Generation of Hematopoietic Stem and Progenitor Cells from Human iPSCs

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Objective 1: Develop a robust, scalable and clinically-relevant culture system for hematopoietic differentiation of human iPSCs To facilitate the development of functional hematopoietic stem cells (HSCs) fom human iPSCs, we 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). Human iPSCs reprogrammed from CD34+CD38- cells of a healthy volunteer (MCND-TENS2) were subjected to hematopoietic differentiation for 21 days using this approach. Under culture conditions that favored mesodermal specification (Day 0 to 3), an adherent monolayer rapidly formed. With the subsequent addition of hematopoietic cytokines (Day 3 to 21), hematopoietic clusters emerged from the monolayer before their eventual release in the supernatant fraction. We systematically characterized cells arising from this system by harvesting supernatant and monolayer populations at regular intervals between day 5 and 21 of differentiation. Hematopoietic cells were characterized by varying expression of CD43, the earliest marker of human hematopoietic commitment, and by gradual acquisition of the pan-hematopoietic marker CD45 (CD43+CD45+/-). In contrast, non-hematopoietic cells formed a distinct population expressing neither markers (CD43-CD45-). 1.1 Characterization of the hematopoietic fraction Hematopoietic differentiation of human iPSCs with STEMdiffTM enables the sequential development of hematopoietic cells with features of primitive wave one-hematopoiesis (peak at day 7), definitive multilineage HSPCs with potent colony formation activity in vitro but limited engraftment potential in vivo (peak at day 12), and definitive erythroid-committed progenitors expressing adult-type globin chains (peak at day 17 to 21). This nearly exclusive shift to definitive erythroid growth in later stages of differentiation could be exploited to facilitate erythroid differentiation of iPSCs established from patients with hemoglobinopathies or various congenital bone marrow failures and anemias affecting early erythropoiesis. 1.2 Characterization of the non-hematopoietic fraction To understand the possible causes underpinning the absence of engraftable HSCs in this system, we examined the cellular constituents of the supportive non-hematopoietic niche. We first identified a prevalent population of phenotypically defined mesenchymal cells throughout differentiation. Further investigation is needed to fully understand whether these cells may offer inhibitory signals that preclude normal developmental switch to third wave definitive hematopoiesis during iPSC differentiation. We also found limited vascular endothelium (VE) production and arterial specification within the non-hematopoietic fraction that may account for the lack of engrafting HSCs in culture. Indeed, recent advances propose that formation of these cells is restricted to arterial vessels during ontogeny. 1.3 Impact of CHIR/SB of hematopoietic and non-hematopoietic development Our study also provides proof-of-principle that this monolayer iPSC differentiation system is readily amenable to simple, clinically applicable modifications to improve hematopoietic output. Quantitative modulation of WNT/-catenin and activin/nodal/TGF signaling pathways by one-time addition of CHIR/SB molecules during mesodermal specification enhanced arterial VE and increased HOXA gene expression and definitive HSC formation. However, consistent with previous studies, this approach alone was insufficient to orchestrate the formation of functional HSCs and additional revisions to this system will be required. This work was published Stem Cell Research (2019). Objective 2: Uncover human HSC-specific superenhancers (SE) and SE-associated genes and pioneer transcription factors for the conversion of iPSCs and somatic cells into functional HSCs A two-pronged approach was developed in FY20. First, we identified HSC-specific super-enhancers (SE), and SE-associated genes and pioneer transcription factors (TF). Second, we are applying data from the identification of HSC-specific SE for the production of functional HSCs by: a) Ectopic expression of pioneer TFs in iPSCs or somatic cells; b) Activating SE and their associated genes with CRISPRa. 2.1 Identification of HSC-specific SE, and SE-associated genes and pioneer TF A total of 873 HSC-specific SE were identified by bioinformatic search for clusters of the enhancer-associated surrogate epigenetic mark, H3K27Ac, derived from CHIP-seq analyses of HSC-enriched CD34+CD38- cells obtained from three healthy individuals. As observed in other cell types, SE in HSCs were found to represent <5% of total enhancers and span genomic regions 10-fold larger than typical enhancers. Because most genes regulated by SE are expected to reside within 50 kb of the SE, we applied a bioinformatic proximity analysis and a cutoff of 50 kb to identify a list of candidate SE-associated genes. Furthermore, because actively transcribed gene are within open chromatin regions, we performed ATAC-seq in CD34+CD38- cells to identify SE-associated genes with an active promoter within detectable ATAC-seq peaks. A total of 594 SE-associated genes were identified. By interrogating published RNA-seq expression datasets derived from human CD34+CD38- cells, we observed a marked increase in median expression of these genes compared to all genes expressed in CD34+CD38- cells. To further validate our data, we performed an unbiased GO analysis of all SE-associated genes. Notably, 13 pathways were identified and all but one were directly related to the regulation of hematopoietic processes. Globally, these data suggest that the curated list of 873 SE identified in our study has biological relevance in human HSCs. Because of their role in controlling cell fate conversion via reprogramming of chromatin and gene regulatory networks, pioneer TFs regulating HSCs could be key to facilitate the production of ex vivo. these key TFs. A relationship between pioneer TF and superenhancers was first demonstrated in mouse ESCs but this notion has not yet been applied to somatic HSCs. We used a 3-step approach: 1) Identification of TF associated with SE using a proximity approach; 2) Identification of TF binding to their associated SE by analysis of TF binding motif sequence enrichment within the SE; 3) Convergence of both group of TF. A total of 34 HSC-associated pioneer TF were identified. 2.2 Conversion of iPSCs and somatic cells into functional HSCs Two approaches are being evaluated for the production of engraftable HSCs from iPSCs or somatic cells. First, a library of 34 pioneer TF were independently cloned into doxycycline-inducible lentiviral vectors for ectopic enforced expression in iPSCs or somatic cells. Transduced cells will be transplanted into immunodeficient animals before or after ex vivo differentiation to evaluate long-term engraftment potential. Pioneer TF that contributed to the generation of engraftable HSCs will be identified by a PCR screening approach and used in a second round of transduction to validate their role in the generation of engraftable HSCs from iPSC or somatic cells. Second, we are evaluating CRISPRa technology for the targeted activation of HSC-specific genes driven by SE. A five-step approach is ongoing: 1) Construction of a 10,000 gRNA lentiviral vector library targeting the 873 SE identified in HSCs; 2) Establishment of a doxycycline-inducible dCas9-p300 iPSC line; 3) Transduction of a validated CRISPRa iPSC clone with sgRNA lentiviral library; 4) Hematopoietic differentiation in the presence of doxycycline; 5) Transplantation of differentiated cells to assess potential to engraft long-term.