Generation of Hematopoietic Stem and Progenitor Cells from Human iPSCs

National Heart, Lung, And Blood Institute

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Abstract

Objective 1: Develop a clinically-relevant culture system for hematopoietic differentiation of human iPSCs 1.1 Culture system development and characterization 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 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. Within the supernatant. hematopoietic cells (CD43+CD45+/-) underwent sequential development 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. To understand the possible causes underpinning the absence of engraftable HSCs in this system, we examined the cellular constituents of the supportive non-hematopoietic (CD43-CD45-) niche. We identified a prevalent population of mesenchymal cells throughout differentiation, but arterial hemogenic endothelium (HE), a distinct subset of vascular endothelium (VE) from which HSCs are specified during development, was largely absent within the supportive monolayer. These observations may account for the lack of engrafting HSCs in culture. This work was published Stem Cell Research 41: 101600 (2019). 1.2 Promoting HE arterial specification during iPSC differentiation To promote arterial identity of HE, we supplemented our monolayer culture system with CHIR99021 (CHIR) and SB431542 (SB) at day 2-3 of iPSC differentiation to modulate activin/nodal/TGF and WNT/-catenin pathways, respectively, and added Ly294002 (LY) from day 3 to day 6 of iPSC differentiation to promote MAPK/ERK signaling pathways. Control cultures contained no CHIR/SB/LY (non-treated), or were supplemented with CHIR/SB or LY only. Addition of CHIR/SB/LY led to a marked increase in percentages and numbers of CD144+CD34hiCD73midCD184+ arterial VE, peaking at day 5 of differentiation. Addition of CHIR/SB/LY decreased overall CD43+/-CD45+ hematopoietic cell numbers but a notable rise in percentages of phenotypically defined definitive HSCs was observed within the hematopoietic population at day 12 of differentiation compared to controls. In CFU assays, the frequency of progenitors with multilineage differentiation capacity significantly increased in the CHIR/SB/LY group. Notably, we observed a 3-fold increase in total CFU numbers from CD34+ cells derived from CHIR/SB/LY cultures compared to control groups in CFU replating assays. Collectively, our results indicate that combined addition of CHIR/SB and LY294002 during human iPSC hematopoietic differentiation enhances formation of arterial VE and phenotypically defined definitive HSCs with self-renewal and multilineage differentiation capacity. This work was submitted to the ASH 2021 Annual Meeting. 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 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 were transplanted into immunodeficient animals before or after ex vivo differentiation to evaluate long-term engraftment potential. Results are pending. 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.