Expression of the Medial HOXA genes is indispensible for self-renewal in human hematopoietic stem cells

Hematopoietic stem cells (HSCs) are able to self-renew, generate all lineages of the blood, reconstitute the hematopoietic system upon bone marrow (BM) transplantation, and thereby cure diseases of the blood and immune system. BM transplantations have successfully treated a wide range of blood-related diseases (i.e., -thalassemia, leukemia, and HIV/AIDS). However, current demand for BM transplantations outstrips the quantity of HSCs available through cord blood banks and BM registries, and further requires finding matching HLA-types between patients and donors. Given these restrictions in quantity and compatibility, there is significant interest in developing alternative sources of transplantable HSCs by differentiating HSCs in vitro from pluripotent stem cells (PSCs), such as embryonic stem cells (ESCs). Unfortunately, lack of understanding of the regulatory mechanisms governing HSC function has prevented their in vitro generation. The focus of my thesis research is on deciphering the molecular blocks preventing the generation of HSCs from ESCs, and identifying key molecular cues to help overcome these blocks. In the course of my research, I identified the medial HOXA genes as critical regulators for human HSC self-renewal, and explored the pathways that regulate the HOXA genes that are dysregulated during ESC differentiation cultures. Our lab developed a two-step differentiation protocol mimicking the environments a developing HSC would encounter in the embryo and successfully generated cells with the human HSC immunophenotype, CD34+CD38-CD45+CD90+GPI80+. However, although ESC-derived cells differentiated into definitive erythroid, myeloid, and T-cells, they displayed impaired self-renewal and engraftment ability compared to hematopoietic stem/progenitor cells (HSPCs) isolated from the fetal liver (FL), the site of the most active HSC expansion and differentiation during human development. Microarray analysis witnessed the overall close molecular correlation between ESC-HSPCs and FL-HSPCs, but revealed the lack of expression of the medial HOXA genes in ESC-derived cells as a critical defect preventing their self-renewal. Knockdown of HOXA5 or HOXA7 in FL-HSPCs recapitulated the self-renewal defects observed in ESC-HSPCs. A six-day stimulation of the retinoic acid (RA) signaling pathway was sufficient to significantly upregulate HOXA gene expression in ESC-HSPCs at day 6. However, HOXA gene expression was not sustained long-term. Microarray and ChIP data further revealed that the regulator of HOXA genes, MLL1, was expressed, but not bound to HOXA genes in ESC-derived cells, suggesting the inability to recruit MLL1 to HOXA genes prevents their activation during ESC differentiation. I identified in the human genome a region of 89% sequence homology to the mouse lincRNA Mistral, which in mice recruits MLL1 to HOXA6/HOXA7 genes. RNA-seq and microarray data showed high expression of putative human MISTRAL in FL-HSCs but not in their differentiated progeny or in ESC-derived hematopoietic cells, and high correlation of HOXA6/A7 expression to Mistral expression in multiple cell types. These data identify insufficiency of HOXA gene expression is a developmental barrier for generating HSCs from pluripotent cells and suggest RA-signaling is a major inductive signal and MISTRAL a novel component of the fetal HSC regulatory machinery that conveys "stemness" in hematopoietic cells