Stem cell transplantation is an important tool to treat several hematological disorders11. Patients with lymphomas or multiple myeloma receive autologous transplantation of mobilized progenitors after receiving high-dose chemotherapy12. The numbers of mobilized progenitors may not be sufficient, especially when repeat autologous transplantations are required13. These data compel us to develop protocols to expand hematopoietic stem cells. Within the fetal liver (FL), hematopoietic stem cells expand dramatically and for this reason, we have focused our efforts on this organ. The HSC population has been highly characterized in the adult and the fetus, and it is clear that in the adult bone marrow (BM), HSCs are primarily non-cycling/quiescent. During development however, the entire hierarchy of blood lineages must grow to serve the needs of the growing embryo. In kind, all HSCs are cycling and the HSC population increases: self renewal of HSCs is evident. The differences in HSCs in the fetus and adult may be attributed to distinct microenvironmental, or niche, signals from the FL and BM. We wish to identify the fetal HSC niche and dissect the mechanisms employed to regulate HSC proliferation.We previously identified a Nestin+mesenchymal stem cell population in the BM that served as a HSC niche. By applying the tools we have used to identify the HSC niche in the BM, we have identified a candidate HSC niche cell in the Fetal Liver (FL), the major site of fetal hematopoiesis. Perhaps not-surprisingly, Nestin+ cells in the FL bear a striking resemblance to those in the BM. Here, we propose to characterize this population with several in vivo and in vitro assays. We will then demonstrate the importance of the FL Nestin+ cells in maintaining HSCs with both in vivo and in vitro approaches. We will use Cre-recombinase to delete a candidate gene, CXCL12, within our Nestin+ population to test for the importance of this chemokine in maintaining HSCs in the FL niche. We will perform RNA-sequencing of FL and BM Nestin+ cells to quantify the differences between BM and FL niches. Comparative transcriptomicsmay give insight into mechanisms by which FL and BM Nestin+ cells regulate HSC self-renewal, quiescence, and differentiation. Further study of the niche will help define how HSCs may be expanded for therapeutic purposes. The identification of niches in the adult and in the embryo allows us a unique opportunity to compare niches where stem cells are quiescent and where they are proliferating in physiological settings and may also give insight into non-physiological growths such as myelodysplastic syndrome. Our preliminary evidence suggests that we have identified a rare cell type in the FL that is enriched for growth factors necessary to support HSCs. By further characterizing this cell population among other potential candidates, we wish to study the mechanisms of HSC expansion.
The optimal source for autologous bone marrow transplantation is mobilized blood;however in up to 40% of individuals, insufficient numbers of stem cells are obtained. The patient's own stem cells need to be expanded for an autologous transplant in these cases, and a novel culture systems that expand hematopoietic stem cells is called for. Our proposal directly addresses the need to expand hematopoietic stem cells by dissecting the processes that expand these cells in the fetal liver.
