation and is virtually never found as a stable abnormality In chronic phase disease. Interestingly p53, which is located on chromosome 17, has specifically been found to frequently exhibit deletions and point mutations in leukemic cells from some CML patients with blast phase disease. 364,365  mutations have also been demonstrated In some CML patients, but  these have proven to be comparatively rare.366 Logs of the RB gene product in a few CML patients in megakaryoblastic crisis was also recently reported.367 n has therefore been suggested that a series of secondary mutations arise In BCR-ABL-positive chronic phase cells because they are less genetically stable than normal cells.  2.3  CELLULAR STUDIES  To have a complete understanding of the pathogenesis of CML, it is important to clearly define not only abnormalities at the genetic level, but also to establish the biological consequences of such changes in terms of abnormalities in the behavior of Ph^-positive cells at different stages of differentiation. In patients with CML during chronic phase, the proportion of mature cells that carry the Ph^-chromosome typically increases progressively over time until  they account for more than 99% of all dividing bone marrow and circulating blood cells.^^S Nevertheless, none of the differentiation processes involved in the production of any of the variety of mature blood cell types seem to be perturbed.369 There are subtle abnormalities of granulocyte and platelet function, and often an increase in platelet size, but these rarely lead to symptomatic c o m p l i c a t i o n s . I n the chronic phase, there is little evidence of tissue Invasion by leukemic cells, and extramedullary hemopoiesis Is not common when the tumor burden Is not excessive. However, the Ph^-positive cells must have a growth advantage over normal bone marrow progenitors, as shown by the general failure to detect mature normal cells In patients with CML, even though very primitive normal cells can be readily demonstrated In many lnstances.370-372 Analysis of terminally differentiating cells In the marrow of CML patients was the first approach to determining the extent of clonal Involvement. This provided Initial evidence for Ph^-positive cells In all of the myeloid llneages.369 Subsequentiy, as fast as assays for different types of committed progenitors became available, these were also used to examine the number of different types of earlier cells and their derlvation.20,372,373 such studies showed that in most instances, the frequency of clonogenic cells In CML bone marrow (relative to all nucleated cells present) was not markedly Increased for any particular lineage of progenitor, although all were usually Ph^-positive (Reviewed In Eaves et ai^^^). However, Increased numbers of all types of clonogenic progenitors (CFU-GM, CFU-E, BFU-E, CFU-MK, CFU-GEMM, CFU-Blast and BL-CFC) have been found In the blood of patients with elevated WBC counts.369,374 since the total volume of bone marrow In an aspirate Is not possible to quantitate, absolute numbers of of Ph^-positive progenitors cannot be determined. However, due to the overall Increased cellularlty In CML marrow. It Is likely that the absolute number of Ph^-positive clonogenic cells (as compared to normal) Is also Increased. The reason for the selective expansion of grsmulocytes In CML patients (with occasionally Increased monocytes and platelets but usually decreased red cell production) In spite of the generalized amplification of all myeloid lineages at the progenitor level. Is not known. However, different mechanisms appear to  control the terminal differentiation of granulocytes, erythrocytes and platelets and these may be differently perturbed by BCR/ABL expression in different cell types.  2.3.1 The Persistence of Normal Stem Cells in CML Although Ph^-negative dividing cells are rarely detected In the blood and bone marrow of CML patients, it is now widely recognized that such cells often do persist long past diagnosis. It was In 1971 when Chervenick and colleagues first demonstrated the presence of Ph^-negative clonogenic cells in some Ph-^-positive CML patients.^^S They suggested that eradication of the Ph^-positive leukemic clone with chemotherapy, and reconstitution with residual normal stem cells might be possible. Subsequent studies 2 4 5 , 3 7 6 - 3 8 2 showed that temporary reduction of the proportion of Ph^-positive marrow metaphases could be achieved by administration of intensive chemotherapy, although Ph-^-positive disease usuédly recurred within a few years and the treatments had an unacceptable risk of mortality. These results did, however, indicate that Ph^-negative stem cells persisted In some patients and could presumably regenerate hemopoiesis if the Ph^-positive clone were eliminated. In agreement with this concept, Dube et al., also demonstrated that Ph^-negative clonogenic cells could be found even i n untreated CML patients at diagnosis, despite the absence of Ph^-negative metaphases in direct marrow or blood preparations.®®®'®®^  Since, around this time, the LTC system for human hemopoiesis had Just been established and seemed to support the generation of clonogenic cells for periods of severed months, it was immediately applied to the investigation of CML. In 1 9 8 3 , Coulombel et al. described results from a series of LTC initiated with bone marrow from a number of CML cases.®70 A consistent observation was that despite the absence of detectable Ph^-negative metaphases or committed progenitors in the CML bone marrow inocula used to initiate the LTC's, Ph^-negative clonogenic cells became demonstrable over a period of severed weeks. Conversely, Ph^-positive clonogenic cells rapidly disappeared, usually after 3 - 4 weeks. However,  these studies did not reveal why Ph^-positive cells should appear to have a growth advantage In vivo and the Ph^-negative cells In vitro. More recently, the use of interferon for the treatment of chronic phase CML patients has shown that complete remission (with complete loss of Ph^positive cells and transition from a clonal to a polyclonal population of hemopoietic cells) can occur.-^^S These results taken together strongly indicate that the Ph^-negatlve (presumably normal) cells persist In the primitive hemopoietic cell compartment of many CML patients even after diagnosis.  A question raised by these findings was whether the Ph^-negative cells seen derive from normal (non-clonal) stem cells, or whether they represent pre-leukemic cells that have not yet acqufred the Ph^-chromosome. Singer et al. (1980) first reported a case of Ph^-posltive CML with a monoclonal pattern of G6PD isoenzyme expression at diagnosis whose marrow cells became Ph^-negative and polyclonal after intensive chemotherapy, suggesting that the Ph^negatlve cells represented the regrowth of residual normal hemopoietic cells.^^S Supporting evidence came from cytogenetic studies of a mosaic Turner syndrome patient (46,XX/45,X) with CML whose Ph ^-positive clone In bone meirrow was documented to originate from a cell of the 45,X lineage, whereas the Ph^-negative clone was originated from the normal 46,XX llneage.^S^ Similar studies of clonogenic cells detected In LTC undergoing a switch to Ph^-negative hemopoiesis also revealed these to be polyclonal.^^1.386,387 Hemopoiesis after IFN treatment has also recently been shown to be polyclonal In some patients as detected by RFLP analysls.388  2.3.2  Regulation of Hemopoiesis In CML  Attempts to further understand the cellular events In CML would not be possible without the understanding of the biological similarities and differences between normal and leukemic stem cells. However, there is very llttie data regarding what happens to the most primitive  hemopoietic cells, since there has been no assay for their quantitation. This section will, therefore, mostly summarize what is known of CML biology at the level of committed progenitors and CFU-GEMM. I have divided this section into two parts: ceU division and cell adhesion.  2.3.2.1a CeU Division Prior to the studies initiated in the early 1970's, it was believed that normal stem cells, even if they existed, were suppressed by inhibitory molecules secreted by CML leukemic cells. A number of inhibitors have been proposed Including chalones, cell-associated inhibitors, isoferritlns and prostaglandin E.®®9-391 However, there is still no definitive evidence to indicate that humoral suppression of normal hemopoiesis plays an Important role in the pathogenesis of CML. More recent efforts have focused on the regulation of proliferation of the leukemic cells.  Cell proliferation is a highly controlled process at both the population and single cell level. At the population level. Important factors are the fraction of cells that are in cycle and the rate of cell loss due to death and/or differentiation. As previously discussed, the clonogenic cells of most lineages are Increased In the circulation of CML patients, and it was believed that this is due to an increase in fraction of cells that are actively engaged In DNA synthesis/mitosis. ®H-thjTiiidine suicide assays, therefore, have been applied to CML progenitors to obtain a relative estimate of the proportion of dividing CML clonogenic progenitors. Moore et al. were the first to document that a higher percenteige of committed progenitors were cycling continuously In CML patients, and these findings were subsequently confirmed by many others.®92,393 Eaves and Eaves also demonstrated that the abnormal in vivo cycling characteristics of Ph^positive progenitor cells could be reproduced In the LTC system. ®94 Normally, high prolfferatlve potential colony-forming cells (BFU-E >8 clusters and CFU-GM >500 cells) located In the adherent layer of LTC alternate between a quiescent and cycling state 7 days and 2-3 days after they are fed with fresh LTC medium and this has been shown to be related to an oscillating  change in the balance between the positive and negative factors active on primitive hemopoietic cells in this system. ^0,395 when primitive Ph^-positive ceUs in LTC adherent layers were examined, they were found to remain in cycle regardless of the time since the last new medium had been added.50.394,395 ^  j^en therefore suggested that primitive CML stem cell clone  might shEire with their immediate progeny a deregulation of mechanisms that msiintain their normal counterparts in a quiescent state.  Another hj^iothesis proposed to explain the expansion of the leukemic clone in CML patients is that leukemic progenitors require fewer cell divisions to give rise to fully differentiated end cells (skip divisions). This model evolved from the observation that maturing cells in CML patients seemed to be larger than normsd.331 More recently. Strife et al, 396 have proposed another model, the discordant maturation model, in which they suggest that there is an increased ratio of mature to primitive myeloid progenitors in CML, as a result of asynchronous maturation. They found that there is a progressive decrease in the relative numbers of more immature myeloid cells emanating from CML core bone marrow samples and a relative increase in more mature myeloid cells (myelocytes, metamyelocytes, bands, and polymorphonuclear granulocytes). These data were interpreted as indicating that the most mature proliferative cells (i.e late blasts, late promyeloc5rtes, and myelocytes) are mainly responsible for most of the expansion of the Ph^-positive population because they may be able to undergo slightly more divisions than thefr normal counterparts. The major argument against this model Is that Ph^positive clonogenic progenitor cells generate norméd-looklng colonies In the same time frame as normal progenitors.  2.3.2.1b Mechanisms of Altered Growth behavior A large number of hemopoietic growth factors and inhibitors have been characterized. Since in CML there is an increased number of hemopoietic cells, especially granulocytic cells, it could be hypothesized that CML cells might possess an increased sensitivity to some growth  factors, or a decreased sensitivity to inhibitors, or there is an overproduction of certain growth factors by the leukemic cells. However, no consistent evidence of abnormal growth factor responsiveness of leukemic granulocytic cells has been demonstrable, although some erythropoletin-independent colonies are found in a proportion of CML patients.®97 QML progenitors also have generally been found to depend on the same combination of growth factors to grow and produce colonies in agar/methylcellulose cultures as normal progenitors.®98 Similarly, differences in the production of G-CSF, GM-CSF, IL-1, IL-3, IL-6 between CML and normal blood cells have not been found in two independent studies of patients in chronic phase.®99.400 although G-CSF transcripts were fotmd to be variably expressed at Increased levels in mature myeloid cells In one study.  Adherent layers established from CML and  normad marrow were édso shown to be similar in terms of thefr mRNA expression and bioactivities of several hemopoietic growth factors including IL-3, G-CSF, GM-CSF, IL-la. IL-ip, and IL-6.®99,400 Therefore, the existence of abnormal autocrine or paracrine mechanisms to explain the unregulated proliferation of CML progenitor cells seems imllkely, although difficult to rule out completely. Since TGF-P Is known to have an Inhibitory effect on the cycling state of normal primitive high-proltferative CFU-GM and BFU-E from the adherent layer of LTC, Cashman et al. also studied the effects of TGF-p on Ph^-positive leukemic cell cycling. However, they found that primitive CML cells in LTC respond to TGF-P in a similar maimer as normal progenltors.402 Moreover, TGF-p was found to present constitutlvely in the adherent layers of both CML and normal cultures.403 These results, suggested the possible role of other growth factors/Inhibitors (as yet to be Identifled) to explain the abnormal cycUng/proltferation characteristics of CML primitive cells. Recently, preliminary studies in the Teny Fox Laboratory have indicated that primitive CML cells may not be sensitive to the inhibitory effect of MIP-la.  A defective role of T cells in the inhibition of leukemic cell growth has also been shown In CML. It is thought to be secondary to the decreased expression of phosphatidyllnositol (PI)llnked leukocyte-function-associated antigen 3 (LFA-3) on CML cells, as LFA-3 Is known to be an  important cytoadhesion molecule by which T cells bind to target cells.  Upadhyaya et al  recently showed that this defect could be corrected by treatment both in vivo and in vitro with IFN-a, which resulted in an increased expression of LFA-3 and possibly recognition by T ceUs.404  Despite the normal to Increased numbers of phenotypically identifiable CDie"*" natural killer (NK) cells in CML patients, these have been found to be profoundly defective in their ability to lyse NK-sensitlve target cells.'*®^ This deficiency could be corrected by lL-2, suggesting that the NK-deflcient activity may be mediated through defective production of IL-2 by T-helper or NK cells.'*06 A population of IL-2-actlvated NK (A-LAK) cells that are nonclonal and highly cytotoxic from CML patients were ldentifled.^07 However, significant diminution of both cytotoxic and proliferative capacities of the A-LAK population seemed to be associated with CML patients in accelerated and blastlc phases.'^^S Decreased production of IL-ip and IFN-y after PMA (IL-ip) and PHA-M (IFN-7Î stimulation, respectively, were also observed from CML blood cells.'*06 Whether inhibitory controls from T-cells, NK cells and A-LAK cells are important in the proliferatlon/dlfierentiatlon of primitive leukemic stem cells remains to be seen.  2.3.2.2 CeU Adhesion Because of the increased number of progenitors in the cfrculation of CML patients and extramedullary sites, it was thought that the leukemic cells may not only have an altered proliferative capacity, but also have a decreased adhesiveness to marrow stromal cells and thefr products, resulting in the massive flux of these cells out of the marrow space. It was observed that CML progenitor cells as assessed by BL-CFC assays are deficient in thefr binding to stromal layers generated in the presence of methylprednlsolone (MP+ stroma) but show an increased capacity to adhere to stroma layers generated In the absence of methylprednlsolone (MPstroma) by comparison to normal BL-CFC.'^09,410 Gordon et al. proposed that the MP+ stroma Is equivalent to normal stromal elements, whereas MP- is similar to the extramedullary  space.  Recently, this group further suggested that this altered adhesiveness of CML  progenitor cells might be due to the deficient expression of a Pl-llnked cell adhesion molecule (CAM) which occurs as a result of an altered phosphorylation pattern in the cell due to activities of the BCR/ABL kinase.'* 11 It is possible that altered adhesiveness of CML progenitor cells might also explain the abnormal proliferation of leukemic cells since it is believed that close Interactions between primitive hemopoietic cells and stromal elements are Important In the regulation of normal stem cell proliferation and dlfferentiation.38.412,413 whether such adhesion molecules have a role In stem cell regulation awaits further studies.  3. THESIS OBJECTIVES  In spite of recent progress In the molecular characterization of a consistent but unique fusion gene between BCR-1 and c-ABL in the neoplastic cells of patients with CML, it has not been possible to analyze the biological sequelae of this abnormality in the very cells believed to be responsible for initiating and maintaining expemsion of the neoplastic clone. This Is due largely because the fact that quantitative and specific assays for such cells In man and methods for their purification have only recentiy become available. My first objective, therefore, was to develop an in vitro assay for CML stem cells using as a starting point the LTC-IC assay. This assay had just been developed for normal human marrow cells and appeared to identify a cell with the appropriate properties. Since It was known that LTC Initiated with CML marrow cells rarely yield significant numbers of Ph^-positive clonogenic progeny after 5 weeks,370 t^is did not appear to be a suitable source of cells for beginning my studies. In contrast, It was known that when light density cells from the blood of CML patients with high WBC count (and elevated clonogenic progenitors) were seeded onto LTC adherent layers established with normal marrow, Ph^-positive hemopoiesis persisted for many weeks.394 j therefore chose to use this system to see if an LTC-IC assay could be developed for Ph^-positive cells. The first requirement was to  examine the relationship between the number of clonogenic cells present after 5 weeks éind the number of CML ceUs initially seeded down to limiting numbers of input cells to evaluate the feasibility of measuring absolute frequencies of leukemic LTC-IC by limiting dilution analysis. These studies form the basis of Chapter V.  My second and longer term objective was then to apply this assay to the characterization (and purification) of very primitive Ph^-positive cells and to compare the results with analogous normal cells. To explore the possibility that even the most primitive Ph^-positive cells would be actively proliferating, whereas such cells In normal Individuals appear to be largely quiescent populations, I chose to examine a number of properties that might be expected to differ between proliferating (or activated) and non-proltferatlng (quiescent) cells. However, also of Interest was the possibility that very primitive Ph^-positive cells might be abnormal in their self-renewal or initial differentiative behaviour in LTC because of the rapid decline in Ph^-positive cells seen in LTC initiated with CML marrow. Therefore I hoped to use the LTC-IC assay to obtain information about these functional properties of primitive Ph^-positive cells (described in Chapter V) as well as characteristics likely to reflect their cycling status in vivo (described in Chapter VI). Because it seemed likely that these efforts would continue to focus on the Investigation of primitive cells In the blood of CML patients with high WBC counts, thus obviating the need for confirming the neoplastic origin of the progenitors examined, I felt I needed to obtain baseline quantitation and characterization data for LTC-IC in the blood of normed individuals as this was not available. Such control studies are outlined in Chapter IV. Similarly, I wanted to extend the phenotypic markers to be evaluated to include Rh-123 uptake studies for which no data were available when the experiments described In this thesis were first begun. An additional series of control studies on the Rh-123 uptake of normal marrow LTC-IC were therefore imderiaken and these are described in Chapter III.  In summaiy, the hypothesis my thesis research was designed to test was that the most primitive Ph^-positive cells in the neoplastic clone responsible for CML would be foimd to resemble an abnormally "turned on" population of normal LTC-IC, characterized by changes expected in rapidly proliferating cells but not causing any significant change in their subsequent differentiative behaviour.  REFERENCES 1.  Golde DW, Takaku F: Hematopoietic Stem Cells. New York, Marcel Dekker, Inc, 1985, 379  2.  Wintrobe MM, Lee OR, Boggs DR, BIthell TC, Foester J , Athens JW, Lukens JN: Origin and development of the blood and blood-forming tissues, in Wintrobe M M (ed): Clinical Hematology (edition 8). Philadelphia, Lea and Febiger, 1981, pp 35  3.  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Chang WC, Hsiao MH, Pattengale PK: Natural killer cell Immunodeficiency in patients with chronic myelogenous leukemia. Cancer Immimol Immunother 10:57, 1991  407. Verfaillle C, Miller W, Kay N. McGlave P: Adherent lymphoklne-actlvated killer cells in chronic myelogenous leukemia: A benign cell population with potent cytotoxic activity. Blood 74:793. 1989 408. Verfaillle C. Kay N, Miller W. McGlave P: Diminished A-LAK cytotoxicity and proliferation accompsmy disease progression in chronic myelogenous leukemia. Blood 76:401. 1990 409. Gordon MY. Dowdlng CR, Riley GP. Goldman J M . Greaves MF: Altered adhesive interactions with marrow stromal of haematopoietic progenitor cells in chronic myeloid leukaemia. Nature 328:342, 1987 410. Gordon MY: Adhesive properties of haemopoletic stem cells. Br J Haematol 68:149, 1988 411. Gordon MY, Atkinson J , Clarke D, Dowdlng CR, Goldman J M , Grlmsley PG, Siczkowski M, Greaves MF: Deficiency of a phosphatidylinosltol-anchored cell adhesion molecule Influences haemopoletic progenitor binding to marrow stroma in chronic myeloid leukemia. Leukemia 5:693, 1991 412. Gordon MY, Riley GP. Clarke D: Heparem sulfate Is necessary for adhesive interactions between human eéirly hemopoietic progenitor ceUs and the extraceUular matrix of the marrow mlcroenvlronment. Leukemia 2:804. 1988 413. Verfaillle CM. McCarthy J B , McGlave PB: Differentiation of primitive human multipotent hematopoietic progenitors into single lineage clonogenic progenitors Is accompanied by alterations in their Interaction with flbronectin. J Exp Med 174:693, 1991  CHAPTER  II  MATERIALS AND METHODS  1. CELLS  1.1  Normal Bone Marrow The normal marrow used In these studies were leftovers from aspirate harvests obtained  with informed consent from normal Individuals donating marrow for autologous or allogeneic marrow transplants after approved of the University of British Columbia Clinical Screening Committee for Research Involving Human Subjects. Cells were originedly collected in TCI99 medium with 100-200 U/mL of preservative free heparin and kept on ice prior to use. Marrow cells were used directly without further processing if the samples were celluleir (>2xlo7 cells per mL) or after removing the buffy coat obtédned by spinning the whole sample at 200g for 4 minutes. For fluorescence-activated cell sorter (FACS) sorts, light density marrow ceUs (< 1.077 g/cm®) were isolated by density gradient centrifugation on Percoll or FicoU-hj^jaque (FH)(Pharmacia Fine Chemicals, Uppsala, Sweden).  Discontinuous Percoll density centrifugation was performed using the method previously described by Sutherland et al. ^ An isosmotic PercoU (Pharmacia Fine Chemicals, Uppsala, Sweden) stock solution was prepsired by mixing the percoll with a lOX concentrated phosphatebuffered saline (PBS) solution to obtain a percoll stock solution with an osmolality of 290±5 mosm and a pH of 7.4. Marrow ceUs were washed twice with 2% FCS, suspended in Iscove's 2% FCS at 2x10^ cells/mL, a mlcrohematocrit determined, and the ceUs then mixed with an appropriate volume of the percoU stock solution to suspend the ceUs In a solution with a density 1.066 g/mL - 1.068 g/mL, as follows:  Volume of Percoll Stock = Solution to be  Density Desired (1.066-1.068 g/cm^) Density ot Percoll Stock Solution  Density of - Iscove's 2% FCS - Desired Density  Volume x of Cells &Wash (mL)  Hematocrit x l - TOD  Added (mL) The cell suspension was then overlayered with 5 mL of Iscove's 2% FCS and centrifuged at 600g for 15 minutes at room temperature. The top layer over the Percoll solution was collected and washed twice in 2% FCS. In some cases, marrow cells were prepared using FH density gradient separation. This was done by overlayerlng the marrow cells on top of the FH solution at a 3:2 ratio In a 50 mL Falcon tube (Becton-Dlcklnson Canada, Ontario). They were then centrifuged at 600g for 30 minutes at room temperature. The Interface layer was then removed with a Pasteur pipette and washed twice with 2% FCS. Light density cells to be stained for sorting were stored at 4°C overnight In Iscove's medium with 50% FCS. Nucleated cell counts were performed using a hemocytometer. 1.2  Normal Blood Blood cells were obtained with Informed consent from normal platelet donors undergoing  platelet/leukapheresis at the Cell Separator Unit of the Vancouver General Hospital. Cells were first centrlfiaged at 200g for 10 minutes to remove excess plasma. They were then further depleted of T cells by Incubation with 2-amlnoethylisothlouronlum-treated sheep red blood cells (2-AET-SRBC) as described earlier by Marsden et al.^ 2-AET-SRBC were prepared by first washing the SRBC with Hank's solution three times and then Incubating at 37°C for 15 minutes with a solution of 0.8 gm AET In water (adjusted to pH 9.0 with NaOH and sterilized by filtration through 0.2^lm filter). The SRBC were then washed three times with Hank's 2% FCS and suspended In 20% FCS to a total volume of 35 mL (approximately 2-4x10^ SRBC/mL). Light density normal blood cells were incubated with 2-AET-SRBC for 30 minutes at 4°C followed by centrlfugation at 4°C on FH to remove the rosetted T cells. Random checking of this procedure showed that less than 2% of the recovered T cell-depleted fi-actions were CD2''" (T cells) by FACScan analysis. All experiments with normal blood cells used the T cell-depleted fraction  obtained in this way. The purpose of removing the T cells was to prevent the spontaneous activation and outgrowth in LTC of Epsteln-Barr virus-transformed B lymphocytes which had previously been shown to otherwise occur at high frequency within 5 weeks in the LTC system.®'^  1.3  CML Blood Blood was also obtained with informed consent from CML patients undergoing routine  hematologic assessment. All patients were in chronic phase and Ph^-positive. Only samples from patients with markedly elevated WBC counts were used, as this allows selection of patients whose cfrculating Ph^-positive progenitors are markedly elevated, (i.e., >100 fold) and hence could be analyzed as If these were pure neoplastic progenitor populations. The age, sex and WBC coimts of the CML patients studied are shown in Table 5. Light density cells were isolated by FH centrifugation. T cell depletion was not performed with CML blood samples because the T cell content of the peripheral blood from CML patients with high WBC counts is already diluted to a few percent or less and development of spontemeous transformants was not encountered.®  Table 5. Characteristics of the CML Patients Studied  Patients  Age (yrs)  Sex  1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27  62 58 56 81 58 61 40 37 36 52 43 47 33 60 50 50 33 56 61 40 58 53 35 53 60 59 32  F M M M M F M M F M M F M M M F M M F M M F M M M F M  WBC (zlO^/L)* 76 190 45 250 88 116 48 62 137 156 262 40 45 110 25 22 37 41 64 436 142 104 316 177 60 144 108  * At the time blood was obtédned for the studies described in this thesis.  2. STAINING  Low density marrow or blood cells were washed twice with Hank's solution with 2% FCS and 0.1% NaN3 (HFN), resuspended at 10^ cells/mL. and then kept on ice before, during and after all sorts. Staining procedures were as follows:  2.1  CD34 Cells to be stained according to their cell surface expression of CD34 were Incubated  with 8G12, a monoclonal IgG j anti-CD34 antibody produced by Dr. Peter Lansdorp in the Teny Fox Laboratory.5 Fab' fragments of 8G12 were either dfrectly labelled with R-phycoeiythrln (PE) as described for OX22 Fab'fragments by McCarthy et al (8G12-PE), or were crossllnked to monoclongd £mtl-PE antibodies by incorporation into tetreunerlc antibody complexes (8G12TC).^''^ For the latter, 50|ag of 8G12 were complexed with 150|xg anti-PE antibodies using 200^g of F(ab')2 fragments of P9 rat anti-mouse IgGj. Control tetramerlc complexes were prepared by mixing equimolar amounts of anti-PE with P9 F(ab')2. All TC were Incubated at 37°C overnight and spun for 10 minutes at 10,000 g before use. For staining, cells were either Incubated with 8G12-PE for 30 minutes at 4°C, then washed twice, or in the experiments where 8G12 TC were used, cells were incubated with 2[ig/mL of RPE for a further 30 minutes at 4°C, and then washed twice.  2.2  Rhodamine-123 (Rh-123) Rh-123 was purchased from Eastman Kodak, Rochester, NY. Cells were labelled by  incubation with 0. l|ig/mL Rh-123 for 20 min at 37°C, then washed twice with HFN. Cells were further incubated with HFN for another 15 min at 37°C, and then washed again, and finally suspended at a concentration of 2xlO®/mL in HFN with 100|ig/mL DNAase 1 (Sigma, StLovils, MO).^ In the experiments where anti-CD34 staining was used together with Rh-123 staining, 8G12 was Included in the second incubation step with Rh-123.0  2.3  HLA-DR  Antl-HLA-DR directly conjugated to PE (antl-HLA-DR-PE) was purchased from BectonDicklnson (Sunnyvale, CA). Cells were labelled by Incubation with HLA-DR PE (1 ^ig per 10^ cells) for 30 minutes at 4°C, then washed twice and suspended in HFN at a concentration of 2x10® cells/mL with 100^lg DNAase 1. ^ In the experiments where double-staining with antiCD34 were needed, 8G12 FITC was included during the same incubation.  3.  3.1  FLOW CYTOMETRYIQ  Set-up Cells were sorted using a Becton-Dlcklnson FACStarP^"^. The principles of the FACS are  illustrated in Figure 6. The FACS consists of three major components: an optical bench, an electronic console, and a computer. The optical bench contains the laser, optical system and fluid assemblies. It provides multiple controls and Indicators for laser power, stream deflection, fluid setting, droplet control and sample differential pressure. Light scatter and fluorescence signals from ceUs in the sample streeim intercepting the laser beam are coUected by means of lenses and detectors that are located at different angles or positions relative to the incident beam, and subsequentiy transformed to electric pulses that are then processed further. Usually cells are passed through the fluid assemblies at a flow rate of 2500-3000 cells/min and detected by the system as a drop packet, containing a number of ceUs determined by the user with a ceU of interest in the center of the packet. In this way, the system is able to balance between purity and recovery, depending on the purpose of Individual sorts. Prior to each sort, the FACS was calibrated using 10M,m fluorescent microsphere standards (Coulter Corp., Hialeah, FL) to ensure that the baseline scatter and fluorescence output was the same for aU experiments performed. Compensation for double-stained specimens was set up in both FITC, PE and Rh-123 with single-stained samples.  FIGURE 6. Principles of FACS  3.2  Sorting Cells with différent properties were sorted into separate fractions depending on thefr size  (FSC), internal granularity (SSC) and fluorescence at up to 3 different wavelengths each corresponding to the eimount of a given antibody or dye bound to the ceU. CeUs to be analyzed or sorted were placed in a sample chamber where they were then pressurized for deUvery by tubing to the flow chamber. In the flow chamber, the sample streeim containing the ceUs is injected into the center of a ceU-free stream of sheath fluid. The sample and coaxial sheath fluid then enter a constriction region, where thefr diameters are reduced and the flow velocity increased. As the ceUs flow through the laser beam, the fluorochrome marker is excited and fluorescence is detected by a detector, resulting in an electrical signal proportional to the fluorescence intensity, which is proportional to the ceUular parameter of Interest. Light scatter by a ceU as It Intersects the laser beam Is also coUected by a detector. The ceU stream containing the ceUs then breaks Into a series of smaU droplets at the droplet breakoff point. If a decision is made to sort that ceU, an electrical voltage is then appUed to the fluid stream Just as a droplet containing the ceU breaks off from the stream. This leaves an electrical charge on the droplets containing the ceUs to be sorted. Droplets may be charged positively or negatively for sorting. The droplet stream containing charged and tmcharged droplets then passes through an electric field between two deflection plates. Positively- or negative-charged ceUs are then deflected towards the coUection tubes containing 50% FCS. In the meantime, the computer system generates profiles of the Ught scatter and fiuorescence of each ceU population sorted, and its proportion of the original total can then be easUy calculated. Since the FACS Inspects each ceU indlvlduaUy, it is particularly useful for the isolation and enrichment of relatively rare ceU types, such as primitive ceUs from marrow and blood.  Enrichment of clonogenic ceUs and LTC-IC relative to unsorted low density ceUs was calculated by dividing the progenitor frequency per 10^ ceUs in the sorted fraction by the progenitor number per 10^ unsorted ceUs. To derive enrichment values from the original blood  sample, It was assumed that 100% of all progenitors were recovered In the low density fraction. ^ Recovery of cells in a given sorted fraction was calculated by multiplying the percentage of nucleated cells retrieved in each fraction by the calculated enrichment. To allow comparisons between fractions in different experiments, the recovery in individual fractions was normalized so that the sum of all fractions In an Individual experiment was 100%.  4. FUNCTIONAL ASSAYS  4.1  Short-term Colony Assays Clonogenic erythropoietic (BFU-E), granulopoietic (CFU-GM), and multillneage (CFU-  GEMM) progenitors were assayed in standard methylcellulose (MC) cultures as previously described. ^ ^ A stock MC culture medium was obtained from the Media Prepeu-ation Service of the Terry Fox Laboratory in botties of 100 mL, each containing 40 mL Of 2.2% methylcellulose (4000cps) diluted in Iscove's medium and supplemented with 30 mL of 100% FCS, 10 mL of 10% deionlzed bovine serum albumin, 1 mL of lO'^M 2-mercaptoethanol (2-ME), 1 mL of Lglutamlne (29.2 mg/mL in distilled water), 2 mL of highly purified human erythropoietin (150 U/mL) and 10 mL of agar-stimulated human leucocyte conditioned medium.  This MC  medium was usually allquoted in volumes of 3 or 5 mL per tube, and then kept frozen prior to use. Normally, low density marrow or blood cells at an appropriate concentration (to j^leld <200 colonies per 1.1 mL dish) were added to the MC In these tubes, thoroughly mixed, and then the contents transferred to 35 mm petrl dishes (Grelner, Nurtlngen, Germany). Sorted cells were usually plated at lower cell concentrations depending on the enrichment of progenitors anticipated from prellmlnciry experiments. All assays were set up In duplicate or quadruplicate 1.1 mL volumes and then Incubated at 37°C in a humidified atmosphere of 5% CO2 in afr. Colonies were scored 18-20 days later, according to established criteria. ^ ^  4.2  LTC-IC Assays LTC-IC were assayed by seeding £in aliquot of test cell suspensions usually Into 35 mm.  Coming tissue culture dishes (Coming Glassworks, Coming, NY) containing Irradiated (1500cGY) normed marrow adherent cells (3x10^ cells per cm^) that had been subcultured from the adherent layer of a 2-4 week old LTC established from normal donors. ^ This Involved plating 8x10® of the Initial unprocessed marrow or marrow buffy coat cells per 2.5 mL LTC. Test marrow cells and CML marrow or blood were added at 8x10® and 1-2x10® cells per 2.5 mL LTC, respectively, and sorted fractions at proportionately lower cell concentrations depending on the degree of enrichment anticipated from preliminary experiments. Low density normal blood cells were plated at higher concentrations (5x10® to 10^ cells per LTC) because of the expected low frequency of LTC-IC In the blood. LTC were kept at 37 °C In an atmosphere of 5%C02 in afr for the first 3-5 days, then switched to 33°C thereafter. They were fed weekly by replacement of the half of the growth medium containing half of the nonadherent ceUs with fresh growth medium (a-medium supplemented with 40 mg/L inositol, 10 mg/L folic acid. 400 mg/L glutamlne, lO'^ 2-ME. 10'® M hydrocortisone sodium hemlsucclnate. 12.5% horse semm. and 12.5% fetal calf semm). Most experiments were performed in 35mm Coming tissue culture dishes, except for limiting dilution smalyses that were done in 96-well flat bottom tissue culture plates. After a total of 5 weeks, the cultures were harvested by trypsinlzation. 1® The nonadherent and adherent ceUs were then washed with 2% FCS and plated In methylceUiflose cultures, as described above. The number of clonogenic cells present in LTC harvested at week 5 (i.e., the sum of the number of BFU-E, CFU-GM, and CFU-GEMM present in both the adherent and nonadherent fractions at this time) was then used to provide a quantitative measure of the number of LTC-IC originally seeded into the LTC. 14,15 Experiments to validate the use of this LTC-IC assay to quantitate normal and CML blood progenitors are described in Chapter IV and V, respectively.  5. LIMITING DILUTION ANALYSIS  To quantitate the absolute frequencies of LTC-IC In samples from normal and CML blood, limiting dilution experiments were performed. Low density blood cells were seeded Into 6 mm wells In 96 well flat bottom Nunclon plates (Nunc, Roskflde, Denmark) with pre-established Irradiated marrow feeders (1x10^ cells per well). In these analyses, each ceU suspension was seeded at 3 or 4 different initial cell concentrations with a mean of 23±1 replicate wells per concentration. After 5 weeks, the nonadherent and adherent cells were suspended by trypslnlzatlon as described above for regular LTC. The total content of each weU was then plated Into a single methylcellulose assay. The proportion of positive and negative wells was determined by the presence or absence of colony formation In each dish. Using Poisson statistics and the weighted mean method, ^ 6.17,18  frequency  of LTC-IC in the starting  sample was then determined by deriving the reciprocal of the concentration of test cells that gave 37% negative cultures as foUows: P (x) = Vi^/éhi P(x=0) = l/e^^ = probability of having no LTC-IC In the sample \i = mean number of LTC-IC per dose From this value and a knowledge of the total number of clonogenic cells produced by a large number of cells In the same Input suspension, the average 5 week output of clonogenic cells per LTC-IC In normal and CML samples was calculated.  6. MAINTENANCE STUDIES  For LTC-IC mgilntenance studies, the entfre contents of primary LTC were harvested after 10 days, 4-5 weeks, and 8 weeks and aUquots used to initiate secondary LTC on new irradiated feeders. These secondary LTC were then maintained for a further 5 weeks prior to harvesting and plating of the cells In methylcellulose assays as Illustrated In Figure 7. The number of  clonogenic cells detected in these assays was used to provide a relative measure of the LTC-IC in the prtmaiy LTC at the time they were harvested. This value was then normalized by the relative number of LTC-IC detected in primary LTC-IC assays of the original cell suspension to yield a percent input value.  INITIATE LTC ASSAY FOR CLONOGENIC CELLS  CSS  10 DAYS  4-5 WKS  8 WKS  1 ^  ASSAY FOR CLONOGENIC CELLS  FIGURE?. Maintenance Studies  7. 4-HC SENSITIVITY STUDIES  Marrow or blood cells from normal and CML samples were resuspended to a final concentration of 2x10^ cells/mL and Incubated with 4-HC (100 tig/mL) for 30 minutes at 37°C with 7% erythrocytes. Cells were washed twice with 2% FCS and depleted of erythroc5rtes by Incubation with NH4CI for 10 minutes at 4°C. They were washed again with Iscove's 2% FCS and assayed for LTC-IC and clonogenic cells at appropriate concentrations. In this set of experiments, LTC-IC function (before and after exposure to 4-HC) was assessed in terms of the clonogenic cell content of assay cultures evaluated after 4 and 8 weeks since previous experiments had reveeded differences in LTC assays of bone marrow samples for autologous transplants when these two time points were compared. 19'20 4.HC was obtained from NOVA Pheu-maceuticals (Baltimore, MD) through the courtesy of the Leukemla/BMT Program of British Columbia, Vancouver General Hospital (for research purpose only).  REFERENCES 1.  Sutherland HJ, Eaves CJ, Eaves AC, Dragowska W, Lansdorp PM: Characterization and partlcd purification of human marrow cells capable of Initiating long-term hematopoiesis in vitro. Blood 74:1563, 1989  2.  Marsden M. Johnsen HE, Hansen PW, Christiansen J E : Isolation of human T and B lymphocytes by E-rosette gradient centrifugation: Characterization of the isolated subpopulations. J Immunol Methods 33:323, 1980  3.  Eaves AC, Cashman J D , Gaboury LA, Kalousek DK, Eaves C J : Unregulated proliferation of primitive chronic myeloid leukemia progenitors in the presence of normal meirrow adherent cells. Proc Natl Acad Sci U S A 83:5306, 1986  4.  Stevenson M, Volsky B, Hedenskog M, Volsl^ DJ: Immortalization of human T lymphocytes after transfection of Epstein-Barr virus DNA. Science 233:980, 1986  5.  Lansdorp PM, Sutherland HJ, Eaves C J : Selective expression of CD45 isoforms on functional subpopulatlons of CD34+ hemopoietic cells from human bone marrow. J Exp Med 172:363, 1990  6.  McCarthy KF, Hale ML, Fehnel PL: Purification and analysis of rat hematopoietic stem cells by flow cytometry. Cytometry 8:296, 1987  7.  Wognum AW, Thomas TE, Lansdorp PM: Use of tetramerlc antibody complexes to stain cells for flow cytometry. Cj^ometry 8:366, 1987  8.  Bertoncello I, Hodgson GS, Bradley TR: Multiparameter analysis of transplantable hemopoietic stem cells: I. The separation and enrichment of stem cells homing to marrow and spleen on the basis of Rhodamine-123 fluorescence. Exp Hematol 13:999, 1985  9.  Udomsakdi C, Eaves C J , Sutherland HJ, Lansdorp PM: Separation of functionally distinct subpopulations of primitive human hematopoietic cells using rhodamlne-123. Exp Hematol 19:338, 1991  10. Shapiro HM: Practical Flow Cytometry (edition 2). New York, Alan R. LIss Inc., 1988, 353 11. Cashman J , Eaves AC, Eaves C J : Regulated proliferation of primitive hematopoietic progenitor cells in long-term human marrow cultures. Blood 66:1002, 1985 12. Eaves CJ, Cashman JD, Eaves AC: Methodology of long-term culture of human hemopoietic cells. J Tissue Culture Methods 13:55, 1991 13. Coulombel L, Eaves AC, Eaves C J : Enzymatic treatment of long-term human marrow cultures reveals the preferential location of primitive hemopoietic progenitors in the adherent layer. Blood 62:291, 1983 14. Sutherland HJ, Lansdorp PM, Henkelman DH. Eaves AC, Eaves C J : Functional characterization of Individual human hematopoietic stem cells cultured at limiting dilution on supportive marrow stromal layers. Proc Nati Acad Sci U S A 87:3584, 1990  15. Sutherland HJ, Eaves AC, Eaves C J : Quantitative assays for human hemopoietic progenitor cells, in Gee AP (ed): Bone Mgirrow Processing and Purging: A Practical Guide. Boca Raton, CRC Press Inc, 1991, pp 155 16. Taswell C: Limiting dilution assays for the determination of immunocompetent cell frequencies. I. Data analysis. J Immunol 126:1614, 1981 17. Porter EH, Berry RJ: The efficient design of transplantable tumour assays. Br J Cemcer 17:583, 1963 18. Fazekas de St.Groth S: The evaluation of limiting dilution assays. J Immtmo Methods 19:R11, 1982 19. Winton EF, Colenda KW: Use of long-term human marrow cultures to demonstrate progenitor cell precursors in marrow treated with 4-hydroperoxycyclophosphamide. Exp Hematol 15:710, 1987 20. Eaves CJ, Sutherland HJ, Udomsakdl C, Lansdorp PM, Szllvassy SJ, Fraser CC, Humphries RK, Bamett M J , Phillips GL, Eaves AC: The human hematopoietic stem cell in vitro and in vivo. Blood Cells(ln press):.  CHAPTER  III  SEPARATION OF FUNCTIONALLY DISTINCT PRIMITIVE HEMOPOIETIC CELLS IN NORMAL HUMAN BONE MARROW USING RHODAMINE-123  1. INTRODUCTION  A hlercirchy of primitive hemopoietic progenitors has been identified in murine bone marrow from comparisons of the properties of cells with long-term repopulating ability and short-term clonogenic capacity. Such studies indicate that spleen colonies detected 12 days cifter transplcmtation of normal mgirrow are derived from cells (CFU-S day 12) that are more primitive than those generating colonies detected after 8 days (CFU-S day 8), and that cells capable of producing CFU-S that generate spleen colonies in secondary recipients (i.e., pre-CFUS) may be more closely related to cells with long-term repopulating ability than either CFU-S day 8 or CFU-S day 12. ^'^ In humans, anedogous in vivo assays are not possible and most efforts to develop quantitative end-points for human repopulating stem cells have focussed on in vitro strategies. Cross comparisons between the properties of cells defined by appropriate in vitro assays of murine cells where direct linkage to repopulating cells can be made will, therefore, be a crucial step in validating any in vitro human stem cell assay devised. Since lympho-myeloid repopiilating cells persist and proliferate in long-term cultures (LTC) initiated with murine bone marrow,^ and the cell responsible for generating sustained hemopoietic activity In this system has been shown to share characteristics with the murine In vivo repopulatlng cell,^ human LTC would seem to be a useful starting point for the development of an assay for human hemopoietic stem cells. Studies with the drug 4-hydroperoxycyclophosphamlde (4-HC) have shown that the cells responsible for producing clonogenic progenitors In human LTC detectable after 4 to 8 weeks. I.e. LTC-lnltlatlng cells (LTC-IC), are relatively insensitive to this drug, a property they share with in vivo repopulatlng cells. In contrast, doses of 4-HC that spare LTC-IC will kill most  directly clonogenic cells.®'^ LTC-IC in normal human marrow have also been shown to differ from their clonogenic progeny in a number of other properties including size and surface antigen expression.  12  Previous studies have demonstrated that the most primitive cells In murine bone marrow differ from most CFU-S and in vitro clonogenic cells In their ability to take up the supravital dye Rhodamlne-123 (Rh-123).®'4'7.13 Murine repopulating stem cells, pre-CFU-S and murine LTC-IC are Rh-123^^^ in contrast to the majority of murine marrow cells which are Rh123bright Including all day-8 CFU-S and 60% of day-12 CFU-S. Rh-123 has been reported to bind specifically to mitochondria of living cells In a variety of cell types without being accumulated In other organelles. 14-17 From these studies It has been concluded that differences In Rh-123 fluorescence reflect either differences in the number of mitochondria per ceU and/or their size or charge. Because of the differential Rh-123 characteristics of primitive murine hemopoietic ceUs, I investigated the potential of Rh-123 to distinguish between recently defined functionally distinct, but closely related, populations of primitive human hemopoietic cells.  2. RESULTS  2.1  Lack of Toxicity of Rh-123 for Clonogenic Cells or LTC-IC To evaluate whether Rh-123 might be toxic to either clonogenic progenitors or LTC-IC  and to select an optimal concentration for staining human marrow ceUs with this dye, an initial series of dose-response staining experiments were performed. Cells were incubated with varying concentrations of Rh-123 from 0.1 to 10 |Ag/ml for 20 minutes and then passed through the FACStarPl^s ungated so they would have the same exposure to the laser as ceUs to be sorted in later experiments. As shown in Figure 8, exposure to doses of up to even 10 M,g/ml were not  toxic under the conditions used. As stairoing was optimal at 0.1 [ig/ml, this dose was used for all subsequent studies.  200  O tr H Z  o o  100-  DOSE (ng/mL)  FIGURE 8. Survival of clonogenic (•) and LTC-IC (•) after exposure to Rh-123 at variable doses. Light density cells were exposed to variable doses of Rh-123 for 20 minutes and then passed through the FACS ungated. Values represent the mean ±SEM percentage of progenitors cells as compared to control from three replicate experiments.  2.2  Uptake of Rh-123 by Clonogenic CeUs and LTC-IC Low density cells were labelled with Rh-123, analyzed, sorted and assayed In clonogenic  and LTC-IC assays. Histogram analysis of each marrow stained revealed two distinct populations. The fluorescence channel that gave maximal separation between these was used to discriminate between Rh-123^"^ and Rh-123^'^ëht cells as there was some variability In the relative fluorescence Intensity of these ceUs In different marrow samples, presumably as a result of variable destalnlng kinetics. Of the nucleated ceUs In the light scatter window selected (Figure 9A), approximately 10% of cells were Rh-123^"^ (Figure 9C) (i.e. 3 to 5% of the total low density cell population), with the majority of cells showing bright Rh-123 staining. When the total number of clonogenic cells was calculated by adding together aU BFU-E, CFU-GM and CFU-GEMM In each fraction, the combined populations were found to be equally distributed between both fractions (Figure 10). However, as shown in Figure 11, more detailed lineage specific analysis revealed the majority (70%) of BFU-E to be present In the Rh-123^^^^ fraction, with CFU-GM approximately equally distributed between the Rh-123^^ëh* and dull fractions. CFU-GEMM when detected were always present in the Rh-123^"^ fraction. Virtually all (94%) of the LTC-IC were present in the Rh-123'^"^^ fraction.  FIGURE 9. Contour plots of forward light scatter versus 90° light scatter (A.) or versus log Rh-123 fluorescence intensity (B.C) of low density cells. A & B, unstained cells; C. Rh- 123-stained cells. B & C. cells in the light scatter window shown in A.  < o  H U-  O  CLONOGENIC CELLS  LTC-IC  FIGURE 10. Expression of Fai-123 on directly clonogenic cells and LTC-IC. The mean progenitor recovery ± SEM from 4 experiments is expressed as a percent of the total number of low density progenitors recovered within the light scatter window shown in 9A; • Rh-123'*"ll; • Rh-123'^'^ê^t.  FIGURE 11. Distribution of BFU-E and CFU-GM in the Rh-123'^"*^ (•) and Rh-123^^êh* fractions (•). Values shown are the mean ±SEM percentages of BFU-E and CFU-GM recovered in each fraction measured in 4 experiments.  2.3  Double-Staining of Human Marrow With CD34 and Rh-123 Because of the observed selectivity of Rh-123 for LTC-IC and the known strong reactivity  of antl-CD34 Mab with LTC-IC,  another series of experiments were undertaken to evaluate  the degree of enrichment of LTC-IC obtained when these two reagents were used in combination. Approximately 1.5 to 4.5% of all light density bone marrow cells were CD34''", and of these, on average 35% were R h - 1 2 3 ^ ^ (range 15-50% In 6 experiments. Figure 12B and C). The CD34+ Rh-123*^^^ cells appeared to have a lower forward light scatter signed as compared to the CD34'*' Rh-123^'^ëht cells (Figure 12E and F). They also showed a relatively higher CD34 fluorescence intensity than the CD34+ Rh-123^'^ght ^eUs (data not shown). By sorting the CD34+ Rhj23dull fraction in the selected light scatter window it was possible to enrich for LTC-IC on average approximately 240-fold as compared to low density bone marrow cells (Table 6). After 5 weeks, 6 clonogenic cells were detected per 100 cells initially placed In LTC, as compared to 0.04 per 100 ceUs in the unsorted light density fraction. About 93% of LTC-IC were present In the CD34+ Rh-123'^"^ ceU fraction. In contrast to LTC-IC, the majority of clonogenic ceUs were found In the CD34+ Rh-123^^ë'^t ceU fraction. The enrichment of these ceUs relative to low density marrow cells was 40-fold and the frequency was 10 per 100 cells as compared to 0.3 per 100 ceUs prior to sorting.  FORWARD LIGHT SCATTER  FIGURE 12. FACS contour plots of anti-CD34 and Rh-123 stained cells in the selected light scatter window (identified in panel D) or gated for CD34 posltivity (window shown in panel C and selected by comparison of staining with anti-CD34 (8G12-PE) versus a control IgG^ antibody {ID3-PE), shown in panels B and A, respectively). The light scattering properties of CD34"'' Rh123°"" cells and CD34+ Rh-123^right ^eiis are shown in 12E and 12F, respectively.  Table 6. Frequency and Enrichment of Hemopoietic Progenitors in the CD34''' Rh-123'^"^ and CD34* Rh-123^'^^^ Fractions of Low Density Normal Human Marrow Cells. Cells Evaluated  % Nucleated CeUs  Clonogenic Cells  Frequency (per 100 ceUs) Marrow (< 1.077 g/cm^)  Enrichment^  LTC-IC  Relative % Recovery'^  0.3 ±0.09  Relative Frequencjr^ (per 100 ceUs)  Enrichment^  Relative % Recovery^  240 ±140  93 ± 3  0.04 ±0.019  CD34+Rh-123^"^  1.0 ±0.8  6.0 ±2.4  23 ± 9  29 ± 14  6.2 ±2.0  CD34+Rh-123^'^éht  1.6 ±0.9  10.5 ± 5.8  38 ± 20  71 ± 14  0.3 ± 0.3  12±  11  7±3  Values shown are the mean ± SEM for 6 experiments. ^  Calculated by dividing the progenitor frequency per 10^ sorted ceUs by the progenitor frequency per 10^ unsorted, low density ceUs in each individual experiment, and then deriving the mean ± SEM of these vedues for the 6 experiments performed.  ^  Calculated by multiplying the percentage of ceUs retrieved In either fraction by the corresponding calcidated enrichment In each experiment, then expressing the value obtcdned as a percent of the total number of progenitors In the CD34''' low density population sorted In that experiment, and then deriving the mean ± SEM of these values for the 6 experiments performed. Recoveries of total Ught density clonogenic ceUs and LTC-IC in the CD34''" fraction In these same experiments was 62 ± 26 and 164 ± 67, respectively.  ^  Measured as the number of clonogenic ceUs present after 5 weeks.  o  3. DISCUSSION Several studies have demonstrated that actively proliferating cells are brightly stained by Rh-123 in contrast to resting cells. Tliese observations have been explained by assuming that proliferating cells contain either more, larger, or more active mitochondria because Rh-123 is known to bind selectively to mitochondria. 14-17 ^y. studies demonstrate that the majority of human clonogenic cells differ from LTC-IC in their much greater uptake of Rh-123. This finding is consistent with the genered observation that primitive hemopoietic cells normally exhibit features of a quiescent population and would thus be anticipated to incorporate relatively low amounts of Rh-123.^9.20 j ^jgo fou^d that many BFU-E (70%) and some CFU-GM (40%) and all CFU-GEMM were in the Rh-123*^"^ fi-action. This is also not surprising, given that a subset of both BFU-E and CFU-GM populations in normal human marrow, like CFU-GEMM, are known to be quiescent.^ 1 Moreover, this subset Includes a larger proportion of cells classified as BFUE than those classified as CFU-GM, perhaps because of the selective exclusion of a late clonogenic erythropoietic progenitor population (I.e. CFU-E) from such inter-lineage comparisons. Interestingly, analogous differences between marrow BFU-E and CFU-GM in their expression of CD45R and CD45RO were also reported.  To evaluate the potential of Rh-123 staining for isolating highly purified populations of LTC-IC, cells were sorted on the basis of both CD34 expression and Rh-123 fluorescence. Because only - 1 % of cells in the light density marrow are CD34"*" Rh-123*^"^, isolation of this fraction yielded a population that was highly enriched for LTC-IC. Dr. Sutherland in the Terry Fox Laboratory, has previously shown that the number of clonogenic ceUs present after 5 weeks in LTC is approximately 4 times the actual number of LTC-IC originally present as qugmtltated by limiting dilution; i.e. each LTC-IC produces an average of 4 clonogenic progenitors detectable 5 weeks later.22 Thus, It can be Inferred that 1 to 2% of the ceUs In the CD34+ Rh-123*^"^ fraction are LTC-IC, a result that Is comparable to the most purified suspensions currently obtainable. 1^' 11 This concentration of LTC-IC represents an overaU enrichment of  approximately 1000-fold by comparison to marrow buffy coat since the density separation step gives roughly a 4-fold enrichment of LTC-IC. It should also be noted that while only a minority of the original population of clonogenic cells are co-purified in the CD34+ Ph-123^^  fraction,  their frequency in this fraction (6%) is still, on average, 4 times higher than that measured for LTC-IC.  In conclusion, my studies show that Rh-123 can be used to stain functionally distinct hemopoietic cells. In a differential and non-toxic fashion. The ease of obtaining highly purified LTC-IC populations in high yield (>90%) when this reagent is combined with anti-CD34 staining suggests its utility for further purification and analysis of the earliest stages of human hemopoietic cell development. Moreover, given the evidence that primitive leukemic cells are actively proliferating,^3.24 Rh.123 staining may also provide a unique approach to the selective Isolation of normal stem cells in leukemic marrow samples.  REFERENCES 1.  Magll MC, Iscove NN, Odartchenko N: Transient nature of early haematopoietic spleen colonies. Nature 295:527, 1982  2.  Hodgson GS, Bradley TR, Radley J M : The organization of hemopoietic tissue as Inferred from the effects of 5-fluorouracil. Exp Hematol 10:26, 1982  3.  BertonceUo I, Hodgson GS, Bradley TR: Multiparameter analysis of treinsplEmtable hemopoietic stem cells: I. The sepgiration and enrichment of stem ceUs homing to marrow and spleen on the basis of Rhodamine-123 fluorescence. Exp Hematol 13:999, 1985  4.  Ploemacher RE, Brons RHC: Separation of CFU-S from primitive ceUs responsible for reconstitution of the bone marrow hemopoietic stem cell compartment foUowIng Irradiation: Evidence for a pre-CFU-S ceU. Exp Hematol 17:263, 1989  5.  Jones RJ, Wagner J E , Celano P, Zlcha MS, Sharkis SJ: Separation of pluripotent haematopoietic stem cells from spleen colony-forming cells. Nature 347:188, 1990 (letter)  6.  Fraser CC, Eaves C J , Szflvassy S J , Humphries RK: Expansion in vitro of retrovlrally marked totipotent hemopoietic stem cells. Blood 76:1071, 1990  7.  v£m der Sluljs JP, de Jong JP, Brons NHC, Ploemacher RE: Marrow repopulating ceUs, but not CFU-S, establish long-term in vitro hemopoiesis on a marrow-derived stromal layer. Exp Hematol 18:893, 1990  8.  Yeager AM, Kalzer H, Santos GW, Saral R, Colvln OM, Stuart RK, Bralne HG, Burke P J , Amblnder RF. Bums WH, FuUer D J , Davis J M , Karp J E , Stratford M, Rowley SD, Sensenbrenner LL, Vogelsang GB. WIngard JR: Autologous bone marrow transplantation In patients with acute nonljnnphocytic leukemia, using ex vivo marrow treatment with 4-hydroperoxycyclophosphamide. N Engl J Med 315:141, 1986  9.  Wlnton EF, Colenda KW: Use of long-term human marrow cultures to demonstrate progenitor ceU precursors in marrow treated with 4-hydropero3qrcyclophosphamlde. Exp Hematol 15:710, 1987  10. Sutherland HJ. Eaves C J , Eaves AC, Dragowska W, Léinsdorp PM: Characterization and pgirtial purification of humem marrow ceUs capable of Initiating long-term hematopoiesis In vitro. Blood 74:1563, 1989 11. Andrews RG, Singer JW, Bemstein ID: Precursors of colony-forming ceUs In humans can be distinguished from colony-forming cells by expression of the CD33 and CD34 antigens and light scatter properties. J Exp Med 169:1721, 1989 12. Lansdorp PM, Sutherland HJ, Eaves C J : Selective expression of CD45 isoforms on functional subpopulations of CD34+ hemopoietic ceUs from human bone marrow. J Exp Med 172:363, 1990 13. Mulder AH, Visser JWM: Separation and functional analysis of bone marrow cells separated by rhodamlne-123 fluorescence. Exp Hematol 15:99, 1987  14. Johnson LV, Walsh ML, Chen LB: Localization of mitochondria in living cells with Rhodamine-123. Proc Natl Acad Sci U S A 77:990, 1980 15. Cohen RL, Mulrhead KA, Gill J E , Waggoner AS, Horan PK: A cyanlne dye distinguishes between cycling and non-cycling fibroblasts. Nature 290:593, 1981 16. James TW, Bohman R: Proliferation of mitochondria during the cell cycle of the human ceU line HL-60. J Cell Physiol 89:256, 1981 17. Darzynklewicz Z, Stalémo-Colco L, Melamed MR: Increased mitochondrial uptake of rhodamlne 123 during lymphocjrte stimulation. Proc Natl Acad Sci U S A 78:2383, 1981 18. Civin CI, Banquerigo ML, Strauss LC, Loken MR: Antigenic analysis of hematopoiesis. VI. Flow C5d;ometric characterization of My-lO-positive progenitor cells In normal human bone marrow. Exp Hematol 15:10, 1987 19. Becker AJ, McCulloch EA, Siminovitch L, Till J E : The effect of differing demands for blood cell production on DNA sjmthesis by hemopoietic colony-forming cells of mice. Blood 26:296, 1965 20. Fauser AA, Messner HA: Proliferative state of human plurlpotent hemopoietic progenitors (CFU-GEMM) In normal individuals and under regenerative conditions after bone marrow transplantation. Blood 54:1197, 1979 21. Cashman J , Eaves AC, Eaves C J : Regidated proliferation of primitive hemopoietic progenitor cells in long-term human marrow cultures. Blood 66:1002, 1985 22. Sutherland HJ, Lansdorp PM, Henkelman DH, Eaves AC, Eaves CJ: Functional characterization of individual human hemopoietic stem cells cultured at limiting dilution on supportive marrow stromal layers. Proc Nati Acad Scl U S A 87:3584, 1990 23. Eaves CJ. Eaves AC: Cell culture studies In CML, In Goldman J M (ed): Ballllere's Clinical Haematology. Vol. 1, #4. Chronic Myeloid Leukaemia. London, Ballllere Tindall, 1987, pp 931 24. Minden MD, Buick RN, McCulloch EA: Separation of blast cell and T-lymphocjd;e progenitors in the blood of patients with acute myeloblastic leukemia. Blood 54:186, 1979  CHAPTER  IV  CHARACTERIZATION OF PRIMITIVE HEMOPOIETIC CELLS IN NORMAL HUMAN PERIPHERAL BLOOD 1. INTRODUCTION  The presence of primitive hemopoietic cells In adult peripheral blood has been recognized for tiiree decades. Initial experiments showed that peripheral blood from a variety of species, including man, was capable of protecting recipients from lethal doses of whole body Irradiation by restoring blood cell formation from circulating donor cells. ^'^ Subsequent studies led to the demonstration and quantitation of specific progenitor populations detected by colony assays. ^"^ This prompted evaluation of leukapheresls harvests as an cdtemative source of cells for therapeutic applications where autologous or allogeneic marrow transplants were not feasible.^'12 More recentiy. Identification of strategies for Increasing the concentration of clonogenic progenitors in the cfrculation has heightened Interest in the potential of peripheral blood harvests for clinical treatment protocols requiring hematologic rescue. 13-16  Under optimized assay conditions, LTC-IC in human marrow can be quantitated by limiting dilution analysis 1^ and have been shown to have several characteristics of quiescent cells. These include a relative insensitivity to 4-hydroperoxycyclophosphamide (4-HC), ^3.19 retention of rhodamine-123 (Rh-123),20 smsdl size,^0,21 ^nd low expression or absence of CD71 (the transferrin receptor)22.23 ^nd H L A - D R ^ l At least a proportion of the LTC-IC in normal adult human marrow demonstrate multipotentiality in the LTC systemic and malntcdn thefr numbers at levels comparable to CRU in analogous murine cultures.^'*'^^ Several years ago. Eaves et al. reported that myeloid clonogenic cells were generated for at least 2 months in LTC cultures Initiated by seeding T cell-depleted light density peripheral blood cells onto irradiated, pre-established allogeneic marrow adherent layers,^6 suggesting the normal presence of some  LTC-IC In the circulation. In this chapter, I undertook studies to determine whether these cells coidd be quantltated using the same conditions and limiting dilution procedure originally developed and applied to the detection of LTC-IC in human marrow^^'^l and then analyze some of their properties.  2. RESULTS  2.1  Quantitation of LTC-IC In Normal Blood In an Initial series of experiments, the number of clonogenic cells present after 5 weeks  In LTC initiated with T cell-depleted suspensions of normal peripheral blood mononuclear cells seeded onto pre-established, irradiated marrow adherent layers was found to be a linear function of the number of cells initially added over a 1000-fold range of Input cell numbers. Results for a representative experiment are shown in Figure 13. Three such dose response experiments also included a series of assay cultures (20-25 per point) which were seeded with limiting numbers of LTC-IC (I.e., about 1 LTC-IC per assay culture). From the proportion of positive and negative assay cultures (containing ^1 clonogenic cell each, or none, respectively,) absolute frequencies of LTC-IC In the original test cell suspension were calculated using Poisson statistics27.28 (pigure 14).  FIGURE 13. Linear relationship between the number of light density (<1.077g/cm®) T celldepleted peripheral blood cells from a representative normal Individual seeded onto preestablished. Irradiated normal marrow feeders and the total number of clonogenic cells detected when these LTC were harvested and assayed in methylcellulose 5 weeks later. The slope of the regression line fitted to this data set Is 0.92 ± 0.09.  FIGUKE 14. Limiting dilution analysis of data from a representative experiment In which decreasing numbers of light-density T cell-depleted normal peripheral blood cells were seeded onto irradiated marrow feeders and the number of clonogenic cells detectable after 5 weeks was then determined. For this experiment, the frequency of LTC-IC in the starting cell suspension (l.e., the reciprocal of the concentration of test cells that gave 37% negative culttires) was 1 per 1.5 X 10^ ceUs (95% confidence limits = 1 per 9.9 x lO'* - 1 per 2.2 x 10^ cells).  From this value and a knowledge of the total number of clonogenic cells produced by a large number of cells of the same input suspension, the average 5 week output of clonogenic cells per LTC-IC in normal blood was calculated. This value was found to be 3.7 ± 1.2,^9 which is similar to the value of 4.3 ± 0.4 that was reported for LTC-IC in normal marrow.  Bulk  measurements of the 5 week clonogenic cell content of assay cultures Initiated with T celldepleted blood samples from other normal adults could then be used to derive absolute LTC-IC per mL values using this average clonogenic output per LTC-IC conversion factor. Table 7 shows the average concentration of LTC-IC In the peripheral blood calculated from values measured on 23 normal adults, together with the average concentration of circulating clonogenic cells (BFU-E plus CFU-GM plus CFU-GEMM) obtained for the same 23 samples. The derived vEdue  of ~3 LTC-IC per mL Is ~75-fold lower than the concentration of circulating clonogenic  cells and hence the frequency of LTC-IC relative to other nucleated cells In the blood (~1 per 2 X 10®) is ~ 100-fold lower than the frequency of LTC-IC relative to other cells in the marrow.  Table 7. guantltatlon of LTC-IC and Clonogenic Cells In Normal Blood Cell Type  Concentration (per mL)  BFU-E  170 ±  20  CFU-GM  51 ±  5  CFU-GEMM  4.6 ±  0.6  LTC-IC  2.9 ±  0.5  Values for Individual samples were cedculated by multiplying the progenitor frequency per 10^ cells by the total nucleated cell recovery after both the T cell-depletion and FH density centrifugation steps and then again by the WBC per mL. Vadues shown are the mean ± SEM of data obtained from 23 different normal Individugds.  2.2  Phenotype of Circulating LTC-IC The distributions of LTC-IC and clonogenic cells in various phenotypically-deflned  subpopulations of the T cell-depleted, light density fraction of normal peripheral blood were then assessed. These were obtained using the FACS to separate cells on the basis of thefr light scattering properties, expression of CD34, HLA-DR, and Rh-123 uptake. As illustrated in Figure 15, even after removal of the T cells from the light density fraction of leukapheresls samples, the frequency of CD34'*' cells was still very low (0.1-0.5%) as compared to normal marrow, where values of 1-4% are typically obtained.^O Most of the CD34"'" cells (defined by the vertical gate shown in Figure 15B) express no or low levels of HLA-DR (Figure 15C), and have low SSC properties (Figure 15E and F). Cells with a CD34+ and HLA-DR^^^ phenotype (defined by the horizontal gate shown in Figure 15C) are found almost exclusively amongst the smallest light density cells (low FSC, Figure 15E).  FIGURE 15. Bivariate contour histograms of light density T cell-depleted normal peripheral blood cells stained with anti-CD34 and a n t l - H L A - D R in the low side scatter window (fraction I + II in Figure 16A) (Panel B) and after gating for C D 3 4 + (Panel C). The light scattering properties of C D 3 4 + H L A - D R 1 O W and C D 3 4 + H L A - D R + cells are demonstrated In Panels E and F, respectively. Unsorted unstained control and irrelevant (1D3) antibody-stained cells are shown in Panels D and A , respectively.  Figure 16 shows the distributions of LTC-IC and clonogenic cells observed when the total light density T cell-depleted fraction of peripheral blood cells was subdivided into 3 populations defined by their light scattering properties: I -low FSC, low SSC; II - Intermediate to high FSC, low SSC; and III - aU remaining cells (i.e. open FSC, intermediate SSC). Although each gated population contained approximately equal numbers of cells, virtually all LTC-IC and most of the clonogenic cells were conslstentiy found In the fraction containing the smallest cells (I). No LTCIC and less than 5% of all clonogenic cells were found In fraction III. Therefore In subsequent sorts, only cells In the low SSC fi-actions (I and II) were analyzed.  I  n  m  F I G U R E 1 6 . Light scatter profiles of T cell-depleted light density normal blood cells (Panel A). The mean ± SEM of the percentages of nucleated cells (open bar), clonogenic cells (stippled bar), and LTC-IC (solid bar) in each sorted fraction are shown in Panel B (n=4). A difference between the recovery of LTC-IC in fraction I and II are significant (p<0.0001).  Figure 17 shows the results of functional assays performed on cells sorted both according to their expression of CD34 and HLA-DR. In this case, only CD34"*' cells were assayed and those defined as HLA-DR^°^ (as In Figure 15C) were ftirther subdivided Into an HLA-DR' and an HLA-DR* population. It can be seen that no LTC-IC and very few directly clonogenic ceUs were HLA-DR+. However, further subdivision of the remaining CD34+, H L A - D R 1 ° ^ cells allowed some differential separation of LTC-IC and directly clonogenic cells, more of the latter (-10% vs -40%) being found In the HLA-DR* fraction.  CO  ^1  CD34+DR"  CD34+DR*  CD34+DR+  FIGURE 17. A representative histogram of 0034"*". light density T-cell depleted normal blood cells (in the previously described low SSC window shown in Figure 16A) double-stained with PE conjugated antl-HLA-DR and sorted into CD34+DR", CD34+DR* and CD34+DR+ fractions (Panel A). The dark histogram In Panel A shows the profile for unstained cells. The mean ± SEM of the percentages of nucleated cells (open bar), clonogenic cells (stippled bar), and LTC-IC (solid bar) in each sorted fraction are shown in Panel B (n=3). Differences between the recoveries of clonogenic cells and LTC-IC in CD34'^DR" and CD34+DR+ fractions are significant (p<0.02).  Enrichment and recovery values for LTC-IC and clonogenic cells, as compared to the imstalned light density T cell-depleted starting population from these experiments, are shown in Table 8. Recovery of LTC-IC in the CD34+, HLA-DR^o^ fraction was >100% in all 5 experiments performed suggesting that all cfrculating LTC-IC are also CD34+, as they are in normal marrow. 21 Because of the initial very low frequency of LTC-IC In peripheral blood. Isolation of cells defined by the same properties as previously applied to meirrow (I.e., density, light scatter, and expression of CD34 and HLA-DR), allowed a greater enrichment (~2,000-fold) of cfrculating LTC-IC to be routinely obtained with the final purity of LTC-IC achievable from normal peripheral blood (Table 8) and marrowl^'20 using these parameters being approximately the same. Recovery of clonogenic cells In these same experiments appeared to be somewhat lower, although on average there was no significant difference from the starting value measured on the unstained sample (Table 8). Failure to detect additional clonogenic cells in the higher FSC/SSC fraction (II and III) due to potential inhibition of thefr colony-forming ability by the presence of Increased numbers of monocjdies was ruled out by mixing experiments (i.e. no reduction of clonogenic cells detected when cells from Fraction I were mixed with cells from Fraction III In a 1:2 ratio; data not shown).  Table 8.  Cell Type Evaluated LTC-IC  Frequency, Enrichment and Recovery of LTC-IC^ and Clonogenic Cells in the CD34''', Light Density Fraction of Normal Peripheral Blood Cells Source unsorted cells  Frequency  % Recovery'l  No. of Exp  0.0022 10.0004  5  oe  DR+  Clonogenic cells  Enrichment^  5  DRlowf  3.7 ± 1.1  1930 ± 470  300 ± 40  2  DR±g  1.0 ± 0.5  540 ± 270  48 ± 36  3  DR-h  2.8 ± 0.5  1470 ± 340  280 ± 17  3  unsorted cells  0.11 ±0.02  DR+  7.6 ± 0.4  65  DR±g  21  ± 2  DR-h  15 ± 4  5 2.7 ±1.2  4  240 ± 40  21  ± 6  4  160 ± 40  38  ± 5  4  ± 22  a  Measured as the number of clonogenic cells present after 5 weeks  b  Frequency of the cell type eveduated (LTC-IC or total clonogenic cells) relative to all nucleated cells In the popidatlon analyzed. Cedculated by dividing the progenitor frequency per 10^ sorted cells by the progenitor frequency per 10^ unsorted, Ught-denslty cells In each Individual experiment, and then deriving the mean ± SEM of these values for the (n) experiments performed. Calculated by multlpl)dng the percentage of cells retrieved In the fraction Indicated by the corresponding calculated enrichment in each individual experiment, and then deriving the mean ± SEM of these vîdues for the (n) experiments performed.  ^  None detected; I.e., <0.15. Enrichment and recovery values therefore not calculated,  f  See text and Figure 15 for gating criteria.  ë  Refers to a subpopulatlon of CD34''" cells defined as fraction 2 in Figure 17A.  ^  Refers to a subpopulation of CD34"'" cells defined as fraction 1 in Figure 17A.  The results of combined staining for CD34 expression and Rh-123 uptake are shown In Figure 18. In this case, no difference was noted between circulating LTC-IC and clonogenic cells in terms of their distribution between the Rh-123^^11 and Ph-I23^^êl^^ fractions with more than 80% of both being found in the Rh-123^"^ fraction. This contrasts with normal marrow, where most of the LTC-IC are also Rh-123^"^ but most of the clonogenic cells are Rh-123^^ël^t thus allowing thefr differential Isolation by sorting. ^0 Nevertheless, the final purity of LTC-IC in the Ught density, T ceU-depleted, CD34+, Rh-123^"ll fraction of normal blood was simUar to that obtainable by appUcation of the same criteria to marrow. This reflects the greater overaU enrichment (~2000-fold, data not shown) achieved with blood versus marrow using these parameters.  RHODAMINE-123  CD34+Rh-123-dull CD34+Rh-123-bright FIGURE 18. A representative histogram of CD34+, light density T cell-depleted normal blood cells double-stained with Rh-123 and sorted Into CD34+Rh-123*^"l^ and CD34-^-123'5'^*>t fractions (Panel A). The dark histogram in Panel A shows the profile for unstained cells. The mean ± SEM of the percentages of nucleated cells (open bar), clonogenic cells (stippled bar), and LTC-IC (solid bar) In each sorted fraction are shown In Panel B (n=3).  2.3  4-HC Sensitivities of Circulating Progenitors Because normal circulating clonogenic cells were known to be a quiescent population*^ 1  and appeeired phenotypicedly to be more similar to LTC-IC In either blood or marrow than to the clonogenic cells found in the marrow, it was of interest to compgu-e the sensitivities of circulating clonogenic cells and LTC-IC to 4-HC, using the same type of treatment protocol that is in widespread clinical use for treating autologous marrow transplemts. In this set of experiments, LTC-IC function (before or after exposure to 4-HC) was assessed In terms of the clonogenic cell content of assay cultures evaluated after 4 and 8 weeks (rather them after 5 weeks as In the experiments described above), since previous experiments had revealed differences In LTC assays of marrow samples for autologous transplants when these two time points were compared. ^^'^^ Results for LTC-IC and clonogenic cells In normal peripheral blood and marrow are shown In Figure 19. A dramatic difference In the effect of a 30 minute exposure at 37°C to lOOfig/mL of 4-HC on the viability of clonogenic cells from blood and marrow Is appsirent (p<0.05). Conversely, normal circulating clonogenic cells and LTC-IC appear to be similar to marrow LTC-IC In their relative resistance to 4-HC. For LTC-IC from both sources, a subtie Increase In 4-HC resistance was noted for LTC-IC defined by the longer clonogenic cell output endpoint (I.e. 8 weeks).  1000  FIGURE 19. Comparison of the number of clonogenic cells émd LTC-IC surviving a 30 minute exposure to 100 Mg/ml of 4-HC at 37°C with 7% erythrocytes present. Values shown are the mean ± SEM of the percentages of clonogenic cells and LTC-IC from normal marrow (open bars) and normal blood (solid bars) as a percent of values for control cells (n=3 for marrow and n=4 for blood cells).  2.4  Differentiative Potential Erpressed by Circulating LTC-IC In LTC Table 9 shows the relative proportions of BFU-E. CFU-GM and CFU-GEMM In the total  clonogenic population of 5 week-old LTC Initiated with circulating LTC-IC of varying purities, and compares these to the relative numbers of these same types of clonogenic cells in the original blood samples. Data for unseparated gmd LTC-IC enriched cell populations from norméd marrow obtained in previous studies^l are also shown In Table 9 for comparison. It can be seen that the differentiative behavior exhibited by LTC-IC in normal blood and marrow is similar and is also not affected by the purity of the LTC-IC In the starting population. In both cases, a significant skewing towards the generation of CFU-GM by comparison to the number of CFU-GM and BFU-E actually found In normal blood or marrow was observed. To some extent this might be expected because all stages of granulopoietic cell differentiation are supported in the LTC system whereas erythropolesls appears to be blocked at the stage of mature BFU-E production.®^ As a result, this latter contribution to total BFU-E numbers in vivo is absent from LTC-derived populations.  Table 9.  Relative Proportions of Different Types of Clonogenic Cells Detected Before and After 5 weeks in LTC (% of total).  Original Progenitor Source  No. of Samples  Clonogenic cells^  BFU-E CFU-GM  LTC-IC"^  CFU-GEMM  BFU-E  CFU-GM  CFU-GEMM  Light  density fraction of normal bloodC  23  74 ± 3  24 ± 2  2.2 ± 0.3  11 ± 2  89 ± 2  0.5 ± 0.2  enriched fraction of normal blood«^  6  72 ± 5  28 ± 5  0.8 ± 0.3  11 ±1  89 ± 2  1.010.6  Normal marrow^  10  3613  6214  1.210.2  912  9112  0.810.3  LTC-IC enriched fraction of normal marrow^  10  2415  7515  0.410.3  913  9014  1.410.8  LTC-IC  Values shown are the meem 1 S E M of proportions measured in standard short-term methylcellulose assays. Values shown are the mean 1 SEM of proportions measured In methylcellidose assays of cells from 5 week-old LTC. Same samples as In Table 7. Data from LTC-IC in fraction I (CD34+, DR") In Figure 17 (n=3) and fraction I (CD34+. Rh123dull) in Figure 18 (n=3). Data for normal marrow from previously published studies.  3. DISCUSSION  In Chapter III, I have described how the LTC system may be used to quemtitate and characterize a very primitive cell in the marrow of normal adults. Key to the interpretation of data obtained with this approach is the use of a competent feeder layer onto which the test cells are seeded so that the endpolnt of hemopoietic activily measured several weeks later is determined solely and quantitatively by the number of primitive hemopoietic cells (LTC-IC) Initially present. In addition, the duration of time allowed to elapse prior to assessment of the hemopoietic cell content of the culture and the level of differentiation of the hemopoietic cells measured are importemt. A minimum of 5 weeks is required for most input clonogenic cells to differentiate and disappear. ^1 Problems may also occur If quantitation of terminally differentiating granulocj^es and macrophages are used as a read-out of input hemopoietic potential since the production of clonogenic cells and their subsequent differentiation into mature progeny in these cultures appear to be differentiy regulated.®® In the present study I have explored the usefulness of the LTC-IC assay originally validated for human marrow cell suspensions, for assessment of primitive hemopoietic cells In normal peripheral blood. An additional requirement, noted previously, was the need to rigorously remove T cells from the mononuclear fraction of peripheral blood samples to be tested for their LTC-IC content in order to circumvent the otherwise frequent spontaneous emergence of rapidly growing EBVtransformed lymphocytes during the 4 to 8 week period required to complete the LTC-IC assay. 2®  My results show that LTC-IC can be reproduclbly quantitated and found to be present, albeit at low levels. In normal adult blood. Similar findings have also recentiy been reported by Dooley and Law.®'^ I have further shown that normal circulating LTC-IC are indistinguishable from normal LTC-IC in normal marrow In terms of the number and types of clonogenic progeny they produce and In terms of their expression of CD34, ability to retain Rh-123, and sensitivity  to 4-HC. Two interesting differences, however, are the apparent smaller size (lower FSC) and lack of detectable expression of HLA-DR by most circulating LTC-IC. Interestingly, although the concentration of circulating LTC-IC cells Is much lower (~75-foId) than the concentration of circulating clonogenic cells, the phenotypic characteristics of these functionally distinguished populations are very similar. Whether circulating LTC-IC simply represent a subset of cells also detectable by standard clonogenic cell assays thus remains unresolved.  Eventually, It might be possible to replace the adherent flbroblast-llke "stromal" cells by a soluble source of the relevant factors they produce, and some success along these lines has recently been reported. ^5 However, other studies have shown that the self-maintenance of LTCIC as well as their ability to generate clonogenic progeny In the LTC system can be fully retained when LTC-IC are co-cultured with murine Sl/Sl fibroblast feeders that do not contain the Steel gene and also do not express detectable G-CSF or IL-6.36 Such feeders, of course, also do not produce any species-specific human factors (e.g., GM-CSF and IL-3) indicating that other, as yet undefined factor(s) must be able to support the medntenance and initial differentiation of human LTC-IC. Clearly, further studies will be required to establish the molecular identity of these factor(s) £md to determine whether a cellular mode of their presentation is important, and to evaluate whether LTC-IC In marrow and blood are similarly regulated by such factors.  In the next chapter I will Investigate whether the LTC-IC assay can also be used to quantitate what appears to be a developmentally analogous primitive neoplastic (Ph^-positive) progenitor In patients with chronic myeloid leukemia (CML).-^^ The present studies thus also serve as an Important baseline for compeirlson with these CML LTC-IC and will likely facilitate future analyses of abnormal properties of neoplastic stem cells In patients with other types of myeloproliferative or myelodysplastic clones.  REFERENCES 1.  Brecher G. Cronklte EP: Post-radiation parabiosis and survival in rats. Proc Soc Exp Biol Med 77:292. 1951  2.  Goodman JW. Hodgson GS: EMdence for stem cells in the peripheral blood of mice. Blood 19:702. 1962  3.  Epstein RB. Graham TC, Buckner CD: Allogeneic marrow engraftment by cross circulation in lethally irradiated dogs. Blood 28:692. 1966  4.  Storb R, Graham TC. Epstein RB. Sale GE. Thomas ED: Demonstration of hemopoietic stem cells in the peripheral blood of baboons by cross circulation. Blood 50:537. 1977  5.  Abrams RA. Glaubiger D. Appelbaum FR, Deisseroth AB: Result of attempted hemopoietic reconstitution using isologous, peripheral blood mononuclear cells: A case report. Blood 56:516, 1980  6.  McCredie KB, Hersh EM, Freireich E J : Cells capable of colony formation in the peripheral blood of man. Science 171:293, 1971  7.  Chervenick PA. Boggs DR: In vitro growth of granulocytic and mononuclear cell colonies from blood of normal Individuals. Blood 37:131. 1971  8.  Clarke B J . Housman D: Characterization of an erythroid precursor cell of high proliferative capacity in normal human peripheral blood. Proc Natl Acad Sci U S A 74:1105. 1977  9.  Korbling M. Dorken B. Ho AD. Pezzutto A. Hunstein W. FUedner TM: Autologous transplantation of blood-derlved hemopoietic stem cells after myeloablative therapy in a patient with Burkitt's lymphoma. Blood 67:529. 1986  10. Reiffers J . Bernard P, David B, Vezon G. Sarrat A. Marit G. Moulinler J . Broustet A: Successful autologous transplantation with peripheral blood hemopoietic cells in a patient with acute leukemia. Exp Hematol 14:312. 1986 11. Juttner CA, To LB, Ho JQK, Bardy PG, Dyson PG, Haylock DN, Kimber RJ: Early Ijnnpho-hemopoletic recovery after autografting using peripheral blood stem cells In acute non-lymphoblastic leukemia. Transplant Proc 20:40, 1988 12. Kesslnger A, Armltage JO, Landmark JD, Smith DM, Weisenburger DD: Autologous peripheral hemopoietic stem cell transplantation restores hemopoietic function following marrow ablative therapy. Blood 71:723, 1988 13. Richman CM. Welner RS, Yankee RA: Increase In circulating stem cells following chemotherapy In man. Blood 47:1031, 1977 14. CUne M, Golde W: MobUlzation of hemopoietic stem ceUs (CFU-C) into the peripheral blood of man by endotoxin. Exp Hematol 5:186, 1977 15. Socinski MA, Cannlstra SA, EUas A, Antman KH, Schnlpper L, Grlflfln JD: Granulocyte-macrophage colony stimulating factor expands the circulating haemopoietic progenitor ceU compartment in man. Lancet 1:1194, 1988  16. Gianni AM, Siena S, Bregnl M, Tarella C, Stem AC, Plleri A, Bonadonna G: Granulocyte-macrophage colony-stimulating factor to harvest circulating haemopoletic stem cells for autotransplantation. Lancet 2:580, 1989 17. Sutherland HJ, Lansdorp PM, Henkelman DH, Eaves AC, Eaves C J : Functional characterization of Individual human hemopoietic stem cells cultured at limiting dilution on supportive marrow stromal layers. Proc Nati Acad Scl U S A 87:3584, 1990 18. Winton EF, Colenda KW: Use of long-term humem marrow cultures to demonstrate progenitor cell precursors In marrow treated with 4-hydroperoxycyclophosphamlde. Exp Hematol 15:710, 1987 19. Eaves C J , Sutherland HJ, Udomsakdl C, Lansdorp PM, Szllvassy SJ, Fraser CC, Humphries RK, Bamett M J , Phillips GL, Eaves AC: The human hemopoietic stem cell In vitro and In vivo. Blood Cells(In press):, 20. Udomsakdl C, Eaves C J , Sutherland HJ, Lansdorp PM: Separation of fimctionally distinct subpopulations of primitive human hemopoietic cells using rhodamine-123. Exp Hematol 19:338, 1991 21. Sutherland HJ, Eaves CJ, Eaves AC, Dragowska W, Lansdorp PM: Characterization and partial purification of human meirrow cells capable of Initiating long-term hematopoiesis Invlti-o. Blood 74:1563, 1989 22. Sutherland HJ, Eaves C J , Eaves AC, Lansdorp PM: Differential expression of antigens on cells that Initiate haemopoiesis In long-term human marrow culture. In Knapp W, Dorken B, Gilks WR, Richer EP. Schmidt RE, Stein H, von dem Borne AEG (eds): Leucocyte Typing IV. White Cell Differentiation Antigens. Oxford, Oxford University Press, 1989, pp 910 23. Lansdorp PM, Dragowska W: Long-term erythropoiesls from constant numbers of CD34''' cells In semm-free cultures Initiated with highly purified progenitor cells from human bone marrow. J Exp Med(ln press):, 24. Fraser CC, Szllvassy SJ. Eaves C J , Humphries RK: Proliferation of totipotent hemopoietic stem cells In vitro with retention of long-term competitive In vivo reconstituting ability. Proc Nati Acad Scl U S A 89:1968, 1992 25. Eaves C J , Cashman J D , Sutherland HJ, Otsuka T, Humphries RK, Hogge DE, Lansdorp PM, Eaves AC: Molecidar analysis of primitive hemopoietic cell proliferation control mechanisms. Ann N Y Acad Scl 628:298, 1991 26. Eaves AC, Cashman JD, Gaboury LA, Kalousek DK, Eaves CJ: Unregulated proliferation of primitive chronic myeloid leukemia progenitors In the presence of normal marrow adherent ceUs. Proc Nati Acad Scl U S A 83:5306. 1986 27. Porter EH, Berry RJ: The efficient design of transplantable tumour assays. Br J Cancer 17:583, 1963 28. Taswell C: Limiting dfiution assays for the determination of Immunocompetent cell fi-equencles. 1. Data analysis. J Immunol 126:1614, 1981  29. Udomsakdi C, Eaves C J , Swolln B, Reid DS, Bamett MJ, Eaves AC: Rapid decUne of chronic myeloid leukemic cells in long-term culture due to a defect at the leukemic stem cell level. Proc Natl Acad Sci U S A(in press):, 30. Clvln CI, Trischmann TM, Fackler MJ, Bemstein ID, Buhring H-J, Campos L, Greaves MF, Kamoun M, Katz DR, Lansdorp PM, Look AT, Seed B, Sutherland DR, Tlndle RW, Uchanska-Ziegler B: Report on the CD34 cluster workshop. In Knapp W, Dorken B, Gllks WR, Rieber EP, Schmidt RE, Stein H (eds): Leucocyte Typing IV. White Cell Differentiation Antigens. Oxford, Oxford University Press, 1989, pp 818 31. Eaves C J , Eaves AC: Cell culture studies In CML, in Goldman J M (ed): Bailliere's Clinical Haematology. Vol. 1, #4. Chronic Myeloid Leukaemia. London, Bailllere Tlndall. 1987, pp 931 32. Coulombel L, Eaves AC, Eaves CJ: Enzjonatlc treatment of long-term humem marrow cultures reveals the preferential location of primitive hemopoietic progenitors in the adherent layer. Blood 62:291. 1983 33. Sutherland HJ. Eaves CJ. Lansdorp PM. Thacker JD, Hogge DE: Differential regulation of primitive human hemopoietic cells in long-term cultures maintained on genetically engineered murine stromal cells. Blood 78:666, 1991 34. Dooley DC, Law P: Detection and quemtitation of long-term culture-Initiating cells In normal human peripheral blood. Exp Hematol 20:156, 1992 35. Brandt J , Srour EF, van Besien K, Briddell RA, Hoffman R: Cytokine-dependent long-term culture of highly enriched precursors of hemopoietic progenitor cells from human bone marrow. J CUn Invest 86:932, 1990 36. Sutherland HJ, Hogge DE, Cook DN, Eaves CJ: Long-term maintenance of highly purified human stem ceUs on humgm and murine fibroblast feeders is not influenced by conditions that either increase or decrease exposure to steel factor. Blood 78 (suppl l):256a, 1991 (abstr)  CHAPTER  V  RAPID DECLINE OF CHRONIC MYELOID LEUKEMIC CELLS IN LONG-TERM CULTURE IS DUE TO A DEFECT AT THE LEUKEMIC STEM CELL LEVEL  1. INTRODUCTION  As reviewed In Chapter 1, CML Is a multl-llneage clonal hemopoietic malignancy characterized by excessive production of granulocytes and the presence In the leukemic cells of a consistent rearrangement of the BCR and ABL genes, tjrplcally memlfested In metaphase preparations as the Philadelphia chromosome (Ph^). ^ The Initial cell tremsformed and hence the origin of the leukemic clone Is believed to be a totipotent hemopoietic cell with lymphoid as well as myeloid differentiation potential since Ph^-positive cells In these lineages are frequentiy demonstrable.^ This has suggested that production of the BCR-ABL kinase In a totipotent hemopoietic cell gives It a selective growth advamtage. Recent experiments Involving retroviral Infection of murine bone meurow cells with BCR-ABL constructs are consistent with thls^-^ although an underljing molecular mechanism has not been determined. In particular, the biological consequences of BCR-ABL kinase expression In very primitive human hemopoietic cells has been difficult to Investigate because methods for their selective Isolation have not been available.  CML patients with elevated white blood cell (WBC) counts show dramatic increases in the number of Ph^-positive clonogenic progenitors in their c i r c u l a t i o n ^ a n d previous studies in the Teny Fox Laboratory have shown that continued production of Ph^-positive clonogenic cells for many weeks can occur at a high level when peripheral blood cells from such patients are cultured on irradiated marrow cell adherent layers established from normal individuals.^  Recently, it was shown that the number of clonogenic cells present after 5 to 8 weeks in similar cultures initiated with normal hemopoietic cells allows the detection of a very primitive class of clonogenic cell precursors that exhibit properties characteristic of cells with long-term in vivo reconstituting potential.®"!'^ These normal human "long-term culture-initiating cells" (LTC-IC) can be quantltated by limiting dilution analysis, which then allows the proliferative potential of individual LTC-IC to also be determined. ^ ^ In this chapter are described experiments that show how this approach can be applied to detect and queintltate leukemic LTC-IC from patients with CML and that these cells exhibit both similarities and differences In their behaviour by comparison to normal LTC-IC.  2. RESULTS  2.1  Assays for CML LTC-IC LTC-IC are defined as cells that give rise to clonogenic hemopoietic cells (i.e., BFU-E,  CFU-GM and/or CFU-GEMM) detectable after a period of initial culture for 5 weeks on competent feeder layers. ^ ^ Such feeders can be provided in a standardized form by subculturing into the LTC-IC assay cultures a fixed number of cells per imlt area using 2 to 6 week-old normal mêirrow-derlved LTC adherent layer cells as a source of feeder cells. The validity of using the clonogenic cell producing property of LTC-IC as an endpolnt for their quantitation depends, however, on the existence of a lineeir relationship between the number of LTC-IC seeded into the cultures and the number of clonogenic cells present 5 weeks later regardless of the input LTC-IC concentration. This was previously demonstrated for assays of LTC-IC in normEd marrow cell suspensions, ^ ^ and in T cell-depleted light density cell suspensions from normal blood.  Previous studies had shown that LTC Initiated with peripheral blood cells from CML patients with high WBC counts (and a marked elevation In circulating Ph^-positive  progenitors)^, when analyzed 4-8 weeks later contained high numbers of exclusively Ph^positlve clonogenic progenitors^ in contrast to LTC initiated with CML marrow.  This  suggested that it might be possible to use CML peripheral blood from such patients as an enriched source of primitive leukemic cells to investigate the relationship between cell input and leukemic clonogenic cell output 5 weeks later. In a series of 8 such experiments (with samples from 8 different patients with high WBC counts) in which the number of light density peripheral blood Input cells was varied from 5 x 10^ to a maximum of 10^ cells per 2.5 ml LTC (in 35 mm tissue culture dishes), the slope of the line relating the logarithm of the Innoculum size (total nucleated cells) to the logarithm of the number of clonogenic cells detected after these LTC had been maintained for 5 weeks was 1.05 ± 0.21 (which is not significantly different from 1.0, p>0.5). The results for a representative patient are Illustrated in Figure 20. Thus, conditions developed for normal LTC-IC appear to also be suitable for the detection and quantitation of Phi-positive LTC-IC.  10"*  10^  Initial Cells per  10^  10  LTC  FIGUKE 20. Linear relationship between the number of light density CML peripheral blood cells seeded Into individual LTC (containing irradiated pre-established normal marrow feeders) and the number of clonogenic cells detected in secondary methylcellulose assays of cells harvested from these primary LTC 5 weeks after their Initiation. Each point represents a single LTC. All points are derived from a single representative experiment using cells from a CML patient with a WBC count of 190 x lO^/L. The slope of the regression line fitted to this data set is 0.81 ± 0.13.  2.2  Measurements of Leukemic LTC-IC In CML Blood and Marrow Because the relative output of clonogenic cells from cfrculating leukemic LTC-IC was  found to be constant imder the assay conditions used even down to limiting numbers of input cells (e.g., see Figure 20), quantitation of absolute leukemic LTC-IC numbers by limiting dilution analysis was possible. For such experiments, blood from CML patients with elevated WBC counts was again used as a highly and selectively enriched source of leukemic LTC-IC. Irradiated normal marrow derived feeders were subcultured into 96 well flat bottom Nunclon plates 11 and then from 50 to 2 x 10^ light density cells added per well In volumes of 100 ^,1 with 23 ± 1 wells per group. Five weeks later, all of the cells in each well were suspended and plated In methylcellulose assay cultures to enable detection of one or more clonogenic cells per well. From the proportion of positive and negative LTC defined in this way, the frequency of LTC-IC In 6 different input samples was calculated using Poisson statistics. ^^^'^^ Results for a representative experiment are shown in Figure 21. From this, the average 5 week output of clonogenic cells per Ph^-positive LTC-IC was then derived in each case. The results of this latter cedculation are shown In Table 10 together with results obtained when the same procedure and assay conditions were used to analyze LTC-IC In normal marrow ^ ^ or blood.  The proliferative  potential of all of these types of LTC-IC as assessed by this 5 week clonogenic cell output endpolnt can be seen to be both similar and relatively constant, providing further support for the use of the LTC-IC assay to quantitate and characterize a very primitive Ph^-positive cell type.  FIGURE 21. Limiting dilution analysis of data from a representative experiment In which decreasing numbers of light density CML peripheral blood cells (from a patient with a WBC count of 21 x 10^/L) were seeded onto irradiated marrow feeders and the cultures then assayed 5 weeks later for the presence (positive cultures) or absence (negative cultures) of >1 clonogenic cell. In this experiment, the frequency of LTC-IC In the suspension assayed (i.e.. the reciprocal of the concentration of cells that gave 37% negative cultures) was 1 per 7.6 x lO'* cells (95% confidence limits = 1 per 5.3 x 10* - 1 per 11.0 x 10"*^).  Table 10. Proliferative Potential of Normal and leukemic LTC-IC No. of clonogenic cells per LTC-IC after 5 weeks* Normal Marrow  4.3 ± 0.4 (5)  Normal Blood  3.7 ± 1.2 (3)  CML Blood  3.1 ± 0.4 (6)  * Mean ±SEM of values from (n) experiments calculated by multiplying the frequency of LTC-IC In each experiment (determined by limiting dilution assays) by the total number of cells plated In all LTC to determine the total number of LTC-IC for that experiment. The total content of clonogenic progenitors In all LTC for an Individual experiment was obtained dfrectiy from clonogenic progenitor assays. Knowledge of the 5 week clonogenic cell output per leukemic CML allows absolute values to be derived from total 5 week clonogenic cell yields measured In cultures Initiated with nonUmltlng Innocula, which are experimentally easier to perform than limiting dilution analyses. LTC-IC values were thus obtained for peripheral blood samples from an additional 20 CML patients, and the concentration of LTC-IC per ml of blood then calculated assuming 100% LTCIC recovery In the light density fraction assayed. ^ The results are shown in Figure 22 together with cfrculating LTC-IC vedues obtained from similar measurements of T cell-depleted, light density peripheral blood samples from a large series of normal Individuals.  In Figure 22A,  LTC-IC concentrations In CML blood are plotted as a function of the WBC count. It can be seen that LTC-IC numbers Increase exponentially such that values >10^-fold higher than normal cfrculating LTC-IC levels are seen In patients with the largest tumor burdens. In Figure 22B, the number of cfrculating LTC-IC in individual CML patients is plotted as a function of the number of cfrculating clonogenic cells (BFU-E plus CFU-GM plus CFU-GEMM per ml) In the same patient. On average, leukemic LTC-IC were found to cfrculate at a 10-fold lower frequency than clonogenic cells although these two pargmieters showed a highly significant association (Spearman's rank correlation coefficient, rs=0.77, p<0.0001, n=26). By comparison the ratio of cfrculating LTC-IC to clonogenic cells in normal blood appears much lower (~1:80).  E  in4.  0) Q.  Q.  O  O  I  •  o  O  10'  10^  WBC per ml  10*  10^  10-»  10**  10=»  10°  10'  Clonogenic Cells per ml  FIGURE 22. LTC-IC concentration (per ml) in the peripheral blood of different CML patients (solid circles) as compared to 23 normal individuals (the open circle in each panel shows the mean ± SEM of 2.9 ± 0.5 LTC-IC per ml measured In these individuals) as a function of the WBC count (per ml)(Panel A), or the peripheral blood clonogenic progenitor (BFU-E + CFUG M + CFU-GEMM) content per ml (Panel B). Absolute LTC-IC values were obtained either dlrectfy by limiting dilution analysis, or indirectly from the total clont^enlc cell output measured at week 5 divided by the average number of clonogenic cells produced per LTC-IC, i.e.. 3 and 4 for CML and normal LTC-IC, respectively, as described in the text. A significant association between the two parameters measured in Panel B is indicated by a Spearmann's rank correlation coefficient rg=0.77 (p<0.0001). 03 00  LTC-IC assays were also performed using CML marrow samples (Table 11). However, in each of these experiments, cytogenetic analyses were performed on the colonies produced from the clonogenic progenitors present after 5 weeks in LTC to distinguish Ph^-positive and Ph^negative LTC-IC as, in contrast to CML blood, Ph^-positive LTC-IC would be anticipated to frequently represent a minority population relative to normal LTC-IC In CML marrow. 13,16 predicted by previous studies, the concentration of Ph^-positive LTC-IC (relative to other nucleated cells) in the 12 CML marrows emalyzed was quite variable and In general markedly reduced, both by comparison to LTC-IC vgdues In control marrows (I.e., ^2.8 ± 1.4 Ph^-positive LTC-IC per 10^ CML marrow cells as compared to 55 ± 12 LTC-IC per 10^ marrow cells from normal individuals, n=13), and by comparison to normal (Ph^-negative) LTC-IC co-existing in the same CML marrows tested (for which a value of 5.4 ±1.2 per 10® cells was obtained).  Table 11. Quantitation of LTC-IC in Normal and CML Marrow *  Clonogenic Cells per 10^ Normal marrow  1400 ± 200  CML marrow  1534 ± 661 (Phl+)  LTC-IC per 10^ 55 ± 12 <2.8 + 1.4(Phl+)  ^ Data provided from Stem Cell Assay Service, Terry Fox Laboratory with the courtesy of Dr. Connie Eaves  2.3  Differential Maintenance of Normal and Leukemic LTC-IC In Culture In previous studies it has been shown that normal marrow LTC-IC are well maintained in  LTC established from a single input innoculimil^' 1® and similar kinetics are seen when highly purified LTC-IC from normal marrow are seeded onto pre-established feeders.  Figure 23  shows the corresponding results obtained when light density peripheral blood cells from CML patients with high WBC counts were seeded onto irradiated human marrow feeders and the number of LTC-IC then followed by harvesting these primary LTC and performing secondary LTC-IC assays (as described in the Methods). For comparison analogous experiments were performed for primary LTC established by seeding light density, T cell-depleted normal peripheral blood or normal marrow buffy coat cells onto pre-established marrow feeders. It can be seen that normal LTC-IC maintenance in such cultures was the same regardless of the source of LTC-IC with no decrease in overall population size during the first 10 days. In contrast the leukemic LTC-IC population showed an immediate and rapid rate of decline down to ~3% of input values within the same initial period during which time the cultures had not been mcinlpulated in any way except to reduce the temperature from 37°C to 33°C.  0.01 r  I  I  1  I  L  0  2  4  6  8  Weeks in Culture FIGURE 23. Differential kinetics of CML (solid symbols) versus normal (open symbols) LTC-IC In LTC Initiated from cells seeded onto Irradiated normal marrow feeders. Values shown are mean ±SEM after normalization of data In Individual experiments by setting LTC-IC values In the primary Innoculum In each experiment to 100%; n=6 for CML (peripheral blood LTC-IC). n=5 for LTC-IC In normal blood (open circles) and n=2 for LTC-IC in normal marrow (open triangles). Open squares show previously published data for LTC-IC in normal unseparated marrow cultured in the absence of pre-established feeders.  3. DISCUSSION  In this report I describe the development and initial use of a quantitative assay for a primitive Ph^-positive cell that meets the definition of a LTC-IC; I.e., a cell that after a minimum period of 5 weeks in culture together with certain marrow adherent cells but in the absence of exogenous growth factors, will have produced detectable clonogenic progenitors. Appropriate purification studies cannot yet rule out the possibility that some LTC-IC (either normal or leukemic) are also detectable in either standard direct clonogenic assays or In the blast colony assays described by Ogawa and colleagues"^^ or Gordon et al.-^^ However, it shoiild be noted that none of these have used such a prolonged interval prior to assessment of clonogenic cell production and, indeed, if this requirement were imposed, would decrease considerably the quoted frequencies of any of these clonogenic cell types. Addltioned operational advantages of the LTC-IC assay eire its relative simplicity, ease of standardization, and applicability to quantitation of primitive hemopoietic cells in primary patient samples. The need for selective identification of psirticular colony subtypes In primsiry or secondary assays Is avoided and the requirement to use subcultured Irradiated normal human marrow feeder layers can be met by substituting murine fibroblasts.  By restricting my initial studies to examination of peripheral blood samples from CML patients with elevated WBC counts, the problem of contaminating normal LTC-IC contributing to the resvdts was circumvented as this proved to be a source of highly enriched Ph^-positive LTCIC (Figure 22 and Eaves et al.^). Ph^-posltlve LTC-IC were found to produce on average, a similar number of clonogenic cell progeny after 5 weeks In LTC as do their normal counterparts In the blood or marrow of normal individuals. However, a number of abnormalities In the CML LTC-IC population were also revealed. First, their distribution between marrow and blood was shown to be grossly altered, even more dreimatlcaUy than Is the case for Ph^-positive clonogenic cells. Both populations increase exponentially In the blood with linear Increases In the WBC  count, but Ph^-positive LTC-IC appear to be present at relatively reduced frequencies In CML marrow whereas Ph^-positive clonogenic cell frequencies in CML marrow are relatively normal.22 Second, in spite of a normal output of clonogenic cell progeny by Ph^-positive LTC-IC and the provision of a pre-established feeder derived from a normal marrow donor, their initial maintenance in the LTC system was highly compromised relative to normal LTC-IC. Whether this Is due to an intrinsic defect in the Ph^-positive LTC-IC that is not subject to extrinsic modulation and/or whether such differences may also prevail in vivo have yet to be determined. However, it is Interesting to speculate that the behavioiar of normal and leukemic LTC-IC In the LTC system may Indicate how these cells behave In vivo under gmalogous conditions of stimulation. One might then expect to see evidence of a growth advantage of the stem cells in the Ph^-positive clone in vivo only when most co-exlstlng normal stem cells were In a quiescent state. The latter might be anticipated to occur In chronic phase CML patients memaged with conventional therapy, but a situation more closely resembling that obtained In LTC might occur In vivo, albeit trémslently, following more Intensive treatment. It Is Interesting to note that clinical experience fits well with these predictions. ^^'^^  In human LTC, primitive normal clonogenic cells In the adherent layer alternate weekly between a quiescent and a dividing state^^ and In murine LTC, It has been possible to demonstrate that extensive proliferation of some long-term totipotent reconstituting cells does occur.26 In LTC initiated with Ph^-positive LTC-IC, their derivative primitive clonogenic progeny divide continuously suggesting a defective but unregulated mechemlsm for Inevitable expansion of the Ph^-positive clone. Thus LTC may serve as an Important model for further dissection of the mechanisms that regulate normal versus CML recovery patterns In vivo.  The present studies also suggest a simple explanation for the previous, apparentiy paradoxical finding that Ph^-positive cells often rapidly decline In LTC Initiated with CML marrow^^ but not with CML blood.^ It Is now clear that the ratio of leukemic to normal LTC-IC  numbers In these two sources of hemopoietic cells may differ over many orders of magnitude. Thus a decline of Ph^-positive clonogenic cells to undetectable levels within the first 5 weeks in a LTC initiated with a CML marrow would be expected if the frequency of leukemic LTC-IC were very low (i.e., less than one in the number of cells used to initiate each culture). Nevertheless, It Is interesting to note that Ph^-positive LTC-IC are also selectively disadvantaged in the LTC system; providing a strong rationale for the continued use of this approach to purge autologous CML marrow autografts. 16'27 Preliminary studies in our laboratory have also shown that Ph^positive LTC-IC have features expected of activated cells28 in contrast to normal LTC-IC which show features of quiescent cells.®The availability of a quantitative assay for a very primitive Ph^-positive cell population should thus make possible a variety of studies to further characterize these cells, obtain a better estimate of the number of leukemic stem cells In individual CML patients, and to devise more effective treatment strategies both in and ex vivo.  REFERENCES 1.  : Balllere's Clinical Haematology: Chronic Myeloid Leukaemia, vol 1 (edition 4). London, BaUlere Tindall, 1987, 208  2.  Flalkow PJ, Gartler SM, Yoshlda A: Clonal origin of chronic myelocjrtlc leukemia In man. Proc Natl Acad Scl U S A 58:1468, 1967  3.  Daley GQ, Van Etten RA. Baltimore D: Induction of chronic myelogenous leukemia In mice by the P210°'^''/^^1 gene of the Philadelphia chromosome. Science 247:824, 1990  4.  Kelllher MA, McLaughlin J , Wltte ON, Rosenberg N: Induction of a chronic myelogenous leukemla-Uke syndrome In mice with v-abl and BCR/ABL. Proc Nati Acad Scl U S A 87:6649, 1990  5.  Eaves C J , Eaves AC: CeU culture studies In CML, In Goldman J M (ed): BaUUere's CUnlcal Haematology. Vol. 1, #4. Chronic Myeloid Leukaemia. London, BalUlere TindaU, 1987, pp 931  6.  Dowdlng CR, Gordon MY, Goldman J M : Primitive progenitor ceUs In the blood of patients with chronic granuloc5mc leukemia. Int J CeU Cloning 4:331, 1986  7.  Eaves AC, Cashman JD, Gaboury LA, Kalousek DK, Eaves C J : Unregulated proliferation of primitive chronic myeloid leukemia progenitors In the presence of normal marrow adherent ceUs. Proc Nati Acad Scl U S A 83:5306, 1986  8.  Sutherland HJ, Eaves CJ, Eaves AC, Dragowska W, Lansdorp PM: Chciracterlzation and peutlal purification of human marrow ceUs capable of Initiating long-term hematopoiesis Invlti-o. Blood 74:1563, 1989  9.  Udomsakdl C, Eaves C J , Sutherland HJ, Lansdorp PM: Separation of functionaUy distinct subpopulations of primitive human hemopoietic ceUs using rhodamine-123. Exp Hematol 19:338, 1991  10. Eaves C J , Sutherland HJ, Udomsakdl C. Lansdorp PM. SzUvassy SJ. Fraser CC. Humphries RK. Bamett MJ, PhlUlps GL, Eaves AC: The human hemopoietic stem ceU In vitro and In vivo. Blood CeUs(In press):, 11. Sutherland HJ, Lansdorp PM, Henkelman DH, Eaves AC, Eaves C J : Functional characterization of Individual humem hemopoietic stem ceUs cultured at Umltlng dfiution on supportive marrow stromal layers. Proc Nati Acad Scl U S A 87:3584, 1990 12. Udomsakdl C, Lansdorp PM, Hogge DE, Reld DE, Eaves AC, Eaves CJ: Characterization and purification of long-term culture-Initiating ceUs (LTC-IC) and clonogenic ceUs from normal peripheral blood. Blood 78 (suppl l):292a, 1991 (absti-) 13. Coulombel L, Kalousek DK, Eaves CJ, Gupta CM, Eaves AC: Long-term marrow culture reveals chromosomaUy normal hemopoietic progenitor ceUs In patients with PhUadelphla chromosome-positive chronic myelogenous leukemia. N Engl J Med 308:1493, 1983 14. TasweU C: Limiting dUution assays for the determination of Immimocompetent ceU frequencies. I. Data analysis. J Immunol 126:1614, 1981  15. Coller HA, Coller BS: Poisson statistical analysis of repetitive subcloning by the limiting dilution technique as a way of assessing hybrldoma monoclonality, in Langone J J , Van Vunakis H (eds): Methods in Enzymology, vol 121. New York, Academic Press, Inc., 1986, pp 412 16. Bamett MJ, Eaves CJ, Phillips GL, Kalousek DK, Klingemann H-G, Lansdorp PM, Reece DE, Shepherd JD, Shaw GJ, Eaves AC: Successful autografting in chronic myeloid leukaemia after maintenance of marrow in culture. Bone Mgirrow Transplant 4:345, 1989 17. Otsuka T, Thacker JD, Hogge DE: The effects of interleukin 6 and interleukin 3 on early hemopoietic events in long-term cultures of human marrow. Exp Hematol 19:1042, 1991 18. Eaves CJ, Cashman J D , Sutherland HJ, Otsuka T, Humphries RK, Hogge DE, Lansdorp PM, Eaves AC: Moleculsir analysis of primitive hemopoietic cell proltferation control mechanisms. Ann N Y Acad Sci 628:298, 1991 19. Sutherland HJ, Eaves CJ, Lansdorp PM, Thacker JD, Hogge DE: Differential regulation of primitive human hemopoietic cells in long-term cultures maintained on genetically engineered murine stromal cells. Blood 78:666, 1991 20. Leary AG, Ogawa M: Blast cell colony assay for umbillced cord blood and adult bone marrow progenitors. Blood 69:953, 1987 21. Gordon MY, Hibbln JA, Keamey LU, Gordon-Smith EC, Goldman J M : Colony formation by primitive haemopoietic progenitors in cocultures of bone marrow cells and stromal cells. Br J Haematol 60:129, 1985 22. Eaves AC, Henkelman DH, Eaves CJ: Abnormad eiythropoiesis in the myeloproliferative disorders: A n analysis of underljdng celluléir and humoral mechanisms. Exp Hematol 8 (suppl 8):235, 1980 23. Goto T, Nlshikorl M, Arlin Z. Gee T, Kempln S, Burchenal J , Strife A, Wlsnlewski D, Lambek C, Littie C, Jhanwar S, Chaganti R, Clarkson B: Growth characteristics of leukemic and normal hemopoietic cells in Ph'+ chronic myelogenous leukemia and effects of intensive treatment. Blood 59:793, 1982 24. Kantarjlan HM. Vellekoop L, McCredie KB, Keating MJ, Hester J , Smith T, Barlogie B, Tmjillo J , Freireich EJ: Intensive combination chemotherapy (ROAP 10) and splenectomy in the management of chronic myelogenous leukemia. J Clin Oncol 3:192, 1985 25. Cashman J , Eaves AC, Eaves C J : Regulated prolfferation of primitive hemopoietic progenitor cells in long-term human mcirrow cultures. Blood 66:1002, 1985 26. Fraser CC, SzUvassy SJ, Eaves C J , Humphries RK: Proliferation of totipotent hemopoietic stem cells In vitro with retention of long-term competitive in vivo reconstituting ability. Proc Nati Acad Sci U S A 89:1968, 1992 27. Bamett MJ, Sutherland HJ, Eaves AC, Hogge DE, Humphries RK, Klingemann H-G, Lansdorp PM, Phillips GL, Reece DE, Shepherd JD, Eaves C J : Human hemopoietic stem cells In long-term culture: Quantitation and manipulation. Bone Marrow Transplant 7 (suppl 1):70, 1991  Udomsakdi C, Eaves C J , Lansdorp PM, Eaves AC: Unique characteristics of primitive neoplastic cells from patients with chronic myeloid leukemia (CML) assessed using the long-term culture-initiating cell (LTC-IC) assay. Blood 78 (suppl l):29a, 1991 (abstr)  CHAPTER  VI  PHENOTYPIC HETEROGENEITY OF PRIMITIVE LEUKEMIC HEMOPOIETIC CELLS IN PATIENTS WITH CHRONIC MYELOID LEUKEMIA CCML)  1. INTRODUCTION  Very little is known about the control of BCR-ABL expression in different primary hemopoietic cell types, particularly progenitor ceUs, or the biological consequences of such expression. The most primitive hemopoietic cells are difBcult to study because they make up such a small proportion of aR the nucleated cells in the blood and marrow. Nevertheless, effects of BCR-ABL expression on their behaviour are of key interest because it is these cells that are believed to be responsible for the initial amplification of the leukemic clone in patients with CML.  Such studies require not only specific, quantitative assays for the relevant cells, but also methods for their characterization and ultimately for their isolation as pure populations. The assay for long-term culture initiating cells (LTC-IC) appears to qualify as one that detects a population more primitive than clonogenic cells and that may overlap with totipotent reconstituting ceUs. ^''^ This assay also detects a functionally analogous Ph^-positive cell present at low frequency in the meurow and in elevated concentrations in the blood of CML patients.^ A characteristic feature of a variety of primitive Ph^-posltlve clonogenic cell types Is that their proliferation is deregulated both In vivo® and in vitro^ under conditions where their normal counterparts are quiescent, suggesting perturbation of a common control mechanism also active on the most primitive normal hemopoietic cells. It might therefore be expected that Ph^-positive LTC-IC would also exhibit properties of cycling cells, in contrast to normal LTC-IC. The present studies were designed to test this prediction. To facilitate the characterization of  Ph^-positive clonogenic cells and LTC-IC, peripheral blood from CML patients with elevated WBC counts was used as starting material. This made laborious genotyping studies (by PCR or cytogenetic analysis of their individual progeny) unnecessary, because of the very marked increase in all types of circulating neoplastic progenitors in such patients, thereby increasing both the number of experiments that cotdd be performed and the precision of the measurements made.  2. RESULTS  2.1  Phenotype analysis of CML LTC-IC To determine the light scattering properties of circulating, Ph^-positive, clonogenic cells  and LTC-IC, light-density blood cells from patients with elevated progenitor concentrations (Table 12) were sorted into three fractions (as Illustrated In Figure 24A) and the results compared with data for normal meirrow'* and blood (Figure 24B). Most of the nucleated cells (~ 85%) in the light density fraction of CML blood have a high SSC (Fraction III) In contrast to the light density cells In normal blood where the proportion of such cells Is much lower (< 40%, see Chapter IV). The mean number of clonogenic cells and LTC-IC recovered In each sorted fraction was determined and expressed as a percentage of the total number of progenitors present In the starting cell suspension of each experiment. As shown In Figure 24B, It can be seen that the majority of both the clonogenic cells and LTC-IC were consistently found in fraction II (i.e., cells with high FSC but low SSC). Cells from this fraction also generally produced more nucleated cells (as well as clonogenic cells) after 5 weeks In LTC (both In the adherent and nonadherent layer) than other fractions on a per cell basis (data not shown). However, a significant proportion of clonogenic cells (~ 15%) and LTC-IC (~ 30%) were present in a population of cells with low FSC and low SSC (fraction I). Some clonogenic cells (~ 5%) were found amongst the cells with a high SSC (fractions III). These findings suggest subtle differences between CML clonogenic cells and LTC-IC In terms of thefr overaU light scattering  properties. This was reinforced by experiments in which fraction II was subdivided further into 2-3 additional fractions. These showed that the clonogenic cells were concentrated in the fractions containing cells with a slightly higher FSC by comparison to the LTC-IC in the same samples (data not shown). The high FSC of cfrculating leukemic clonogenic cells differs markedly from the FSC typical of clonogenic cells In the cfrculation of normal individuals (p<0.05, see also Chapter IV), but is very similar to the majority of clonogenic cells in normal marrow.'* Since very few progenitors were present in fraction III, only cells in fractions I and II were analyzed in all subsequent sorts.  Table 12.  Frequency of Primitive Progenitors in the CML Patients Studied  Patient No.  Clonogenic CeUs/mL  LTC-lC/mL^  1  45  29,000  17,000  2  156  704,000  266,000  3  137  72.000  14,000  4  62  82,000  7,200  5  262  1,060,000  145,000  6  104  161,000  8,300  7  110  86,000  1,300  8  142  344,000  12,500  9  436  1,090,000  10,000  Mean + SEM  ^  WBC/L (x 109)  162 + 40  403,000 + 145.000  Absolute LTC-IC numbers calculated as described in the Methods  52,000 + 31,000  FORWARD LIGHT SCATTER 100  o ILL  O  n  m  FIGURE 24. Distribution according to light scatter characteristics of total cells (representative sample - Panel A), and clonogenic cells (open bars) and LTC-IC (solid bars) (combined results for paUents -Panel B) in the light density fraction of CML blood. Results for fractions I, H and III shown In Panel B are as defined in Panel A. Error bars in Panel B indicate the mean + ISEM of values obtained on each of 5 patients studied individually.  Figure 25 shows representative distributions of light density normal and CML blood cells gated for low SSC after two colour staining for expression of CD34 and HLA-DR, or expression of CD34 and uptake of Rh-123. A much larger proportion of light density CML blood cells express CD34 than is the case for normal blood cells in the same light scatter window (compare Panels D and F with C and E). The CML cells also contained a higher proportion of cells that expressed readily detectable levels of HLA-DR or that retained Rh-123 by comparison to normal blood. Figures 26 and 27 show the results obtained when the CD34''", SSC^°^ cells were sorted according to their expression of HLA-DR (Figure 26) or Rh-123 uptake (Figure 27) and then analyzed functionally for clonogenic cell or LTC-IC content. It can be seen that most of the neoplastic clonogenic cells In CML blood like most of the clonogenic cells In normal marrow express readily detectable levels of HLA-DR and take up Rh-123.'*^'® In this respect, however, they both differ markedly from normal circulating clonogenic cells, of which very few show a DR^^g^ or mi-123^^è^^ phenotype (Chapter IV). Further subdivision of the CD34+ DR^^g^ fraction of CML blood cells Into DR"*" and DR"*"*" subpopulations, as defined in Figure 25B, revealed the presence of clonogenic cells in both (Tables 13 and 14). A small proportion (~10%) of leukemic clonogenic progenitors were found in the DR^°^ or R h - 1 2 3 ^ ^ subpopulatlons of CD34'*' CML blood cells. Although none of these were specifically genotyped, it is very unlikely that significant numbers in either phenotyplcally defined group were residual normal progenitors since the latter, even if present at normal levels would have accounted for < 10% of the progenitors In the DR^°w or Rh-123^"^ fractions (Table 14).  GREEN FLUORESCENCE D  RHODAMINE-123 FIGURE 25. Bivariate contour plots of a single représentative sample of normal (Panels A, C & E) and CML (Panel B. D & F) light density blood ceUs in the low SSC window (fractions I and II In Figure 24A). CD34+ ceUs were subdivided into CD34+DR^°^ and C D 3 4 + D R ^ ^ subpopulaUons as shown In Panels C and D, or CD34+Rh-123<^"ll and CD34-^-123^'^^<^ populations as shown In Panels E and F. Unstained cells are shown In Panels A and B.  CD34+DR'OW  CD34+DR*^'9*I  FIGURE 26. Distribution of neoplastic clonogenic ceUs (Panel A) and LTC-IC (Panel B ) within the CD34"'" fraction of circulating CML cells subdivided (as shown in Figure 25D) according to their expression of HLA-DR (solid bars). The mean progenitor recovery + ISEM is expressed as a percent of the total number of light density progenitors recovered within the low S S C fraction shown in Figure 24A from studies of 4 different patients. For comparison, previously obtained analogous results for normal marrow^ (open bars, n=6) and normal blood (Chapter IV) (stippled bars, n=3) are also included in this figure. Differences between the recoveries of CML versus normal blood clonogenic cells and CML versus normal blood LTC-IC in the CD34'''DR1°^ and CD34+DR^*g^ fractions are significant (p<0.001 for clonogenic cells and p<0.05 for LTC-IC, respectively). A difference between the recoveries of normal marrow and CML clonogenic cells is not significant (p>0.4).  CD34+Rh-123^""  CD34+Rh-123^^'^^^^  CD34+Rh-123d""'  CD34+Rh-123*^"g^i*  FIGURE 27. Distribution of leukemic clonogenic cells {Panel A) and LTC-IC (Panel B) within the CD34+ fraction of circulating CML cells subdivided (as shown in Figure 25F) according to their uptake of Rh-123 (solid bars). The mean progenitor recovery + 1 S E M is expressed as a percent of the total number of light density progenitors recovered within the low SSC fraction shown in Figure 24A from studies of 4 different patients. For comparison previously obtained analogous results for normal marrow (Chapter III) (open bars, n=6) and normal blood (ChapterlV) (stippled bars, n=3) are also Included in this figure. Differences between CML versus normal blood clonogenic cells and CML versus normal blood LTC-IC in the CD34+Rh-123*^"^ and CD34+Rh123bnght fractions are significant (p<0.0001 for clonogenic cells and p<0.001 for LTC-IC. respectively).  When the sorted CML cells were assayed for LTC-IC, the majority (-75%) were also present amongst the CD34'*' DR^ë^ cells (Figure 25D). This is also in contrast to normal LTCIC, the majority of which in either blood (-100%) or marrow (~55%) express little or no HLADR."* Thus isolation of CD34+ D R ^ g ^ populations of ceUs from the peripheral blood of CML patients greatly exaggerates the already significant elevation in progenitor numbers observed in the original blood sample (Table 13). As noted for the circulating clonogenic cells in the CML samples studied, a proportion of the LTC-IC (In this case, -30%), was foimd in the CD34"'' DR^°^ fraction. Because of the marked elevation in total LTC-IC numbers in these samples, the number of CD34+ DR^^^ LTC-IC was also greatly (>1000x) in excess of normal values for CD34+ DRIOW  LTC-IC in the circulation (see Table 14). Similarly, most of the LTC-IC In the CML blood  samples were Rh-123^^ë^* (Figure 27B) In contrast to the LTC-IC In either the blood or marrow of normal Individuals (Chapter III and IV). However, on average -20% of the circulating LTC-IC In patients with CML were found to have a Rh-123*^"^ phenotype, of which < 1% would be anticipated to be residual normal LTC-IC even if these were still present at normal levels.  It can be seen from Table 12 that the initial frequencies of the clonogenic cells and LTCIC In the CML blood samples studied, although elevated, were quite variable both on a volume and on a per nucleated cell basis. Similarly, variability was also encountered after these progenitors were separated into vEirlous subpopulatlons as shown In Table 13 for light density, CD34+, DR^ow or D R ^ g ^ cells. However, on average the purity of LTC-IC In the CD34+ DR^o^ and CD34+ DR"*" fractions was 12% and 10%, respectively. This is - 5 to 6-fold higher than the purest populations of normal LTC-IC (1-2%) thus far Isolated from normal blood or marrow samples.®'1^ Corresponding values for the frequency of clonogenic cells in the CD34+ DR^°^ and CD34''' DR"*" fractions were 10% and 20%. Most of both types of progenitors were recovered in these experiments (73% of the clonogenic cells and 129% of the LTC-IC, Table 13).  Table 13:  Frequency, Enrichment and Recovery of Primitive CML Progenitors from CBSL Blood After Density Centrlfugation and Isolation of Subpopulations of 0034+ SSC^°^ Cells.  Source of Progenitors  Clonogenic CeUs Frequency per 10®^ Enrichment^ Recovery^ -  C D 3 4 + DRIOW e  104,000± 23,000  120± 50  CD34+ DR+ e  192,000+  230±  C D 3 4 + DR++ «  105,000± 44,000  63,000  Enrichment*^  Recovery^  542 + 395  -  -  367,000± 291,000  1,200± 7 0 0  2 4 ± 11  5 1 ± 22  312,000± 242,000  990± 630  70 ± 39  14± 5  43,000± 21,000  320 + 200  35+  -  1,750± 9 4 0  Unprocessed blood  LTC-IC^  130  80 ± 30  8±  2  Frequency per 10^^  31  Measured as total clonogenic ceU output in LTC after 5 weeks (on pre-estabUshed irradiated normal marrow feeder layers) Relative to aU nucleated ceUs in the population emalyzed Calculated by dividing the frequency per 1 0 ^ sorted ceUs by the progenitor frequency per 10® unsorted, Ught-density ceUs in each individual experiment, emd then deriving the meem ± SEM of these values for the four experiments performed Calculated by multiplying the percentages of ceUs retrieved in the fraction indicated by the corresponding calculated enrichment in each indlviduzd experiment, and then deriving the mean ± SEM of these values for the experiments performed, assuming 1 0 0 % recovery after centrlfugation on FH. Defined in Figure 25D.  Table 14.  Concentration of Various PhenotypicaUy Defined Subpopulations of CML Blood (n=4) and Comparison to Normal Values (n»5) CML  Subpoptilation Evaluated CD34+DR1OW C  CD34+DR+ ^  CD34+DR++ ^  ^ ^ ^ ^ ^ ^ g  Normal^  Patient No.  % of Total CD34+ CeUs  Clonogenic CeUs per mL  LTC-IC per mL^  % of Total CD34+ CeUs  Clonogenic CeUs per mL  LTC-IC per mL  1 2 3 4 Mean + SEM  17 13 10 28 17±4  2.900 62,600 5,400 16,400 21.800 ± 13,900  2,900 45,200 1,300 5,000 13,600 ± 10,500  84 ± 3  264 + 52  2.4 + 0.4  1 2 3 4 Mean + SEM  65 72 28 39 51± 10  25.000 507,000 24.000 42,000 150.000 ± 119.000  12,600 190,100 4,500 2,200 52,300 ± 46,000  16 ±3^  34  ± 18^  1 2 3 4 Mean + SEM  18 14 62 33 32 ± 11  520 134.000 42.000 2.400 50.300 ± 24.000  510 27,000 8,600 36 8,900 ± 6,200  16 ±3*"  34  ± 18^  oè  From previous data (Chapter IV) Absolute vîdues calculated as described in the Methods (Chapter II) No or low HLA-DR fluorescence as defined previously in Chapter IV Represented CD34+DR^ë''^ ceUs In CML blood that show moderately-positive HLA-DR fluorescence, as defined in Figure 25D Represented CD34+DR*^g*^ ceUs in CML blood that show strongly-positive HLA-DR fluorescence, as defined in Figure 25D Combined data of CD34+ DR+ and CD34+ DR^"*" fractions of normal blood (Chapter IV) Not detected  CD  2.2  Sensitivity of CML Progenitors to 4-Hyclroperoxycyclophosphamide (4-HC) I have previously shown that LTC-IC In normal blood, like LTC-IC In normal marrow, are  relatively resistant to 4-HC, as are circulating clonogenic cells, whereas clonogenic cells In normal marrow are more 4-HC-sensltive (Chapter IV). Recent clinical findings indicate that reconstitution of hematopoiesis with Ph^-negative cells can be achieved In some CML patients receiving 4-HC-treated autologous marrow transplants. ^ ^ This suggests that transplantable Ph^-positive stem cells may be more sensitive to 4-HC than normal stem cells. It was therefore of Interest to evaluate the 4-HC sensitivity of Ph^-positive clonogenic cells and LTC-IC and compare these to normal clonogenic cells and LTC-IC. In this series of experiments, LTC-IC function was assessed in terms of the clonogenic cell content of LTC evaluated after 4 and 8 weeks (rather than after 5 weeks, as in the studies described above), since previous experiments had revealed differences In the 4-HC sensitivity of normal LTC-IC measured by these two different endpoints.^  Results for light density CML blood cells exposed to 100 |ig/ml of 4-HC under standard transplant exposure conditions (I.e., 2 x 10^ cells/ml with 7% red cells for 30 minutes at 37°C) are shown In Figure 28, together with previous data for normal progenitors tested using the same procedures and reagents. Circulating leukemic clonogenic cells and clonogenic cells in normal marrow were similarly reduced (to ~ 10% of initial numbers) by this treatment. Circulating leukemic LTC-IC appeared only sllghtiy more resistant and were signiflcantiy more sensitive (p < 0.05) than normal LTC-IC from any source.  TIME IN CULTURE (WEEKS)  FIGURE 28. Survival of neoplastic clonogenic cells (Day 0) and LTC-IC (4 and 8 week clonogenic cell output endpoints) after a brief exposure to 100 jig/ml 4-HC (30 minutes at 37°C in the presence of 7% red blood cells, cells at 2 x 10^ cells/ml). Results for the neoplastic progenitors (solid bars showing mean + ISEM for 4 different patients) are shown for comparison together with previously obtained results for normal marrow (open bars, n=6) and normal blood (stippled bars, n=3) progenitors treated using the same conditions (see Chapter II). A difference between normal marrow and CML clonogenic cells is not significant (p>0.5).  2.3  Differentiative Potential of CML LTC-IC Previous studies have shown that the relative numbers of different types of clonogenic  progenitors present in 5 week-old LTC provides a consistent average overall measure of the differentiative behaviour of LTC-IC assayed imder standard LTC conditions.'*'To assess whether this parameter is altered In Ph^-positive LTC-IC, the ratio of BFU-E, CFU-GM and CFUGEMM numbers before and after LTC of light density CML blood cells was assessed. As shown in Table 15, after 5 weeks In LTC the proportion of progenitors identified as CFU-GM increased as documented previously for LTC-IC In the blood and marrow of normal Individuals (Chapter IV) and this remained constant for £in additional 3 weeks (data not shown). Differences between the differentiative capacities of CML versus normal marrow and blood LTC-IC were not significant (p>0.2).  Table 15.  Relative Numbers of Different Types of Clonogenic Cells Present in CML Blood and Produced by LTC-IC in CBiIL Blood after 5 Weeks in LTC  Origin of Samples  No. of Samples  CML blood  17  65+3  34 + 3  1.3 + 0.2  15 + 3  83 + 3  1.2 + 0.4  23  74 + 3  24 + 2  2.2 + 0.3  11 +2  89 + 2  0.5 + 0.2  Normal marrow^ 10  36 + 3  62 + 4  1.2 + 0.2  9+2  91 +2  0.8 + 0.3  Normal blood^  Clonogenic Cells BFU-E CFU-GM CFU-GEMM  From Chapter FV From Sutherland et al.  BFU-E  LTC-IC CFU-GM CFU-GEMM  3. DISCUSSION  This report describes a number of features of two functionally distinguished classes of primitive Ph^-positive cells : clonogenic cells and LTC-IC. Both are defined by quantitative assays that measure developmental potential using standeirdlzed culture conditions and specific progeny output endpoints, thus allowing comparison with similarly defined normal cells. Because normal (Ph^-negative) LTC-IC persist In many CML patients (and to a much lesser extent, normal clonogenic ceUs) (Chapter V and  ^^), a source of ceUs that would be  reproduclbly, signiflcantiy, and preferentially enriched for neoplastic progenitors was sought In order to avoid the need for laborious genotyping studies. This was achieved by focussing on the progenitors present in peripheral blood samples from patients with high WBC counts in whom both progenitor populations are markedly elevated (Chapter V). Thus normal cells, even if present in such samples at normal levels, would have remained well below the limit of detectabillty in any of the separation experiments performed.  These results show that both the neoplastic clonogenic ceUs and LTC-IC from patients with CML are phenotypically slmfiar to one another with respect to size (FCS), expression of CD34 and HLA-DR, uptake of Rh-123 and sensitivity to 4-HC. In both cases most had a phenotype expected of proliferating or activated cells (i.e., high FSC, high expression of HLA-DR, high Rh-123 uptake and relative sensitivity to 4-HC), although subtie differences between circulating neoplastic clonogenic cells (more activated) and LTC-IC (less activated) were consistentiy noted. This predominant, "abnormal" phenotype amongst the neoplastic progenitors is essentiaUy the opposite of that previously shown for the majority of clonogenic ceUs and LTC-IC In the circulation of normal adults and shared by the majority of LTC-IC in normal marrow (i.e., low FSC, low expression of HLA-DR, low Rh-123 uptake and relative insensitivity to 4-HC). These findings thus confirm and extend previous evidence that neoplastic clonogenic cells in CML blood are CD34'*", HLA-DR"*"!^-  and are in agreement with the  reported difference In size and HLA-DR phenotype of normal and neoplastic LTC-IC that co-exist In CML marrow.  Such observations are consistent with the likelihood that many Ph^-positive  LTC-IC, like their clonogenic progeny wUl be found to be actively proliferating cells regardless of their location in the CML patient (in blood or marrow)® Direct measurements of the cycling status of LTC-IC are not yet available, but should allow this prediction to be formally tested.  It is of interest to note that a substantial proportion (10-30%) of the circulating progenitors in the blood of the patients analyzed In this study showed features characteristic of their counterparts in normal Individuals. As noted above, it is very unlikely that these represented co-existing normal elements, since the numbers Involved would have required the latter to have been greatiy Increased in the circulation, a possibility not supported by existing data. 14,18,19 Accordingly one might anticipate that the turnover of all classes of primitive neoplastic progenitors in CML patients is increased, although not maximally so, with greater numbers of quiescent cells occurring amongst the Ph^-positive LTC-IC population than amongst their clonogenic progeny. Alternatively, It Is possible that the phenotypic markers examined may not necessarily be strictiy regulated In concordance with changes In cell cycle status.  By limiting dilution analysis, I previously showed in Chapter V that the total number of clonogenic cells produced on average by Ph^-positive and normal LTC-IC after 5 weeks In LTC is the same, cdthough in the same cultures the maintenance of normal LTC-IC Is much better. Another functional endpolnt of LTC-IC behaviour examined here Is provided by analysis of the relative numbers of the different types of clonogenic progeny produced. The present studies show that the ratio of eiythroid-restricted, granuloc5d;e-macrophage-restricted, and multi-ltneage clonogenic cells produced by Ph^-positive LTC-IC Is Indistinguishable from that previously documented for normed LTC-IC from all sources thus far analyzed (Chapter V and Sutherland et al. 1^ These findings reinforce the concept that the LTC-IC assay detects a functionally homogeneous population of primitive hemopoietic cells and that the processes underlying  commitment to specific lineages are not substantiaUy afiected by the presence of the BCR-ABL gene.  In summary, 1 have shown that a majority of the most primitive neoplastic progenitors that are currently detectable in patients with CML differ from their cotmterparts In normal individuals with respect to a number of functionaUy related properties. These differences are suggestive of a deregulation in the control of ceU proliferation In CML at the level of the ceUs InltiaUy responsible for maintenance and expansion of the Ph^-positive clone without alteration of their commitment to, or early differentiation down, each of the hemopoietic lineages. These findings may provide a potentially useful theoretical framework for future analysis of the mechanism of BCR-ABL-induced multi-lineage disease. On the other hand, from a practical viewpoint, the demonstration that, on average, as many as 30% of the neoplastic LTC-IC are phenotyplcaUy indistinguishable from normal LTC-IC does not auger weU for the selectivity of this type of approach to Isolate primitive normal stem cells from CML marrow for use in autologous tremsplantation protocols. This latter finding Invites further investigation of the basis of the observed phenotypic heterogeneity of apparentiy functionaUy sImUar primitive neoplastic ceUs. The procedures described here, which edlow populations of these ceUs to be readUy obtained at 10-20% purity and in high )rleld should provide a useful starting point for such studies.  REFERENCES 1.  Bamett MJ, Eaves CJ, Phillips GL, Kalousek DK, Kllngemann H-G, Lansdorp PM, Reece DE, Shepherd JD, Shaw GJ, Eaves AC: Successful autograftlng In chronic myeloid leukaemia after maintenance of marrow In culture. Bone Marrow Transplant 4:345, 1989  2.  Eaves CJ, Sutherland HJ, Udomsakdl C, Lansdorp PM, Szllvassy SJ, Fraser CC, Humphries RK, Bamett MJ, Phillips GL, Eaves AC: The human hemopoietic stem cell In vitro and In vivo. Blood Cells(ln press):,  3.  Fraser CC, Szllvassy SJ, Eaves C J , Humphries RK: Proliferation of totipotent hemopoietic stem cells In vitro with retention of long-term competitive In vivo reconstituting ability. Proc Nati Acad Scl U S A 89:1968, 1992  4.  Sutherland HJ, Eaves CJ, Eaves AC, Dragowska W, Lansdorp PM: Characterization and partial purification of human marrow cells capable of Initiating long-term hematopoiesis invltiro. Blood 74:1563, 1989  5.  Udomsakdl C, Eaves C J , Swolln B, Reld DS, Bamett MJ, Eaves AC: Rapid decline of chronic myeloid leukemic ceUs In long-term culture due to a defect at the leukemic stem cell level. Proc Nati Acad Scl U S A (In press):,  6.  Eaves C J , Eaves AC: Cell culture studies In CML, In Goldman J M (ed): BalUlere's Clinical Haematology. Vol. 1, #4. Chronic Myeloid Leukaemia. London, Ballllere Tindall, 1987, pp 931  7.  Eaves AC. Cashman J D , Gaboury LA, Kalousek DK, Eaves C J : Unregulated proliferation of primitive chronic myeloid leukemia progenitors In the presence of normal marrow adherent ceUs. Proc Nati Acad Scl U S A 83:5306, 1986  9.  Udomsakdl C, Eaves CJ, Sutherlcmd HJ, Lansdorp PM: Separation of functionaUy distinct subpopulations of primitive human hemopoietic ceUs using rhodamine-123. Exp Hematol 19:338, 1991  10. Lansdorp PM, Sutherland HJ, Eaves CJ: Selective expression of CD45 Isoforms on functional subpopulations of CD34+ hemopoietic ceUs from human bone marrow. J Exp Med 172:363, 1990 11. Sutherland HJ, Lansdorp PM, Henkelman DH, Eaves AC, Eaves C J : Functional characterization of Individual human hemopoietic stem ceUs cultured at Umltlng dUution on supportive marrow stromal layers. Proc Nati Acad Scl U S A 87:3584, 1990 12. Carlo-SteUa C, MangonI L, Plovanl G, Almlcl C, Garau D, Caramatti C, RlzzoU V: In vitro marrow purging in chronic myelogenous leukemia: Effect of medbsfcmilde and recombinant granulocyte-macrophage colony-stimulating factor. Bone Marrow Transplant 8:265, 1991 13. Winton EF, Colenda KW: Use of long-term human msirrow cultures to demonstrate progenitor ceU precursors in marrow treated with 4-hydroperoxycyclophosphamide. Exp Hematol 15:710, 1987 14. Coulombel L, Kalousek DK, Eaves CJ, Gupta CM, Eaves AC: Long-term marrow culture reveals chromosomaUy normal hemopoietic progenitor ceUs in patients with PhUadelphla chromosome-positive chronic myelogenous leukemia. N Engl J Med 308:1493, 1983  15. Kalousek DK. Eaves CJ. Eaves AC: In-vltro cjrtogenetlc studies of haemopoietic malignancies. Cancer Surv 3:439, 1984 15. Katz FE, Watt SM, Martin H, Lam G, Capellaro D, Goldman J M , Greaves MF: Co-ordlnate expression of BI.3C5 and HLA-DR antigens on haemopoietic progenitors from chronic myeloid leukaemia. Leuk Res 10:961, 1986 16. Robak T, Nolasco I, Hlbbln JA, Goldman J M : Antigenic characteristics of circulating CFU-GM in chronic granulocytic leukemia resemble those of CFU-GM from normal marrow and differ from those of normal blood. Blood, 1985 17. Verfaillle CM, Miller WJ, Boylan K, McGlave PB: Selection of benign primitive hemopoietic progenitors in chronic myelogenous leukemia on the basis of HLA-DR antigen expression. Blood 79:1003, 1992 18. Singer JW. Fialkow PJ. Steinmann L. Najfeld V, Stein SJ. Robinson WA: Chronic myelocjdlc leukemia (CML): failure to detect residual normal committed stem cells In vitro. Blood 53:264. 1979 19. Dube ID. Gupta CM. Kalousek DK. Eaves C J . Eaves AC: Cytogenetic studies of early myeloid progenitor compartments in Ph^-positive chronic myeloid leukaemia (CML). I. Persistance of Ph^-negative committed progenitors that are suppressed from differentiating in vivo. Br J Haematol 56:633, 1984 20. Srour EF, Brandt J E , Leemhuls T, Ballas CB, Hoffman R: Relationship between cj^oklne-dependent cell cycle progression and MHC class II antigen expression by human CD34+HLA-DR- bone marrow cells. J Immunol 148:815, 1992  CHAPTER  VII  SUMMARY AND FUTURE DIRECTIONS  The hemopoietic system comprises a hierarchy of cell types in different stages of development whose regulated proliferation, self-renewed and differentiation allows a variety of mature, relatively short-lived end cells to be maintained at constant levels or to be temporarily produced in greater numbers, as required. The most primitive hemopoietic cells in adult marrow are capable of extensive self-renewal, as well as differentiation to all myeloid and lymphoid lineages. Quantitative assays for human totipotent hemopoietic cells that measure directly the long-term in vivo repopulating potential eire not cvirrentiy available. Therefore efforts along this line have focussed on possible In vitro alternatives. Recent experiments have shown that murine cells with long-term in vivo reconstituting potential have unique properties also shared by cells that can generate In vitro colony-forming cells in LTC for more than 4 weeks, in contrast to cells with short-term in vivo reconstituting potential which overlap more extensively with In vitro colony-forming cells (reviewed In Chapter I). It has also been demonstrated that a primitive hemopoietic cell type In normal humsin marrow can be maintained In the same type of LTC that support the self-renewed of transplantable totipotent hemopoietic cells of murine origin. 1'® Such findings suggest that the cells responsible for Initiating long-term hemopoiesis In the LTC system (LTC-IC) are closely related to. If not Identical with, the most primitive hemopoietic cells In normal humgm marrow. The LTC-IC assay might thus also be expected to serve as a basis for developing analogous quantitative methods for investigating perturbations of stem cell behaviour In diseases believed to originate In this compartment.  CML Is probably the best studied example of a hemopoietic disease believed to result from the "transformation" of a plurlpotent stem cell. It Is imlque In that almost all patients carry the same abnormal chromosome (and underljdng molecular genetic change) In their  leukemic cells which usually dominate the whole hemopoietic system by the time the disease becomes symptomatic. In this thesis, I have shown that the LTC-IC assay can be used as method for quantltating a Ph^-positive cell type that has the same defining functional properties as cells Identified as LTC-IC in normal marrow; I.e., production of clonogenic progenitors for at least 5 weeks when co-cultured on competent marrow fibroblasts. As described In Chapter V, the total number of clonogenic ceUs produced under these conditions (using Irradiated normal marrow LTC adherent layers as feeders) was shown to be linearly related to the number of CML blood ceUs originally added down to limiting numbers of LTC-IC. Accordingly, it was possible to use limiting dfiution analysis techniques to determine the output of clonogenic ceUs per leukemic LTC-IC and hence rapidly derive the absolute concentration of leukemic LTC-IC present In the peripheral blood of a léu"ge number of patients. This revealed that the peripheral blood content of leukemic LTC-IC Increases exponentially with the WBC count, as previously shown for clonogenic ceUs.* In contrast, the number of Ph^-positive LTC-IC, relative to other ceU types. In CML marrow appears to be decreased, on average, to less them 5% of the frequency of LTC-IC In normed marrow. In contrast, Ph^-positive clonogenic cell frequencies In CML marrow eire relatively normal or even sUghtly increased. These findings cleeirly demonstrate a unique biological behaviour of leukemic LTC-IC and suggest changes at the level of this very early ceU population that may account for Its particular faUure to expand emd be retedned in the marrow. Because it is difficult to quemtitate the toted ceUuleuity of the meirrow; I.e., toted body content of marrow ceUs, it is difilcult to determine the total number of leiikemic LTC-IC In a given patient. However, It Is clear that a significant contribution wUl be made by the circulating LTC-IC in patients with particularly elevated WBC counts, as weU as other possible extrameduUary sites of hemopoiesis (e.g. spleen and fiver). In patients with a less obvious burden of leukemic ceUs (e.g. peripheral WBC counts <5 x lO^^/L), the leukemic LTC-IC population (stem cells?) may weU not exceed the number of co-existing normal LTC-IC, a prediction of considerable Importance for the design of therapeutic modafities with fittie discriminatory capablUty.  The availability of an assay for Ph^-positive LTC-IC (and clonogenic progenitors) made it possible to undertake characterization studies to allow these cells to be compéired with one another as well as with their counterparts in normal marrow and blood. In these studies, I focussed on several so-called "activation" markers (such as Rh-123, HLA-DR, cell size and 4-HC sensitivity) based on the premise that the cycling of very primitive leukemic cells In CML may be deregulated.® Accordingly, such cells would be expected to differ from their counterparts in normal individuals which show features of quiescent cells. As part of this approach It was, however, first useful to obtain further Information about the phenotype of LTC-IC in normal marrow and to obtcdn base-line data for LTC-IC in normal blood. As described in Chapter III, I demonstrated that the majority of LTC-IC in normal marrow differ from clonogenic cells In their lower uptake of Rh-123, which would be consistent with a quiescent status. Since only 1% of light density normal meirrow cells are CD34''' Rh-123*^"^, isolation of this fraction provides a relatively simple, reproducible and rapid procedure for obtaining a population that Is as highly enriched for LTC-IC as is possible with other strategies.® This procedure should therefore be useful for further biological studies of normal LTC-IC.  Since most of my studies utilized CML blood as a selectively enriched source of primitive leukemic progenitors, and yet little was known about the phenotype of circulating LTC-IC In normal steady-state Individuals, I undertook a detailed study of these cells also. I first established that the number of clonogenic cells present after 5 weeks In LTC Initiated by seeding light density T cell-depleted normal blood cells onto Irradiated normal marrow feeders was, as for other sources of LTC-IC, linearly related to the input number of normal blood cells over a wide range of cell concentrations. This, In turn permitted the quantitation of circulating LTC-IC by limiting dilution analysis. Using this approach, I found the concentration of LTC-IC in the circulation of normal adults to be 2.9 + 0.5 per ml. This Is ~75-fold lower than the concentration of circulating clonogenic cells and represents a frequency of LTC-IC relative to  other nucleated cells that Is ~ 100-fold lower than that measured in normal marrow. Characterization of the LTC-IC In normal blood reveeded most of them to be small (low FSC), CD34+, Rh-123^"^, HLA-DR', 4-HC-resistant cells. Isolation of the light-density, CD34+ and either HLA-DR'or Rh-123*^"^ fraction of normal blood yielded a population of cells that were 0.5-1% LTC-IC, a purity comparable to the most enriched populations of human marrow LTC-IC reported to date (Chapter IV). I also confirmed previous data indicating that this phenotype shared by circulating clonogenic cells, consistent with their known quiescent state.  Chapter VI describes the results of studies aimed at phenotypic characterization of leukemic LTC-IC and clonogenic cells. These Eire summarized in Tables 16 and 17. Circulating Ph^-positive clonogenic cells were found to have very similar characteristics to clonogenic cells In normal marrow, and thus differ from normal circulating clonogenic cells. Interestingly, the majority although not all of the circulating leukemic LTC-IC showed a phenotype expected of activated/proliferating cells. Taken together, my data predict that, for the various sources and types of cells exemilned, normal circulating LTC-IC and clonogenic cells would be the most quiescent population, with leukemic clonogenic cells (of either blood or marrow origin) and normal marrow clonogenic cells being the most actively proliferating population. Leukemic LTCIC differ markedly from normal circulating LTC-IC and appear to be Intermediate between LTCIC and clonogenic cells In normal marrow (Figure 29).  Table 16.  Properties of Normal and CML LTC-IC  Normal Marrow  Normal Blood  CML  1/2x10*  ~3/ml  Î exponentially with WBC  small (low FSC) CD34+ HLA-DR-/+ Rh.l23dull 4-HC-reslstant  very small CD34+ HLA-DRRh.l23dull 4-HC-reslstant  large (high FSC) CD34+ HLA-DR+/++ Rh.l23brlght 4-HC-sensitive  Differentiative potential in LTC (BFU-E:CFU-GM)  1:9  1:9  1:9  Self-medntenance in LTC (% of input after 10 days)  100%  100%  3%  FYequency Predominant phenotype  Proliferative potential in LTC (CFC/LTC-IC)  Table 17. Properties of Normal and CML Clonogenic Cells  Frequency Predominant phenotype  Normal Marrow  Normal Blood  CML  1/10-^  225 + 27 per ml  Î exponentially with WBC  large (high FSC) CD34+ HLA-DR++ Rh-123brtght 4-HC-sensitive  small (low FSC) CD34+ HLA-DR-/+ Rh-123duU 4-HC-resistant  large (high FSC) CD34+ HLA-DR++ Rh.iasbright 4-HC-sensitive  Normal Blood LTC-IC Normal Blood Clonogenic cells Normal Marrow LTC-IC CML LTC-IC Normal Marrow Clonogenic cells CML Clonogenic cells  gUIESCENT PHENOTYPES  ACTIVATED PHENOTYPES  FIGURE 29. Proposed Phenotypic Organization of Normal and CML Primitive Cells  In addition to analyzing these properties of early leukemic cell types In CML, I edso undertook studies to examine their proliferative, dlfferentiative and self-maintenance capacities In the LTC system. A major Impetus for this study was derived from earlier observations that the number of Ph^-positive clonogenic cells usually decreases rapidly In LTC Initiated with CML marrow, whereas, even In the same cultures, Ph^-negative clonogenic cell numbers may be maintained. As noted above, the limiting dilution experiments used to enable the quantitation of Ph^-positive LTC-IC In CML blood (and hence In the marrow) revealed marked differences In their numbers In these two sites. The relative paucity of Ph^-positive LTC-IC in the marrow (compared to Ph^ clonogenic cells and co-existing normal LTC-IC) would thus appear to be a major contributing factor to the differential kinetics of Ph-^-positive and normal clonogenic cells typical of LTC-lnltiated with CML marrow. On the other hand, when the number of LTC-IC was assessed after varying periods of time In LTC, leukemic LTC-IC showed an Immediate and rapid decline such that at the end of the first 10 days only 3% of the Input number remained. This Is In marked contrast to the relatively constémt number of LTC-IC maintained In cultures of either  normal marrow or blood cells. Moreover, this defective self-medntenance of LTC-IC occurred in spite of a normal output of clonogenic cell progeny by Ph^-positive LTC-IC as shown by the limiting dilution experiments (Chapter V). Subsequent studies also showed that the distribution of different types of daughter clonogenic cells by CML LTC-IC was also normal (Chapter VI). Thus defective seff-malntenance of CML LTC-IC appears to be a uniquely altered function of these cells.  The molecular mechanism(s) responsible for the defective self-maintenance of CML LTCIC Is not known, but various possibilities can be considered. For example, CML LTC-IC may have a different growth factor requirement or, alternatively, CML LTC-IC may be less able to respond to localized growth factor due to possible deficiencies in their adherence properties. Such a mechanism would be expected to be of greater significance if the stromal cell-derived growth factors responsible for self-maintenance were cell-bound (as has been shown to be the case for some forms of Steel factor^ or CSF-l).® Such a possibility would also require that the factors regulating clonogenic cell production be different from the factors regulating LTC-IC seffmalntengmce. Recent data for normal LTC-IC Is In fact consistent with this prediction.^'  If the defective self-maintenance of CML LTC-IC in LTC is mediated by a growth factor deficiency, then it might be possible to alter this parameter by Increasing the concentration of appropriate growth factors in the cultures. To test this hypothesis, I have recentiy begun a series of experiments using various alternative feeder layers. Of particular Interest are a series of genetically engineered murine fibroblast lines, which produce a variety of human hemopoietic growth factors including IL-3, IL-6, G-CSF, GM-CSF and IL-7 which have been generated by Dr. Hogge in the Terry Fox Laboratory. Murine S l / S l fibroblasts are also of Interest since studies using normal marrow LTC-IC have demonstrated that such feeders are able to support the selfmaintenance, proltferation and differentiation of normal LTC-IC as well as human mgirrow feeders. Whether these murine feeder cells can also support CML LTC-IC and whether this  function may be altered when such cells are engineered to produce specific human growth factors is stiU not known. Because murine fibroblasts that do not produce any of the factors known to infiuence normal human LTC-IC maintenance are equivalent In their supportive abfilty to human marrow feeders, It seems Ukely that there are additional, as yet undefined factors with this function. The effects of Inhibitors such as M l P - l a and TGF-p on the activation/cycling state of the highly enriched LTC-IC populations firom both normal and CML blood can also be dlrectiy tested using the purification technics described in this thesis.  Other avenues of Investigation that would now be of Interest could exploit the possible use of retroviral vectors to transfer the BCR-ABL gene into purified normal LTC-IC and then observe the phenotypic, proliferative and self-maintenance changes obtained in the infected cells. 11 Whether the activated phenotypes (i.e., HLA-DR+, Rh-123+, high FSC and 4-HCsensltive) of CML LTC-IC would occur as a result of such Introduced BCR-ABL expression could then be evaluated. Also of Interest would be the transplantation Into Immunocompromised mice of human ceUs transduced with a vector.  Subsequent studies of the characteristics of LTC-IC  generated In these mice might also help to estabUsh the role of BCR-ABL In the development of a clinical picture In these animals of CML.  In summary, the studies described in this thesis have contributed to the development of a new, quantitative assay for a leukemic ceU type present in CML patients that appear to be more primitive than any other type of progenitor yet characterized. Functioned studies of the proliferative, dlfferentiative and self-maintenance behaviour of these ceUs reveeded only the latter parameter to be abnormal. Examination of their number and phenotype and additional baseline measurements for LTC-IC in normal marrow and blood predict that leukemic LTC-IC wlU be found to have an abnormally elevated turnover rate In vivo. These baseline studies with normal LTC-IC should also facfiltate future analysis of stem ceU regulatory defects In other myeloproliferative and myelodysplastic diseases. FinaUy, the development of methods to obtain  highly enriched populations of very primitive Ph^-positive cells should be valuable for further Investigation of the molecular alterations they may show and thereby lead to new strategies for therapy.  REFERENCES 1.  Fraser CC, Eaves C J , Szllvassy SJ, Humphries RK: Expansion in vitro of retrovlrally marked totipotent hematopoietic stem cells. Blood 76:1071, 1990  2.  Fraser CC, Szllvassy SJ, Eaves C J , Humphries RK: Proliferation of totipotent hematopoietic stem cells in vitro with retention of long-term competitive in vivo reconstituting ability. Proc Natl Acad Sci U S A 89:1968, 1992  3.  Sutherland HJ, Lansdorp PM, Henkelman DH, Eaves AC, Eaves C J : Functional characterization of individual human hematopoietic stem cells cultured at limiting dilution on supportive marrow stromed layers. Proc Natl Acad ScIU S A 87:3584, 1990  4.  Eaves C J , Eaves AC: Cell culture studies in CML, in Goldman J M (ed): Bailliere's Clinical Haematology. Vol. 1, #4. Chronic Myeloid Leukaemia. London, Bailllere Tlndall, 1987, pp931  5.  Eaves AC, Cashman JD, Gaboury LA, Kalousek DK, Eaves CJ: Unregulated proliferation of primitive chronic myeloid leukemia progenitors In the presence of normal marrow adherent cells. Proc Natl Acad Sci U S A 83:5306, 1986  6.  Sutherland HJ, Eaves CJ, Eaves AC, Dragowska W, Lansdorp PM: Characterization and partial purification of human marrow cells capable of initiating long-term hematopoiesis in vitro. Blood 74:1563, 1989  7.  Anderson DM, Lyman SD, Baird A, Wignall J M , Eisenman J , Ranch C, March C J , Boswell HS, Glmpel SD, Cosman D, Williams DE: Molecular cloning of mast cell growth factor, a hematopoietin that Is active in both membrane boimd and soluble forms. Cell 63:235, 1990  8.  Sherr C J : Colony-stimulating factor-1 receptor. Blood 75:1, 1990  9.  Sutherland HJ, Eaves CJ, Lansdorp PM, Thacker JD, Hogge DE: Differential regulation of primitive human hematopoietic cells In long-term cultures maintcdned on genetically engineered murine stromal cells. Blood 78:666, 1991  10. Sutherland HJ, Reld D, Eaves CJ: Steel factor synerglzes with IL-3 and G-CSF to replace marrow feeders in promoting differentiation of highly purified human hematopoietic cells. Exp Hematol(ln press):, (abstr) 11. Hughes PFD, Thacker JD, Hogge D, Sutherland H J , Thomas TE, Lansdorp PM, Eaves CJ, Humphries RK: Retroviral gene transfer to primitive normal and leukemic hematopoietic cells using clinically applicable procedures. J Clin lnvest(in press):, 12. Dick J E : Immune-deficient mice as models of normal and leukemic human hematopoiesis. Cancer Cells 3:39, 1991  

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