UBC Theses and Dissertations
Studies of hemopoietic stem cell behaviour in vitro Humphries, Richard Keith
Although the key role played by stem cells in maintaining hemopoiesis is well recognized, mechanisms that determine stem cell behaviour (e.g. self-renewal) have remained poorly defined. Historical precedent has illustrated the usefulness of in viitro colony assays in facilitating the investigation of various primitive hemopoietic progenitor classes with restricted differentiation and proliferative potential. In particular, such assays have made possible the definition of the sequential nature of a series of events that early erythropoietic cells undergo and suggested properties that might be anticipated to characterize colonies derived from stem cells proliferating and differentiating in vitro. The present studies were undertaken to test the hypothesis that very large, late maturing erythroid colonies previously noted on occasion in routine erythroid colony assays were, in fact, derived from progenitors with the self-renewal and multiple myeloid differentiation properties associated with stem cells. Initial experiments showed that cells capable of yielding macroscopic sized erythroid colonies were present at low frequency in normal marrow but became the predominant erythropoietic cell type after 2 to 3 weeks in flask culture. Macroscopic erythroid colony formation in assays of cells from either source were shown to have: 1) identical very late maturation kinetics (onset of hemoglobinization after 1 week of initial colony growth), 2) the same high erythropoietin requirements, and 3) similar responsiveness to factors present in media conditioned by mitogen stimulated spleen cells. Optimization of these 2 classes of stimulant yielded an assay that was linear down to very low cell concentrations (less than 5 x 103 cells/ml). This made possible the cytological analysis of macroscopic erythroid colonies under conditions where overlap with other colony types was minimal. Such studies revealed that macroscopic colonies contained, in addition to erythroid cells, megakaryocytes ( > 90% of colonies) and granulocyte cells (30% of colonies) including cells of the eosinophil lineage. Such colonies were also found to contain cells capable of macroscopic spleen colony formation in irradiated mice, the conventional assay for mouse hemopoietic stem cells (on average 1 CFU-S per colony of flask culture origin, and 0.3 CFU-S per colony in assays of fresh marrow). Direct evidence for self-renewal was obtained from replating experiments using irradiated feeders to optimize plating efficiency. Mixed colonies of macroscopic size were regularly demonstrable in replating assays after 1, and even 2, generations of mixed colony formation indicating up to 6 self-renewal divisions during colony formation. By comparison to flask culture cells, the extent of self-renewal exhibited by cells in fresh marrow yielding macroscopic erythroid colonies was found to be 5-fold lower. Finally, the in vitro expression of stem cell self-renewal behaviour was investigated in individual colonies. The number of stem cells generated, when assessed either by CFU-S or replating assays was found to vary markedly. This variation was not accounted for by errors of detection or by variations in colony size. Such findings are similar to previous data on the CFU-S content of individual spleen colonies and provide the first evidence that the type of variation in stem cell self-renewal observed in vivo also occurs in vitro where microenvironmental factors are unlikely to be contributing factors. These results are consistent with a model of stem cell self-renewal in which intrinsic cellular factors play the key role in influencing the decision of individual cells to self-renew.
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