1. Objective 3.1: Understanding the ontogeny of definitive hematopoiesis During vertebrate embryonic development, multiple waves of hematopoiesis take place and are defined as either primitive or definitive. Only the intra-embryonic definitive wave provides hematopoietic stem cells (HSCs) with long-term repopulating capacity. However, most in-vitro differentiation systems developed to generate blood have successfully replicated the primitive wave, but none have been able to produce long-term repopulating HSCs. To further understand the ontogeny of definitive hematopoiesis, we have initiated basic developmental studies in collaboration with Dr. Catherine Porcher (Oxford University). Using a published in vitro differentiation system, we have identified a population of hemogenic endothelium (the precursors of HSCs). We showed that these cells do not express markers of arterial, venous, or lymphatic identity, suggesting a very early, uncommitted hemogenic endothelial population, similar to what has been reported in the primitive wave of hematopoiesis. Because hematopoietic stem cells arise within an arterial niche, and arterial endothelium contains signaling molecules important for hematopoietic and endothelial development (VEGF, Notch), we hypothesized that intra-embryonic hemogenic endothelium was different to yolk sac hemogenic endothelium due to its arterial identity. A recent publication at the single-cell level confirms that early hematopoietic progenitors in the dorsal aorta maintain the expression of arterial genes. We therefore focused on developing conditions to differentiate cells towards an arterial niche. We found that treating the cells with high levels of VEGFA led to a block in Runx1 activation in Flk-1+ hemogenic endothelium at Day 5.5 and therefore an abrogation of hematopoiesis. We also saw an increase in Dll4 ligand, an arterial marker that leads to downstream Notch signaling and further arterial differentiation. We found that the block in Runx1 activation was Notch independent. By assaying a panel of genes for arterial identity and specification, we were able to show that cells begin to acquire a more fully developed arterial program after two to three days after being replated. In FY17, we will continue developing optimal culture conditions for promoting endothelial differentiation into arterial endothelium, and for inducing the arterial endothelium towards activation of Runx1 and differentiation into definitive HSCs. Given the important role that hypoxia plays in the arterial specification of cells, as well as its critical role in the maintenance of the stemness of hematopoietic stem cells in the bone marrow, we will explore the role of hypoxia in the development of arterial endothelium and in the regulation of hematopoietic stem cell development as these cells arise in-vitro. Other signaling factors will be investigated, including TGF-beta, Wnt, BMP, and cAMP. 2. Objective 3.2: Development of a culture system for hematopoietic differentiation of normal human iPSCs We have established a novel system for de novo generation of easily accessible suspension human hematopoietic cells (CD45+CD34+) from iPSCs. Up to 60% of iPSC-differentiated cells have a CD45+CD34+ phenotype. These cells form colonies in clonogenic progenitor assays, albeit at reduced capacity compared to primary CD34+ cells. However, they failed to home to the bone marrow of immuno-deficient (NSG) animals and did not result in long-term engraftment after transplantation. To understand differences in engraftment potential between bona fide HSCs and iPSC-derived HSCs, we have conducted single cell RNA Seq experiments comparing both cell populations. Bioinformatic comparative expression analysis is underway to pinpoint genes or pathways that may be deregulated in iPSC-derived hematopoietic cells. 3. Objective 3.3: Differentiation of genetically corrected iPSCs derived from patients with inherited bone marrow failure syndromes into transplantable HSCs We have obtained original and genetically corrected iPSC lines derived from individuals with inherited bone marrow failure syndromes (Fanconi Anemia and Diamond-Blackfan Anemia) from the laboratories of Dr. Juan Carlos Izpisua-Belmonte (Salk Institute) and Dr. MJ Weiss (St. Jude Childrens Research Hospital), respectively. In FY17, culture conditions for optimal growth of these lines are will be optimized and the differentiation protocol developed for normal iPSCs will be evaluated for hematopoietic differentiation of patient-derived iPSCs.
| Townsley, Danielle M; Scheinberg, Phillip; Winkler, Thomas et al. (2017) Eltrombopag Added to Standard Immunosuppression for Aplastic Anemia. N Engl J Med 376:1540-1550 |
| Ito, Sawa; Barrett, A John; Dutra, Amalia et al. (2015) Long term maintenance of myeloid leukemic stem cells cultured with unrelated human mesenchymal stromal cells. Stem Cell Res 14:95-104 |
| Glait-Santar, Chen; Desmond, Ronan; Feng, Xingmin et al. (2015) Functional Niche Competition Between Normal Hematopoietic Stem and Progenitor Cells and Myeloid Leukemia Cells. Stem Cells 33:3635-42 |
