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Coinhibition of activated p38 MAPKα and mTORC1 potentiates stemness maintenance of HSCs from SR1‐expanded human cord blood CD34 + cells via inhibition of senescence

Abstract The stemness of ex vivo expanded hematopoietic stem cells (HSCs) is usually compromised by current methods. To explore the failure mechanism of stemness maintenance of human HSCs, which were expanded from human umbilical cord blood (hUCB) CD34+ cells, by differentiation inhibitor Stem Regenin 1 (SR1), an antagonist of aryl hydrocarbon receptor, we investigated the activity of p38 mitogen‐activated protein kinase α (p38 MAPKα, p38α) and mammalian target of rapamycin complex 1 (mTORC1), and their effect on SR1‐expanded hUCB CD34+ cells. Our results showed that cellular senescence occurred in the SR1‐expanded hUCB CD34+ cells in which p38α and mTORC1 were successively activated. Furthermore, their coinhibition resulted in a further decrease in hUCB CD34+ cell senescence without an effect on apoptosis, promoted the maintenance of expanded phenotypic HSCs without differentiation inhibition, increased the hematopoietic reconstitution ability of multiple lineages, and potentiated the long‐term self‐renewal capability of HSCs from SR1‐expanded hUCB CD34+ cells in NOD/Shi‐scid/IL‐2Rγnull mice. Our mechanistic study revealed that senescence inhibition by our strategy was mainly attributed to downregulation of the splicesome, proteasome formation, and pyrimidine metabolism signaling pathways. These results suggest that coinhibition of activated p38α and mTORC1 potentiates stemness maintenance of HSCs from SR1‐expanded hUCB CD34+ cells via senescence inhibition. Thus, we established a new strategy to maintain the stemness of ex vivo differentiation inhibitor‐expanded human HSCs via coinhibition of multiple independent senescence initiating signal pathways. This senescence inhibition‐induced stemness maintenance of ex vivo expanded HSCs could also have an important role in other HSC expansion systems.

2 cells were cultured ex vivo with HGFs along with 0.1% DMSO (vehicle) or LY and/or Rapa. After 7 days, uncultured cells and their expanded progeny were stained with Alexa Fluor 488 -linked antibodies against p-P38 or p-S6. The expression of p-P38 (a) and p-S6 (b) in uncultured and expanded CD34 + cells were determined by flow cytometry (n = 3 for all). *** p < 0.001, ** p < 0.01.

Supplementary Figure S3. Coinhibition of activated p38α and mTORC1
signaling pathways further inhibit senescence in ex vivo expanded CD34 + cells.

Supplementary Tables
A total of 1000, 3000, and 5000 uncultured hUCB CD34 + cells or a fraction of the final culture equivalent to 1000, 3000, and 5000 starting hUCB CD34 + cells were transplanted into sublethally irradiated (230 cGy) NOG mice via the tail vein within 24 h after irradiation. After 16 weeks, the mice were sacrificed, and the engraftment of donor cells in bone marrow was determined by flow cytometry. For secondary engraftment, half of the bone marrow cells of both femurs and tibiae from primary recipient mice were collected and transplanted into sublethally irradiated secondary recipient NOG mice for another 16 weeks. Reconstitute mice was determined by the Supplementary Table S1. Summary of primary and secondary NOG engraftment 13 presence of greater than 0.1% of hCD45 + mCD45cells in recipient bone marrow.

Supplementary Table S2. Poisson statistics for primary engraftment. Poisson
statistics was applied to the data in supplementary table S1. SRC frequency and statistical differences between various groups were determined using L-Calc software.
The number of SRC in 10 5 hUCB-derived CD34 + cells is calculated.