Investigations of Hematopoietic Stem Cells and Their Age-Associated Alterations

Abstract: The hematopoietic stem cell (HSC) has and continues to be extensively investigated, and represents by far the most studied somatic stem cell. Development of various experimental technologies, including fluorescence activated cell sorting (FACS), have been crucial for research advancements within the hematopoietic field. However, utilization of flow cytometry techniques put high demands on study design, execution, and data analysis. In article I, we addressed a number of important aspects of flow cytometry-based experiments, particularly for experiments evaluating cells present at low frequencies, such as HSCs, and/or of limited sample amounts. Specifically, we highlighted the importance of different types of positive and negative controls. We also presented a 17-parameter analysis design for simultaneous investigation of numerous different mature and immature human hematopoietic cell populations, including HSCs, megakaryocyte/erythrocyte progenitors (MEPs), granulocyte/macrophage progenitors (GMPs), common lymphoid progenitors (CLPs), CD11b+ myeloid cells, CD19+ B cells, CD4+ T cells, and CD8+ T cells, in one bone marrow sample. Lastly, we discussed data visualization alternatives that allow for appropriate presentation of analyzed results.Serial transplantation is considered the gold standard approach for in vivo evaluation of long-term HSC capacity. Despite the extensive use of this methodology, such experiments are not conducted uniformly between laboratories. In article II, we therefore compared the two most common strategies for serial transplantation (serial transplantation of whole bone marrow [wBM] versus serial transplantation of purified HSCs). We revealed that donor-derived cells were not evenly distributed among separate bones within individual mice three months after transplantation – a time point that is frequently used for readout of bone marrow engraftment and/or isolation of cells for serial transplantations. We showed that such unequal distribution could impede isolation and serial transplantation of wBM with representative chimerism levels. Serial transplantation of purified HSCs would however correct for uneven chimerism levels. Furthermore, serial transplantation of wBM was associated with a relative lymphoid skewing, likely as a result of long-lived lymphoid-restricted non- HSCs. Again, serial transplantation of purified HSCs is preferential as it ensures evaluation of lineage output from the designated candidate HSC population. We also demonstrated that serial transplantation of purified HSCs enable distinction of long-term effects (occurring in secondary recipients) from short-term effects (presented in primary recipients), which may be hindered when wBM is transplanted. Therefore, we highly recommend utilization of purified HSCs for serial transplantation purposes. We also presented a proposed serial transplantation design that would maximize the chances to correctly evaluate long-term HSC competence.Lastly in this thesis, we investigated the effects of aging on human and murine hematopoietic stem and progenitor cells (HSPCs). Such investigations are important for understanding of the underlying mechanisms that cause age-associated alterations of the hematopoietic system, including immune impairments, and increased incidences of anemia and myeloid malignancies. Murine studies indicate that at least some age-associated hematologic changes are linked to alterations occurring in the most immature hematopoietic compartments. Such studies in humans are sparse, as well as conflicting, but necessary to establish species-conserved and species-specific agingpatterns, and to confirm mice as relevant model organisms for investigations of aging hematopoiesis. In article III, we revealed that humans and mice demonstrate several similar hematologic aging patterns, including increased HSC frequencies, and lymphoid differentiation impairments. Both species also exhibited prominent transcriptional lineage-skewing patterns of aged HSCs toward enrichments of megakaryocytic/erythroid signatures, while genes involved in lymphoid specification were markedly downregulated. In conclusion, these results confirmed several HSC aging similarities across the human-to-mouse species barrier, as well as demonstrated novel lineage-skewing patterns within aging HSPCs.

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