Improving hematopoietic differentiation from human induced pluripotent stem cells by the modulation of Hippo signaling with a diarylheptanoid derivative.

BACKGROUND: The diarylheptanoid ASPP 049 has improved the quality of adult hematopoietic stem cell (HSC) expansion ex vivo through long-term reconstitution in animal models. However, its effect on hematopoietic regeneration from human induced pluripotent stem cells (hiPSCs) is unknown. METHOD: We utilized a defined cocktail of cytokines without serum or feeder followed by the supplementation of ASPP 049 to produce hematopoietic stem/progenitor cells (HSPCs). Flow cytometry and trypan blue exclusion analysis were used to identify nonadherent and adherent cells. Nonadherent cells were harvested to investigate the effect of ASPP 049 on multipotency using LTC-IC and CFU assays. Subsequently, the mechanism of action was explored through transcriptomic profiles, which were validated by qRT-PCR, immunoblotting, and immunofluorescence analysis. RESULT: The supplementation of ASPP 049 increased the number of phenotypically defined primitive HSPCs (CD34+CD45+CD90+) two-fold relative to seeded hiPSC colonies, indicating enhanced HSC derivation from hiPSCs. Under ASPP 049-supplemented conditions, we observed elevated HSPC niches, including CD144+CD73- hemogenic- and CD144+CD73+ vascular-endothelial progenitors, during HSC differentiation. Moreover, harvested ASPP 049-treated cells exhibited improved self-renewal and a significantly larger proportion of different blood cell colonies with unbiased lineages, indicating enhanced HSC stemness properties. Transcriptomics and KEGG analysis of sorted CD34+CD45+ cells-related mRNA profiles revealed that the Hippo signaling pathway is the most significant in responding to WWTR1/TAZ, which correlates with the validation of the protein expression. Interestingly, ASPP 049-supplemented HSPCs upregulated 11 genes similarly to umbilical cord blood-derived HSPCs. CONCLUSION: These findings suggest that ASPP 049 can improve HSC-generating protocols with proliferative potentials, self-renewal ability, unbiased differentiation, and a definable mechanism of action for the clinical perspective of hematopoietic regenerative medicine.

Iron-loaded transferrin potentiates erythropoietin effects on erythroblast proliferation and survival: a novel role through transferrin receptors.

Red blood cell production, or erythropoiesis, is a proliferative process that requires tight regulation. Erythropoietin (Epo) is a glycoprotein cytokine that plays a major role in erythropoiesis by triggering erythroid progenitors/precursors of varying sensitivity. The concentration of Epo in bone marrow is hypothesized to be suboptimal, and the survival of erythroid cells has been suggested to depend on Epo sensitivity. However, the key factors that control Epo sensitivity remain unknown. Two types of transferrin receptors (TfRs), TfR1 and TfR2, are known to play a role in iron uptake in erythroid cells. Here, we hypothesized that TfRs may additionally modulate Epo sensitivity during erythropoiesis by modulating Epo receptor (EpoR) signaling. Using an Epo-sensitive UT-7 (UT7/Epo) erythroid cell and human erythroid progenitor cell models, we report that iron-loaded transferrin, that is, holo-transferrin (holo-Tf), synergizes with suboptimal Epo levels to improve erythroid cell survival, proliferation, and differentiation. This is accomplished via the major signaling pathways of erythropoiesis, which include signal transducer and activator of transcription 5 (STAT5), mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK), and phosphoinositide-3-kinase (PI3K)/AKT. Furthermore, we found that this cooperation is improved by, but does not require, the internalization of TfR1. Interestingly, we observed that loss of TfR2 stabilizes EpoR levels and abolishes the beneficial effects of holo-Tf. Overall, these data reveal novel signaling properties of TfRs, which involve the regulation of erythropoiesis through EpoR signaling.

Ex vivo expansion and functional activity preservation of adult hematopoietic stem cells by a diarylheptanoid from Curcuma comosa.

Hematopoietic stem cells (HSCs, CD34+ cells) have shown therapeutic efficacy for transplantation in various hematological disorders. However, a large quantity of HSCs is required for transplantation. Therefore, strategies to increase HSC numbers and preserve HSC functions through ex vivo culture are critically required. Here, we report that expansion medium supplemented with ASPP 049, a diarylheptanoid isolated from Curcuma comosa, and a cocktail of cytokines markedly increased numbers of adult CD34+ cells. Interestingly, phenotypically defined primitive HSCs (CD34+CD38-CD90+) were significantly increased under ASPP 049 treatment relative to control. ASPP 049 treatment also improved two functional properties of HSCs, as evidenced by an increased number of CD34+CD38- cells in secondary culture (self-renewal) and the growth of colony-forming units as assessed by colony formation assay (multilineage differentiation). Transplantation of cultured CD34+ cells into immunodeficient mice demonstrated the long-term reconstitution and differentiation ability of ASPP 049-expanded cells. RNA sequencing and KEGG analysis revealed that Hippo signaling was the most likely pathway involved in the effects of ASPP 049. These results suggest that ASPP 049 improved ex vivo expansion and functional preservation of expanded HSCs. Our findings provide a rationale for the use of ASPP 049 to grow HSCs prior to hematological disease treatment.