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.

The Effects of PP2A Disruption on ER-Mitochondria Contact and Mitochondrial Functions in Neuronal-like Cells.

Mitochondria-associated membranes (MAMs) regulate several cellular processes, including calcium homeostasis and mitochondrial function, and dynamics. While MAMs are upregulated in Alzheimer's disease (AD), the mechanisms underlying this increase remain unknown. A possible mechanism may include dysregulation of protein phosphatase 2A (PP2A), which is reduced in the AD brain. Furthermore, PP2A has been previously reported to modulate MAM formation in hepatocytes. However, it is unknown whether PP2A and MAMs are linked in neuronal cells. Here, to test the correlation between PP2A and MAMs, we inhibited the activity of PP2A to mimic its low levels in AD brains and observed MAM formation, function, and dynamics. MAMs were significantly increased after PP2A inhibition, which correlated with elevated mitochondrial Ca2+ influx and disrupted mitochondrial membrane potential and mitochondrial fission. This study highlights the essential role PP2A plays in regulating MAM formation and mitochondrial function and dynamics for the first time in neuronal-like cells.