The cellular organization and relationships among precursors that initiate embryonic angiogenesis and hematopoiesis in the human have yet to be characterized. Here, we identify a subpopulation of primitive endothelial-like cells derived from human embryonic stem cells (hESCs) that express PECAM-1, Flk-1, and VE-cadherin, but not CD45 (CD45negPFV cells), and that are uniquely responsible for endothelial and hematopoietic development. Molecular profiling of CD45negPFV cells is consistent with endothelial and hematopoietic competency. Clonal isolation demonstrates that the CD45negPFV population includes bipotent cells with endothelial and hematopoietic capacity. We suggest that human hematopoiesis and endothelial maturation originate exclusively from a subset of embryonic endothelium that possesses hemangioblastic properties and offers a model system to study these lineage relationships in the human.

Despite the need for alternative sources of human hematopoietic stem cells (HSCs), the functional capacity of hematopoietic cells generated from human embryonic stem cells (hESCs) has yet to be evaluated and compared with adult sources. Here, we report that somatic and hESC-derived hematopoietic cells have similar phenotype and in vitro clonogenic progenitor activity. However, in contrast with somatic cells, hESC-derived hematopoietic cells failed to reconstitute intravenously transplanted recipient mice because of cellular aggregation causing fatal emboli formation. Direct femoral injection allowed recipient survival and resulted in multilineage hematopoietic repopulation, providing direct evidence of HSC function. However, hESC-derived HSCs had limited proliferative and migratory capacity compared with somatic HSCs that correlated with a distinct gene expression pattern of hESC-derived hematopoietic cells that included homeobox (HOX) A and B gene clusters. Ectopic expression of HOXB4 had no effect on repopulating capacity of hESC-derived cells. We suggest that limitations in the ability of hESC-derived HSCs to activate a molecular program similar to somatic HSCs may contribute to their atypical in vivo behavior. Our study demonstrates that HSCs can be derived from hESCs and provides an in vivo system and molecular foundation to evaluate strategies for the generation of clinically transplantable HSC from hESC lines.

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Transplanted murine bone marrow (BM) progenitor cells recruit to the injured pancreas and induce endogenous beta cell proliferation to improve islet function. To enrich for analogous human progenitor cell types that stimulate islet regeneration, we purified human BM based on high-aldehyde dehydrogenase activity (ALDH(hi)), an enzymatic function conserved in hematopoietic, endothelial, and mesenchymal progenitor lineages. We investigated the contributions of ALDH(hi) mixed progenitor cells or culture-expanded, ALDH-purified multipotent stromal cell (MSC) subsets to activate endogenous programs for islet regeneration after transplantation into streptozotocin-treated NOD/SCID mice. Intravenous injection of uncultured BM ALDH(hi) cells improved systemic hyperglycemia and augmented insulin secretion by increasing islet size and vascularization, without increasing total islet number. Augmented proliferation within regenerated endogenous islets and associated vascular endothelium indicated the induction of islet-specific proliferative and pro-angiogenic programs. Although cultured MSC from independent human BM samples showed variable capacity to improve islet function, and prolonged expansion diminished hyperglycemic recovery, transplantation of ALDH-purified regenerative MSC reduced hyperglycemia and augmented total beta cell mass by stimulating the formation of small beta cell clusters associated with the ductal epithelium, without evidence of increased islet vascularization or Ngn3(+) endocrine precursor activation. Thus, endogenous islet recovery after progenitor cell transplantation can occur via distinct regenerative mechanisms modulated by subtypes of progenitor cells administered. Further, understanding of how these islet regenerative and pro-angiogenic programs are activated by specific progenitor subsets may provide new approaches for combination cellular therapies to combat diabetes.

The molecular basis governing functional behavior of human hematopoietic stem cells (HSCs) is largely unknown. Here, using in vitro and in vivo assays, we isolate and define progenitors versus repopulating HSCs from multiple stages of human development for global gene expression profiling. Accounting for both the hierarchical relationship between repopulating cells and their progenitors, and the enhanced HSC function unique to early stages of ontogeny, the human homologs of Hairy Enhancer of Split-1 (HES-1) and Hepatocyte Leukemia Factor (HLF) were identified as candidate regulators of HSCs. Transgenic human hematopoietic cells expressing HES-1 or HLF demonstrated enhanced in vivo reconstitution ability that correlated to increased cycling frequency and inhibition of apoptosis, respectively. Our report identifies regulatory factors involved in HSC function that elicit their effect through independent systems, suggesting that a unique orchestration of pathways fundamental to all human cells is capable of controlling stem cell behavior.