Year 1The objectives of our proposal are the isolations of blood-forming and heart-forming stem cells from human embryonic stem cell (hESCs) cultures, and the generation of monoclonal antibodies (mAbs) that eliminate residual teratogenic cells from transplantable populations of differentiated hESCs. For isolation of progenitors, we hypothesized that precursors derived from hESCs could be identified and isolated using mAbs that label unique combinations of lineage-specific cell surface molecules. We used hundreds of defined mAbs, generated hundreds of novel anti-hESC mAbs, and used these to isolate and characterize dozens of hESC-derived populations. We discovered four precursor types from early stages of differentiating cells, each expressing genes indicative of commitment to either embryonic or extraembryonic tissues. Together, these progenitors are candidates to give rise to meso-endodermal lineages (heart, blood, pancreas, etc), and yolk sac, umbilical cord and placental tissues, respectively. Importantly, we have found that cells of the meso-endodermal population give rise to beating cardiomyocytes. We are currently enriching cardiomyocyte precursors from this population using cardiac-specific genetic markers, and are assaying the putative progenitors using electrophysiological assays and by transplantation into animal hearts (a test for restoration of heart function). In addition, we established in vitro conditions that effectively promote hESC-differentiation towards the hematopoietic (blood) lineages and isolated populations that resemble hematopoietic stem cells (HSCs) in both surface phenotype as well as lineage potentials, as determined by assays in vitro. We have generated hESC-lines that express the anti-apoptotic gene BCL2, and have found that these cells produce significantly greater amounts of hematopoietic and cardiac cells, because of their increased survival during culturing and sorting. We are currently isolating hematopoietic precursors from BCL2-hESCs and will test their ability to engraft in immunodeficient mice, to examine the capacity of hESC-derived HSCs to regenerate the blood system. Finally, we have utilized the novel mAbs that we prepared against undifferentiated hESCs, to deplete residual teratogenic cells from differentiated cultures that were transplanted into animal models. We discovered that following depletion teratoma rarely formed, and we expect to determine a final cocktail of mAbs for removal of teratogenic cells from transplantation products this year.
Year 2The main objective of our proposal is to isolate therapeutic stem cells and progenitors from human embryonic stem cells (hESCs) that give rise to blood and heart cells. Our approach involves isolation of differentiated precursor subset of cells using monoclonal antibodies (mAbs) and cell sorting instruments, and subsequent characterization of their respective hematopoietic and cardiomyogenic potential in culture as well as following engraftment into mouse models of disease. In addition, we aim to develop mAbs that specifically bind to undifferentiated hESCs for removal of residual teratoma-initiating cells from therapeutic cell preparations, to ensure transplantation safety.
We have made substantial advancement towards achieving these goals. First, we discovered that the initial differentiation of hESCs occurs through only 4-5 different progenitor types, of which one is destined to give rise to heart lineages. We purified this population using three novel cell surface markers, and found a significant enrichment of cardiomyocyte clones in colony formation assays that we developed. This subset also expressed particularly high levels of cardiac genes and was receptive to further differentiation into beating cardiomyocytes or vascular endothelial cells. When transplanted into immunodeficient mice these progenitors differentiated into ventricular myocytes and vascular endothelial cells. In the coming year we will perform transplantation experiments to evaluate whether they improve the functional outcome of heart infarction in hearts of mice. Second, we have optimized cell culture conditions and cell surface markers to sort hematopoietic progenitors derived from hESCs. We have also begun to transplant these populations into immunodeficient mouse recipients to identify blood-reconstituting hematopoietic populations. Third, we identified 5 commercial and 1 custom mAbs that are specific to human pluripotent cells (hESCs and induced pluripotent cells). We are currently testing the capacity of combinations of 3 pluripotency surface markers to remove all teratoma-initiating cells from transplanted differentiated cell populations. In summary, we expect provide functional validation of the blood and heart precursor populations that we identified from hESCs by the end term of this grant.
Year 3The main objective of our proposal is to isolate therapeutic stem and progenitor cells derived from human embryonic stem cells (hESCs) that can give rise to blood and heart cells. Our approach involves developing differentiation protocols to drive hematopoietic (blood) and cardiac (heart) development of hESCs, then to identify and isolate stem/progenitor cells using monoclonal antibodies (mAbs) specific to surface markers expressed on blood and heart stem/progenitor cells, and finally to characterize their functional properties in vitro and in vivo. In addition, we sought to develop mAbs that specifically bind to undifferentiated hESCs for removal of residual teratoma (tumor)-initiating cells from therapeutic preparations, to ensure transplantation safety.
We have made substantial progress toward achieving these goals. First, we discovered that the initial differentiation of hESCs occurs through only 4-5 different progenitor types, of which one is destined to give rise to heart lineages. We purified this population using four novel cell surface markers (ROR2, PDGFRα, KDR, and CD13), and found a significant enrichment of cardiomyocyte clones in colony formation assays that we developed. This subset also expressed particularly high levels of cardiac genes and was receptive to further differentiation into beating cardiomyocytes or vascular endothelial cells. When transplanted into immunodeficient mice these progenitors differentiated into ventricular myocytes and vascular endothelial cells. We have also successfully developed a human fetal heart xenograft model to test hESC-derived cardiomyocyte stem/progenitor cells in human heart tissue for engraftment and function.
Second, we have optimized cell culture conditions and cell surface markers to sort hematopoietic progenitors derived from hESCs. In doing so, we have mapped the earliest stages of hematopoietic specification and commitment from a bipotent hematoendothelial precursor. Our culture conditions drive robust hematopoietic differentiation in vitro but these hESC-derived hematopoietic progenitors do not achieve hematopoietic engraftment when transplanted in mouse models. Furthermore, we overexpressed the anti-apoptotic protein BCL2 in hESCs, and discovered a significant improvement in viability upon single cell sorting, embryoid body formation, and in cultures lacking serum replacement. Moving forward, we feel the survival advantages exhibited by this BCL2-expressing hESC line will improve our chances of engrafting hESC-derived hematopoietic stem/progenitor cells.
Third, we identified a cocktail of 5 commercial and 1 novel mAbs that enable specific identification of human pluripotent cells (hESCs and induced pluripotent cells). We have found combinations of 3 pluripotency surface markers that can remove all teratoma-initiating cells from differentiated hESC and induced pluripotent stem cell (iPSC) populations prior to transplant. While these combinations can vary depending on the differentiation culture, we have generated a simple, easy-to-follow protocol to remove all teratogenic cells from large-scale differentiation cultures.
In summary, we accomplished most of the goals stated in our original proposal. We successfully achieved cardiac engraftment of an hESC-derived cardiomyocyte progenitor using a novel human heart model of engraftment. While we unfortunately did not attain hematopoietic engraftment of hESC-derived cells, we are exploring a strategy to address this. Our research has led to four manuscripts: one on the protective effects of BCL2 expression on hESC viability and pluripotency (published in PNAS, 2011), another describing markers of pluripotency and their use in depleting teratogenic potential in differentiated PSCs (accepted for publication in Nature Biotechnology), and two submitted manuscripts, one describing a novel xenograft assay to test PSC-derived cardiomyocytes for functional engraftment and the other describing the earliest fate decisions downstream of a PSC.