Ojective 1: Develop approaches for expansion of hematopoietic stem cells (HSCs) Attempts to improve hematopoietic reconstitution and engraftment potential of ex vivoexpanded hematopoietic stem and progenitor cells (HSPCs) have been largely unsuccessful due to the inability to generate sufficient stem cell numbers and to excessive differentiation of the starting cell population. Experience from in vitro studies indicates that control of HSPC self-renewal and differentiation in culture remains difficult. Protocols that are based on hematopoietic cytokines (e.g. thrombopoietin TPO, stem cell factor SCF) have failed to support reliable amplification of immature stem cells in culture, suggesting that alternative cytokines or additional factors are required and highly desirable. ELTROMBOPAG. Eltrombopag is a novel TPO agonist with an intrinsic ability to expand HSPC in vivo given observed clinical benefits in patients with aplastic anemia. We have cultured human CD34+ cells for up to 21 days in our standard culture medium supplemented with Eltrombopag or with the combination Eltrombopag + TPO. We found that Eltrombopag was not superior to TPO for HSPC expansion in vitro. However, the combination TPO + Eltrombopag favored expansion of total CD34+ cells, platelets and myeloid progenitors better than TPO or Eltrombopag alone. Transplantation of these expanded cells in immune-deficient mice, the gold standard for evaluation of the in vivo repopulating potential of these cells, will determine the clinical utility of this drug for in vitro expansion of HSCs. NOTCH PATHWAY AND HYPOXIA. The primary mediators of hypoxic adaptation are hypoxia-inducible factors (HIF), a family of transcription factors composed of two subunits, an oxygen-labile subunit that rapidly stabilizes in response to low O2 tensions (HIF-1 and HIF-2), and a subunit (HIF-1) that is constitutively expressed. In immunoprecipitation assays, HIF-1 physically interacted with the intracellular domain of Notch, a critical component for the maintenance of undifferentiated stem and progenitor cell populations, providing a striking molecular link between hypoxia and stemness. Given this recent evidence of a convergence of pathways involved in hypoxia sensing and stem cell maintenance, we are investigating the possibility of stem cell expansion under hypoxic conditions by activation of the canonical Notch signaling pathway using Delta-1 ligand. We showed that culture of human CD34+ cells for 21 days in the presence of Delta-1 ligand under hypoxic conditions maintains the CD34+ phenotype in 25% of cultured cells compared to <1% in the absence of Delta-1 ligand. Transplantation of these expanded cells in immune-deficient mice will determine the clinical utility of this approach for in vitro expansion of HSCs. NOVEL PATHWAYS FOR HSC EXPANSION. During homeostasis the HSC pool is maintained at a relatively constant level. In contrast, several murine studies have shown that during hematopoietic stress, such as transplantations, HSCs can and will self-renew extensively, suggesting that HSCs are exposed in vivo to specific factors/signals that promote their self-renewal and amplification. We have transplanted human CD34+ cells into immune-deficient mice and showed evidence of extensive self-renewal in vivo. To identify novel factors/pathways involved in HSC expansion, we are comparing gene expression/methylation patterns between HSC at steady state (before transplant) and after transplant, using RNA-Seq, CHIP-Seq and metabolomics approaches. These studies will have a significant impact on the global understanding of human HSC self-renewal and could lead to the development of novel approaches for HSC expansion in vitro. Objective 2: Develop approaches for differentiation of iPSCs into HSCs With the development of induced pluripotent stem cell (iPSC) technologies emerged the concept of generating iPSCs from an individual patient, correcting the genetic defect using gene specific targeting for safe integration of the therapeutic transgenes, and differentiating the disease-free iPSCs into transplantable HSCs. The hematopoietic system serves as a perfect opportunity to cure diseases using these types of approaches. Decades of HSCT performed in the clinic provide a roadmap for clinical use and the fact that the hematopoietic system constantly regenerates itself provides an opportunity to replenish a patients blood system with new or corrected cells. The proof-of-principle of using iPSC technologies to cure hematopoietic disorders attributed to a genetic defect has been performed in a humanized sickle-cell anemia mouse model. Proof-of-concept was also demonstrated in human cells by generating iPSCs from patients with various hematologic disorders and correcting them to generate disease-free hematopoietic cells. These studies indicate that iPSC technologies could provide a novel long-awaited treatment option for patients with life-threatening bone marrow failure syndromes. Currently used protocols for iPSC differentiation into HSCs can generally be divided into two main categories: those that co-culture stem cells with stromal layers (e.g. OP9), and those that culture stem cells in suspension to form embryoid bodies (EBs). However, these protocols remain inefficient at producing the quantity and quality of HSCs required for clinical applications. On the basis of recent data demonstrating that definitive HSCs are generated from a unique population of endothelial cells known as hemogenic endothelium (HE), we have established a novel system for de novo generation of transplantable HSCs from iPSCs. Human iPSCs derived from normal individuals are cultured in the presence of a cytokine combination that favors development of an adherent layer of HE in vitro. Over a period of 12-14 days, these cells further differentiate to produce and release cells in suspension. Up to 70% of these cells have a CD45+CD34+ phenotype compared to 10-15% CD45+CD34+ using current co-culture or EB-based protocols. We have characterized these cells further and demonstrated a subpopulation with the most defined HSC phenotype described (CD34+CD38-CD45RA-CD90+CD49f+Rholo). The ability of these cells for establish hematopoiesis in vivo in underway. Future work will focus on the application of this technology to patients with life-threatening bone marrow failure syndromes.

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