Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Identification and functional analysis of genes regulating ES cell pluripotency Hsu, Lien 2006

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-ubc_2006-0506.pdf [ 4.55MB ]
Metadata
JSON: 831-1.0092716.json
JSON-LD: 831-1.0092716-ld.json
RDF/XML (Pretty): 831-1.0092716-rdf.xml
RDF/JSON: 831-1.0092716-rdf.json
Turtle: 831-1.0092716-turtle.txt
N-Triples: 831-1.0092716-rdf-ntriples.txt
Original Record: 831-1.0092716-source.json
Full Text
831-1.0092716-fulltext.txt
Citation
831-1.0092716.ris

Full Text

Identification and Functional Analysis of Genes Regulating ES Cell Pluripotency By Lien Hsu National Chung-Hsing University, 2002 A thesis submitted in fulfillment of the requirements for the degree of M A S T E R OF SCIENCE in THE F A C U L T Y OF G R A D U A T E STUDIES (Genetics) THE UNIVERSITY OF BRITISH C O L U M B I A June 2006 © Lien Hsu, 2006 populations showing increased expression. Functional studies, including embryoid body (EB) formation and colony forming cell (CFC) assays, as well as assessment of cell survival and proliferation rates, were done. Of note, G b x 2 and Inhbb were shown to partially rescue the differentiation potential of ESC following LIF removal, as well as to alter cell lineage differentiation and EB morphology. In addition, ESC over-expressing Inhbb or Cxcll showed differences in proliferation rate. Ankrdl influenced the plating efficiency of ESC. This work lays the foundation for further studies into the molecular mechanisms that regulate ESC "sternness". Moreover, these studies may provide clues for the development of effective strategies to direct the differentiation of ESC into functional end cells of a specific type for use in cell replacement and cancer therapies. 111 A B S T R A C T Embryonic stem cells (ESC) are a pluripotent cell population with the capacity to undergo symmetrical self-renewing divisions, as well as to differentiate into cells of all three embryonic germ layers both in vitro under appropriate culture conditions and in vivo. Recent gene expression profiling studies have begun to identify the key genes defining the mouse ESC phenotype. In addition, comparisons of ESC gene expression profiles with those of differentiated cells, or various types of adult stem cells, have been done in an attempt to identify a set of critical ESC regulatory genes. However, these studies have been limited by a lack of related functional data for the ESC, correspondent to the expression analysis. In order to more accurately identify important regulatory genes a global gene expression profiling study on undifferentiated and early differentiated ESC was carried out at the BC Cancer Research Center (BCCRC) to identify those genes whose loss of expression correlates with loss of stem cell function. Further comparison of the BCCRC R l ESC dataset with a similar R l ESC data set generated through the Stem Cell Network (SCN) allowed us to identify candidate genes whose expression either significantly decreased in the first 18 hours following removal of leukemia inhibitory factor (LIF) or which continued decreasing over 72 hours. An additional comparison was carried out to identify over-lapping genes amongst 3 different ESC lines: R l , J l , V6.5 (all from SCN). Eight candidate genes, present in all data sets and fulfilling additional criteria, were identified through these analyses: Bcl3, Klf5, Empl, Inhbb, Cxcll, Gbx2, Ankrdl, and 8430410A17Riken. Their expression patterns were confirmed using quantitative real time PCR during ESC differentiation. Lenti-viral vectors for over-expression of these candidates were generated and used to obtain ESC ii Abstract T A B L E OF CONTENTS 11 Table of Contents iv List of Tables vi List of Figures vii List of Abbreviations viii Acknowledgements ix CHAPTER 1 INTRODUCTION 1 1.1 Stem Cells 1 1.1.1 What is a Stem Cell? 1 1.1.2 Totipotency and Pluripotency 1 1.1.3 Multipotent adult stem cells 2 1.1.4 Types of Stem Cells 4 1.2 Embryonic Stem Cells 5 1.2.1 Embryonic Stem Cell Cultures 5 1.2.2 Differentiation of Embryonic Stem Cells in vitro 7 1.2.3 The Cell Cycle of Mouse Embryonic Stem Cells: It's Role in Preventing Differentiation 9 1.2.4 Assays for Measuring Murine ESC pluripotency 9 1.2.5 Embryonic Stem Cells for Regenerative Medicine 11 1.3 Molecular Mechanisms Maintaining Mouse ESC in Their Undifferentiated State 13 1.3.1 Leukemia Inhibitory Factor and STAT3 Activation 13 1.3.2 Oct-4 and Nanog in ESC pluripotency 16 1.4 Analysis of Gene Function 18 1.5 ESC gene expression profiling studies 19 1.6 Hypothesis and Specific Aims 21 CHAPTER 2 METHODS AND MATERIALS 23 2.1 Gene Array Analyses 23 2.1.1 Comparisons of the BCCRC and SCN Microarray datasets 23 2.1.2 Constructing Gene lists for Preliminary Candidate Identification 23 2.1.3 Gene Ontology (GO) Analysis to narrow the candidate list 25 2.2 Growth of Mouse Embryonic Stem Cells and Mouse Embryonic Fibroblasts (MEF) 25 iv 2.3 Embryoid Body formation in suspension culture 26 2.4 Harvest of Embryoid Bodies from Methylcellulose Cultures 27 2.5 RNA Isolation, cDNA Synthesis, and Quantitative RT-PCR (RT-qPCR) 28 2.6 Synthesis of Full-length cDNAs for Vector Construction 30 2.7 TOPO Cloning and Generation of the Lentiviral Constructs 31 2.8 Transfection of 293 T cells for Lentivirus Production 33 2.9 ESC Infection, Flow Cytometry, and Fluorescence Activated Cell Sorting 34 2.10 Functional Analyses of the ESC 35 2.10.1 CFC analysis and ESC proliferation 35 2.10.2 Analysis of differentiation potential 36 2.11 Statistics 37 CHAPTER 3 RESULTS 38 3.1 Gene Array Analyses and Validation 38 3.1.1 Analyses of the ESC Microarray Data 38 3.1.2 Additional Analyses to Identify Candidates for Functional Studies 41 3.2 Validation of Gene Expression Patterns to Finalize the Candidate List 46 3.3 Lentivirus Generation and Validation of Candidate Over-expression 48 3.4 Functional analysis 56 3.4.1 CFC analysis and ESC proliferation 56 3.4.2 Analysis of differentiation potential 58 3.5 The interaction between candidates and LIF-signaling pathway 65 CHAPTER 4 DISCUSSION 67 4.1 Microarray analyses and R l ESC line 67 4.2 Criteria for Identifying Candidate Genes 67 4.3 Advantages and Disadvantages of the Over-expression Strategy 69 4.4 Alternative to lentivirus: Electroporation and Knock-in 70 4.5 ESC Transduction Results 72 4.6 Hypotheses and Future Directions 75 4.6.1 Gbx2: a possible regulatory gene of ESC pluripotency 75 4.6.2 Inhbb: a possible regulatory gene of ESC pluripotency 77 4.6.3 Ankrdl: a possible anti-apoptosis gene which may also promote cardiac cell differentiation in ESC 80 4.6.4 Cxc l l : a tumorigenesis gene presumably accelerating ESC proliferation 82 4.7 Final Conclusions 82 REFERENCES 84 LIST OF TABLES Table 2.1 Primer sequences for RT-qPCR validation 29 Table 2.2 Primer sequences used to amplify whole coding regions of candidates 30 Table 3.1 Comparisons of the ESC microarray data 39 Table 3.2 The candidates identified through the various analyses 44 Table 3.3 Relative expression of candidate genes in transduced ESC or EB 54 Table 3.4 Percentage reduction in GFP expression in day two EB suspension cultures 56 vi L I S T O F F I G U R E S Figure 1.1 Day 5 embryoid body cultured in M C 8 Figure 1.2 The LIF-STAT3 signaling pathway promotes ESC self-renewal 14 Figure 2.1 Schematic diagram of the lentiviral vector 32 Figure 3.1 Correlation between gene arrays 40 Figure 3.2 Venn diagrams for the comparisons amongst the various ESC libraries 43 Figure 3.3 RT-qPCR validation of expression level changes for the 8 selected candidates during ESC differentiation 47 Figure 3.4 Analysis of GFP expression following ESC infection with the indicated lenti-viral vectors 51 Figure 3.5 Analysis of GFP expression on the day of sorting 52 Figure 3.6 Analysis of GFP expression post-sorting 54 Figure 3.7 RT-qPCR validation to confirm over-expression 55 Figure 3.8 CFC assay in the presence of LIF 57 Figure 3.9 CFC assay in the absence of LIF 57 Figure 3.10 Growth curves for the various ESC lines 60 Figure 3.11 Primary EB formation assay 61 Figure 3.12 Cell numbers per day 5 EB 61 Figure 3.13 Secondary EB formation 62 Figure 3.14 RT-qPCR analysis of germ layer marker genes in day 5 EB 64 Figure 3.15 The expression of Oct4 and Nanog in Inhbb over-expressing cells by RT-qPCR 66 vii LIST OF A B B R E V I A T I O N S BC Cancer Research Center BCCRC Bcl3 B-cell leukemia/lymphoma 3 BMP Bone morphogenetic protein CFC Colony forming cell cPPT Central polypurine tract D M E M Dulbecco's modified Eagle's medium DMSO Dimethyl sulfoxide EB Embryoid body EC Embryonic carcinoma E F l - a Elongation factor 1 alpha E G Embryonic germ cell ERK Extracellular signal-regulated kinase ESC Embryonic stem cells FACS Fluorescence-activated cell sorting FBS Fetal bovine serum Fgf2 Fibroblast growth factor 2 GO Gene Ontology HBS Hank's balanced salt solution HIV-1 Human immunodeficiency virus type 1 hrGFP Humanized renilla green fluorescent protein ICM Inner cell mass I16st Interleukin 6 signal transducer IMDM Iscove's modified Dulbecco's medium JAK Janus kinase Klf4 Kruppel-like factor 4 LIF Leukemia inhibitory factor MACS Magnetic-activated cell sorting MAS 5.0 Microarray Suite 5.0 M C Methylcellulose MEF Mouse embryonic fibroblast MGI Mouse Genome Informatics M T G Monothioglycerol Osmr Oncostatin M receptor PBS Phosphate buffered saline PF PBS containing 2% FBS RA Retinoic acid RT Reverse transcription StemCell Network SCN Stat3 Signal transducer and activator of transcription 3 Socs3 Suppressor of cytokine signaling-3 TGF Transforming growth factor V C M Virus containing media WPRE Woodchuck hepatitis virus posttranscriptional regulatory element viii A C K N O W L E D G E M E N T S I w o u l d l i k e t o e x p r e s s m y a p p r e c i a t i o n t o m y s u p e r v i s o r , D r . C h e r y l D H e l g a s o n , f o r h e r s u p e r v i s i o n a n d i n s p i r a t i o n , a n d f o r g i v i n g m e t h e o p p o r t u n i t y t o b e i n v o l v e d i n s u c h a n i n t e r e s t i n g p r o j e c t . I w o u l d l i k e t o t h a n k D r . K e i t h H u m p h r i e s a n d D r . C h r i s O n g f o r b e i n g m y s u p e r v i s o r y c o m m i t t e e m e m b e r s a n d f o r t h e i r w o n d e r f u l s u g g e s t i o n s . M o r e o v e r , I w o u l d a l s o l i k e t o a c k n o w l e d g e p e o p l e f r o m t h e H e l g a s o n l a b f o r t h e i r g r e a t h e l p . i x C H A P T E R 1 I N T R O D U C T I O N 1.1 Stem Cells 1.1.1 What is a Stern Cell? S t e m c e l l s a r e d e f i n e d b y t w o p r o p e r t i e s : 1 ) t h e c a p a c i t y f o r s e l f - r e n e w a l w h i c h m e a n s t h a t t h e y a r e c a p a b l e o f m a k i n g c o p i e s o f t h e m s e l v e s , o f t e n t h r o u g h o u t t h e l i f e o f t h e o r g a n i s m ; a n d 2 ) u n d e r t h e r i g h t c o n d i t i o n s , t h e a b i l i t y t o d i f f e r e n t i a t e i n t o o n e o r m o r e s p e c i a l i z e d c e l l t y p e s . D i f f e r e n t f r o m m o s t c e l l s o f t h e b o d y , s u c h a s h e a r t c e l l s a n d n e r v e c e l l s , a s t e m c e l l i s u n c o m m i t t e d o r n o t f a t e d t o c o n d u c t a s p e c i f i c f u n c t i o n u n t i l i t r e c e i v e s a p p r o p r i a t e s i g n a l s t o d e v e l o p i n t o a s p e c i a l i z e d c e l l . In vivo, s t e m c e l l s a r e i m p o r t a n t f o r t h e g e n e r a t i o n o f t i s s u e s a n d o r g a n s d u r i n g d e v e l o p m e n t a n d f o r t h e i r m a i n t e n a n c e a n d r e p a i r i n t o a d u l t h o o d . F o r t h i s r e a s o n , t h e y h a v e b e e n p r o p o s e d a s a s o u r c e o f c e l l s f o r t h e r e p l a c e m e n t o f d e f e c t i v e t i s s u e ( / ) . 1.1.2 Totipotency and Pluripotency M a n y o f t h e t e r m s u s e d t o d e f i n e s t e m c e l l s d e p e n d o n t h e b e h a v i o r o f t h e c e l l s i n t h e i n t a c t o r g a n i s m in vivo, u n d e r s p e c i f i c l a b o r a t o r y c o n d i t i o n s in vitro, o r a f t e r t r a n s p l a n t a t i o n in vivo, o f t e n t o a t i s s u e t h a t i s d i f f e r e n t f r o m t h e o n e f r o m w h i c h t h e s t e m c e l l s w e r e d e r i v e d . F o r e x a m p l e , t h e f e r t i l i z e d e g g i s s a i d t o b e t o t i p o t e n t — f r o m t h e L a t i n totus, m e a n i n g e n t i r e — b e c a u s e i t h a s t h e p o t e n t i a l t o g e n e r a t e a l l t h e c e l l s a n d t i s s u e s t h a t m a k e u p a n e m b r y o , i n c l u d i n g p l a c e n t a a n d g e r m c e l l s , a n d t h a t s u p p o r t i t s d e v e l o p m e n t in utero. A c e l l ' s t o t i p o t e n c e i s p r e s e r v e d u p t o a n d i n c l u d i n g t h e 4 - c e l l 1 stage; in the step from 4 to 8 cells, the cells lose their totipotency and are then referred to as pluripotent. Most scientists use the term pluripotent to describe stem cells that can give rise to cells derived from all three embryonic germ layers—mesoderm, endoderm, and ectoderm. These three germ layers are the embryonic source of all cells of the body. All of the many different kinds of specialized cells that make up the body are derived from one of these germ layers. "Pluri"—derived from the Latin plures—means several or many. Thus, pluripotent cells have the potential to give rise to several types of cells, a property observed in the natural course of embryonic development and under certain laboratory conditions. Unipotent stem cell, a term that is usually applied to a cell in adult organisms, means that the cells in question are capable of differentiating along only one lineage. "Uni" is derived from the Latin word unus, which means one. Also, it may be that the adult stem cells in many differentiated, undamaged tissues are typically unipotent and give rise to just one cell type under normal conditions. This process would allow for a steady state of self-renewal for the tissue. However, if the tissue becomes damaged and the replacement of multiple cell types is required, pluripotent stem cells may become activated to repair the damage (2, 3). Unlike embryonic stem cells, there are no isolated adult stem cells that are capable of forming all cells of the body. 1.1.3 Multipotent adult stem cells Multipotent stem cells can give rise to several cell types, although they are limited in number compared to those generated from pluripotent stem cells. Hematopoietic cells are an example of a multipotent stem cell. They can differentiate into several 2 t y p e s o f b l o o d c e l l s , b u t c a n n o t d e v e l o p i n t o b r a i n c e l l s (4). S i m i l a r l y , m e s e n c h y m a l s t e m c e l l s h a v e t h e c a p a c i t y t o d i f f e r e n t i a t e i n t o a v a r i e t y o f c o n n e c t i v e t i s s u e c e l l s i n c l u d i n g b o n e , c a r t i l a g e , t e n d o n , m u s c l e a n d a d i p o s e t i s s u e . T h e s e m u l t i p o t e n t c e l l s h a v e b e e n i s o l a t e d f r o m b o n e m a r r o w a n d f r o m o t h e r a d u l t t i s s u e s i n c l u d i n g s k e l e t a l m u s c l e , f a t a n d s y n o v i u m ( 5 ) . T h e e v i d e n c e f o r m u l t i p o t e n c y i s g o o d a l t h o u g h u s u a l l y d e r i v e d f r o m s i t u a t i o n s o f s e v e r e t i s s u e d a m a g e . F o r e x a m p l e , i n t h e s m a l l i n t e s t i n e , t h e r e a r e f o u r c l a s s e s o f m a t u r e d i f f e r e n t i a t e d c e l l s ( a b s o r p t i v e , g o b l e t , P a n e t h , a n d e n t e r o e n d o c r i n e c e l l s ) . T h e c o n c e p t o f a m u l t i p o t e n t s t e m c e l l p r o d u c i n g a l l f o u r t y p e s w a s p r o p o s e d b y C h e n g a n d L e b l o n d (6). M u l t i p o t e n t s t e m c e l l s w o u l d p r e s u m a b l y r e s e m b l e t h e o r i g i n a l e m b r y o n i c r u d i m e n t f o r t h e t i s s u e i n q u e s t i o n , w h i c h w i l l p r o d u c e t h e a p p r o p r i a t e m i x t u r e o f c e l l t y p e s i n t h e c o u r s e o f n o r m a l d e v e l o p m e n t . D e s p i t e t h e u n d o u b t e d e x i s t e n c e o f s o m e c e l l s t h a t c a n s h o w m u l t i p o t e n t b e h a v i o r f o l l o w i n g t i s s u e d a m a g e , t h e r e i s a l s o e v i d e n c e t h a t , w h e r e t i s s u e d a m a g e i s l o w o r n o n e x i s t e n t , m o s t s t e m c e l l s a r e u n i p o t e n t , p r o d u c i n g j u s t o n e t y p e o f d i f f e r e n t i a t e d c e l l . O n e q u e s t i o n i s w h e t h e r d i f f e r e n t i a t e d c e l l s c a n b e a l t e r e d o r c a u s e d t o b e h a v e i n a n y w a y o t h e r t h a n t h a t i n w h i c h t h e y h a v e b e e n n a t u r a l l y c o m m i t t e d ? I n t e r e s t i n g l y , r e c e n t s t u d i e s h a v e d e m o n s t r a t e d t h a t h e m a t o p o i e t i c s t e m c e l l s m a y b e a b l e t o a d o p t c e r t a i n n o n h e m a t o p o i e t i c p h e n o t y p e s , s u c h a s e n d o t h e l i a l , n e u r a l , o r s k e l e t a l m u s c l e p h e n o t y p e s , w i t h o u t e n t i r e l y l o s i n g t h e i r i n i t i a l h e m a t o p o i e t i c i d e n t i t y ( 7 , 8). T h u s , i t ' s a l s o p r o p o s e d t h a t t r a n s d i f f e r e n t i a t i o n c a n , i n c e r t a i n c o n d i t i o n s , b e a p a r t i a l r a t h e r t h a n a c o m p l e t e e v e n t . S u c h o b s e r v a t i o n s e n c o u r a g e f u r t h e r i n v e s t i g a t i o n i n t o t h e p h e n o m e n o n o f a s t e m c e l l s i m u l t a n e o u s l y e x p r e s s i n g p h e n o t y p i c f e a t u r e s o f t w o 3 distinct cell fates (9). There is also continuing research to see if it is possible to make multipotent cells into pluripotent types. 1.1.4 Types of Stem Cells Embryonic stem cell (ESC): ESC are derived from a group of cells called the inner cell mass (ICM), which is part of the early 3.5-day murine or 4- to 5-day human embryo called the blastocyst. These cells possess properties of both the ICM and ectoderm-like cells {10). Under appropriate culture conditions, ESC retain the capacity to contribute to all cell lineages, except the placenta, when reimplanted back into a blastocyst. This potential, combined with their ease of genetic manipulation and selection, has revolutionized many fields by facilitating the ability to generate transgenic, chimeric, and "knockout" mice for gene function studies in vivo (11-16). Once removed from the blastocyst, the cells of the ICM can be cultured into ESC. These ESC are not themselves embryos. In fact, evidence is emerging that these cells do not behave in the laboratory as they would in the developing embryo—that is, the conditions in which these cells develop in culture are likely to differ from those in the developing embryo. Embryonic germ cell (EG): E G cells are isolated from the primordial germ cells of the gonadal ridge of the 5- to 10-week fetus in human or around 8.5 days post coitum in mouse. Later in development, the gonadal ridge develops into the testes or ovaries (and the primordial germ cells give rise to eggs or sperm. ESC and E G are pluripotent, but they are not identical in their properties and characteristics. Human ESC form relatively flat, compact colonies that easily dissociate into single cells in trypsin or in C a 2 + - and Mg -free medium, whereas human E G cells form tight, more spherical colonies that are 4 refractory to routine dissociation methods, but which more closely resemble the morphology of mouse ES and E G cell colonies. In addition, although ES and E G cell lines both demonstrate a remarkable developmental potential, there appears to be differences between mouse ES and E G cell lines as a result of genomic imprinting (17). Adult stem cell: An adult stem cell is an undifferentiated (unspecialized) cell found in a differentiated (specialized) tissue that renews itself and can differentiate to yield all of the specialized cell types of the tissue from which it originated. Adult stem cells usually divide to generate progenitor or precursor cells, which then differentiate or develop into "mature" cell types that have characteristic shapes and specialized functions, e.g., muscle cell contraction or nerve cell signaling. Sources of adult stem cells include bone marrow, blood, the cornea and the retina of the eye, brain, skeletal muscle, dental pulp, liver, skin, the lining of the gastrointestinal tract, and pancreas (18-24). 1.2 Embryonic Stem Cells 1.2.1 Embryonic Stem Cell Cultures The first pluripotent cell lines to be established were embryonic carcinoma (EC) cell lines, derived from the undifferentiated compartment of murine and human germ cell tumors (25). These cells could be expanded continuously in culture and could also be induced to differentiate into derivatives of all three embryonic germ layers (26). Murine EC cells can also contribute extensively to all the normal tissues of chimeric mice generated by the injection of EC cells into mouse blastocysts (27). However, being 5 cancer-derived and usually aneuploid, EC cells are not suitable for clinical application, although they have proven to be a very useful model system in the laboratory. In 1981, murine ESC were first isolated. The studies of EC cells from mice and human have helped establish parameters for growing and assessing ESC. The techniques for culturing mouse ESC from the ICM of pre-implantation blastocysts were first reported 20 years ago (10, 28), and versions of these standard procedures are used today in laboratories throughout the world. ESC cultures have been established from the early embryos of fish, birds, and a variety of mammals. Generating cultures of mouse or human ESC that remain in a proliferating, undifferentiated state is a multi step process. First, the ICM of a preimplantation blastocyst around 4 to 5 days post-ovulation is removed from the trophectoderm that surrounds it. The small plastic culture dishes used to grow the cells contain growth medium supplemented with fetal calf serum, and are sometimes coated with a "feeder" layer of non dividing cells. The feeder cells are often mouse embryonic fibroblast (MEF) cells that have been inactivated so they will not divide. Mouse ESC can be maintained in an undifferentiated state in vitro on feeder layers of mitotically inactivated M EF or on gelatinized dishes without feeder layers if the cytokine leukemia inhibitory factor (LIF) (29) is added to the culture medium (30, 31). Human ESC do not respond to LIF. Second, after several days to a week, proliferating colonies of cells are removed and dispersed into new culture dishes, each of which also contains a ME F feeder layer. Under these in vitro conditions, the ESC aggregate to form colonies. Some colonies consist of dividing, non-differentiated cells; in other colonies, some cells may be differentiating. It 6 is difficult to maintain human ESC in dispersed cultures where cells do not aggregate, although mouse ES cells can be cultured this way. Depending on the culture conditions, it may also be difficult to prevent the spontaneous differentiation of mouse or human ESC. In the third step required to generate ESC lines, the individual, non-differentiating colonies are dissociated and re-plated into new dishes, a step called passage. This re-plating process establishes a "line" of ESC. The line of cells is "clonal" if a single ESC generates it. Following some version of this fundamental procedure, human and mouse ESC can be grown and passaged for two or more years, through hundreds of population doublings, while maintaining a normal complement of chromosomes, a so called euploid karyotype (32, 33). 1.2.2 Differentiation of Embryonic Stem Cells in vitro The most common method for initiating differentiation in culture is the formation of 3-D spherical structures in suspension culture, termed embryoid bodies (EBs) (Fig. 1.1) due to their similarity to post-implantation embryonic tissue in vivo. These aggregated structures contain derivatives of all three embryonic germ layers (34, 35). Their formation is a prerequisite for the generation of most mature somatic cell lineages as ESC differentiated in monolayer culture alone form large quantities of an endodermal-like cell which is identified by ESC colonies flattening and developing a 'rough' appearance. It should be noted however, that EB differentiation does not reconstitute the full array of embryonic development, having no form of polarity or 'body plan'. As such, ESC cannot form viable embryos in vitro. EB formation can be achieved in a number of ways following the withdrawal of LIF and/or the feeder layer (36). Cells can be transferred to 7 suspension culture at high density (37) or to methylcellulose (MC)-containing medium (38), to form EBs of a range of sizes and shapes. Alternatively 'hanging drop' cultures provide a more controlled method of generating single EBs from a defined cell number in individual droplets of culture medium (39, 40). The length of EB culture is dependent upon the ultimate target cell type and EB differentiation appears to correlate well temporally with the post-implantation development of embryos (36). Mesodermal and ectodermal precursors form within a few days, whereas some endodermal cell types may benefit from more extended culture time (up to 10 days) to a stage where most EBs have cavitated and become cystic (34, 35). Figure 1.1 Day 5 embryoid body cultured in M C The controlled differentiation of mouse ESC into near homogeneous populations of both neurons and skeletal muscle cells that can survive and function in vivo after transplantation has been reported (41, 42). Addition of retinoic acid (RA) and dimethyl sulfoxide (DMSO) has been shown to give rise to direct differentiation of G A B A expressing neurons and skeletal myoblasts (43). These results provided evidence that the 8 specific cell type formed (whether it be muscle, neuronal, or other cell types) can be controlled in vitro. Further, these results demonstrated that E S C can provide a source o f multiple differentiated cell types that can be used for transplantation. 1.2.3 The Cell Cycle of Mouse Embryonic Stem Cells: It's Role in Preventing Differentiation Like the cells of the epiblast in the preimplantation mouse embryo, mouse E S C in vitro have an unusual cell cycle. Specifically, the G l checkpoint does not appear to operate in proliferating epiblast and E S C (31, 44). This may explain why it has not been possible to induce quiescence—withdrawal from the cell cycle to a G l or GO state—in undifferentiated E S C . However, i f E S C begin to differentiate by forming E B , cyclin D expression increases, the G l phase of the cell cycle becomes longer, and the rate of cell division slows (44). This can occur i f L IF or feeder layers are withdrawn from mouse E S C cultures. Then, cell division continues for only a few days as the process of differentiation begins. Perhaps constant cell proliferation somehow inhibits cell differentiation, and once the signals for cell division are removed, differentiation can occur (45). 1.2.4 Assays for Measuring Murine E S C Pluripotency The various in vivo and in vitro assays used to assess the effects of culture conditions and genetic manipulations on E S C properties are described below. (1) Blastocyst injection: The gold standard for verifying the pluripotent E S C state involves injection of defined numbers of E S C (typically 10-15) into a mouse 9 blastocyst (46). Pluripotent ESC can contribute to the developing blastocyst, and thus derivatives of all germ layers, in the resulting chimeric progeny. (2) Teratoma formation: This is a second in vivo assay to assess the ability of ESC to differentiate into derivatives of all three germ layers. Although not widely used for murine ESC, it is the only assay that can be used to assess the pluripotency of human ESC. (3) Primary embryoid body formation assay: This assay measures ESC differentiation potential when cells are cultured using differentiation conditions as described in section 1.2.2. The number of EB formed can be used to quantify the differentiation potential of the ESC and it has been demonstrated previously that this assay correlates closely with the blastocyst injection assay (47). In addition, RT-PCR can be applied to the EB to determine if the ESC have differentiated into all three germ layers. (4) Colony forming cell (CFC) assay: This assay measures the ability of ESC to adhere to the plate and form new colonies. (5) Secondary embryoid body assay: When primary EBs are dissociated and replated in secondary culture, cells that fail to commit and differentiate in the primary culture, yet retain pluripotency and the capacity to undergo cell division, grow into new EBs, termed secondary EBs. The number of secondary EBs reflects the ESC capacity to maintain an undifferentiated state and provides insight into ESC self-renewal capacity (48). This assay has been widely used as a precise method of measuring self-renewal ability (49, 50). 10 1.2.5 Embryonic Stem Cells for Regenerative Medicine It has been found that mouse ESC can contribute to every tissue in the adult mouse. These types of experiments cannot be done with human tissues, even though human ESC lines are currently being studied and several research teams are working to determine whether or not they possess the same properties as mouse ESC. Although the potential of ESC in transplantation medicine is vast, before any clinical application of ESC can succeed there are a number of obstacles that must be overcome. Firstly, no approach to the differentiation of ESC has yet yielded a 1 0 0 % pure population of mature progeny. It will be essential to avoid implanting undifferentiated ESC or inappropriate cell lineages because of the risk of teratoma formation or further perturbation of tissue function. Alternatively, therefore there must be an efficient means to purify the required differentiated cell population. Methods such as fluorescence-activated cell sorting (FACS) or magnetic-activated cell sorting (MACS) allow such purification but are dependent on the cell type of interest expressing a surface marker that can be recognized by a fluorescent or magnetic micro bead-tagged antibody and, to be fully effective, the marker needs to be absolutely cell-type specific. In many cases, such a marker is not presently available, and sorting methods then rely on genetic modification of the ESC with a marker gene under the control of a lineage-specific promoter. Alternatively, cells could be transduced with a drug-resistance gene instead of a marker, to allow for preferential selection of subpopulations. However, enriching specific cell types by these means is less attractive for therapeutic applications because it involves genetic modifications, which may have an undesirable and unpredictable outcome and 11 which may impair the ability of cells to undergo the full repertoire of lineage differentiation. Despite difficulties in obtaining homogeneous populations, functional derivatives of all three primary germ layers have been successfully produced from ESC. Differentiated dopamine-producing neurons were found to incorporate into the striatum and improve performance in behavioral tests when grafted into mice with Parkinson's disease (51, 52). ESC-derived motor neurons have been grafted into chick embryo spinal cords and are shown to survive and differentiate correctly in vivo (53). Flkl- expressing, ES cell-derived vascular progenitor cells were able to differentiate into endothelial cells, after grafting into tumor-bearing mice. The grafted cells were incorporated into the developing vasculature of the tumor, demonstrating an ability to contribute to adult neo-vascularization (54). Pancreatic cells produced by culture in selective media and supplemented with fibroblast growth factor 2 (FGF2), formed islet-like structures and, upon transplantation into diabetic mice, improved survival and body weight maintenance (55). In vitro derived hepatocyte progenitors were found to undergo proper differentiation into parenchymal cells in the liver of recipient animals (56, 57). Finally, as with all transplants, there is a risk that allogeneic ESC-derived implants could be rejected by the host. Although the immunogenicity of the transplant can be contained through the lifelong use of immunosuppressive drugs, they are associated with many unpleasant side effects and render the patient extremely susceptible to infection. The production of autologous ESC by somatic nuclear transfer is one possible approach although it is technically challenging and still subject to ethical concerns. The amenability of ES cells to genetic modification may provide a means to reduce their 12 immunogenicity. This could be achieved by the insertion of immunosuppressive molecules such as Fas ligand, or by deleting immunoreactive molecules such B7 antigens (58, 59). More ambitiously, foreign major histocompatability complex genes could be replaced by the recipient's histocompatibility genes, increasing the immunocompatability of the cells. 1.3 Molecular Mechanisms Maintaining Mouse ESC in Their Undifferentiated State 1.3.1 Leukemia Inhibitory Factor and STAT3 Activation The LIF/STAT3 (signal transducer and activator of transcription 3) signaling pathway plays a critical role in maintaining mouse ESC, whereas the mitogen-activated protein kinase pathway antagonizes ESC self-renewal partly because of a negative feedback on Janus kinase (JAK) activity (60, 61). LIF is produced by feeder cells and allows mouse ESC in vitro to continue proliferating without differentiating (62). LIF exerts its effects by binding to a two-part receptor complex that consists of the LIF receptor and the gp 130 receptor. The binding of LIF triggers the activation of STAT3 (Fig. 1.2), a necessary event in vitro for the continued proliferation of mouse ESC (60, 63, 64). Recent evidence indicates that two transcription factors, STAT3 and Oct-4, may interact and perhaps affect the function of a common set of target genes (65). In vivo, signaling through the gpl30 receptor is not necessary for normal, early embryonic development but is required to maintain the epiblast during diapause. After gastrulation, LIF signaling and STAT3 activation promote the differentiation of specific cell lineages such as the myeloid cells of the hematopoietic system or astrocyte precursor cells in the central nervous system (66). 13 The self-renewal of mouse ESC also appears to be influenced by SHP-2 and signal-regulated kinase activity. SHP-2 is a tyrosine phosphatase that interacts with the intracellular (amino terminus) domain of the gpl30 receptor. E R K (extracellular regulated kinase) is one of several kinds of enzymes that become activated when the gpl30 receptor and other cell-surface receptors are stimulated. Both E R K and SHP-2 are components of a signal-transduction pathway that counteracts the proliferative effects of STAT3 activation. Therefore, i f E R K and SHP-2 are active, they inhibit ESC self-1 4 renewal (60) (Fig. 1.2). It is possible that some of the components of signaling pathways in cultured mouse ESC are unique to these cells. For example, mouse ESC in vitro express high amounts of a modified version of an adapter protein, Gabl (Fig. 1.2). The unusual form of Gabl that occurs in ESC may suppress interactions of specific receptors with the Ras-ERK signaling pathway (32). In fact, inhibition of the ERK-activating enzyme M E K actually enhances self-renewal, implying that there is a pro-differentiative effect of ERK activation (60). ESC homozygous for a Shp-2 mutation (Shp-2A46-110) demonstrate LIF hypersensitivity and increased LIF-stimulated phosphorylation of STAT3 (60). Yanjun Li et.al detected 41 genes whose expression was modified by LIF in Shp-2A46-110ES cells by micro-array (67). The two most significantly down-regulated genes, suppressor of cytokine signaling-3 (SOCS-3) and Kruppel-like factor 4 (Klf4), were subsequently shown to have roles if over-expressed in ESC. SOCS-3 over-expression resulted in an increased capacity to differentiate to hematopoietic progenitors, rather than to self-renew. In contrast, ESC over-expressing Klf4 had a greater capacity to self-renew based on secondary EB formation. Klf4-transduced day 6 EBs expressed higher levels of Oct-4, consistent with the notion that Klf4 promotes ESC self-renewal. These findings verify the negative role of SOCS-3 in LIF signaling and provide a novel role for Klf4 in ES cell function (67). cYes, a member of the Src family of non-receptor tyrosine kinases, is highly expressed in mouse and human ESC. In 2004, Cecilia et al. demonstrated that cYes kinase activity is regulated by LIF and serum and is down-regulated when cells differentiate (68). A synergistic effect on differentiation is observed when ESC are 15 cultured with a Src family inhibitor and low levels of RA (69). Src family kinase inhibition does not interfere with LIF induced JAK/STAT3 or p42/p44 mitogen-activated protein kinase phosphorylation (68). The Src family has also been implicated in the maintenance of ESC by the fact that LIF activates the Src family member Hck (69), and expression of kinase active mutants of Src and Hck can maintain ESC in an undifferentiated state when LIF concentrations are reduced but not absent (69, 70). Together the results suggest that the activation of the Src family is important for maintaining mouse and human ESC in an undifferentiated state and may represent a third, independent pathway, downstream of LIF in mouse ESC. 1.3.2 Oct-4 and Nanog in ESC pluripotency One of the hallmarks of an undifferentiated, pluripotent cell is the expression of the Pou5fl gene, which encodes the transcription factor Oct-4 (also called Oct-3 or Oct-3/4). Oct-4 is an essential regulator required throughout blastocyst development and the formation of the ICM in vivo (71) maintaining its pluripotency (65). Oct-4 is also expressed in the primordial germ cells of mice and in primitive ectoderm (72, 73). In vitro studies of ESC have shown that undifferentiated proliferating mouse and human (74). ESC express Oct-4 and developmental regulation is effected by varying OCT4 expression levels. As is the case with inner cell mass and epiblast cells in vivo, Oct-4 expression in vitro is required to maintain the pluripotent, undifferentiated state of ESC. The repression of Oct-4 induces trophectodermal differentiation and if Oct-4 expression is artificially increased, mouse ESC differentiate into primitive endoderm and mesoderm, whereas intermediate Oct-4 levels are required for the maintenance of pluripotency of ESC in 1 6 vitro (65). Oct-4 can act as a repressor or activator of transcription depending on the presence of its cofactors (75). S o x 2 is a potential co-activator for Oct-4 (76). Candidate targets of Oct-4 and S o x 2 include F g f 4 and Handl, which are associated with maintenance and differentiation of trophoblasts (75, 77). In fact, the overall impact of Oct-4 may be to prevent the expression of genes that are required for differentiation (78). In mouse ESC, Oct-4 expression and increased synthesis of Gabl may help suppress induction of differentiation. Another homeodomain protein, Nanog, also plays a central role in directing ESC self-renewal and maintaining pluripotency. Although most closely related to members of the Nkx family of homeodomain proteins, Nanog is not itself a member of this family as it lacks sequence motifs characteristic of the Nkx family (79). It was initially identified by a strategy involving the direct selection of cDNAs, from an ESC cDNA library, that were capable of maintaining ESC self-renewal in the absence of the normally obligatory LIF stimulation (80) and it was subsequently demonstrated that ESC over-expressing Nanog could self-renew in completely defined media without LIF (81). 1 7 1.4 Analysis of Gene Function T h e f i r s t r e p o r t o f g e n e t i c m a n i p u l a t i o n o f E S C d e m o n s t r a t e d t h a t v e c t o r s d e r i v e d f r o m r e t r o v i r u s e s c a n i n f e c t E S C a n d t h a t t h e i n t e g r a t e d v i r u s ( p r o v i r u s ) i s t r a n s m i t t e d t h r o u g h t h e g e r m l i n e (82, 83). F u r t h e r m o r e , i t w a s s h o w n t h a t r e t r o v i r a l v e c t o r s a r e a b l e t o i n f e c t p r e i m p l a n t a t i o n e m b r y o s , g i v i n g r i s e t o t r a n s g e n i c a n i m a l s t h a t t r a n s m i t t h e p r o v i r a l D N A t o o f f s p r i n g (84-87). H o w e v e r , f u r t h e r a n a l y s i s r e v e a l e d t h a t b o t h i n f e c t e d E S C a n d p r e i m p l a n t a t i o n e m b r y o s l a c k s i g n i f i c a n t p r o v i r u s t r a n s c r i p t i o n . T w o m a j o r m e c h a n i s m s h a v e b e e n i d e n t i f i e d f o r r e t r o v i r u s s i l e n c i n g (88): t r a n s - a c t i n g f a c t o r s t h a t b i n d t o t h e v i r a l p r o m o t e r s i n t h e l o n g t e r m i n a l r e p e a t s ( L T R s ) ; a n d m e t h y l a t i o n o f t h e i n t e g r a t e d r e t r o v i r a l g e n o m e a n d f l a n k i n g h o s t D N A s e q u e n c e s . H u m a n i m m u n o d e f i c i e n c y v i r u s t y p e 1 ( H I V - 1 ) i s o n e o f t h e b e s t - s t u d i e d c o m p l e x r e t r o v i r u s e s . I t h a s t h e a b i l i t y t o i n f e c t n o n - d i v i d i n g c e l l s p r e s u m a b l y b y i m p o r t o f t h e v i r a l D N A t h r o u g h t h e n u c l e a r p o r e a n d s u b s e q u e n t i n t e g r a t i o n i n t o t h e h o s t g e n o m e (89). L e n t i v i r a l v e c t o r s d e r i v e d f r o m s i m i a n i m m u n o d e f i c i e n c y v i r u s ( S I V ) h a v e n o w b e e n g e n e r a t e d i n s e v e r a l l a b o r a t o r i e s (90). C h a r a c t e r i z a t i o n o f t h e s e v e c t o r s h a s i n d i c a t e d t h a t t h e y a r e s i m i l a r t o t h o s e d e r i v e d f r o m H I V - l o r H I V - 2 w i t h r e s p e c t t o t h e i n s e r t i o n o f t r a n s g e n e s i n n o n - p r o l i f e r a t i n g c e l l s . H o w e v e r , i t i s b e c o m i n g c l e a r t h a t S I V v e c t o r s p e r f o r m b e t t e r t h a n H I V - 1 v e c t o r s i n s i m i a n c e l l s (91); t h u s t h e y m a y p r o v i d e a v a l i d a l t e r n a t i v e t o H I V - 1 - b a s e d v e c t o r s , a t l e a s t i n t h e e a r l y p h a s e s o f t h e c l i n i c a l t e s t i n g o f l e n t i v i r u s v e c t o r s . V e c t o r s d e r i v e d f r o m l e n t i v i r u s e s c a n t r a n s d u c e a b r o a d s p e c t r u m o f t e r m i n a l l y d i f f e r e n t i a t e d , n o n - d i v i d i n g c e l l s a s w e l l a s h e m a t o p o i e t i c s t e m c e l l s o f m u l t i p l e m a m m a l i a n s p e c i e s . A l e x a n d e r e t . a l (92) s h o w t h a t u n l i k e t r a d i t i o n a l o n c o r e t r o v i r a l v e c t o r s , e x p r e s s i o n o f t r a n s g e n e s i n t r o d u c e d b y l e n t i v i r a l v e c t o r s i n t o 1 8 murine or human ESC is not silenced. Moreover, transgenic expression is not "shut o f f during differentiation, and the transgene is expressed in multiple tissues of chimeric animals generated by transfer of lentivector-transduced ESC in blastocysts. Thus, lentiviral vectors now are widely used for gene expression studies in ESC. 1.5 ESC gene expression profiling studies Gene expression profiling to determine regulatory factors and signaling pathways present in ESC has been performed extensively in recent years (93-101). It has been hypothesized that the undifferentiated state is conferred on various stem cell populations through the use of similar molecular mechanisms. Initial support for this hypothesis came from experiments comparing the gene expression profiles of multiple stem cell populations (95, 96, 101). However, further analysis of available data indicated minimal overlap between different published stem cell-associated gene sets (102, 103). Furthermore, there is no consensus regarding candidate genes that may be important for conferring ESC properties by comparing different adult stem cell lines. The discrepancy observed amongst these studies likely arises in large part from significant differences in the strategies used to identify the stem cell profile. However, true differences in stem cell biology probably also exist among stem cells taken from different tissues (e.g., ESC, neural stem cells, or hematopoietic stem cells). Some studies have relied on comparison of ESC to terminally differentiated tissues (97, 100, 101) to establish a list of genes enriched in ESC. However, this strategy is likely to miss genes involved in pluripotency that are transiently turned off early during the differentiation process but not uniquely expressed in the stem cell population. Another strategy used has been to compare stem 1 9 cell populations arising from the same tissue source across species. An example is the comparison of mouse with human ESC. Although mouse and human ESC are both derived from preimplantation blastocysts, they differ in responsiveness to extrinsic signals and in expression of surface markers. For example, LIF cannot sustain self-renewal of human ESC, even in the presence of serum, suggesting the existence of other signaling pathways essential for self-renewal in human ESC (33). The evidence that human and mouse ESC share a common core molecular program is also somewhat conflicting (93, 97, 100). Despite the number of studies already carried out, there does not appear to be consensus regarding candidate genes that may be important for conferring ESC properties. In part, this lack of agreement may be attributed to a paucity of correlative functional data and thus uncertainly about whether the expression profiles being compared are from similar populations of cells. As a first step in attempting to correlate gene expression profiles with functional measures of ESC pluripotency, the ability of differentiating ESC to give rise to chimeric mice, EB, and CFC was assessed over a time course (time 0 - 120 hours) (47). This work revealed that measures of ESC pluripotency decrease rapidly during the first 24 hours following LIF removal and are almost completely gone by 72 hours. Based on these observations the BCCRC group selected three time points were selected on which to carry out gene expression profiling using Affymetrix arrays: 1) undifferentiated ES cells; 2) 18 hours of differentiation - an early time point when significant decreases in pluripotency are observed; and 3) 72 hours when virtually all potential is lost. Genes differentially expressed during differentiation were identified 20 using the following criteria: 1) the difference in expression between two time points was at least 2-fold; 2) the gene was expressed in all three replicates; and 3) it was expressed in at least one of the time points as determined by Microarray Suite (MAS) 5.0. Additionally, "decreased" genes that were scored as absent in the denominator of the fold change and "increased" genes scored as absent in the numerator of the fold change were removed. Moreover, the difference in expression had to be statistically significant (p<0.05, in parametric Welsh-ANOVA t-test). Using these criteria 472 unique genes were identified as significantly differentially expressed (274 decreased, 194 increased and 4 both decreased and increased) during early ESC differentiation. Unigene and RefSeq ID were used in the analysis to exclude redundant genes included in the array probe sets. Bioinformatic validation revealed the expected changes in numerous genes. In addition, results for selected genes were further verified using RT-PCR. Taken together, this work suggests that many of the candidates, which have not previously been implicated in maintaining ESC pluripotency, are likely to have important functions in ESC. However, functional analysis of such large gene lists is not a feasible task for most laboratories. 1.6 Hypothesis and specific aims The long-term objective of this research was to identify critical regulatory genes that can be used to enhance our efforts to utilize ESC as an unlimited supply of functional, differentiated end cells (i.e. beta cells; neurons; hematopoietic cells) for cell replacement therapy. We postulated that genes implicated in the maintenance of the pluripotent state would be significantly down-regulated in the differentiated population. In this study I sought to identify candidate genes and determine their role in regulating ESC 21 p r o l i f e r a t i o n a n d d i f f e r e n t i a t i o n b y o v e r - e x p r e s s i o n t e c h n i q u e s u s i n g l e n t i v i r u s . T h e s p e c i f i c a i m s w e r e : A i m 1: T o i d e n t i f y c a n d i d a t e g e n e s o f i n t e r e s t f o r f u n c t i o n a l s t u d i e s A i m 2: T o v a l i d a t e c a n d i d a t e g e n e e x p r e s s i o n c h a n g e s d u r i n g d i f f e r e n t i a t i o n A i m 3: T o d e t e r m i n e i f g e n e t i c a l l y a l t e r i n g e x p r e s s i o n l e v e l s o f c a n d i d a t e g e n e s i n f l u e n c e s t h e c a p a c i t y o f E S C t o p r o l i f e r a t e , s e l f - r e n e w a n d / o r d i f f e r e n t i a t e . 22 C H A P T E R 2 M E T H O D S A N D M A T E R I A L S 2.1 Gene Array Analyses 2.1.1 Comparisons of the B C C R C and S C N Microarray datasets In total, 4 microarray datasets were used in the present study: the BCCRC R l ESC data and 3 datasets (Rl, Jl and V6.5 ESC lines) generated by members of the SCN (http://www.stemcellnetwork.ca). The BCCRC microarray was done using the mouse GeneChip (MG) U74v2 chips which were hybridized on a GeneChip System (Affymetrix) at the Genome Science Centre, BC Cancer Agency, Vancouver, British Columbia, Canada. The SCN microarrays were done using the mouse GeneChip (MG) MOE430 chips. For the comparisons amongst the datasets all three time points from the BCCRC data (0, 18, and 72 hours) were used, while times 0, 18, and 96 hours were used from the SCN data. Fifteen well-known, highly expressed ESC genes were selected to determine the correlation amongst these datasets. The first comparison was carried out using the two R l ESC datasets. The fold change was calculated between 0 and 18 hours. The other 3 comparisons, R l vs. J l , R l vs. V6.5 and Jl vs. V6.5 were carried out using the SCN data in the same way as the first comparison. 2.1.2 Constructing Gene lists for Preliminary Candidate Identification Affymetrix Micro Array Suite 5.0 (MAS 5.0) software was used to generate absolute expression estimates (Absence/Presence calls). The threshold to determine the present (P) 23 or absent (A) calls were used at default values: alphal=0.04, Alpha2=0.06 and Tau=0.015. Raw data (CEL-files) obtained from MAS 5.0 was then normalized and analyzed in GeneSpring® software version 6.2 (Silicon Genetics, Redwood City, CA). Data were normalized as follows: Values below 0.01 were set to 0.01 and each measurement was then divided by the 50th percentile of all measurements in that sample. Each gene was divided by the median of its measurements in all samples. If the median of the raw values was below 10 then each measurement for that gene was divided by 10. The previous BCCRC study (47) showed that the pluripotent potential of R l ESC decreased significantly during the first 18 hours following LIF removal. Therefore, to identify genes related to this process the following criteria were applied for analysis of the BCCRC gene expression data: 1) Fold change thresholds were set to identify those genes with expression changes of at least 5 fold down-regulated between Ohr and 18hr and at least 2 fold down-regulated between 18hr and 72hr; 2) Genes had to be present in all triplicate chips of a specific ESC stage (i.e. 0 hr). A second candidate list was generated through analysis of the ESC expression profiling data available through the SCN. First, the R l ESC LIF-removal differentiation data was analyzed using the following criteria to identify down-regulated genes: 1) Fold change thresholds were set to identify those genes that are at least over 4 fold down-regulated between Ohr and 18hr and at least 2 fold down-regulated between 18hr and 96hr; 2) Genes had to be present in all triplicate chips of a specific ESC stage (i.e. 0 hr). A third candidate list was generated by identifying commonly expressed, down-regulated genes present in the R l , V6.5 and Jl ESC lines induced to differentiate by LIF removal. The following criteria were used to identify these genes: 1) Fold change 24 thresholds were set to identify those genes that are at least 3 fold down-regulated between Ohr and 18hr and at least 2 fold down-regulated between 18hr and 96hr; 2) Genes had to be present in all triplicate chips of a specific ESC stage (i.e. 0 hr). 2.1.3 Gene Ontology (GO) Analysis to narrow the candidate list In order to narrow the gene lists to a manageable number of candidates, we focused on those candidates that fell into the following GO annotations: 1) Transcription factors; 2) Well known function related to cell proliferation, apoptosis or cell cycle; 3) Unknown genes. The NetAffx Analysis Center (http://www.affymetix.com/analysis/index.affx) was used to correlate GeneChip array results with array design and annotation information. For further information and cited published papers for some candidate genes, another online resource, Mouse Genome Informatics (MGI) (http://www. informatics. i ax.org/), was used. MGI provides integrated access to data on the genetics, genomics, and biology of the laboratory mouse that was used for the GO analysis. 2.2 Growth of Mouse Embryonic Stem Cells and Mouse Embryonic Fibroblasts (MEF) MEF were maintained at 37°C humidified air with 5% CO2 in Dulbecco's modified Eagle's medium (DMEM) (all reagents obtained from StemCell Technologies Inc. [STI], Vancouver, British Columbia, Canada, unless otherwise indicated) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 ug/ml streptomycin, and 100 uM monothioglycerol (MTG; Sigma, Oakville, Ontario, Canada). Prior to use as feeder layers, MEF were trypsinized using 0.25% trypsin and 1 mM E D T A (T/E) (Invitrogen 25 Life Technologies, Burlington, Ontario, Canada) for 5 minutes until cells detached. T/E activity was quenched with D M E M supplemented with 10% F B S , the cells were centrifuged at 1,200 rpm for 7 minutes and then suspended in 2 ml of medium. M E F were exposed to 60 Gy from an x-ray source, plated at a density of 2 x 10 6 and rested over night prior to use. R l E S C , frozen at passage 17 at a density of 10 6 cells per vial, were thawed in a 37°C water bath. D M S O was washed away with 10% F B S D M E M by centrifugation and then E S C were plated on the tissue culture dishes with an irradiated M E F feeder. R l E S C . were routinely maintained at 37°C humidified air with 5% CO2 on a prepared layer of irradiated M E F and fed daily with a complete change of E S C maintenance medium consisting of high-glucose D M E M supplemented with 15% ESC-tested F B S , 0.1 m M nonessential amino acids, 2 m M glutamine, 1,000 U / m l L IF , 100 U / m l penicillin, 100 (ig/ml streptomycin, and 100 u M M T G . Cells were passaged every second day by generating a single-cell suspension using T/E treatment for 5 minutes until cells detached from the culture vessel surface. T /E activity was quenched with D M E M supplemented with 10% F B S , the cells were centrifuged at 1,200 rpm for 7 minutes and suspended in E S C maintenance medium. E S C were routinely as seeded at 5 x 105 per 100 mm T C plate and were not maintained for more than two weeks. 2.3 Embryoid Body formation in suspension culture R l E S C were thawed and cultured for two passages (96 hours) on irradiated M E F in E S C maintenance media. To prepare E S C for differentiation, they were harvested and washed as described above, resuspended in E S C maintenance medium, and preplated on 26 tissue culture plates for 1 hour at 37°C, 5% CO2 to deplete contaminating MEF. The nonadherent ESC were then discarded and the loosely adherent ESC were collected by gently washing the surface of the tissue culture plate. Cells were pelleted by centrifugation, and viable cell numbers were determined. The frequency of contaminating MEF in the undifferentiated (day 0) ESC samples was estimated to be less than 0.2% based on cell size during counting. The preplated ESC were suspended at a density of 105 cells per 10ml in 100mm Petri-style culture dishes (Falcon) in liquid differentiation medium consisting of Iscove's modified Dulbecco's medium (IMDM), 15% FBS selected for its ability to support ESC differentiation, 2 mM glutamine, 150 uM M T G , and cultured at 37°C, 5% C 0 2 . At various times during the differentiation in liquid culture (0, 6, 12, 18, 24, 36, 48, and 72 hours), non-and loosely-adherent EB were harvested and allowed to settle to the bottom of a 50-ml conical tube for approximately 10 minutes. The supernatant, containing mainly single cells, was removed, and the spontaneously pelleting EB fraction was collected by centrifugation at 1,200 rpm for 7 minutes. EB were disrupted by incubation in T/E for 3 minutes at room temperature followed by passage through a 21-gauge needle to achieve single-cell suspensions. The cells were washed with 10% D M E M and then centrifuged again to collect the cell pellets. All cell pellets were disrupted in 1 ml Trizol (Invitrogen) and stored at -20° prior to RNA isolation. 2.4 Harvest of Embryoid Bodies from Methylcellulose Cultures Regardless of the age of the EBs in the primary differentiation cultures, the initial stages of the harvest are the same. First, each culture dish is flooded with 1 ml of medium 27 (IMDM plus 2% differentiation FBS) and gently mixed using a pipetter with a 1 ml tip. The contents of 2-3 dishes are transferred into a 14 ml round-bottom polystyrene tube. Each dish is washed with 1 ml more of medium to ensure all EBs are collected. The tube is mixed and centrifuged at 1200 rpm (300 g) for 10 minutes and the supernatant is carefully removed so as not to disturb the loose pellet. EB are disrupted with 3 ml of Trypsin-EDTA (2-3 minutes; RT) or Collagenase (1 hour; 37°C) depending upon the age of the EBs. Single cell suspensions are prepared by passing the mixture through a 21g needle 2-3 times. Finally, 5% FBS in IMDM is added and the suspension centrifuged at 1200 rpm for 5-8 minutes. 2.5 R N A Isolation, cDNA Synthesis, and Quantitative R T - P C R (RT-qPCR) RNA was isolated using Trizol™ according to manufacturer's protocols. The samples were then treated with DNase I (amplification grade) prior to reverse transcription (RT) according to the manufacturer's recommendations (Invitrogen). Complementary DNA (cDNA) was generated by RT with random primers and the Superscript II enzyme in the presence of lui RNase inhibitor (Invitrogen) in a 20ul reaction volume. The RT reaction was incubated at 42°C for 50 min followed by 15 min at 70°C. The cDNA was stored at -20°C for subsequent Q-PCR analysis. Transcripts were quantified by real-time PCR using the ABI apparatus and were detected with SYBR Green (Power SYBR Green PCR Master Mix, Applied Biosystems) as the flurochrome. Primer sequences, shown in Table 2.1, were designed to flank exons. Relative expression changes were determined with the 2"AACT method (104) and the housekeeping glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene transcript was 28 used to normalize the results. PCR efficiency was tested for each primer pair with a dilution series of cDNA to make sure that the efficiency was appropriate for the 2"AACT method (i.e. 95% or above). To identify amplification of any contaminating genomic DNA and ensure the specificity and integrity of the PCR product, melt curve analyses were performed on all PCR products. No products were obtained with real-time PCR from RNA samples when reverse transcription was omitted. Samples without template were included for each primer pair to identify contamination. Table 2.1 Primer sequences for RT-qPCR validation Gene Forward Primer Reverse Primer Product Size (bp) Gapdh A A C T T T G G C A T T G T G G A A G G A T G C A G G G A T G A T G T T C T G G 130 Bcl3 G C T G T T C T G C T G C T C C T G T C A G G C T G T T G T T C T C C A C 100 Empl A C T G G C T G G T C T C T T T G T G G T T G C G T A A T C T G C A A C C A T C 92 Socs3 G A G A T T T C G C T T C G G G A C T A A A C T T G C T G T G G G T G A C C A T 129 Ifil6 G C A G T G C A G A C A G G C A G A C G C C T T G C T C T T T T T C A C T C 90 C x c l C T T G A A G G T G T T G C C C T C A G A A G G G A G C T T C A G G G T C A A G 99 Riken cDNA 1700110N18 C T T A C A G C A T T C G G C A T A C G T T G C T T G A T T T C C T T T C T G G A 97 Inhbb C G A G A T C A T C A G C T T T G C A G T G G T T G C C T T C A T T A G A G A C G 84 Klf5 C T G A A A C A C G C G C A C C A C G G A G C T G A G G G G T C A G A T A C T 119 Riken cDNA 8430410A17 G G T T A A G A A G G A G C C C A A G G T T G G G T G A G T C A G G G A C T T C 96 Tgml A G A A G C A C A G A T T G G C G A A C T C C G A T C C A G G A T G T A C A G G 86 Gbx2 A G A C G G C A A A G C C T T C T T G T T G A C T C G T C T T T C C C T T G C 113 Ankrdl A A A G C G A G A A A C T G C G A G A G G G C T C C T T C A C A A C T G G A A C 148 29 2.6 Synthesis of Full-length cDNAs for Vector Construction Those candidates that showed the expected down-regulation of expression in the R T -q P C R analysis were selected for further study. Gene sequences for primer design to allow amplification of the full length c D N A were obtained from the N C B I Reference Sequences database (http://www.ncbi.nlm.nih.gov/RefSeq/). Primers were chosen using the Primer3 software (http://www.broad.mit.edu/cgi-bin/primer/primer3_www.cgi). The primers were designed to include an A s c l restriction site ( G G C G C G C C ) at the 5' end and a Pac l restriction site ( T T A A T T A A ) at the 3' end to allow directional cloning. Synthesis of the primers was performed at Invitrogen Life Technologies, Canada. Primer sequences are shown in Table 2.2. The specificity of the P C R primers was confirmed using B L A S T searches (http://www.ncbi.nlm.nih.gov/genome/seq/MmBlast.html'). Table 2.2 Primer sequences used to amplify whole coding regions of candidates Gene Forward Primer (GGCGCGCC. . . . ) Reverse Primer ( T T A A T T A A . . . . ) Product Size (bp) Klf5 T A G A C A T G C C C A G T T C G A C A C G C T C G C T C A G T T C T G G T 676 Riken A17 A T G T G C G G G C G A A C G T C C C T A G C T G T T A G G C T T C T T 983 Bcl3 A T G G A C G A G G G G C C C G T G T C A G C T G C T T C C T G G A G C 1339 Empl G A G A G G A C C A G A C C A G C A C C T C C C A G G T T A G G T T G C T T 669 Inhbb C G A G A T C A T C A G C T T T G C A G C C C A A T G G A A C A A C C A C A C T 904 Gbx2 A C G G G G A C T T T T C G T C T C T C G G A G G T G C C T C T G A G T T C T G 1204 Ankrdl G A C T C A C G G C T G C C A A C C A G C T C C T G T T G A G T C T C T T T T 1016 Cxc l l G A C T C C A G C C A C A C T C C A A C A G G T G C C A T C A G A G C A G T C T 369 30 A l l P C R reactions were carried out in a total volume of 25 ul consisting of: P C R Hi f i Taq polymerase buffer 2.5ul, 50mM M g S 0 4 0.5ul, lOOmM dNTP l u l , D M S O 2.5ul (final concentration is 10%), Primer M i x l u l , Taq Hi f i polymerase 0.25u.1, c D N A sample (generated from E S C R N A ) l u l , and d H 2 0 16.25ul. Reactions were carried out in a BioRad M J thermocycler using the following program: 95°C for 4 min, then 40 cycles of 95°C for 30sec, 58°C for 30sec, 70°C for lmin , and final step 70°C for 10 min. 2.7 T O P O Cloning and Generation of the Lentiviral Constructs Appropriate-sized bands for the wanted candidate genes were isolated using a Qiagen Gel Extraction kit i f more than one band was observed; otherwise, the P C R sample was directly TOPO-cloned into the pCR2.1-TOPO vector using the T O P O cloning kit (Invitrogen). Ligations were carried out by mixing l u l T O P O vector, 3ul fresh P C R product, l u l salt solution and l u l sterile water for 5 minutes at room temperature. Next, 2ul of the T O P O Cloning reaction was added to a vial of One Shot Chemically Competent E.coli (DH5a) and incubated on ice for 30 min. The bacteria were heat-shocked for 45 seconds at 42 °C without shaking. After that, the tubes were immediately transferred to ice for at least 1 min then 250ul of room temperature S O C medium was added and cultures were incubated with shaking for 1 hour at 37°C. Finally, 75ul bacteria were spread and grown overnight at 37°C on L B plus ampicillin (50ug/ml) agar plates. Several colonies were picked from each transformation and grown in 2ml liquid L B plus ampicillin overnight at 37°C. Plasmid D N A was isolated by MiniPrep. Plasmid D N A was then digested with two restriction enzymes - one within and one outside of the 31 cDNA sequence to confirm integration of the correct fragment. Appropriate cloning of the cDNA was confirmed by sequencing at UBC (http://naps.msl.ubc.ca/dnalims.html). The cDNA fragments were liberated from the TOPO vector by AscI-PacI digestion, gel-purified as described above, and directionally cloned in lentiviral vector #KA391 (Fig. 2.1). This vector contains an EFl-alpha promoter to drive gene expression, as well as an IRES-GFP cassette to allow sorting of infected cells. Ligations were transduced into commercial TOP-10 cells, grown, and plasmid DNA was isolated as above. Successful lentivector constructs were confirmed by digestion with AscI and Pad. Figure 2.1: Schematic diagram of the lentiviral vector Insert here 32 2.8 Transfection of 293 T cells for lentivirus production V-SVG (an envelope construct), REV (regulatory viral protein construct), and delta R (packaging construct), all provided by Dr. Connie Eaves and originally obtained from Dr. Philippe Leboulch (Harvard Medical School and Brigham & Women's Hospital) were expanded and then purified by MidiPrep (HiSpeed Plasmid Midi Kit; Qiagen) before transfection. Twenty-four hours prior to transfection, 293 T cells were plated at 5 x 106 cells per 10 cm tissue culture dish in 7 ml of 10% D M E M . Cells were incubated at 37°C overnight and fresh media was added 2-4 hrs before transfection. Solutions required for transfection were prepared during this time and included: 1) 2x Hank's Balanced salt solution (HBS) prepared by diluting lOx HBS with water and adjusting the pH to 7.05-7.10 with IN NaOH; 2) 2.5M CaCl 2 prepared by dissolvingl8.37 grams CaCl 2 in 50 ml H?0. Both solutions were sterilized using a 0.22um filter prior to use. For each 10cm2 dish of 293 T cells, lOug vector, 6.5ug delta R, 2.5ug R E V and 3.5ug V S V G were diluted together in a volume of 450 ul of dH20 in a 50 ml polypropylene tube. Next, 50 ul of 2.5M CaCl2 was added dropwise to the DNA mixture and the solution was allowed to sit for 2-3 minutes. Next, this DNA- CaCb mixture was added drop-wise, while gently mixing, to a 50 ml tube containing an equivalent volume of 2x HBS yielding a total volume of 1 ml. This mixture was allowed to sit for 2-5 minutes and then this DNA/CaPCU suspension was added slowly to the 7 ml of media in the dish of 293T cells. The media was then gently swirled to evenly distribute the suspension over the cells. After 24 hours of culture, the media was changed to a volume of 5.0 ml; the cells were cultured for an additional 24 hours before the supernatant was collected. This step was repeated once more. The two supematants were pooled, filtered 33 through a 0.45 urn low protein binding Millipore filter, and then the virions were concentrated by ultra-centrifugation at 25,000 rpm for 90 minutes. The pellets were resuspended in 1ml ESC maintenance media containing 5% DNase and were mixed at very low speed for 1 hour prior to freezing at -80°C until needed. These protocols, as well as the ESC infection, were all carried out in the Level I I I facility. 2.9 ESC Infection, Flow Cytometry, and Fluorescence Activated Cell Sorting R l ESC, passaged three times prior to infection, were plated at 106 cells per 100mm tissue culture dish on MEF. 48 hours after the final passage, ESC were harvested, pre-plated to remove contaminating MEF, plated at 4 x 105 ESC per 60mm gelatinized tissue culture dish, and cultured for an additional 24 hours. Infection of these R l ESC was carried out by adding 300ul of viral supernatant plus lul protamine sulfate (5 ug/ml) as the DNA carrier to each dish. ESC were cultured for 24 hours, fresh media was added and the cells were cultured for an additional 24 hours prior to harvest to assess GFP expression by flow cytometry. ESC were harvested and diluted in phosphate buffered saline (PBS) containing 2% FBS (PF). Cells were washed once by centrifugation with PF and then 7-amino actinomycin D (7AAD 1:1000), which is used to distinguish between live and dead cells, was added. Cells were incubated for 20 minutes at room temperature in the dark and ESC were then analyzed by flow cytometry using a FACSCalibur flow cytometer and CELLQuest software (BD Pharmingen). Viable cells were gated based on 7AAD staining and uninfected ESC were used to determine appropriate gating for quantification of GFP expression. 34 The transduced ESC were harvested and plated on MEF feeder layers for 7 days to expand before sorting. On the day of sorting, single cell suspensions of ESC were harvested and pre-plated for 1 hour to deplete contaminating MEF. The nonadherent ESC were then discarded and the loosely adherent ESC were collected by gently washing the surface of the tissue culture plate. Harvested ESC were spun down and then suspended at 107 ESC/ml in PF. GFP + ESC were sorted using a Becton Dickinson FACS-Vantage Flow Cytometer/Cell Sorter. Sorted ESC populations, as well as the unsorted ESC, were expanded on gelatinized dishes until sufficient numbers of cells were obtained to allow freezing 5 or 6 vials of each population at a density of 106 cells per vial. The freezing media consisted of 90% FBS and 10% DMSO. 2.10 Functional Analyses of the ESC ESC were thawed onto gelatinized plates and passaged twice prior to setting up the various functional assays outlined below. 2.10.1 C F C analysis and ESC proliferation To determine the colony forming cell (CFC) frequency within a population, single cell suspensions of ESC were plated at various densities (500 to 20,000 cells per gelatinized 60-mm gridded tissue culture dish) in ESC media with and without LIF. Cells were cultured for 5-6 days at 37°C and were then microscopically enumerated. Three independent experiments were performed. The CFC plating efficiency was calculated by dividing the total number of colonies by the number of cells plated multiplied by 100. 35 CFC were also generated in the absence of LIF for 5-6 days as outlined above to determine the effects of the various genes on maintenance of ESC pluripotency. Plating efficiencies were calculated as outlined above. Growth curves were generated for each of the ESC lines by plating 5 x 104 ESC in triplicate in 24-well plates. Cells were cultured for 2 days in ESC maintenance media, harvested, counted, and replated at the same cell density. This process continued for a total of 6 days. Projected cell numbers for each of the days were calculated by multiplying the cell number from the previous day by the fold change in cell numbers during the subsequent two days of culture. Results are expressed as the mean ± SD of triplicate determinations. 2 . 1 0 . 2 Analysis of differentiation potential To determine the efficiency of EB formation, single-cell suspensions were prepared as outlined above. Three thousand cells were plated in triplicate 35-mm Petri-style dishes in ESC differentiation methylcellulose and were cultured for 5-6 days in humidified chambers at 37°C. EB numbers were determined microscopically and then the EB were harvested for analysis of gene expression. In addition, RT-qPCR was carried out to examine expression levels of selected germ layer markers: Fgf5, Soxl7, Foxa2, Nestin and Brachyury. Three independent experiments were performed. The EB formation efficiency was calculated by dividing the total number of EB formed by the number of cells plated multiplied by 100. Meanwhile, EBs were harvested and disrupted into single cell suspensions. Viable cell numbers were determined using trypan blue exclusion and the number of cells per EB was determined by dividing cell number by the number of EB. 36 The secondary EB formation assay was carried out in duplicate as follows: ESC were differentiated in suspension cultures (IMDM differentiation media) in the absence of LIF for 18 hours. The non-and loosely attached EBs were harvested by gently rinsing the plates several times, single cell suspensions were prepared, and the EB assay was set up as outlined above. 2.11 S t a t i s t i c s For microarray analyses, the Pearson correlation between two datasets was calculated using Excel software. For statistical analysis of the functional assays, the lvalue - Meanl outliers were excluded using the Z-score formula as follows: Z = J . The & SD range of Z could vary dependent on the sample numbers. In each individual CFC or EB experiment, 3 plates as triplicate were set up for reducing technical error. Therefore, if Z> 1.15 (in the case of N=3) the value was considered to be an outlier and was excluded. After removing all the outliers, the average of the three trials was calculated, as well as the standard deviation. Significant differences were calculated with t-test (tails: 2, type: 1) statistics using Excel software. 37 C H A P T E R 3 R E S U L T S 3.1 Gene Array Analyses and Validation 3.1.1 Analyses of the ESC Microarray data Analysis of the BCCRC R l ESC gene expression profiles following differentiation induced by LIF removal has been previously described (47). Members of the SCN carried out a similar gene expression profiling study on murine ESC using microarray analyses to detect gene expression changes in 3 different ESC lines (Rl, Jl and V6.5) at 18 and 96 hours following LIF removal. The differentiation conditions varied from ours in the following ways: 1) DMEM-based medium instead of IMDM-based medium was used for EB formation and 2) ESC were cultured for one passage without MEF prior to EB formation rather than just pre-plating for lhour to get rid of the MEF. We first compared the two ESC datasets by determining the correlation between the two R l ESC results for 15 well-known ESC genes (Table 3.1) at 18 hours after LIF removal. In addition, to verify if the gene expression changes observed in R l ESC are general and more broadly observed two other ESC lines, Jl (105) and V6.5, were also compared with the SCN Rl data. As shown in Figure 3.1, even with different GeneChip use (MG U74v2 and MOE430) and slightly different culture conditions, the two R l ESC datasets were highly correlated (r = 0.84). In contrast, correlation coefficients of 0.68, 0.61 and 0.47 were obtained for the R l vs. J l , R l vs. V6.5 and Jl vs. V6.5 comparisons respectively. These results suggest the absence of a shared gene expression profile among different ESC lines. 38 Table 3.1: Comparisons of the ESC microarray data. The gene array results are the fold change between 0 and 18 hour following LIF removal. B C C R C Array SCN Arrays Gene name R l ESC line R l ESC line J l ESC line V6.5 ESC line Stat3 0.51 0.75 0.54 0.77 Osmr 0.4 0.66 1.08 0.18 I16st 0.62 0.81 1.00 0.52 Piml 1.08 1.21 0.99 0.74 Socs3 0.22 0.18 0.13 0.26 Akp2 1.24 1.08 1.08 1.46 Oct-4 1.09 0.81 0.72 0.78 Sox2 0.9 1.22 0.94 0.67 Fgf4 1.39 1.04 0.86 0.88 Nanog 0.82 0.83 0.78 0.52 Dppa3 1.06 1.12 1.10 0.77 Esgl 1.02 0.97 0.94 0.90 Klf4 0.32 0.37 0.29 0.26 Nestin 0.97 0.90 0.84 0.84 Promininl 1.27 1.51 0.75 0.76 39 Figure 3.1: Correlation between gene arrays. Pearson's correlation analysis was done on the relative fold change values obtained from the gene-array fold changes (n = 15). a) R l vs. R l ; b) R l vs. J l ; c) R l vs. V6.5; d) Jl vs. V6.5. The regression line is shown. The linear correlation coefficient = r. a) r=0.84 b) r=0.68 0.00 0.50 1.00 R1 (SCN) 1.50 2.00 40 LIF binds to the gpl30 receptor that leads to activation of the transcription factor Stat3 (32). Stat3 was clearly detected in undifferentiated ESC, and LIF removal led to decreased Stat3 expression with the most pronounced effect during the first 18 hours in all datasets. This was also seen for two other genes involved in the LIF/gpl30 pathway, the Oncostatin M receptor (Osmr) and the interleukin 6 signal transducer (I16st) except in the Jl ESC line. Two Stat3-induced genes, Socs3 and Klf4, were also reduced in all datasets. However, a known Stat3 target gene, Piml(7(3(5), was not decreased in either the R l or Jl ESC lines, but was slightly reduced in the V6.5 dataset (Table 3.1). Taken together, these observations confirm that the LIF/gpl30/Stat3 pathway was rapidly shut down after LIF removal. In contrast, two other ESC regulatory genes that function independent of the LIF signaling pathway, Oct-4 and Nanog, were not consistently changed in all cell lines. For example, Oct-4 was slightly increased in the BCCRC dataset but decreased to a small extent in all SCN datasets. Similarly, Nanog was not significantly decreased from 0 to 18 hour in either R l or Jl ESC (Table 3.1). 3.1.2 Additional Analyses to Identify Candidates for Functional Studies Previous studies showed that the pluripotent potential of R l ESC decreases significantly during the first 18 hours following LIF removal and is almost gone at 72 hour (47). This loss of pluripotency was similarly observed using three assays: blastocyst injection, CFC assay and EB formation assay. Therefore, to identify genes related to this process the following criteria were applied for analysis of the BCCRC gene expression data: 1) Fold change thresholds were set to identify those transcripts that decreased at least 5 fold between Ohr and 18hr and at least 2 fold between 18hr and 72hr; and 2) Genes had to be present in all triplicate chips of a specific ESC stage (i.e. 0 hr). A Venn diagram 41 of this analysis is shown in Figure 3.2a. There were 23 genes fitting these criteria (Table 3.2). A second candidate list was generated through analysis of the R l ESC LIF-removal differentiation expression profiling data available through the SCN using the following criteria to identify down-regulated genes: 1) Fold change thresholds were set to identify those transcripts that decreased at least 4 fold between Ohr and 18hr and at least 2 fold down-regulated between 18hr and 96hr; and 2) Genes had to be present in all triplicate chips of a specific ESC stage (i.e. 0 hr). A Venn diagram of this analysis is shown in Figure 3.2b. Of the 8 genes in this group (Table 3.2) only Empl and Bcl3 were also present in the group of 23 genes identified through analysis of the BCCRC data. The third, and final, candidate list was generated by identifying commonly expressed, down-regulated genes present in the R l , V6.5 and Jl ESC lines induced to differentiate by LIF removal. This gene list was generated based on the assumption that genes similarly down-regulated in all 3 ESC lines are more likely to represent genes related to "sternness". The following criteria were used to identify these genes: 1) Fold change thresholds were set to identify those transcripts that decreased at least 3 fold between Ohr and 18hr and at least 2 fold between 18hr and 96hr; and 2) Genes had to be present in all triplicate chips of a specific ESC stage (i.e. 0 hr). A Venn diagram of this analysis is shown in Figure 3.2c. Five candidates met these criteria (Table 3.2). It should be noted that the criteria for identifying candidate genes varied from one analysis to the next. This variation was necessary to ensure reasonable numbers of candidates were obtained in each analysis since the criteria used to generate the first gene list was too stringent for the others. 42 Figure 3.2: Venn diagrams for the comparisons amongst the various ESC libraries. Candidate genes were selected from the BCCRC R l data set (a), the SCN R l dataset (b) and from a comparison amongst the V6.5, Jl and R l ESC datasets (c) using the indicated criteria. a) 5 fold decreased from 0 to 18hr b) 2 fold decreased 4 fold decreased from 0 to 18hr from 2 fold decreased from 18to96hr Present in ESC Present in ESC C) 3 and 2 fold decreased from 0-18 and 18~96hr respectively for J l 3 and 2 fold decreased from 0—18 and 18~96hr respectively for V6.5 3 and 2 fold decreased from 0—18 and 18~96hr respectively for R l (SCN) 43 Table 3.2: The candidates identified through the various analyses. Gene Title Gene Symbol G O Biological Process Description G O Molecular Function Description Top 23 down-regulated genes identified through analysis of the BCCRC microarray data glycoprotein 49 B Gp49b (Lilrb4) hematology: hematopoietic anomalies mast cell membrane receptor with two immunoreceptor tyrosine-based inhibitory motifs (ITIM), constitutively inhibits mast cell activation-secretion induced by stem cell factor (SCF), a tissue-derived cytokine that also regulates mast cell development fibulin 2 Fbln2 Protein like: EGF domain calcium ion binding, protein binding tenascin C Tnc unknown unknown B-cell leukemia/lymphoma 3 Bcl3 regulation of transcription, DNA-dependent D N A binding, transcription factor activity chemokine (C-C motif) ligand 2 Ccl2 Chemotaxis; inflammatory response; immune response signal transduction G-protein-coupled receptor binding; cytokine activity; protein binding; chemokine activity chemokine (C-C motif) ligand 9 Ccl9 Chemotaxis; immune response; signal transduction cytokine activity; chemokine activity thrombomodulin Thbd Pregnancy; blood coagulation; embryonic development; negative regulation of coagulation lectin_c;sugar binding;9.8e-06 suppressor of cytokine signaling 3 Socs3 SH2;intracellular signaling cascade;4.7e-08 tenascin C Tnc unknown unknown suppressor of cytokine signaling 3 Socs3 regulation of cell growth; signal transduction; intracellular signaling cascade serine (or cysteine) proteinase inhibitor, clade B, member 2 Serpinb2 serine-type endopeptidase inhibitor activity; plasminogen activator activity actin, alpha 2, smooth muscle, aorta Acta2 (actin alpha2) cytoskeleton organization and biogenesis structural constituent of cytoskeleton about skeleton serine (or cysteine) proteinase inhibitor, clade F, member 1 Serpinfl endopeptidase inhibitor activity; serine-type endopeptidase inhibitor activity matrix gamma-carboxyglutamate (gla) protein Mglap calcium ion binding serine (or cysteine) proteinase inhibitor, clade E, member 1 Serpinel regulation of angiogenesis serine-type endopeptidase inhibitor activity; protein binding; plasminogen activator activity chemokine (C-X-C motif) ligand 1 Cxc l l regulation of cell cycle; inflammatory response; immune response; cell growth and/or maintenance cytokine activity; chemokine activity growth factor activity biglycan Bgn increased or decreased average body size, or adipose tissue abnormalities manifesting after birth About skeleton epithelial membrane protein 1 Empl cell growth 44 interferon, gamma-inducible protein 16 I f i l 6 regulation of transcription, DNA-dependent; immune response Mediated in apoptosis pathway interleukin 1 receptor-like 1 I l l r l l D N A methylation defense response D N A binding; receptor activity; transmembrane receptor activity; interleukin-1 receptor activity; N -methyltransferase activity matrix metalloproteinase 3 Mmp3 proteolysis and peptidolysis; collagen catabolism metalloendopeptidase activity; stromelysin 1 activity; calc ium ion binding; peptidase activity; metallopeptidase activity; zinc ion binding; hydrolase activity R I K E N c D N A 170011 ON 18 gene 1700110N18Ri k unknown Homolog : Hs colon carcinoma related protein gremlin 1 G r e m l cysteine knot superfamily 1, B M P antagonist 1 embryonic pattern specification embryonic l imb morphogenesis serum deprivation response Sdpr GO:0005624 - membrane fraction Candidates identified through analysis of the SCN microarray data (Rl) epithelial membrane protein 1 E m p l cell growth B-cel l leukemia/lymphoma 3 Bc l3 regulation o f transcription, DNA-dependent D N A binding transcription factor activity R I K E N c D N A 2210409E12 gene 2210409E12Rik gastrulation brain homeobox 2 Gbx2 regulation of transcription, DNA-dependent axon guidance D N A binding transcription factor activity 2-cell-stage, variable group, member 1 Tcstvl zinc finger protein 352 Zfp352 kinesin light chain 2 K l c 2 motor activity microtubule motor activity M u s musculus transcribed sequences G-patch;nucleic acid binding; 1.9e-15 Top 5 down-regulated genes overlapping in the analysis of the SCN V6.5, J l and R l ESC lines B-cel l leukemia/lymphoma 3 Bcl3 t "ranscription, regulation o f ranscription, DNA-dependent D N A binding, transcription factor activity R I K E N c D N A 843041 OA 17 gene 843041 OA 17R ik inhibin beta-B Inhbb i growth 1 i lormone activity ?rowth factor activity Transcribed locus transglutaminase 1, K polypeptide T g m l i ( 1 1 jrotein modif ication, ] jrganogenesis, peptide cross- j inking, protein metabolism, ceratinization ; Drotein-glutamine gamma-*lutamyltransferase activity, calcium ion binding, acyltransferase activity, transferase ictivity, metal ion binding 45 3.2 Validation of Gene Expression Patterns to Finalize the Candidate List The bioinformatics analyses described above revealed 33 different genes of potential interest (Table 3.2). In order to narrow this list to a manageable number of candidates for functional studies, we focused on those that fell into the following gene ontology (GO) annotations: 1) Transcription factors; 2) directly or indirectly involved in cell-cycle control and cell proliferation; 3) involved in intracellular signal transduction, cell-cell signaling, or response to external stimuli or 4) Unknown genes. Of the initial 33 candidates, 12 fell into these three categories. We next carried out reverse transcription-quantitative PCR (RT-qPCR) validation of expression levels of these 12 candidates during an ESC differentiation time course experiment with samples collected from suspension cultures at 0, 6, 12, 18, 24, 36, 48, and 72 hours. Although Socs3 was within this list of 12 candidates, it was excluded from further study since its role in ESC has been described (67). Tgml, 1700110N18Rik and 2210409E12Rik were up-regulated after 24 hour (data not shown) and did not correlate with the microarray results, so they were removed from the list. Primers for Ifil 6, although validated for specificity using a BLAST search, amplified more than one product. Therefore, elimination of these genes resulted in a short-list of 7 candidates for which roles in ESC maintenance or differentiation have not previously been reported: Bcl3, Klf5, Empl, Cxcll, Gbx2, RikenA17, and Inhbb (Fig. 3.3). Ankrdl was selected as an additional candidate, despite being absent from our gene lists, due to its dramatically decreased expression during the first 18 hours of differentiation (Fig. 3.3) and its role as a transcription factor. Although the RT-qPCR results suggest that expression of Cxcll and Klf5 slightly increased from 18 to 72 hours, the changes were too small to be considered of significance. 46 Figure 3.3: RT-qPCR validation of expression level changes for the 8 selected candidates during ESC differentiation. Duplicate RT-qPCR analyses were carried out at the indicated time points using RNA isolated from a single time course experiment. Gapdh was used as the endogenous control. —*— RkenA17 - * — &np1 —4— Inhbb — x— BcB 1.20 n 1 0 10 20 30 43 50 60 70 80 Hours of differentiation —*—Klf5 —•—CxcM —i—Gbx2 Ankrdl 1.40 - i — 1.20 -0.20 J Hours of differentiation 47 In addition to the expression data, there are compelling biological reasons for selecting these candidates. Interestingly, expression of Gbx2 has been shown to decrease during EB formation, as well as during RA and DMSO-induced differentiation, although the functional significance of this observation has not been determined (107). Empl is highly expressed in human ESC and was present on both R l ESC gene lists. Bcl3, B-cell leukemia/lymphoma 3, was a very interesting candidate since it was the only one that was detected in all three gene sets. Klf5 is defined as a transcription factor and related to Klf4, which plays a role in ESC (67). Inhbb is a member of the TGF-0 superfamily; this family is thought to play roles in the self-renewal and differentiation of human and mouse ESC (108). 843041 OA 17Riken was the only one categorized as "unknown" in our gene lists. Furthermore, it is also down regulated during ESC differentiation induced by DMSO or RA/LIF (communication from Clive Glover). Cxcll was selected since it has growth factor activity and is in the extracellular space. In addition, Cxcll is also highly expressed in human ESC. 3.3 Lentivirus Generation and Validation of Candidate Over-expression PCR amplified cDNA sequences corresponding to each of the candidates were TOPO cloned into a lenti-viral vector containing IRES-GFP for cell selection. It should be noted that it was not possible to confirm appropriate amplification of Bcl3, which is a putative protein at present, due to the lack of complete sequence data. Thus, this candidate was excluded from the study. TOPO cloning of the PCR products was used for two reasons: 1) The PCR products will have single 3' adenine overhangs. Since the plasmid vector (pCR2.1-TOPO) is supplied linearized with single 3'-thymidine overhangs the cloning 48 efficiency is increased; and 2) The restriction enzyme sites required for directional cloning into the lentivectors will be more efficiently cut within the TOPO vector. All TOPO vectors containing inserts were sent to Nucleic Acid Protein Service Unit at UBC for sequence validation. All validated inserts were then cloned into the parental lentiviral vector. The lentivector used included the posttranscriptional regulatory element from woodchuck hepatitis virus (WPRE) (109) and the central polypurine tract (cPPT) (110, 111) which increase transgene expression and transduction efficiency, respectively. More importantly, the cellular polypeptide chain elongation factor 1 alpha (EFl-a) promoter (112) was shown to be active in both undifferentiated mouse ESC and their differentiated derivatives (51) and to function better than other promoters (113). For example, compared to C M V and RSV, it has been shown that in undifferentiated ESC the E F l a promoter was highly effective, while C M V had moderate activity and RSV had no activity by looking at the expression of humanized renilla green fluorescent protein (hrGFP) in murine ESC. After 3 months in culture, hrGFP expression was unchanged for the E F l a promoter and decreased for C M V (114). Transient transfection of the viral vectors into 293T cells is a convenient way to over-express and obtain both cellular and extracellular (secreted or membrane) viral proteins. The 293 cell line is a human renal epithelial cell line transformed by the adenovirus E l A gene product. The 293T cell line is a derivative that also expresses the SV40 origin and early promoter region. This cell line is highly transfectable using the Ca3 ( P O ^ transfection protocol with efficiencies of up to 50% being attainable. The virions, collected at 24 and 48 hours post-transfection, were concentrated by ultra-centrifugation prior to use. A small titration experiment was carried out to estimate viral titers in the 49 supernatant: lOul or 40ul of the control lentiviral concentrated virus containing media (VCM), added to R l ESC along with lul protamine sulfate, resulted in 0.44% and 1.29% GFP + cells respectively. Since the empty lenti vector control yielded the lowest GFP + frequency, its titer was used to establish the infection protocol. The viral titer was calculated by multiplying the starting cell number times the percentage of positive cells. This yielded an approximate titer of 1.61 x 104/ ml for the control vector. Based on this estimate, the infection of the R l ESC was carried out by adding 300ul of viral supernatant plus lul protamine sulfate (5ug/ml) to each dish. It should be noted that the other vectors yielded different titers, and hence infection efficiencies, most likely due to differences in the size and sequence of the inserted gene. ESC were cultured for 24 hours, fresh media was added and the cells were cultured for an additional 24 hours prior to harvest to assess GFP expression by FACS. ESC lines were expanded for another 7 days before sorting to presumably get rid of transient gene expression, thus resulting in isolation of stable, polyclonal populations (Figure 3.4). On the day of sorting, the frequency of GFP + ESC ranged from a low of 5% for the control vector to a high of 40% for RikenA17 (Figure 3.5). Sorted, as well as unsorted, ESC populations were expanded on gelatinized dishes for another 4-6 days until sufficient numbers of cells were obtained for freezing. GFP + expression was not assessed during this expansion time. 50 Figure 3.4: Analysis of GFP expression following ESC infection with the indicated lenti-viral vectors. - • - L e n t i - " - C x c l l -A -Emp1 -^<^Gbx2 Ankrdl -•—RikenA17 —t— Inhbb Klf5 60 i 0 -I 1 1 day2 day5 day9 days post infection 51 Figure 3.5: Analysis of GFP expression on the day of sorting. The percentage of GFP ESC was assessed by flow cytometry on day 9 post-sorting. The percentage indicates the frequency of GFP + ESC in the viable cell gate. WT Lenti Cxcl l 10° 101 10 2 10 3 10 4 io° io 1 102 103 id 4 io° io 1 102 i o 3 104 Empl RikenA17 Klf5 Ankrdl Gbx2 Inhbb , ^ GFP 52 The frequency of GFP + cells was next assessed, following thawing, over a culture period of 24 days on gelatinized dishes in ESC maintenance media. There was no gene whose GFP + population reached the expected 100%. Moreover, a small degree of gene shut down was observed in every transduced ESC line from day 2 until day 8 (Figure 3.6). Expression in all transduced ESC lines appeared stable thereafter. These "stabilized" transduced cell lines were used for further RT-qPCR and functional analyses. In order to determine if GFP expression corresponded with expression of the candidate gene, RT-qPCR was carried out on undifferentiated ESC, as well as on day 5 EB. All candidates, except Klf5 (not shown), were expressed at higher levels in the infected cell populations than in the wild type or control vector-infected ESC (Table 3.3; Figure 3.7). Klf5 therefore was excluded from further study. There is a good correlation between GFP expression levels and the relative fold increase in gene expression. RT-qPCR analyses of expression levels revealed much higher relative levels of expression of Gbx2 and Inhbb in day 5 transduced EB compared to the relative expression in transduced ESC (Figure 3.7). Relative expression of Empl and Ankrdl was reduced slightly in transduced EB compared to levels in transduced ESC, while Cxcll and RikenA17 showed pronounced reductions in relative expression levels. It has been reported that gene shut down occurs in differentiating ESC infected with lentivirus vectors (d). Therefore, we carried out two days of EB formation in liquid suspension cultures without LIF to address this question. This analysis indicated a dramatically decreased expression of GFP in all ESC lines with the levels of reduction ranging from 13% to 29% (Table 3.4). 53 Figure 3.6: Analysis of GFP expression post-sorting. Table 3.3: Relative expression of candidate genes in transduced ESC or EB (Candidate ESC vs. Lenti ESC; Candidate EB vs. Lenti EB) Candidate % GFP+ Ratio compared to Lenti Transduced ESC Transduced EB Inhbb 62% 203.14 476.65 Cxcl l 52% 63.52 17 Empl 44% 8.17 8.08 Gbx2 38% 8.85 16.6 Ankrdl 33% 15.3 8.06 RikenA17 18% 9.86 1.18 54 Figure 3.7: RT-qPCR validation to confirm over-expression. After sorting, ESC were expanded for 4-6 days. Samples were then collected for RT-qPCR and for freezing at the same time. EB samples (day 5 of differentiation) were collected in two individual experiments. All samples were stored in Trizol prior to RNA isolation and RT-qPCR analysis. Expression levels of a) Inhbb, b) Gbx2, c) Empl, d) Ankrdl, e) RikenA17, and f) Cxcll in transduced ESC and day 5 EB are shown. Open bars = lentiviral control; Closed bars = indicated candidate. All lentiviral and candidate gene expression levels were normalized to WT ESC and EB respectively. a) Inhbb b) Gbx2 450.00 400.00 350.00 300.00 250.00 200.00 150.00 100.00 50.00 0.00 Tranduced ES Transduced EB Tranduced ES Transduced EB c) Empl d) Ankrdl Tranduced ES Transduced EB Tranduced ES Transduced EB e) RikenA17 f) Cxcll Tranduced ES Transduced EB Tranduced ES Transduced EB 55 Table 3.4: Percentage reduction in GFP expression in day two EB suspension cultures Candidate and Lentivector only control Percentage of gene shut-down Ranking (from highest to lowest) Lenti 29% Cxcll 29% RikenA17 23% Empl 20% Inhbb 19% Ankrdl 18% Gbx2 13% 3.4 Functional analysis 3.4.1 C F C analysis and ESC proliferation CFC assays were carried out by plating ESC at various densities (500 to 2000 cells per gelatinized 60-mm gridded tissue culture dish) in ESC media with LIF. On day 5 the colonies were counted microscopically. The initial experiment revealed that 1000 input cells were optimal and thus this condition was used for further experiments. No significant differences in CFC frequencies were observed between the controls and any of the ESC lines expressing the candidate genes (Fig. 3.8), suggesting that none of the candidates have an effect on plating efficiency in the presence of LIF. As expected, in the absence of LIF, the frequency of CFC dropped to approximately 2/3 of the numbers observed in the presence of LIF, regardless of the input cell line (Fig. 3.9). Interestingly, Ankrdl significantly (p<0.05) increased CFC numbers (Mean ± SD = 131.78 ± 3.14) over 2 fold compared with WT (57.24 ± 3.14) or Lenti-vector only (67.5 ± 0.71) controls suggesting that Ankrdl may play a role in maintaining the plating efficiency of undifferentiated ESC in the absence of LIF. 56 Figure 3.8: CFC assay in the presence of LIF. R l ESC were plated at a density of 103 per dish and cultured in ESC media for 5 days. Colony numbers were counted. The mean ± SD of duplicate determinations is shown. 250.0 200.0 150.0 100.0 50.0 0.0 I WT Lenti Emp1 Cxcll Gbx2 Ankrdl RikenA17 Inhbb O u o U 1 I Figure 3.9: CFC assay in the absence of LIF. R l ESC were plated at a density of 10 per dish and cultured in ESC media minus LIF for 5 days. Colony numbers were counted. The mean ± SD of 3 separate experiments is shown. Statistical analysis was carried out using the students t-test where * indicates p<0.05 versus WT and Lenti control levels. 180 160 140 120 100 80 60 40 20 0 WT Lenti Emp1 CxcM Gbx2 Ankrdl RikenA17 Inhbb 57 To further assess self-renewal, growth curves were generated for each of the ESC lines by plating 2 x 104 ESC in triplicate in 24-well plates. Cells were cultured for 2 days in ESC maintenance media, harvested, counted, and replated at the same cell density. This process continued for a total of 6 days and cumulative cell numbers were calculated by multiplying the cell number from the previous day by the fold change in cell number during the subsequent two days of culture. Interestingly, ESC over-expressing Inhbb showed a significantly reduced rate of proliferation, while increased expression of Cxcl l significantly increased the proliferation rate (Fig. 3.10). No significant differences were observed with any of the other candidates. 3.4.2 Analysis of differentiation potential To determine the efficiency of EB formation, 3000 ESC were plated in triplicate 35-mm Petri-style dishes in ESC differentiation methylcellulose and were cultured for 5-6 days in humidified chambers at 37°C. EB numbers were determined microscopically and then the EB were harvested for analysis of cell numbers and gene expression. The frequency of EB formation in all cases was approximately 2-5% which is similar to what has been reported previously for this ESC line (115). No statistically significant differences were detected between WT ESC and the Lenti control as expected (Fig. 3.11). In keeping with its apparent ability to maintain the plating efficiency of ESC in the absence of LIF, ESC over-expressing Ankrdl showed a statistically significant, though modest, reduction in EB formation compared to WT and Lenti. Modest, though not statistically significant, reductions were observed with ESC 58 expressing Empl , Cxc l l , and Gbx2. In contrast, a slight increase in EB formation was observed with cells expressing Inhbb. Analysis of cell numbers per EB revealed no significant differences between WT and lenti-viral control ESC lines (mean ± SD: 354 ±18 and 352 ± 46 respectively; Fig. 3.12). The number of cells per EB was significantly increased in ESC expressing Inhbb (461 ± 21; p<0.05) and significantly reduced in those expressing Gbx2 (191 ± 68; p<0.05). Taken together, the cumulative results suggest that Gbx2, Ankrdl and Inhbb may play important roles in maintaining ESC pluripotency and regulating their capacity to differentiate. To further assess the role of these candidates, ESC were differentiated in suspension cultures in the absence of LIF for 18 hours. The resulting EB were harvested, single cell suspensions were prepared, and secondary EB assays were carried out. Surprisingly, Gbx2 and Inhbb showed a significantly (2 fold) higher secondary EB formation capacity (Fig. 3.13). Ankrdl also increased secondary EB formation by almost 50%, but the increase was not statistically significant. In conclusion, Gbx2 and Inhbb may maintain the ability of ESC to give rise to secondary EB following differentiation. 59 Figure 3.10: Growth curves for the various ESC lines. R l ESC were plated at a density of lxlO 4 cells per plate, cultured for 2 days, and then harvested and counted. The same number of cells was then re-plated so that the cells were in culture for a total of 6 days. Cumulative cell numbers for each candidate (solid line) are shown compared to those for the lentiviral control (dashed line). The results are the mean ± SD of triplicate determinations expressed as log value using 2 as the base. Statistical analysis was carried out using the students t-test where * indicates p<0.01 versus control levels, a) Inhbb b) Cxcll 10.0 ' 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 • * DayO c) Ankrdl Day2 Day4 Day6 14.0 DayO Day2 d) RikenA17 Day 4 Day6 DayO Day2 Day4 Day6 DayO Day2 Day4 Day6 Days in culture 60 Figure 3.11: Primary EB formation assay. R l ESC were plated at 3 x 10 ESC per 35mm Petri dish in methylcellulose and cultured for 5 days. The EB numbers were counted under the microscope. Results are the mean ± SD of four experiments. Statistical analysis was carried out using the students t-test where * indicates p<0.05 versus WT and Lenti control levels. 160.00 3 140.00 ~ 120.00 PQ S 100.00 !Z3 S3 80.00 60.00 40.00 20.00 0.00 X WT Lenti Empl Cxcll Gbx2 Ankrdl RikenA17 Inhbb Figure 3.12: Cell numbers per day 5 EB. EBs were harvested on day 5 and disrupted into single cell suspensions. Viable cell numbers were determined using trypan blue exclusion and the number of cells per EB was determined by dividing cell number by the number of EB. Results are the mean ± SD of triplicate determinations. Statistical analysis was carried out using the students t-test where * indicates p<0.05 versus control levels. 600.00 • 500.00 400.00 • ca hj iSi 300.00 • TS 200.00 • I 100.00 • 0.00 WT Lenti Gbx2 Irhbb 61 Figure 3.13: Secondary E B formation. E S C were cultured for 18 hours in I M D M differentiation medium. E B were harvested, disrupted with T /E , and plated in methylcellulose to assess E B formation. The mean ± SD of duplicate determinations is shown. Statistical analysis was carried out by t-test where * indicates p<0.05 versus W T and Lenti control levels. 250.00 200.00 ~ 150.00 3 Q. £ O O § 100.00 % m LU 50.00 0.00 * T * j 1 1 T 1 — i — 1 WT Lenti Gbx2 Ankrdl Inhbb We next sought to determine whether over-expression of the candidates altered the ability of E S C to differentiate along specific lineages by analyzing the expression of selected marker genes. Fgf5 is expressed in pluripotent cells of the primitive ectoderm at 5.25 days post coitum and expression of Fgf5 continues in the primitive ectoderm until the onset of gastrulation (116, 117). Sox l7 and Foxa2 are both markers of definitive endoderm formation. Loss of S O X 17 results in a severe reduction in the amount of definitive endoderm. Soxl7-nul l definitive endoderm has reduced viability and proliferation, resulting in occupation of the gut by visceral endoderm-like cells. In the mouse, Foxa2 (previously known as HNF3P) , a factor expressed in the anterior region of the primitive streak, in endoderm and the early liver, is essential for the development of 62 prospective foregut and midgut endoderm (775). Nestin is abundant in ES-derived progenitor cells that have the potential to develop into neuroectodermal, endodermal and mesodermal lineages. Although it remains unclear what factors regulate in vitro and in vivo expression of nestin, it has been reported that nestin expression may indicate multi-potentiality and regenerative potential (119). Brachyury (T-gene) expression occurs in both early stage mesoderm and its epithelial progenitor, and then becomes restricted to the notochord. This expression pattern correlates with the tissues affected in the T-gene mutant, and indicates that the T gene has a direct role in the early events of mesoderm formation and in the morphogenesis of the notochord (120). As shown in Figure 3.14, Inhbb over-expression induced a statistically significantly higher level of Fgf5 expression suggesting enhanced differentiation along the ectoderm lineage. Inhbb also induced slightly higher levels of expression for FOXA2, SOX17 and Nestin, while expression of the mesoderm marker Brachyury was modestly reduced. In contrast, Gbx2 expressed statistically lower levels of FoxA2, Brachyury and Nestin, suggesting a general impairment in differentiation that corresponds with the reduced cell numbers per EB. 63 Figure 3.14: R T - q P C R analysis of germ layer marker genes in day 5 E B . R N A samples were collected from two separate E B assays, R N A was isolated and reverse-transcribed, and q P C R was done in duplicate. Relative expression levels (normalized against wi ld type) for a) Fgf5; b) FoxA2; c) Sox l7 ; d) Brachyury; and e) Nestin were determined for each population. Results are the mean ± SD of 2 independent differentiation experiments. a)Fgf5 b) F o x A 2 3.00 2.50 2.00 1.50 1.00 0.50 0.00 X I Lenti 6rp1 Cxch Gbx2 Ankrdl Riken Inhbb C) Sox17 4.50 4.00 3.50 3.00 2.50 2.00 1.50 1.00 0.50 0.00 2.00 -r 1.80 1.60 1.40 1.20 1.00 0.80 0.60 0.40 0.20 0.00 - I — 1 — 1 — h - J—'—h — 1 — 1 —h Lenti Brp1 Cxch Gbx2 Ankrdl Riken Inhbb d ) Brachyury 2.50 2.00 1.50 1.00 0.50 0.00 Lenti Brp1 CxcM Gbx2 Ankrdl Riken Inhbb Lenti Emp1 Cxch Gbx2 Ankrdl Riken Inhbb e) Nestin 6.00 5.00 4.00 3.00 2.00 1.00 0.00 Lenti Emp1 Cxch Gbx2 Ankrdl Riken Inhbb 64 3.5 The interaction between candidates and LIF-signaling pathway, Oct4 or Nanog. Since two of our candidates, Gbx2 and Inhbb, showed significant effects in several of our assays, we next asked whether their expression is similarly altered in multiple ESC differentiation conditions. With the assistance of Clive Glover, expression of these two candidates was examined following DMSO or RA+LIF-induced ESC differentiation. Gbx2 (normalized value: 4.206), as well as Inhbb (normalized value: 4.62), were down-regulated significantly in RA plus LIF as compared to ESC in LIF (Gbx2: 0.992; Inhbb: 0.837) implying that LIF does not regulate expression of either transcript. In addition, they were also down regulated in DMSO (Gbx2: 0.366; Inhbb: 0.637) providing further evidence that they are likely regulated by differentiation rather than LIF-withdrawal. In addition, neither Gbx2 nor Inhbb over-expression alter the levels of Stat3 or Socs3 in ESC or EB (data not shown). In conclusion, Gbx2 and Inhbb are likely not to be related to, or dependent on, the LIF signaling pathway. Similarly, Gbx2 over-expression did not alter the levels of either Oct-4 or Nanog (data not shown). Surprisingly, Inhbb over-expressing ESC showed significantly decreased levels of Oct-4, but not Nanog, compared to the WT and lenti controls (Fig. 3.15). Moreover, decreased expression of Oct4 was also observed in day 5 EB generated from Inhbb over-expressing ESC (Fig. 3.15). 65 F i g u r e 3 . 1 5 : T h e e x p r e s s i o n o f O c t 4 a n d N a n o g i n I n h b b o v e r - e x p r e s s i n g c e l l s b y R T -q P C R . a ) O c t - 4 a n d N a n o g e x p r e s s i o n i n I n h b b o v e r - e x p r e s s i n g E S C . b ) O c t - 4 e x p r e s s i o n i n I n h b b o v e r - e x p r e s s i n g E B . R e s u l t s a r e t h e m e a n ± S D o f 2 i n d e p e n d e n t d i f f e r e n t i a t i o n e x p e r i m e n t s a) b) Oct-4 and Nanog expression f - 1 4 -Oct n Nanog 1.2 ™ 0.8 0.6 T3 5 0.4 OJ > '•w IP 0.2 Lenti-ESC Inhbb-ESC Transduced ESC I _i Q 1.2 1.0 0.8 0.6 •w 1 T3 "O" 0.4 > 0.2 0.0 Oct4 expression Lenti-EB Inhbb-EB Transduced EB at day5 6 6 C H A P T E R 4 DISCUSSION 4.1 Microarray analyses and R l ESC line Two of the three gene lists were generated using the R l ESC line. The reasons for this are two-fold. First, several mouse ESC lines have been tested for their ability to produce completely ESC-derived mice at early passage numbers using the tetraploid embryo aggregation technique. The R l ESC line (from 129/Sv x 129/Sv-CP embryos) (121) produced live offspring, which were completely ES cell-derived as judged by isoenzyme analysis and coat color, at a high frequency. These cell culture-derived animals were normal, viable, and fertile. As such, this line is a very good representative of a pluripotent ESC line. Secondly, the previous BCCRC study (47) correlated functional measures of pluripotency with changes in gene expression in this ESC line, thus suggesting that the changes are biologically relevant and not simply reflective of culture variables. Interestingly, 6 of the 8 candidates analyzed came from the R l ESC gene lists. 4.2 Criteria for Identifying Candidate Genes In order to narrow down the gene list to a manageable number for functional studies, we exclusively focused on down-regulated genes based on the following biological assumptions. First, it is likely that genes implicated in maintenance of pluripotency are upstream of differentiation-induced genes and therefore need to be down-regulated to allow initiation of differentiation. In addition, many up-regulated genes are likely to be responsible for specific cell lineage differentiation and thus not directly related to pluripotency. Finally, although it is possible that important regulatory genes are up-regulated during differentiation, no such genes have been found in ESC yet. Taken 67 together, it seemed more likely that we would identify genes implicated in pluripotency by focusing on those that are down-regulated during early differentiation. It is possible that the stringent criteria used to generate the gene lists could exclude some candidates with important regulatory roles. For example, neither Oct-4 nor Nanog fell into any of the gene lists. Oct-4 expression changed only slightly throughout the 72-hour differentiation time course (0 to 18 hours = 1.09; 18 to 72 hours = 1.01) in the BCCRC R l microarray data and did not correspond with the loss of pluripotency. Although Nanog continually decreased throughout differentiation it decreased only one fifth in the first 18 hours and thus was not considered significant using our criteria. It is important to remember that all data analyzed reflect only differentiation induced by loss of LIF signaling. It is possible that this approach is biased toward identification of genes related to LIF signaling and excludes the ones (e.g. Oct-4, Nanog) that function independently from the LIF signaling pathway. An additional concern is whether the changes in mRNA expression levels truly reflect the loss of ESC pluripotency. Since previous studies showed that the frequency of successful chimeric mouse generation using R l ESC dropped from 100 to 20 percent within 18 hours following LIF removal, criteria were used to identify genes that were at least 3 to 4 fold down-regulated for both R l gene lists. Although such a strategy may be effective to limit the number of candidate genes identified, it is likely to miss some that may be important in maintaining ESC pluripotency. In order to partially address this concern, a third gene list, focused on over-lapping genes in multiple ESC lines, was generated with less stringent criteria. Two of the final candidates, Inhbb and RikenA17, were selected from this gene list. 68 4.3 Advantages and Disadvantages of the Over-expression Strategy There are two strategies for assessing gene function in E S C - introduction of foreign genes via transduction or infection and deletion of the gene/transcript of interest by gene targeting or R N A i knock-down (122, 123). Assessment of the effects of such genetic manipulations on the maintenance o f E S C pluripotency and differentiation potential allows some insight into gene function. The over-expression strategy was selected for these studies for several reasons. First, we reasoned that over-expression of genes that are normally down-regulated during differentiation might sustain pluripotency in the absence of L I F . We therefore anticipated that, in either the E B or C F C functional assays, E S C over-expressing candidate genes might have an enhanced ability to self-renew while differentiation might be suppressed. Secondly, by sustaining the expression of transcripts that are normally down regulated during differentiation, we anticipated that relative levels of expression would increase during this time, thus allowing us to detect effects of these candidates on the differentiation process. In this regard, the usefulness of gene knockdown would be restricted since the expression levels of the candidates of interest typically decrease during differentiation. A final consideration is whether over-expression or knockdown of a single target gene is likely to rescue or disrupt the entire "sternness" program. This seems highly unlikely and thus only partial effects w i l l be seen using either approach. In an ideal situation, studies would be done using both of these different strategies in parallel. Nonetheless, such an approach was beyond the scope of these studies for the number of candidates that were examined. Successful examples of using the over-expression technique to identify new genes in E S C are Socs3 and Kl f4 (67). 69 Theoretically, self-renewal and differentiation can be considered as two opposing options for a cell. We therefore assumed that i f a gene induces differentiation, it must inhibit the self-renewal ability of E S C and vice versa. A s such, over-expressing a gene required for self-renewal is likely to increase or sustain higher levels of self-renewal ability while reducing/preventing differentiation. This assumption is based on one candidate gene having one specific function in E S C . However, this is not always the case. For example, forced over-expression of Oct4 does not render E S cells free of their dependence on L I F . Rather, conditional up-regulation of Oct4 expression in E S cells demonstrates that the increased levels of expression produce primitive mesoderm and endoderm, reminiscent of the transient up-regulation as primitive endoderm initially differentiates in the blastocyst (124). Thus it is important to keep in mind that the transcript level in an E S C is likely to be as important as whether it is present or absent. 4.4 Alternatives to lentivirus: Electroporation and Knock-in Studies conducted in the eighties, particularly by the groups of Smithies and Capecchi, demonstrated that mammalian cells have the enzymatic apparatus necessary for recombination between an incoming D N A and the homologous sequence present in the chromosomes, even i f this was relatively rare compared with the random integration of this same D N A (125, 126). Many researchers now rely on knockin mice or cell lines to study gene function. A knockin mouse or cell line is generated by targeted insertion of the transgene at a selected locus. For example, the stem cell leukaemia gene S C L , also known as T A L - 1 , encodes a basic helix-loop-helix transcription factor expressed in erythroid, myeloid, megakaryocyte and hematopoietic stem cells. To be able to make use 70 of the unique tissue-restricted and spatio-temporal expression pattern of the S C L gene, a knock-in mouse line containing the tTA-2S tetracycline transactivator under the control of S C L regulatory elements was generated (127). The S C L knock-in mouse represents a powerful tool for studying normal and malignant hematopoiesis in vivo. The insert is flanked by D N A from a non-critical locus, and homologous recombination allows the transgene to be targeted to that specific, non-critical integration site. In this way, a researcher has complete control of the genetic environment surrounding the overexpression cassette and the D N A wi l l not incorporate itself into multiple locations which might occur using viral vectors. In addition, site-specific knockins result in a more consistent level of expression of the transgene from generation to generation because it is known that the overexpression cassette is present as a single copy. Also , because a targeted transgene is not interfering with a critical locus, the researcher can be more certain that any resulting phenotype is due to the exogenous expression of the protein. Although the generation of a knockin does avoid many of the problems of a traditional transgenic approach, the disadvantages of using this procedure are the requirements of more time to assemble the vector and to identify E S C that have undergone homologous recombination. Electroporation is another way to insert a D N A vector of interest. Electroporation is a significant increase in the electrical conductivity and permeability of the cell plasma membrane caused by an externally applied electrical field. It is usually used as a way of introducing a foreign gene in mammalian cells. The unnatural increase in permeability is theoretically explained as a process of formation of very small openings (pores) in the plasma membrane during which the extracellular compounds have a chance to get inside 71 t h e c e l l . I f t h e s t r e n g t h o f e l e c t r i c a l field a n d d u r a t i o n o f e x p o s u r e t o i t a r e p r o p e r l y c h o s e n , t h e p o r e s f o r m e d b y t h e e l e c t r i c a l p u l s e r e s e a l a f t e r a s h o r t p e r i o d o f t i m e . H o w e v e r , e x c e s s i v e e x p o s u r e o f l i v e c e l l s t o e l e c t r i c a l f i e l d s c a n a l s o c a u s e a p o p t o s i s o r n e c r o s i s , t h e p r o c e s s e s t h a t r e s u l t i n c e l l d e a t h . A s w i t h t h e k n o c k - i n a p p r o a c h i t i s l i k e l y t h a t t h e v e c t o r o n l y i n s e r t s a t o n e i n t e g r a t i o n s i t e i n t h e t a r g e t c e l l s . H o w e v e r , t h e r e i s n o c o n t r o l o v e r t h e s i t e o f i n t e g r a t i o n o r t h e l e v e l s o f e x p r e s s i o n a t t a i n e d . T h i s n e c e s s i t a t e s s c r e e n i n g o f c l o n e s t o i d e n t i f y t h o s e w i t h a p p r o p r i a t e e x p r e s s i o n l e v e l s a n d p a t t e r n s . O n e p r i m a r y c o n c e r n r e g a r d i n g t h e u s e o f t h e l e n t i v e c t o r i s t h e i n s t a b i l i t y o f t h e i n t e r n a l r i b o s o m a l e n t r y s i t e ( I R E S ) c a s s e t t e . I n c o n s i s t e n c i e s i n c o - e x p r e s s i n g t w o t r a n s g e n e s l i n k e d b y a n I R E S e l e m e n t i n a s i n g l e b i c i s t r o n i c t r a n s c r i p t h a v e b e e n s h o w n . I n m a n y i n s t a n c e s , o n l y t h e ( f i r s t ) g e n e t r a n s c r i b e d u p s t r e a m o f t h e I R E S i s e x p r e s s e d s t r o n g l y . M o r e o v e r , w h e n G F P i s p o s i t i o n e d b e h i n d t h e I R E S , i t m a y n o t f l u o r e s c e a s s t r o n g l y a s i t w o u l d d i r e c t l y b e h i n d a p r o m o t e r (<5). R e g a r d l e s s , w e o b s e r v e d G F P e x p r e s s i o n u s i n g a l l o f t h e c o n s t r u c t e d v e c t o r s . 4.5 E S C T r a n s d u c t i o n R e s u l t s T h e u s e o f l e n t i v i r u s v e c t o r s i n E S C h a s b e e n r e p o r t e d t o r e s u l t i n s u p e r i o r l o n g - t e r m m a i n t e n a n c e o f e x p r e s s i o n i n b o t h e x t e n d e d c u l t u r e a n d d i f f e r e n t i a t i o n c o n d i t i o n s . F o r t h e m a j o r i t y o f o u r c a n d i d a t e s w e o b s e r v e d h i g h l e v e l s o f s u s t a i n e d e x p r e s s i o n t h r o u g h o u t d i f f e r e n t i a t i o n . I n t e r e s t i n g l y , t h e r e l a t i v e e x p r e s s i o n l e v e l s o f R i k e n A 1 7 a n d C x c l l b o t h s h o w e d a p r o n o u n c e d r e d u c t i o n i n t h e E B ( F i g u r e 3 . 7 ) s u g g e s t i n g t h a t t h e r e m i g h t b e s h u t d o w n o f e x p r e s s i o n f r o m t h e s e t w o v e c t o r s . S i n c e w e d i d n o t a s s e s s i n t e g r a t i o n n u m b e r s i n t h i s s t u d y , i t i s p o s s i b l e t h a t t h e r e w e r e o n l y s i n g l e i n t e g r a t i o n s o f 7 2 each of these vectors into a site where they were susceptible to altered chromatin configurations and thus reduced expression. An alternative possibility is that ESC over expressing either of these candidates could not differentiate into EB or did not survive for the five-day period of differentiation, and therefore it would appear as if expression were reduced. Determining the frequency of cells present in the EB that still retain the lentiviral integration could be used to assess this possibility. It is interesting to note that 3 of the 4 genes showing high levels of continued expression are the ones that yielded the most interesting results in the functional studies. Based on this observation caution must be used in interpreting the lack of effect observed with RikenA17 and Cxcll until the reasons for reduced expression are determined. One additional concern is that the ESC populations used were not 100% GFP + (Fig. 3.6). Three reasons are proposed to account for the apparent impurity of the sorted populations, although it is likely that some combination of all of these was involved. 1) Sorting was carried out too early after infection and thus there was still significant levels of transient gene expression. In support of this possibility we observed a reduction in GFP expression following thawing. It would have been necessary to determine GFP expression during the post-sort expansion phase to determine for certain that there was still transient gene expression. 2) The gate for collection of the GFP + population was too low. In order to obtain the highest expressors, only the top 10% (i.e. the brightest) GFP + cells should be collected. However, to recover sufficient numbers of cells the gate was set to collect the top 60% which increases the likliehood of including some transiently expressing cells, as well as 73 those that have low copy number or a higher propensity for gene shut-down due to the site of viral integration. 3) The long half-life of the GFP protein might have also been a problem. It is possible that some ESC that had already turned off viral gene expression were included in the sorted population based simply on the continued presence of the protein. Regardless of the reasons that resulted in working with impure ESC populations, the consequences for interpretation of the results of the functional studies must be kept in mind. For example, if only 10% of the population was GFP + (and thus expressing the candidate), a maximal theoretical difference in the functional read-out would only be 10%o. This may result in false-negative results for some candidate-expressing ESC populations with the lower GFP + frequencies. Interestingly, Gbx2 and Ankrdl had the lowest GFP + frequencies at 38% and 33%> respectively (Table 3.3) but both of them exhibited significant functional effects in the various assays. It is therefore likely that more pronounced effects will be seen with these, and other, candidates after re-sorting. In all cases we obtained over-expression of the candidate genes by at least 5 fold compared to WT. Although it was not assessed in our study, it has been proven that integration of lentivirus takes place relatively rapidly following transduction of ESC (128) and in the lentivirus-transduced clones, the GFP expression level was largely proportional to the number of vector copies. This suggests that the expression level from the lentivirus vector is relatively independent of the position where the provirus is integrated and is primarily dependent on the number of proviral copies (128). We did not carry out Southern blot analysis on the ESC populations to determine copy number or integration site. We assumed that the viral titers were sufficiently high to yield polyclonal 74 populations. Numerous reports have demonstrated successful over-expression studies in ESC using polyclonal cell lines (67, 129). 4.6 Hypotheses and Future Directions: Gbx2, Inhbb and Ankrdl From our functional studies, Ankrdl, Gbx2 and Inhbb showed significant effects on different aspects of ESC biology. Literature surveys, in parallel with the interpretation of our results, were used to generate the following hypotheses for the possible functions of these genes in ESC. 4.6.1 Gbx2: a possible regulatory gene of E S C pluripotency We observed that ESC over-expressing Gbx2 yielded smaller EB with reduced expression of various lineage markers. In addition, these ESC gave rise to more secondary EB. Taken together these results suggest that increased expression of Gbx2 may favor self-renewal over differentiation. It has been reported recently that Gbx2 was one of 18 transcriptors bound by Oct4 and Nanog in both mouse and human ESC by investigating the Oct4 and Nanog binding circuitries conserved in pluripotent cells from these two mammalian species (130). This finding highlights the importance of Gbx2 in ESC and supports a role for Gbx2 in ESC pluripotency. Interestingly, it has also been reported that Gbx2 is an important downstream target of the myeloblastosis oncogene (Myb) and that it contributes to hematopoietic lineage choice and differentiation in a signal transduction-dependent fashion (131). However, there are no reported studies looking at the effect of Gbx2 on ESC differentiation into hematopoietic cells. Our observation that Brachyury expression is reduced in EB expressing Gbx2 suggests that it may in fact impair hematopoietic differentiation in this 75 system. It is therefore of interest to determine the efficiency of hematopoietic cell differentiation using these Gbx2 over-expressing ESC. Interestingly, interleukin 6 (IL-6) is a downstream target of the Gbx2 homeobox gene (132) in prostate cancer cells. Interleukin 6, like LIF, signals through the gpl30 receptor. Homodimerization of gpl30 alone, in response to the cytokine IL-6 and a soluble form of IL-6 receptor (sIL-6R) can also support the derivation and propagation of ES cell lines (133, 134). The Janus kinase-signal transducers and activators of transcription signaling pathway transmits IL-6-mediated signals from cell surface receptors to the target genes in the nucleus and is critical in mediating cellular growth and differentiation (135, 136). It has been demonstrated that ectopic expression of IL-6 stimulated prostate cancer cell growth accompanied by activation of the Stat3 signaling pathway (137). Thus, it is of interest to determine whether increased expression of Gbx2 results in up-regulation of IL-6 expression and responsiveness, resulting in activation of Stat3 and increased maintenance of ESC pluripotency. Examination of the BCCRC R l microarray data revealed that IL-6 was called present in only one of the three genechips for the undifferentiated ESC and absent in all differentiated samples. Since IL-6 expression was not detected in Gbx2 over-expressing ESC or EB by RT-qPCR (data not shown), it is likely that Gbx2 indirectly increases IL-6 expression and that whatever the factor responsible for this is not present in ESC. Another hypothesis is that Gbx2 blocks differentiation by regulation of the ESC cell cycle in which the G l checkpoint is lost. In support of this hypothesis, when neurogenesis begins, the expression of Gbx2 is detected as progenitor cells exit the cell cycle (138). In addition, Waters et al. found that in mice homozygous for a Gbx2 76 hypomorphic allele (Gbx2 neo) the cell division regulator cyclin D2 is down-regulated and cellular proliferation is reduced in select cells (139). Thus, it will be of interest to further assess the cell cycle in ESC over-expressing Gbx2. 4.6.2 Inhbb: a possible regulatory gene of E S C pluripotency The transforming growth factor (TGF) beta (P) superfamily of ligands can be divided into the TGF P (140), BMP, and activin subgroups based on sequence. In the activin subgroup, four mammalian isoforms of the activin/inhibin p -subunit have been identified: PA, PB, PC, and PE. Research has focused on the more widely expressed pA and PB isoforms, which assemble into the active ligand dimers activin A (PApA), activin B (pBpB), activin AB (pApB), inhibin A (apA), and inhibin B (apB). Though the p-subunits share a high (63%) sequence identity, they are differentially regulated during development and throughout ovarian follicle selection and maturation (141). Inhibin P-B (Inhbb), our candidate, is the B subtype of the P subunit (PB). The BCCRC microarray study showed that Inhba (PA) was also significantly-decreased from 0 to 18hr, but increased from 18 to 72hr during EB differentiation. Therefore, it was not included in this functional study. In addition, increased expression of Inhbb, using our lentivirus in ESC, is likely to induce the formation of either Activin B or Inhibin B. However, since the a subunit is expressed at low levels, and is thus likely limiting in ESC (142) it is more likely that the presence of additional p subunits would result in the presence of increased activin B rather than activin AB or inhibinB. Western blot for activin B is feasible and would validate this hypothesis. 77 By 3.5 days, only the ICM cells express activin. In addition, transcripts for Inhbb and Inhba are detectable at the morula stage, with Inhbb mRNA persisting into the blastocyst. When ESC are induced into differentiate, levels of activin fall dramatically (142). Numerous studies have suggested that activin B is a key player in mesodermal patterning. Activin B was identified as a potential regulator of these early developmental decisions based on its capacity to induce mesoderm and endoderm formation in Xenopus animal caps in vitro (62). In keeping with this possibility we observed that over-expression of Inhbb in ESC resulted in slightly increased expression of two endoderm markers, Foxa2 and Soxl7, in the EB. However, no difference in Brachyury expression, a mesoderm layer marker, was observed in the RT-qPCR results. Moreover, expression of Bmp4, another mesodermal marker, was similarly expressed in control and Inhbb over-expressing EB (data not shown). Thus, our ESC studies suggest a possible role for Inhbb in endoderm, but not mesoderm, formation in ESC differentiation cultures. It has been found that activin B is a ligand for the activin A receptor, type IC (ALK7). ALK7 is an orphan receptor serine/threonine kinase expressed in neuroendocrine tissues including pancreatic islets (143). The combination of activin receptor IIA (ActRIIA) and ALK7, preferred by activin AB and activin B but not by activin A, is responsible for activin-mediated secretion of insulin from the pancreatic beta cell line, MIN6. In contrast, all activins activate a combination of ActRIIA and activin A receptor, type IB (ALK4) with various levels of potency. Thus, variation in activin signaling through type I receptors is dependent upon homo- and heterodimeric assembly of activin isoforms. Thus, the differential combination of receptor heterodimers mediates variation in activin isoform signaling (143). Furthermore, all the type I receptors (ALK) are downstream of 78 the type II receptors and they are able to propagate the phosphorylation signal via specific downstream mediators such as Smads which translocate into the nucleus and activate transcription of other genes. Activin signaling through ALK4 results in the activation of Smad2 and Smad3 which both transduce a signal to the transcription factor, FOXH1. In our study, the over-expression of Inhbb likely induces the activation of Smad2 and Smad3 over normal threshold and thus induces FOXH1. It is therefore of interest to determine if there is activated FOXH1 in our Inhbb-expressing ESC line. Another ligand for ALK4 is Nodal. The Nodal signaling pathway is known to play an important role in endoderm formation, which is similar to the role proposed here for Inhbb. Nodal, TDGF1 (teratoma-derived growth factor 1, also known as Cripto) and Cerberus 1 are the main components of the Nodal signaling pathway (114). No expression of ALK4 was detected in the BCCRC array data, although the Activin A receptor, type 1 was expressed. If these observations are confirmed, it would suggest that Inhbb may function through ALK7 and not ALK4. Taken together our functional studies suggest that Inhbb partially rescues the differentiation potential in the absence of LIF, but not in the presence of LIF. The significantly increased cell numbers per EB suggest that Inhbb also plays a role in regulating ESC differentiation. Inhbb may play a role in re-directing differentiation as suggested by increased Fgf5 expression, as well as decreased Oct4 expression. Oct4 has been shown to repress gene expression by neutralizing the transcriptional activator, Foxd3. Foxd3 is a member of the Forkhead Box (Fox) family which can bind to the promoters of, and activate transcription of, other members of this family including Foxal and Foxa2, which are critical for the embryonic development of endodermal foregut 79 organs. Guo et al reported that Oct4 can repress the expression of Foxal and Foxa2 through an interaction with the DNA binding domain of Foxd3. The observation in our studies that Oct4 expression is reduced is in keeping with our finding that Foxa2 expression is increased. This report suggests that Oct4 could prevent the differentiation of ESC lineages by acting as a co-repressor of lineage-specific transcription factors like Foxd3. At present the mechanism by which Inhbb regulates Oct4 expression is not clear. Interestingly, a recent study demonstrated that activin A induces the expression of Oct4 in human ESC (144). Although contrary to our findings, these results provide evidence that the TGFp family regulates Oct4 expression. It will be important to confirm Oct4 protein levels in the Inhbb over-expressing ESC. In addition, blocking the receptors for Inhbb, such as ALK4 and ALK7, will provide important clues into the role of Inhbb in ESC. Such studies could be carried out using SB-431542 which is a potent and specific inhibitor of type I activin receptor-like kinase receptors ALK4, ALK5, and ALK7 (145). 4.6.3 Ankrdl : a possible anti-apoptosis gene which may also promote cardiac cell differentiation in ESC Ankrdl, also named cardiac ankyrin repeat protein (CARP), encodes a nuclear co-regulator for cardiac gene expression, lies downstream of the cardiac homeobox gene Nkx 2.5, and is an early marker of the cardiac muscle cell lineage (146). CARP, ankrd-2/Arpp, and DARP, are three members of a conserved gene family, referred to here as MARPs (muscle ankyrin repeat proteins). The expression of MARPs is induced upon injury and hypertrophy (CARP), stretch or denervation (ankrd2/Arpp), and during recovery following starvation (DARP), suggesting that they are involved in muscle stress 80 response pathways and filament systems (147). The fact that Ankrdl plays an important role in stress response pathways may explain the increased plating efficiency of ESC in the absence of LIF when they are over-expressing this transcript. Interestingly, ESC may represent an alternative source of functionally intact cardiomyocytes for treatment of cardiovascular diseases. However, this requires cardiac-specific differentiation of stem cells and the selection of pure lineages consisting of early embryonic cardiomyocytes. Therefore, an understanding of the basic mechanisms of heart development is essential for selective differentiation of ESC into cardiac cells. It has been reported that the development of cardiac cells from ESC is regulated by several soluble factors and signaling molecules together with cardiac specific transcription factors such as the zinc-finger GAT A proteins and Nkx-2.5. GATA-4 and Nkx-2.5 seem to be essential for heart development (148). Since Ankrdl is the main downstream target gene of Nkx-2.5, it would be interesting to know whether Ankrdl could direct ESC differentiation into cardiomyocytes. Previous studies suggested that Ankrdl is a novel pro-apoptotic protein. Suppression of Ankrdl expression results in cell proliferation in several mammalian cell lines, while over-expression of Ankrdl leads to apoptosis and inhibition of proliferation (149). Furthermore, compared with controls treated with an empty vector or with antisense cDNA, the ectopic expression of Ankrdl led to reduced colony formation and to enhanced apoptotic cell death in hepatoma cells (8). Subsequently, Park et al. found that the ectopic expression of Ankrdl in H9c2 cells increased the resistance to hypoxia-induced apoptosis (67). Together, these observations suggest that the role of Ankrdl in 81 apoptosis is likely to be cell-type specific. In keeping with the effects of Ankrdl on ESC, it may have an anti-apoptotic role. 4.6.4 Cxcll: a tumorigenesis gene presumably accelerating E S C proliferation Cxcll was the only gene which significantly accelerated ESC proliferation rates. It has been shown previously that over-expression of Cxcll in melanocytes is associated with enhanced growth, ability to form tumors in nude and SCID mice, and enhanced metastatic capacity in melanoma tumors (150-152). ESC, like cancer cells, are able to propagate indefinitely and have an extremely high proliferation rate compared with normal somatic cells. Although the mechanisms for this are not clear, our finding suggests that Cxcll might be involved. 4.7 Final conclusions Previous studies have focused on bioinformatics approaches to identify genes related to "sternness". Alternatively, they have used manipulation of known signaling pathways in ESC to identify genes that might play a role in maintaining the pluripotent state (95, 96). Moreover, earlier studies have attempted to identify important regulatory genes based on assessment of secondary EB formation or lineage differentiation markers to assess self-renewal or differentiation potential without determining if the identified genes are common to various ESC lines (67, 153). This study is distinct from previous studies in that we used existing ESC gene expression profiles, some of which were validated with functional correlates, and carried out binoinformatics comparisons to identify candidates of interest. We then validated 82 expression patterns and carried out an array of functional studies on ESC engineered to over-express each of the candidates. Our approach was validated by the experimental observations which indicated that almost all of the candidates selected had some effect on ESC properties. They were involved in regulating proliferation, differentiation, or self-renewal, depending on the candidate. Further validation for our approach comes from recent functional studies that showed the majority of these genes are involved in pathways known to regulate ESC pluripotency (95, 108, 154). In our study, Gbx2 and Inhbb both showed significant effects on ESC pluripotency. In addition, Ankrdl and Cxcll influenced ESC growth and/or proliferation. In summary, 4 out of 6 candidate genes selected based on our criteria appear to have some role in regulating ESC pluripotency. Further functional analyses will provide important insights into the roles that each of these molecules play in maintaining ESC pluripotency. Such studies will also provide us with important insights into the mechanisms required to maintain ESC populations in an optimal pluripotent state and to differentiate them along defined lineage pathways for cell replacement therapy. This is a critical step in exploiting ESC for therapeutic goals which could have long term benefits for numerous human beings. 83 References Zandstra, P. W., and Nagy, A. (2001) Stem cell bioengineering. Annu Rev Biomed Eng3, 275-305. Slack, J. M . (2000) Stem cells in epithelial tissues. Science 287, 1431-3. Chandross, K. J. a. M . (2001) Chapter: Plasticity of adult bone marrow stem cells., Mattson, M.P. and Van Zant, G. eds. (Greenwich, CT: JAI Press). Balsam, L. B., and Robbins, R. C. (2005) Haematopoietic stem cells and repair of the ischaemic heart. Clin Sci (Lond) 109, 483-92. Barry, F. P. (2003) Mesenchymal stem cell therapy in joint disease. Novartis Found Symp 249, 86-96; discussion 96-102, 170-4, 239-41. Yu, X., Zhan, X., D'Costa, J., Tanavde, V. M . , Ye, Z., Peng, T., Malehorn, M . T., Yang, X., Civin, C. I., and Cheng, L. (2003) Lentiviral vectors with two independent internal promoters transfer high-level expression of multiple transgenes to human hematopoietic stem-progenitor cells. Mol Ther 7, 827-38. Cogle, C. R., Yachnis, A. T., Laywell, E. D., Zander, D. S., Wingard, J. R., Steindler, D. A., and Scott, E. W. (2004) Bone marrow transdifferentiation in brain after transplantation: a retrospective study. Lancet 363, 1432-7. Hao, H. N., Zhao, J., Thomas, R. L. , Parker, G. C , and Lyman, W. D. (2003) Fetal human hematopoietic stem cells can differentiate sequentially into neural stem cells and then astrocytes in vitro. JHematother Stem Cell Res 12, 23-32. Udani, V. M . (2006) The continuum of stem cell transdifferentiation: possibility of hematopoietic stem cell plasticity with concurrent CD45 expression. Stem Cells Devl5, 1-3. Martin, G. R. (1981) Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci USA 78, 7634-8. Thomas, K. R., and Capecchi, M . R. (1987) Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell 51, 503-12. Rajewsky, K., Gu, H., Kuhn, R., Betz, U. A., Muller, W., Roes, J., and Schwenk, F. (1996) Conditional gene targeting. J Clin Invest 98, 600-3. Nagy, A., and Rossant, J. (1996) Targeted mutagenesis: analysis of phenotype without germ line transmission. J Clin Invest 97, 1360-5. Marth, J. D. (1996) Recent advances in gene mutagenesis by site-directed recombination. J Clin Invest 97, 1999-2002. Lewis, J., Yang, B., Detloff, P., and Smithies, O. (1996) Gene modification via "plug and socket" gene targeting. J Clin Invest 97, 3-5. Jasin, M . , Moynahan, M . E. , and Richardson, C. (1996) Targeted transgenesis. Proc Natl Acad Sci USA 93, 8804-8. Kanatsu-Shinohara, M . , Inoue, K., Lee, J., Yoshimoto, M . , Ogonuki, N., Miki, H., Baba, S., Kato, T., Kazuki, Y., Toyokuni, S., Toyoshima, M . , Niwa, O., Oshimura, M . , Heike, T., Nakahata, T., Ishino, F., Ogura, A., and Shinohara, T. (2004) Generation of pluripotent stem cells from neonatal mouse testis. Cell 119, 1001-12. 84 (18) Ferrari, G., Cusella-De Angelis, G., Coletta, M . , Paolucci, E. , Stornaiuolo, A., Cossu, G., and Mavilio, F. (1998) Muscle regeneration by bone marrow-derived myogenic progenitors. Science 279, 1528-30. (19) Gussoni, E. , Soneoka, Y., Strickland, C. D., Buzney, E. A., Khan, M . K., Flint, A. F., Kunkel, L. M . , and Mulligan, R. C. (1999) Dystrophin expression in the mdx mouse restored by stem cell transplantation. Nature 401, 390-4. (20) Kocher, A. A., Schuster, M . D., Szabolcs, M . J., Takuma, S., Burkhoff, D., Wang, J., Homma, S., Edwards, N. M . , and Itescu, S. (2001) Neovascularization of ischemic myocardium by human bone-marrow-derived angioblasts prevents cardiomyocyte apoptosis, reduces remodeling and improves cardiac function. Nat Med 7, 430-6. (21) Orlic, D., Kajstura, J., Chimenti, S., Jakoniuk, I., Anderson, S. M . , L i , B., Pickel, J., McKay, R., Nadal-Ginard, B., Bodine, D. M . , Leri, A. , and Anversa, P. (2001) Bone marrow cells regenerate infarcted myocardium. Nature 410, 701-5. (22) Alison, M . R., Poulsom, R., Jeffery, R., Dhillon, A. P., Quaglia, A., Jacob, J., Novelli, M . , Prentice, G., Williamson, J., and Wright, N. A. (2000) Hepatocytes from non-hepatic adult stem cells. Nature 406, 257. (23) Lagasse, E. , Connors, H., Al-Dhalimy, M . , Reitsma, M . , Dohse, M . , Osborne, L. , Wang, X., Finegold, M . , Weissman, I. L. , and Grompe, M . (2000) Purified hematopoietic stem cells can differentiate into hepatocytes in vivo. Nat Med 6, 1229-34. (24) Theise, N. D., Nimmakayalu, M . , Gardner, R., Illei, P. B., Morgan, G., Teperman, L. , Henegariu, O., and Krause, D. S. (2000) Liver from bone marrow in humans. Hepatology 32, 11-6. (25) Andrews, P. W. (2002) From teratocarcinomas to embryonic stem cells. Philos Trans R Soc Lond B Biol Sci 357, 405-17. (26) Kleinsmith, L. J., and Pierce, G. B., Jr. (1964) Multipotentiality Of Single Embryonal Carcinoma Cells. Cancer Res 24, 1544-51. (27) Illmensee, K., and Mintz, B. (1976) Totipotency and normal differentiation of single teratocarcinoma cells cloned by injection into blastocysts. Proc Natl Acad Sci USA 73, 549-53. (28) Evans, M . J., and Kaufman, M . H. (1981) Establishment in culture of pluripotential cells from mouse embryos. Nature 292, 154-6. (29) Gough, N. M . , Williams, R. L. , Hilton, D. J., Pease, S., Willson, T. A., Stahl, J., Gearing, D. P., Nicola, N. A., and Metcalf, D. (1989) LIF: a molecule with divergent actions on myeloid leukaemic cells and embryonic stem cells. Reprod FertilDev 7,281-8. (30) Smith, A. G. (1991) Culture and differentiation of embryonic stem cells. Journal of Tissue Culture Methodology 13, 89-94. (31) Williams, R. L. , Hilton, D. J., Pease, S., Willson, T. A., Stewart, C. L. , Gearing, D. P., Wagner, E. F., Metcalf, D., Nicola, N. A., and Gough, N. M . (1988) Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature 336, 684-7. (32) Smith, A. G. (2001) Embryo-derived stem cells: of mice and men. Annu Rev Cell Dev Biol 77,435-62. 85 (33) Thomson, J. A., Itskovitz-Eldor, J., Shapiro, S. S., Waknitz, M . A., Swiergiel, J. J., Marshall, V. S., and Jones, J. M . (1998) Embryonic stem cell lines derived from human blastocysts. Science 282, 1145-7. (34) Abe, K., Niwa, H., Iwase, K., Takiguchi, M . , Mori, M . , Abe, S. I., Abe, K., and Yamamura, K. I. (1996) Endoderm-specific gene expression in embryonic stem cells differentiated to embryoid bodies. Exp Cell Res 229, 27-34. (35) Leahy, A., Xiong, J. W., Kuhnert, F., and Stuhlmann, H. (1999) Use of developmental marker genes to define temporal and spatial patterns of differentiation during embryoid body formation. J Exp Zool 284, 67-81. (36) Keller, G. M . (1995) In vitro differentiation of embryonic stem cells. Curr Opin Cell Biol 7, 862-9. (37) Doetschman, T. C., Eistetter, H., Katz, M . , Schmidt, W., and Kemler, R. (1985) The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium. J Embryol Exp Morphol 57,27-45. (38) Wiles, M . V., and Keller, G. (1991) Multiple hematopoietic lineages develop from embryonic stem (ES) cells in culture. Development 111, 259-67. (39) Wobus, A. M . , Wallukat, G., and Hescheler, J. (1991) Pluripotent mouse embryonic stem cells are able to differentiate into cardiomyocytes expressing chronotropic responses to adrenergic and cholinergic agents and Ca2+ channel blockers. Differentiation 48, 173-82. (40) Boheler, K. R., Czyz, J., Tweedie, D., Yang, H. T., Anisimov, S. V. , and Wobus, A. M . (2002) Differentiation of pluripotent embryonic stem cells into cardiomyocytes. Circ Res 91, 189-201. (41) Wobus, A. M . , Grosse, R., and Schoneich, J. (1988) Specific effects of nerve growth factor on the differentiation pattern of mouse embryonic stem cells in vitro. BiomedBiochim Acta 47, 965-73. (42) Shani, M . , Faerman, A., Emerson, C. P., Pearson-White, S., Dekel, I., and Magal, Y. (1992) The consequences of a constitutive expression of MyoDl in ES cells and mouse embryos. Symp Soc Exp Biol 46, 19-36. (43) Dinsmore, J., Ratliff, J., Deacon, T., Pakzaban, P., Jacoby, D., Galpern, W., and Isacson, O. (1996) Embryonic stem cells differentiated in vitro as a novel source of cells for transplantation. Cell Transplant 5, 131^43. (44) Savatier, P., Lapillonne, H., van Grunsven, L. A., Rudkin, B. B., and Samarut, J. (1996) Withdrawal of differentiation inhibitory activity/leukemia inhibitory factor up-regulates D-type cyclins and cyclin-dependent kinase inhibitors in mouse embryonic stem cells. Oncogene 12, 309-22. (45) Weissman, I. L. (2000) Stem cells: units of development, units of regeneration, and units in evolution. Cell 100, 157-68. (46) Gossler, A., Doetschman, T., Korn, R., Serfling, E. , and Kemler, R. (1986) Transgenesis by means of blastocyst-derived embryonic stem cell lines. Proc Natl Acad Sci US A 83, 9065-9. (47) Palmqvist, L. , Glover, C. H., Hsu, L. , Lu, M . , Bossen, B., Piret, J. M . , Humphries, R. K., and Helgason, C. D. (2005) Correlation of murine embryonic stem cell gene expression profiles with functional measures of pluripotency. Stem Cells 23, 663-80. 86 (48) Choi, K., Kennedy, M . , Kazarov, A., Papadimitriou, J. C , and Keller, G. (1998) A common precursor for hematopoietic and endothelial cells. Development 125, 725-32. (49) Chan, R. J., Johnson, S. A., Li , Y. , Yoder, M . C , and Feng, G. S. (2003) A definitive role of Shp-2 tyrosine phosphatase in mediating embryonic stem cell differentiation and hematopoiesis. Blood 102, 2074-80. (50) Paling, N. R., and Welham, M . J. (2005) Tyrosine phosphatase SHP-1 acts at different stages of development to regulate hematopoiesis. Blood 105, 4290-7. (51) Chung, S., Andersson, T., Sonntag, K. C , Bjorklund, L. , Isacson, O., and Kim, K. S. (2002) Analysis of different promoter systems for efficient transgene expression in mouse embryonic stem cell lines. Stem Cells 20, 139-45. (52) Nishimura, F., Yoshikawa, M . , Kanda, S., Nonaka, M . , Yokota, H. , Shiroi, A., Nakase, H. , Hirabayashi, H., Ouji, Y., Birumachi, J., Ishizaka, S., and Sakaki, T. (2003) Potential use of embryonic stem cells for the treatment of mouse parkinsonian models: improved behavior by transplantation of in vitro differentiated dopaminergic neurons from embryonic stem cells. Stem Cells 21, 171-80. (53) Wichterle, FL, Lieberam, I., Porter, J. A., and Jessell, T. M . (2002) Directed differentiation of embryonic stem cells into motor neurons. Cell 110, 385-97. (54) Yurugi-Kobayashi, T., Itoh, FL, Yamashita, J., Yamahara, K., Hirai, H. , Kobayashi, T., Ogawa, M . , Nishikawa, S., Nishikawa, S., and Nakao, K. (2003) Effective contribution of transplanted vascular progenitor cells derived from embryonic stem cells to adult neovascularization in proper differentiation stage. Blood 101, 2675-8. (55) Lumelsky, N., Blondel, O., Laeng, P., Velasco, I., Ravin, R., and McKay, R. (2001) Differentiation of embryonic stem cells to insulin-secreting structures similar to pancreatic islets. Science 292, 1389-94. (56) Ishizaka, S., Shiroi, A., Kanda, S., Yoshikawa, M . , Tsujinoue, H., Kuriyama, S., Hasuma, T., Nakatani, K., and Takahashi, K. (2002) Development of hepatocytes from ES cells after transfection with the HNF-3beta gene. Faseb J16, 1444-6. (57) Yin, Y. , Lim, Y. K., Salto-Tellez, M . , Ng, S. C , Lin, C. S., and Lim, S. K. (2002) AFP(+), ESC-derived cells engraft and differentiate into hepatocytes in vivo. Stem Cells 20, 338-46. (58) Walker, P. R., Saas, P., and Dietrich, P. Y. (1997) Role of Fas ligand (CD95L) in immune escape: the tumor cell strikes back. J Immunol 158, 4521-4. (59) Harlan, D. M . , and Kirk, A. D. (1999) The future of organ and tissue transplantation: can T-cell costimulatory pathway modifiers revolutionize the prevention of graft rejection? Jama 282, 1076-82. (60) Burdon, T., Chambers, I., Stracey, C , Niwa, H., and Smith, A. (1999) Signaling mechanisms regulating self-renewal and differentiation of pluripotent embryonic stem cells. Cells Tissues Organs 165, 131-43. (61) Schmitz, J., Weissenbach, M . , Haan, S., Heinrich, P. C , and Schaper, F. (2000) SOCS3 exerts its inhibitory function on interleukin-6 signal transduction through the SHP2 recruitment site of gpl30. J Biol Chem 275, 12848-56. 87 (62) Rathjen, P. D., Toth, S., Willis, A., Heath, J. K., and Smith, A. G. (1990) Differentiation inhibiting activity is produced in matrix-associated and diffusible forms that are generated by alternate promoter usage. Cell 62, 1105-14. (63) Matsuda, T., Nakamura, T., Nakao, K., Arai, T., Katsuki, M . , Heike, T., and Yokota, T. (1999) STAT3 activation is sufficient to maintain an undifferentiated state of mouse embryonic stem cells. Embo J18, 4261-9. (64) Niwa, H. , Burdon, T., Chambers, I., and Smith, A. (1998) Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3. Genes Dev 12, 2048-60. (65) Niwa, H., Miyazaki, J., and Smith, A. G. (2000) Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat Genet 24, 372-6. (66) Kishimoto, T., Taga, T., and Akira, S. (1994) Cytokine signal transduction. Cell 76, 253-62. (67) Li , Y., McClintick, J., Zhong, L. , Edenberg, H. J., Yoder, M . C , and Chan, R. J. (2005) Murine embryonic stem cell differentiation is promoted by SOCS-3 and inhibited by the zinc finger transcription factor Klf4. Blood 105, 635-7. (68) Anneren, C , Cowan, C. A., and Melton, D. A. (2004) The Src family of tyrosine kinases is important for embryonic stem cell self-renewal. J Biol Chem 279, 31590-8. (69) Ernst, M . , Gearing, D. P., and Dunn, A. R. (1994) Functional and biochemical association of Hck with the LIF/IL-6 receptor signal transducing subunit gpl30 in embryonic stem cells. Embo J13, 1574-84. (70) Boulter, C. A., Aguzzi, A., Williams, R. L. , Wagner, E. F., Evans, M . J., and Beddington, R. (1991) Expression of v-src induces aberrant development and twinning in chimaeric mice. Development 111, 357-66. (71) Nichols, J., Zevnik, B., Anastassiadis, K., Niwa, H., Klewe-Nebenius, D., Chambers, I., Scholer, H., and Smith, A. (1998) Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell 95, 379-91. (72) Pesce, M . , Gross, M.K. , and Scholer, H.R. (1998) In line with our ancestors: Oct-4 and the mammalian germ. Bioessays 20, 722-32. (73) Scholer, H. R., Ruppert, S., Suzuki, N., Chowdhury, K., and Gruss, P. (1990) New type of POU domain in germ line-specific protein Oct-4. Nature 344, 435-9. (74) Reubinoff, B. E. , Pera, M . F., Fong, C. Y. , Trounson, A., and Bongso, A. (2000) Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol 18, 399-404. (75) Niwa, H. (2001) Molecular mechanism to maintain stem cell renewal of ES cells. Cell Struct Funct 26, 137-48. (76) Ambrosetti, D. C , Basilico, C , and Dailey, L. (1997) Synergistic activation of the fibroblast growth factor 4 enhancer by Sox2 and Oct-3 depends on protein-protein interactions facilitated by a specific spatial arrangement of factor binding sites. Mol Cell Biol 17, 6321-9. (77) Avilion, A. A., Nicolis, S. K., Pevny, L. H., Perez, L. , Vivian, N., and Lovell-Badge, R. (2003) Multipotent cell lineages in early mouse development depend on SOX2 function. Genes Dev 17, 126-40. 88 (78) Pesce, M . , Wang, X., Wolgemuth, D. J., and Scholer, H. (1998) Differential expression of the Oct-4 transcription factor during mouse germ cell differentiation. Mech Dev 71, 89-98. (79) Lints, T. J., Parsons, L. M . , Hartley, L. , Lyons, I., and Harvey, R. P. (1993) Nkx-2.5: a novel murine homeobox gene expressed in early heart progenitor cells and their myogenic descendants. Development 119, 969. (80) Chambers, I., Colby, D., Robertson, M . , Nichols, J., Lee, S., Tweedie, S., and Smith, A. (2003) Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 113, 643-55. (81) Ying, Q. L. , Nichols, J., Chambers, I., and Smith, A. (2003) BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell 115, 281-92. (82) Robertson, E., Bradley, A., Kuehn, M . , and Evans, M . (1986) Germ-line transmission of genes introduced into cultured pluripotential cells by retroviral vector. Nature 323, 445-8. (83) Stewart, C. L. , Ruther, U., Garber, C , Vanek, M . , and Wagner, E. F. (1986) The expression of retroviral vectors in murine stem cells and transgenic mice. J Embryol Exp Morphol 97 Suppl, 263-75. (84) Jaenisch, R., Fan, H., and Croker, B. (1975) Infection of preimplantation mouse embryos and of newborn mice with leukemia virus: tissue distribution of viral DNA and RNA and leukemogenesis in the adult animal. Proc Natl Acad Sci U S A 72, 4008-12. (85) Jahner, D., and Jaenisch, R. (1980) Integration of Moloney leukaemia virus into the germ line of mice: correlation between site of integration and virus activation. Nature 287, 456-8. (86) Jahner, D., Haase, K., Mulligan, R., and Jaenisch, R. (1985) Insertion of the bacterial gpt gene into the germ line of mice by retroviral infection. Proc Natl Acad Sci US A 82, 6927-31. (87) van der Putten, H., Botteri, F. M . , Miller, A. D., Rosenfeld, M . G., Fan, H., Evans, R. M . , and Verma, I. M . (1985) Efficient insertion of genes into the mouse germ line via retroviral vectors. Proc Natl Acad Sci US A 82, 6148-52. (88) Cherry, S. R., Biniszkiewicz, D., van Parijs, L. , Baltimore, D., and Jaenisch, R. (2000) Retroviral expression in embryonic stem cells and hematopoietic stem cells. Mol Cell Biol 20, 7419-26. (89) Pfeifer, A. V., I. M . (2001), Vol. 1, Lippincott-Raven, Philadelphia. (90) Negre, D., Duisit, G., Mangeot, P. E. , Moullier, P., Darlix, J. L . , and Cosset, F. L. (2002) Lentiviral vectors derived from simian immunodeficiency virus. Curr Top Microbiol Immunol 261, 53-74. (91) Negre, D., Mangeot, P. E. , Duisit, G., Blanchard, S., Vidalain, P. O., Leissner, P., Winter, A. J., Rabourdin-Combe, C , Mehtali, M . , Moullier, P., Darlix, J. L . , and Cosset, F. L. (2000) Characterization of novel safe lentiviral vectors derived from simian immunodeficiency virus (SIVmac251) that efficiently transduce mature human dendritic cells. Gene Ther 7, 1613-23. (92) Pfeifer, A., Ikawa, M . , Dayn, Y., and Verma, I. M . (2002) Transgenesis by lentiviral vectors: lack of gene silencing in mammalian embryonic stem cells and preimplantation embryos. Proc Natl Acad Sci USA 99, 2140-5. 89 (93) Bhattacharya, B . , Miura , T., Brandenberger, R., Mejido, J., Luo, Y . , Yang, A . X . , Joshi, B . H . , Ginis, I., Thies, R. S., Amit , M . , Lyons, I., Condie, B . G . , Itskovitz-Eldor, J. , Rao, M . S., and Puri, R. K . (2004) Gene expression in human embryonic stem cell lines: unique molecular signature. Blood 103, 2956-64. (94) Brandenberger, R., Wei , H . , Zhang, S., L e i , S., Murage, J., Fisk, G . J., L i , Y . , X u , C., Fang, R., Guegler, K . , Rao, M . S., Mandalam, R., Lebkowski, J., and Stanton, L . W . (2004) Transcriptome characterization elucidates signaling networks that control human ES cell growth and differentiation. Nat Biotechnol 22, 707-16. (95) Ivanova, N . B . , Dimos, J. T., Schaniel, C. , Hackney, J. A . , Moore, K . A . , and Lemischka, I. R. (2002) A stem cell molecular signature. Science 298, 601-4. (96) Ramalho-Santos, M . , Yoon , S., Matsuzaki, Y . , Mull igan, R. C. , and Melton, D . A . (2002) "Sternness": transcriptional profiling of embryonic and adult stem cells. Science 298, 597-600. (97) Sato, N . , Sanjuan, I. M . , Heke, M . , Uchida, M . , Naef, F. , and Brivanlou, A . H . (2003) Molecular signature of human embryonic stem cells and its comparison with the mouse. Dev Biol 260, 404-13. (98) Sharov, A . A . , Piao, Y . , Matoba, R., Dudekula, D . B . , Qian, Y . , VanBuren, V . , Falco, G . , Martin, P. R., Stagg, C. A . , Bassey, U . C , Wang, Y . , Carter, M . G . , Hamatani, T., Aiba , K . , Akutsu, H . , Sharova, L . , Tanaka, T. S., Kimber, W . L . , Yoshikawa, T., Jaradat, S. A . , Pantano, S., Nagaraja, R., Boheler, K . R., Taub, D. , Hodes, R. J., Longo, D . L . , Schlessinger, D . , Keller, J., Klotz , E . , Kelsoe, G . , Umezawa, A . , Vescovi, A . L . , Rossant, J., Kunath, T., Hogan, B . L . , Curci , A . , D'Urso, M . , Kelso, J. , Hide, W. , and K o , M . S. (2003) Transcriptome analysis o f mouse stem cells and early embryos. PLoS Biol 1, E74. (99) Sperger, J. M . , Chen, X . , Draper, J. S., Antosiewicz, J. E . , Chon, C. H . , Jones, S. B . , Brooks, J. D . , Andrews, P. W. , Brown, P. O., and Thomson, J. A . (2003) Gene expression patterns in human embryonic stem cells and human pluripotent germ cell tumors. Proc Natl Acad Sci USA 100, 13350-5. (100) Ke l ly , D . L . , and Rizzino, A . (2000) D N A microarray analyses of genes regulated during the differentiation of embryonic stem cells. Mol Reprod Dev 56, 113-23. (101) Tanaka, T. S., Kunath, T., Kimber, W . L . , Jaradat, S. A . , Stagg, C. A . , Usuda, M . , Yokota, T., Niwa , H . , Rossant, J., and K o , M . S. (2002) Gene expression profiling of embryo-derived stem cells reveals candidate genes associated with pluripotency and lineage specificity. Genome Res 12, 1921-8. (102) Fortunel, N . O., Otu, H . H . , N g , H . H . , Chen, J., M u , X . , Chevassut, T., L i , X . , Joseph, M . , Bailey, C , Hatzfeld, J. A . , Hatzfeld, A . , Usta, F. , Vega, V . B . , Long, P. M . , Libermann, T. A . , and L i m , B . (2003) Comment on " 'Sternness': transcriptional profiling of embryonic and adult stem cells" and "a stem cell molecular signature". Science 302, 393; author reply 393. (103) Evsikov, A . V . , and Solter, D . (2003) Comment on " 'Sternness': transcriptional profiling of embryonic and adult stem cells" and "a stem cell molecular signature". Science 302, 393; author reply 393. (104) Livak, K . J., and Schmittgen, T. D . (2001) Analysis of relative gene expression data using real-time quantitative P C R and the 2(-Delta Delta C(T)) Method. Methods 25, 402-8. 90 ;i05) L i , E . , Bestor, T. H . , and Jaenisch, R. (1992) Targeted mutation of the D N A methyltransferase gene results in embryonic lethality. Cell 69, 915-26. '106) Shirogane, T., Fukada, T., Muller, J. M . , Shima, D . T., H ib i , M . , and Hirano, T. (1999) Synergistic roles for Pim-1 and c-Myc in STAT3-mediated cell cycle progression and antiapoptosis. Immunity 11, 709-19. ;i07) Chapman, G . , Remiszewski, J. L . , Webb, G . C , Schulz, T. C , Bottema, C. D . , and Rathjen, P. D . (1997) The mouse homeobox gene, Gbx2: genomic organization and expression in pluripotent cells in vitro and in vivo. Genomics 46, 223-33. [108) Valdimarsdottir, G . , and Mummery, C. (2005) Functions of the TGFbeta superfamily in human embryonic stem cells. Apmis 113, 773-89. T09) Zufferey, R., Donello, J. E . , Trono, D . , and Hope, T. J. (1999) Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors. J Virol 73, 2886-92. [110) Zennou, V . , Petit, C , Guetard, D . , Nerhbass, U . , Montagnier, L . , and Charneau, P. (2000) HIV-1 genome nuclear import is mediated by a central D N A flap. Cell 101, 173-85. '111) Follenzi, A . , Ai l les , L . E . , Bakovic, S., Geuna, M . , and Naldini , L . (2000) Gene transfer by lentiviral vectors is limited by nuclear translocation and rescued by HIV-1 pol sequences. Nat Genet 25, 217-22. '112) Mizushima, S., and Nagata, S. (1990) p E F - B O S , a powerful mammalian expression vector. Nucleic Acids Res 18, 5322. '113) Gropp, M . , Itsykson, P., Singer, O., Ben-Hur, T., Reinhartz, E . , Galun, E . , and Reubinoff, B . E . (2003) Stable genetic modification of human embryonic stem cells by lentiviral vectors. Mol Ther 7, 281-7. T14) Zeng, X . , Miura , T., Luo, Y . , Bhattacharya, B . , Condie, B . , Chen, J. , Ginis , I., Lyons, I., Mejido, J., Puri, R. K . , Rao, M . S., and Freed, W . J. (2004) Properties of pluripotent human embryonic stem cells BG01 and B G 0 2 . Stem Cells 22, 292-312. ; i 15) Dang, S. M . , Kyba , M . , Perlingeiro, R., Daley, G . Q., and Zandstra, P. W . (2002) Efficiency of embryoid body formation and hematopoietic development from embryonic stem cells in different culture systems. Biotechnol Bioeng 78, 442-53. '116) Haub, O., and Goldfarb, M . (1991) Expression of the fibroblast growth factor-5 gene in the mouse embryo. Development 112, 397-406. ^ l 17) Hebert, J. M . , Boyle, M . , and Martin, G . R. (1991) m R N A localization studies suggest that murine FGF-5 plays a role in gastrulation. Development 112, 407-15. 118) Levinson-Dushnik, M . , and Benvenisty, N . (1997) Involvement of hepatocyte nuclear factor 3 in endoderm differentiation of embryonic stem cells. Mol Cell Biol 17, 3817'-22. [119) Wiese, C , Rolletschek, A . , Kania, G . , Blyszczuk, P., Tarasov, K . V . , Tarasova, Y . , Wersto, R. P., Boheler, K . R., and Wobus, A . M . (2004) Nestin expression-a property of multi-lineage progenitor cells? Cell Mol Life Sci 61, 2510-22. T20) Wilkinson, D . G . , Bhatt, S., and Herrmann, B . G . (1990) Expression pattern of the mouse T gene and its role in mesoderm formation. Nature 343, 657-9. 91 (121) Nagy, A., Rossant, J., Nagy, R., Abramow-Newerly, W., and Roder, J. C. (1993) Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. Proc Natl Acad Sci U S A 90, 8424-8. (122) Call, L. M , Moore, C. S., Stetten, G., and Gearhart, J. D. (2000) A cre-lox recombination system for the targeted integration of circular yeast artificial chromosomes into embryonic stem cells. Hum Mol Genet 9, 1745-51. (123) Wiles, M . V., Vauti, F., Otte, J., Fuchtbauer, E. M . , Ruiz, P., Fuchtbauer, A., Arnold, H. H. , Lehrach, H., Metz, T., von Melchner, H. , and Wurst, W. (2000) Establishment of a gene-trap sequence tag library to generate mutant mice from embryonic stem cells. Nat Genet 24, 13-4. (124) Palmieri, S. L. , Peter, W., Hess, H., and Scholer, H. R. (1994) Oct-4 transcription factor is differentially expressed in the mouse embryo during establishment of the first two extraembryonic cell lineages involved in implantation. Dev Biol 166, 259-67. (125) Smithies, O., Gregg, R. G., Boggs, S. S., Koralewski, M . A., and Kucherlapati, R. S. (1985) Insertion of DNA sequences into the human chromosomal beta-globin locus by homologous recombination. Nature 317, 230-4. (126) Wong, E. A., and Capecchi, M . R. (1986) Analysis of homologous recombination in cultured mammalian cells in transient expression and stable transformation assays. Somat Cell Mol Genet 12, 63-72. (127) Bockamp, E. , Antunes, C , Maringer, M . , Heck, R., Presser, K., Beilke, S., Ohngemach, S., Alt, R., Cross, M . , Sprengel, R., Hartwig, U., Kaina, B., Schmitt, S., and Eshkind, L. (2006) Tetracycline-controlled transgenic targeting from the SCL locus directs conditional expression to erythrocytes, megakaryocytes, granulocytes and c-kit expressing lineage negative hematopoietic cells. Blood [Epub ahead of print]. (128) Hamaguchi, I., Woods, N. B., Panagopoulos, I., Andersson, E. , Mikkola, H., Fahlman, C , Zufferey, R., Carlsson, L. , Trono, D., and Karlsson, S. (2000) Lentivirus vector gene expression during ES cell-derived hematopoietic development in vitro. J Virol 74, 10778-84. (129) Ueki, K., Kadowaki, T., and Kahn, C. R. (2005) Role of suppressors of cytokine signaling SOCS-1 and SOCS-3 in hepatic steatosis and the metabolic syndrome. Hepatol Res. (130) Loh, Y. H., Wu, Q., Chew, J. L. , Vega, V. B., Zhang, W., Chen, X., Bourque, G., George, J., Leong, B., Liu, J., Wong, K. Y., Sung, K. W., Lee, C. W., Zhao, X. D., Chiu, K. P., Lipovich, L. , Kuznetsov, V. A., Robson, P., Stanton, L. W., Wei, C. L. , Ruan, Y., Lim, B., and Ng, H. H. (2006) The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells. Nat Genet 38, 431-40. (131) Kowenz-Leutz, E. , Herr, P., Niss, K., and Leutz, A. (1997) The homeobox gene GBX2, a target of the myb oncogene, mediates autocrine growth and monocyte differentiation. Cell 91, 185-95. (132) Gao, A. C , Lou, W., and Isaacs, J. T. (2000) Enhanced GBX2 expression stimulates growth of human prostate cancer cells via transcriptional up-regulation of the interleukin 6 gene. Clin Cancer Res 6, 493-7. 92 (133) Nichols, J., Chambers, I., and Smith, A. (1994) Derivation of germline competent embryonic stem cells with a combination of interleukin-6 and soluble interleukin-6 receptor. Exp Cell Res 215, 237-9. (134) Yoshida, K., Chambers, I., Nichols, J., Smith, A., Saito, M . , Yasukawa, K., Shoyab, M . , Taga, T., and Kishimoto, T. (1994) Maintenance of the pluripotential phenotype of embryonic stem cells through direct activation of gpl30 signalling pathways. Mech Dev 45, 163-71. (135) Schindler, C , and Darnell, J. E. , Jr. (1995) Transcriptional responses to polypeptide ligands: the JAK-STAT pathway. Annu Rev Biochem 64, 621-51. (136) Darnell, J. E. , Jr. (1997) STATs and gene regulation. Science 277, 1630-5. (137) Lou, W., Ni, Z., Dyer, K., Tweardy, D. J., and Gao, A. C. (2000) Interleukin-6 induces prostate cancer cell growth accompanied by activation of stat3 signaling pathway. Prostate 42, 239-42. (138) Nakagawa, Y., and O'Leary, D. D. (2001) Combinatorial expression patterns of LIM-homeodomain and other regulatory genes parcellate developing thalamus. J Neurosci 21,2111-25. (139) Waters, S. T., and Lewandoski, M . (2006) A threshold requirement for Gbx2 levels in hindbrain development. Development 133, 1991-2000. (140) Zandstra, P. W., Le, H. V., Daley, G. Q., Griffith, L. G., and Lauffenburger, D. A. (2000) Leukemia inhibitory factor (LIF) concentration modulates embryonic stem cell self-renewal and differentiation independently of proliferation. Biotechnol Bioeng69, 607-17. (141) Muttukrishna, S., Fowler, P. A., Groome, N. P., Mitchell, G. G., Robertson, W. R., and Knight, P. G. (1994) Serum concentrations of dimeric inhibin during the spontaneous human menstrual cycle and after treatment with exogenous gonadotrophin. Hum Reprod 9, 1634-42. (142) Albano, R. M . , Groome, N., and Smith, J. C. (1993) Activins are expressed in preimplantation mouse embryos and in ES and EC cells and are regulated on their differentiation. Development 117, 711-23. (143) Tsuchida, K., Nakatani, M . , Yamakawa, N., Hashimoto, O., Hasegawa, Y. , and Sugino, H. (2004) Activin isoforms signal through type I receptor serine/threonine kinase ALK7. Mol Cell Endocrinol 220, 59-65. (144) Xiao, L., Yuan, X., and Sharkis, S. J. (2006) Activin A maintains self-renewal and regulates FGF, Wnt and BMP pathways in human embryonic stem cells. Stem Cells. (145) Inman, G. J., Nicolas, F. J., Callahan, J. F., Harling, J. D., Gaster, L. M . , Reith, A. D., Laping, N. J., and Hill, C. S. (2002) SB-431542 is a potent and specific inhibitor of transforming growth factor-beta superfamily type I activin receptor-like kinase (ALK) receptors ALK4, ALK5, and ALK7. Mol Pharmacol 62, 65-74. (146) Zou, Y., Evans, S., Chen, J., Kuo, H. C , Harvey, R. P., and Chien, K. R. (1997) CARP, a cardiac ankyrin repeat protein, is downstream in the Nkx2-5 homeobox gene pathway. Development 124, 793-804. (147) Miller, M . K., Bang, M . L. , Witt, C. C , Labeit, D., Trombitas, C , Watanabe, K., Granzier, H. , McElhinny, A. S., Gregorio, C. C , and Labeit, S. (2003) The muscle ankyrin repeat proteins: CARP, ankrd2/Arpp and DARP as a family of titin filament-based stress response molecules. J Mol Biol 333, 951-64. 93 (148) Sachinidis, A., Fleischmann, B. K., Kolossov, E. , Wartenberg, M . , Sauer, H., and Hescheler, J. (2003) Cardiac specific differentiation of mouse embryonic stem cells. Cardiovasc Res 58, 278-91. (149) Liu, B., Liu, Y., Chen, J., Wei, Z., Yu, H., Zhen, Y., Lu, L. , and Hui, R. (2002) CARP is a novel caspase recruitment domain containing pro-apoptotic protein. Biochem Biophys Res Commun 293, 1396-404. (150) Balentien, E. , Mufson, B. E. , Shattuck, R. L. , Derynck, R., and Richmond, A. (1991) Effects of MGSA/GRO alpha on melanocyte transformation. Oncogene 6, 1115-24. (151) Singh, R. K., Gutman, M . , Radinsky, R., Bucana, C. D., and Fidler, I. J. (1994) Expression of interleukin 8 correlates with the metastatic potential of human melanoma cells in nude mice. Cancer Res 54, 3242-7. (152) Schadendorf, D., Moller, A., Algermissen, B., Worm, M . , Sticherling, M . , and Czarnetzki, B. M . (1993) IL-8 produced by human malignant melanoma cells in vitro is an essential autocrine growth factor. J Immunol 151, 2667-75. (153) Meyn, M . A., 3rd, Schreiner, S. J., Dumitrescu, T. P., Nau, G. J., and Smithgall, T. E. (2005) SRC family kinase activity is required for murine embryonic stem cell growth and differentiation. Mol Pharmacol 68, 1320-30. (154) Minchiotti, G. (2005) Nodal-dependant Cripto signaling in ES cells: from stem cells to tumor biology. Oncogene 24, 5668-75. 94 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0092716/manifest

Comment

Related Items