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

• Published in: Stem cell research & therapy Vol. 15; no. 1; p. 60
• Main Authors: Thongsa-Ad, Umnuaychoke, Wongpan, Anongnat, Wongkummool, Wasinee, Chaiwijit, Phaewa, Uppakara, Kwanchanok, Chaiyakitpattana, Gorawin, Singpant, Passanan, Tong-Ngam, Pirut, Chukhan, Amnat, Pabuprappap, Wachirachai, Wongniam, Sirapope, Suksamrarn, Apichart, Hongeng, Suradej, Anurathapan, Usanarat, Kulkeaw, Kasem, Tubsuwan, Alisa, Bhukhai, Kanit
• Format: Journal Article
• Language: English
• Published: England BioMed Central Ltd 03-03-2024; BioMed Central; BMC
• Subjects: Adherent cells; Analysis; Animal models; Antibodies; CD34 antigen; CD45 antigen; CD73 antigen; Cell differentiation; Cell self-renewal; Cord blood; Cytokines; Diagnostic reagents industry; Diarylheptanoid; Endothelium; Ethylenediaminetetraacetic acid; Flow cytometry; Gene expression; Hematopoietic stem cells; Hemopoiesis; Hippo signaling pathway; Human induced pluripotent stem cells; Humidity; Immunoblotting; Immunofluorescence; Kinases; Long-term care of the sick; Pluripotency; Primitive hematopoietic stem and progenitor cells; Progenitor cells; Regenerative medicine; RNA; Signal transduction; Stem cells; Transcriptomics; Umbilical cord; Canada; Human induced pluripotent stem cells; Primitive hematopoietic stem and progenitor cells; Diarylheptanoid; Hippo signaling pathway
• ISSN: 1757-6512; 1757-6512
• DOI: 10.1186/s13287-024-03686-4

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.
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.
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.
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.