Objective 1: Fibroblast- or mononuclear cell (MNC)-differentiated erythroblasts from DBA and FA patient have been successfully reprogrammed in collaboration with the NHLBI iPSC core facility. Reprogramming FA cells has proven to be challenging and efforts in the past year have centered on enhancing efficiency of reprogramming of these cells. Progressive loss of HSPCs in FA patients has been attributed in part to a heightened p53/p21 axis that restricts the cell cycle to G0/G1 in response to accumulation of unresolved DNA damage and replicative stress. Silencing of p53 was previously shown to rescue HSPC defects in FA models in vitro and in vivo. A recent study reported low-efficiency generation of integration-free FA-iPSCs by electroporation of fibroblasts with episomal plasmid vectors expressing both reprogramming factors and p53 shRNA. Here we sought to develop an improved and streamlined reprogramming strategy for FA by transduction of readily accessible FA MNCs with commercially available non-integrating reprogramming Sendai Viruses (SeV2), combined with a more adaptable inhibition of the p53 pathway by addition of variable concentrations of a small molecule during reprogramming. Approximately 100,000 MNCs from an FA patient with confirmed biallelic mutations in FANCA were cultured for 10 days under hypoxic conditions (5% O2) favoring erythroid expansion. Erythroid-expanded FA cells were transduced with SeV2 harboring four standard reprogramming transcription factors (SOX2, OCT4, KLF4 and c-MYC). Cells were subsequently cultured under hypoxia for at least 30 days in a reprogramming cocktail supplemented or not with 0.3uM, 0.6M or 1.2M cyclic pifthrin- (cPFT), a reversible small molecule inhibitor of p53-dependent gene transcription. In the absence of cPFT, no FA-iPSC clones were obtained, consistent with reported reprogramming barriers in FA cells. In contrast, several iPSC colonies were generated with cPFT-treated FA cells, albeit at much slower kinetics (30 days) compared to MNCs without FA mutations (20 days). The overall efficiency of reprogramming was 0.01%, with a trend toward enhanced efficiencies in cultures containing the highest concentration of cPFT (1.2M). To investigate the need for p53 inhibition beyond the reprogramming period, half of the FA-iPSC clones were passaged and further cultured in 5% O2 with cPFT 1.2M, whereas the remaining clones were similarly treated but without p53 inhibitor. None of the FA-iPSC clones that were maintained in cPFT-containing cultures following reprogramming survived further passaging. In contrast, when cPFT was removed after reprogramming, all iPSC clones could stably undergo repeated passages provided that hypoxia (5% O2) was maintained. Overall, this work provides a practical approach for successful reprogramming of FA peripheral blood cells based on commercially available non-integrating SeV2 reprogramming strategies supplemented with p53 inhibition. Objective 2: All selected DBA and FA patients have a known mutation and include single DNA base pair changes, small or large deletions. Novel CRISPR/Cas9-based genome editing approaches are used for genetic correction. Two general homology-directed repair (HDR) approaches are investigated, including targeted gene addition at the endogenous gene locus and correction of point mutations or deletions. With current approaches, only low levels (<1%) of targeted integration via HDR have been reported in human iPSCs, necessitating a time-consuming selection process to identify the few corrected iPSCs clones. Moreover, targeted gene addition and correction of larger mutations (>50bp) rely on electroporation of bulky exogenous homologous DNA donor template which results in pronounced cytotoxicity to iPSCs. Therefore, new strategies to advance the current state of CRISPR/Cas9-mediated targeted gene delivery are being explored. Specifically, delivery of gRNA, Cas9 nuclease and DNA templates for HDR using adeno-associated virus (AAV) are investigated. Multiple novel AAV serotypes have been generated in our laboratory and are investigated for optimization of transduction of human iPSCs and efficiency of genome editing. Objective 3: Efficient and clinically relevant methodologies to derive transplantable autologous HSPCs from human iPSCs ex vivo remain unavailable. To facilitate the development of functional HSPCs from human iPSCs, we previously developed a simple, monolayer-based, chemically-defined, and scalable differentiation protocol requiring no replating or embryoid body (EB) formation (commercially available as STEMdiffTM Hematopoietic Kit, Stem Cell Technologies). During the first 3 days, mesodermal specification is induced using morphogens (bFGF, BMP4, VEGF 10ng/mL) and, for the subsequent 18 days, cells are further differentiated into HSPCs with the addition of hematopoietic cytokines. As previously described by our group, this differentiation system recapitulates the successive waves of hematopoiesis during development and leads to robust production of immunophenotypic HSC-like cells (CD34+CD38-CD90+CD45RA-CD49f+). However, these cells do not result in efficient, long-term engraftment in immunodeficient (NSG) mouse models. To identify possible causes for the lack of durable repopulating potential of iPSC-derived HSPCs in this system, we characterized the supportive monolayer from which HSPCs arise during vitro differentiation. Observations in the developing embryo indicate that definitive HSPCs arise in the dorsal aorta from hemogenic endothelium (HE) in close association with an arterial vascular endothelial niche. The Notch pathway plays a key role in arterial and HSC differentiation; in the embryo, only HE populations found in arterial regions with active Notch signaling through Delta-like ligand 4 (Dll4) and Jagged 1 (Jag1) lead to HSPCs with repopulating potential. Recent studies have also shown that modulation of mesodermal patterning through repression and activation of Activin/Nodal and Wnt/-catenin pathways, respectively, promotes arterial programs and definitive hematopoiesis. In contrast to observations in embryos, we found that the supportive monolayer in the STEMdiffTM in vitro differentiation system has limited percentages of arterial HE (CD43-CD45-CD34hiCD144+CD73-Dll4+) and arterial endothelium (CD43-CD45-CD34hiCD144+CD73midCD184+), and overabundance of stromal cells (CD43-CD45-CD34-CD144-). This provides a possible explanation for the lack of engraftment potential of iPSC-derived HSPCs in this system. To restrict stromal development and further promote differentiation and maintenance of a supportive arterial endothelial niche, we modified the standard differentiation protocol by addition of CHIR99021 (CHIR) and SB431542 (SB) during the mesodermal stage of differentiation (days 2-3) to activate Wnt/-catenin and block of Activin/Nodal signaling, respectively. Given that VEGF acts upstream of the Notch pathway during arterial endothelial differentiation, we also increased the concentration of VEGFA 20-fold throughout differentiation (200ng/mL). Our results showed that mesodermal patterning alone was insufficient to repress stromal production and maintain an endothelial niche. However, increased VEGF concentrations, alone or in combination with CHIR/SB, markedly reduced stromal differentiation and enhanced arterial endothelium formation compared to the standard system. Importantly, combination treatments also led to significantly higher percentages of arterial HE at days 5 and 7. Current assessment of these treatments on the hematopoietic potential of the system is ongoing, and include NSG mouse transplantations. Overall, our data indicate that commercially available technologies can be further modified and improved to move closer to chemically-defined and scalable HSPC differentiation protocols.
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