Fibroblast-Derived Human iPSC Exhibits Superior Haematopoietic Potential over Human ESC during Haematopoietic Differentiation

Haematopoietic stem cells (HSC) and macrophages hold promise for cell-based therapy. Induced pluripotent stem cells (iPSC) offer an alternative to human embryonic stem cells (hESC) for generating haematopoietic cells in vitro, sidestepping ethical concerns. However, precise comparisons of the developmental process and productivity between iPSC and hESC during haematopoietic differentiation are limited, and producing sufficient HSC for clinical use remains challenging. We introduce a refined, simplified protocol that is xeno-, serum-, and feeder-free for differentiating fibroblast-derived human iPSC (NHDF-iPSC) and the hESC-H9 clone (H9-ESC) using the STEMdiff™ Hematopoietic kit, with differentiation extended by in-house cytokine addition. We demonstrate that NHDF-iPSC recapitulate the haematopoietic differentiation of H9-ESC, forming CD31+CD144+CD34+ haemogenic endothelia (HE) as intermediates, and producing CD34+CD43+CD45+/- haematopoietic stem and progenitor cells (HSPC). This protocol facilitates the production of CD34+ HSPC over an extended period and enhances the yield of HSC from NHDF-iPSC-derived HE three-fold. Interestingly, our results demonstrated that NHDF-iPSC outperformed H9-ESC by exhibiting superior differentiation capabilities, resulting in a higher abundance of HE and greater haematopoietic cell output (e.g., HSPC and HSC) upon cytokine stimulation. This phenomenon is presumably due to the higher expression of RUNX1 in NHDF-iPSC-derived HE (three-fold) as observed in our study, which may lead to a more productive endothelial-to-haematopoietic transition process and potentially facilitate the efficient production of haematopoietic cells. These CD34+ haematopoietic cells mature into 25F9+CD45+ macrophages, which exhibit comparable functions to those derived from hESC. Together, our results underscore the potential of iPSCs as a sustainable source for deriving HSC and macrophages for cell-based therapies.