Generation of Hematopoietic Stem and Progenitor Cells from iPSCs

National Heart, Lung, And Blood Institute

Investigators

Linked publications, trials & patents

Abstract

Purpose and Scope The overarching objective for this fiscal year was to establish a clinically scalable pipeline for generating engraftable hematopoietic stem cells (HSCs) from patient-derived induced pluripotent stem cells (iPSCs) as a regenerative autologous therapy for Fanconi anemia (FA). This goal encompassed three integrated research components: 1. Optimization of hematopoietic differentiation protocols using healthy-donor human iPSCs. 2. Development of a "correct-then-reprogram" strategy to produce patient-specific FA iPSCs, aiming to derive engraftable HSCs. 3. Translation of differentiation protocols to nonhuman primate (NHP) models for preclinical safety and efficacy assessment. Human iPSC Hematopoietic Differentiation (Healthy-Donor Lines) We benchmarked a three-dimensional embryoid body (EB) differentiation protocol recently validated to achieve sustained multilineage engraftment from iPSC-derived CD34+ cells, conducting direct comparisons against a two-dimensional (2D) monolayer platform known for rapid production of HLF+ HOXA+ hematopoietic progenitors, but previously lacking evidence of durable engraftment. These complementary methods respectively serve as a functional standard for assessing in vivo chimerism and a scalable, cGMP-compatible production platform. NSG xenograft studies using EB-derived cells from the KOLF2.1J line yielded human CD45+ cell engraftment in the bone marrow (~3.4%) at 16 weeks. Concurrently, we optimized input parameters such as initial iPSC cell numbers to enhance differentiation efficiency and cellular viability. These datasets establish robust differentiation protocols, quality control measures, and in vivo assays directly applicable to FA patient-derived iPSCs. FA Patient Gene Correction and iPSC Generation This component aimed to generate corrected FA iPSCs to integrate into the optimized differentiation pipeline described above, enabling the production of engraftable autologous HSCs. Employing a “correct-then-reprogram” strategy, we targeted primary T cells and fibroblasts from a representative FA patient, utilizing CRISPR-Cas9 ribonucleoproteins (RNPs) and single-stranded donor templates to correct FANCA mutations (c.2957delA and c.2316G>C), followed by Sendai virus (SeV)-mediated reprogramming with OCT4, SOX2, KLF4, and MYC (OSKM). The SeV vector avoids genomic integration, providing a transgene-free iPSC generation method. Sanger sequencing analyses showed that allele frequencies of the pathogenic c.2957delA mutation decreased significantly following editing (from ~35% in unedited T cells to <20% and as low as ~2% with certain guides). Despite robust initial colony formation after SeV reprogramming, iPSC-like clones typically failed to stabilize beyond day 15. Fibroblast-derived cells displayed a promising decrease in the mutant allele (29% pre-edit to 11% at day 15), reflecting selective expansion of corrected cells. Given FA cells’ heightened sensitivity to double-strand breaks (DSBs), we are prioritizing FA-adapted culture conditions (hypoxia at 5% O₂, antioxidant supplementation, chromatin remodeling support with sodium butyrate, and transient p53 inhibition via Pifithrin-α). We are currently evaluating potential dominant-negative effects associated with the c.2316G>C allele and plan to transition to base-editing strategies, which avoid nuclease-induced DSBs, as supported by preclinical data. Nonhuman Primate Translational Studies To mitigate risks associated with clinical translation, we established rhesus macaque iPSC lines and initiated EB-based hematopoietic differentiation studies. Successful reprogramming of CD34+ cells from two rhesus macaques (DJ2L, NWi) produced multiple validated pluripotent clones expressing surface markers SSEA-4 and TRA-1-81/TRA-1-60 (>90% positivity). Comparative trials evaluated differentiation efficiency under variable conditions, including Day 0 versus Day −1 initiation, Matrigel support, and media selection (PluriStem vs. MACS Brew). Day 0 initiation consistently yielded superior CD34+CD45+CD90+ populations, and MACS Brew demonstrated enhanced differentiation efficiency over PluriStem. Initial xenografts into NSG mice provided preliminary evidence of nonhuman primate CD45+ cell presence in peripheral blood at four weeks post-transplant. Protocol refinements included adjustments to EB swirling speed (70 rpm to 67 rpm) to optimize EB sizing, consistent with human differentiation standards, and evaluation of alternative ROCK inhibitors. Further differentiation is temporarily paused to perform qPCR-based molecular profiling at multiple stages of differentiation (days 3, 7, 9, and 11) to better inform future optimization. Key Methods and Datasets Across project components, we employed flow cytometry-based immunophenotyping (markers: CD34, CD43, CD45, CD90, CD45RA, CD144, CD31, DLL4), xenotransplantation in immunodeficient mouse models, CRISPR-Cas9 genome editing, SeV-mediated reprogramming, optimized hypoxia-based culture conditions, and quantitative PCR-based molecular analyses. The validated EB-based multilineage engraftment protocol and monolayer protocols enriched for HLF+ HOXA+ progenitors provide foundational frameworks for further optimization. Limitations and Next Steps Primary limitations include intrinsic FA cellular fragility during reprogramming and intolerance to DSBs during genome editing. Immediate future directions encompass: 1. Finalizing the transition of EB-based differentiation cues into scalable 2D monolayer formats to achieve durable HSC engraftment. 2. Completing genotyping and stabilizing gene-corrected FA iPSC clones under optimized hypoxia, antioxidant, and p53-modulated conditions. 3. Enhancing NHP differentiation protocols through precise EB size control, improved late-stage cell survival, and molecular trajectory mapping via qPCR. Collectively, these steps bridge patient-specific gene correction efforts with established differentiation protocols and preclinical in vivo validation, laying the groundwork for clinical translation in FA and related inherited bone marrow failure syndromes.