Restoration of inflammatory and transcriptional axes via temporal TNFα and CDK8/19 modulation enhances hemogenic specification from human iPSCs Free

Generating hematopoietic stem and progenitor cells (HSPCs) from human induced pluripotent stem cells (iPSCs) holds transformative promise for transfusion medicine, immunotherapy, and regenerative transplantation. However, current differentiation protocols remain inefficient, largely due to poor specification of hemogenic endothelial cells (HECs)—a transient, developmentally critical population that gives rise to HSPCs via the endothelial-to-hematopoietic transition (EHT). We hypothesized that the absence of key inflammatory and transcriptional cues during this transition impairs HEC emergence and downstream HSPC formation. ObjectiveTo identify developmental bottlenecks during in vitro hematopoietic differentiation from iPSCs and functionally restore missing extrinsic and intrinsic pathways to enhance definitive HEC and multipotent HSPC production. Methods Definitive hematopoiesis was modeled using a protocol capable of producing transplantable HSPC. We constructed a high-resolution multimodal single-cell atlas (paired scRNA-seq and scATAC-seq) from human iPSC-derived mesoderm, HECs, HSPCs, stromal, and vascular endothelial cell polpulations. These in vitro profiles were systematically compared to publicly available primary human embryonic hematopoietic datasets to identify conserved versus dysregulated signaling and transcriptional factors. Ligand–receptor network inference revealed active and missing pathways across developmental stages. To test causality, we applied time-resolved interventions at the mesoderm-to-HEC transition (Day 4) using either recombinant TNFα or a selective CDK8/19 inhibitor. Hematopoietic differentiation was assessed by flow cytometry (CD34⁺CD43^- at Day 9; CD34⁺CD45⁺ at Day 17) and Colony-Forming Unit (CFU) assays. ResultsComparative signaling analysis uncovered 34 active ligand–receptor interactions in human embryonic tissues, including robust TNFα-NFκB activity during HEC specification. This inflammatory axis was notably absent during in vitro differentiation. Supplementing TNFα during HEC emergence significantly increased RUNX1⁺CD34⁺ HECs (~1.4-fold, p < 0.01) and CD34⁺CD45⁺ HSPCs (~1.5-fold, p < 0.05), without compromising multilineage potential in CFU assays.Multimodal profiling revealed dysregulated CDK8/19 activity in vitro, characterized by altered enhancer accessibility at key hematopoietic loci (RUNX1, GATA2, STAT1, MYC, HIF1A, E2F1) and induction of stress and interferon-response genes. CDK8/19, a core Mediator kinase, regulates lineage transitions through context-dependent transcriptional control. Short-term CDK8/19 inhibition at the mesoderm–HEC juncture rescued epigenetic accessibility at hematopoietic enhancers and significantly improved HEC and HSPC yields (~2-fold, p < 0.01), with preserved multilineage functionality in CFU assays.Notably, the effects of TNFα and CDK8/19 inhibition suggest convergence on a shared bottleneck involving early inflammatory and transcriptional priming, rather than acting through additive mechanisms. ConclusionsThis study reveals a critical developmental failure during in vitro hematopoiesis protocols: the absence of essential extrinsic and intrinsic cues required for robust hemogenic specification. Temporal TNFα signaling and CDK8/19 inhibition serve as independent but convergent interventions that reestablish inflammatory and transcriptional axes necessary for definitive hematopoietic emergence. These mechanistically guided strategies not only enhance HSPC derivation from iPSCs but also establish a translational framework for engineering functional blood lineages relevant to transfusion medicine and regenerative therapy.