DECIPHERING THE INSTRUCTIONS FOR VERTEBRATE HSC SPECIFICATION AND AMPLIFICATION.
Hematopoietic stem cells (HSCs) are an important population of cells that continuously produce and replace blood and immune cells over the course of our lifetimes. These rare, self-renewing cells are the key element of bone marrow transplants, which are used to treat a variety of conditions including many forms of leukemia and solid tumors. Understanding how hematopoietic stem cells are made during embryonic development is important because it could teach us how to make such cells in the laboratory, and possibly allow circumvention of immune compatibility issues between donor and host. In this research we will perform genetic comparisons of how HSCs are generated in a diverse array of vertebrate embryos to determine the conserved core components of the hematopoietic niche. These results will be validated functionally, then translated to human pluripotent stem cells where we will use our new knowledge to instruct HSCs in vitro, something which is not currently possible.
Understanding how hematopoietic stem cells (HSCs) are made during embryonic development is important because it could teach us how to make and amplify such cells in the laboratory. We will perform genetic comparisons of how HSCs are generated in a diverse array of vertebrate embryos to determine the conserved core components of the hematopoietic niche. These results will be translated to human stem cell populations where we will use our new knowledge to instruct and amplify HSCs in vitro, feats which are not currently possible.
The creation of human induced pluripotent stem cells (hiPSCs) holds great promise for new cell-based therapies, including bone marrow transplants (BMTs). These cells have the potential to generate any tissue type, and can be generated in a patient-specific manner. Thus, hiPSCs hold the promise of cellular replacement therapies without the risk of immune rejection. For use in BMTs, however, hiPSCs must be coaxed to differentiate into hematopoietic stem cells (HSCs), the rare cells responsible for the long-term, curative effects of BMT. This is currently not possible, due to a lack of understanding of the cues required to generate HSCs in vivo.
Insight into the factors needed to instruct and amplify HSCs will be used to provide similar factors at similar timepoints to differentiate hiPSCs into HSCs. Our research will thus lead to great improvements in stem cell therapies to better meet the needs of patients in California.
Despite progress in identifying the cellular composition of hematopoietic stem/progenitor cell (HSPC) niches, little is known about the molecular requirements of HSPC support. To address this issue, we used a panel of recognized HSPC-supportive stromal lines and less-supportive counterparts originating from embryonic and adult hematopoietic sites. Through comprehensive transcriptomic meta-analyses, we identified 481 mRNAs and 17 microRNAs organized in a network implicated in paracrine signaling. Further inclusion of 15 stromal samples demonstrated that this mRNA subset was predictive of the HSPC support. Our gene set contains most of the known HSPC regulators but also a number of novel ones, such as Pax9 and Ccdc80, as validated by functional studies in zebrafish embryos. In sum, our approach has identified the core molecular network required for HSPC support. These cues together with a searchable web resource will inform ongoing efforts to instruct HSPC ex vivo amplification and formation from pluripotent precursors.
The molecular characterization of hematopoietic stem cell (HSC) microenvironments (also termed niches) is a fundamental goal in the field of stem cell biology and regenerative medicine. Using an ensemble of systems biology approaches, we have established shared molecular commonalities in HSC niches from distinct temporal and spatial locations, both in the adult vertebrate animal and in the developing embryo. Our studies have identified several genes that are predicted to be important niche factors. Experimentally, we have validated the importance of many of these factors, and are now working to better understand their functions. To investigate if the molecular pathways identified in the mouse model were conserved in other vertebrate species, we validated the importance of several predicted key regulators by functional studies in the zebrafish embryo. In addition to extracting the molecular core dedicated to HSC support, we also identified specific gene signatures active in the embryonic sites of HSC emergence. More recently, we have extended these approaches to a comparison of gene signatures in the murine AGM region, the birthplace of mammalian HSCs. Using sensitive laser microdissection techniques, coupled with microarray analyses and our proven bioinformatics approaches, we now have assembled new data sets for functional testing. As described in our progress report, we have discovered a variety of factors specifically expressed by the ventral mesenchyme underlying the hemogenic endothelium of the dorsal aorta. We hope that these will include novel mediators of the endothelial to hematopoietic transition (EHT), which we will translate to our human pluripotent cell approaches over the next year.