Inhibition of Both Activated p38 MAPK and mTOR C1 Potentiates the Effect of SR1 on Promotion of Hematopoietic Stem Cell Expansion Ex Vivo

Hematopoietic stem cells (HSCs) transplantation has been widely investigated as standard treatment for various hematological disorders. However, a prominent problem is relatively insufficient numbers of transplantable stem cells, which limits the success of HSCs transplantation. Although the clinical study with cord blood grafts indicated that a 2-fold ex vivo expansion of HSCs would have a profound clinical impact, past HSCs expansion systems were difficult to get HSCs with long term hematopoietic reconstitution ability. Thus, stemness maintenance of HSCs during ex vivo expansion would be a very important aim to us. Our previous studies showed that inhibition of either activated P38 MAPK£¨p38£© or mTOR C1 were identified as two novel ways to enhance long term hematopoietic reconstitution of ex vivo expanded mouse bone marrow (BM) HSCs in part by inhibiting HSCs senescence. In addition, inhibition of the aryl hydrocarbon receptor (AHR) with an antagonist, SR1, had been reported to promote ex vivo expansion of human HSCs by inhibiting HSCs differentiation. We supposed that the stemness of SR1 expanded HSCs could been damaged owing to activation of senescent and/or apoptotic signal pathways. Thus, it is of a great interest to determine whether a combination of inhibition of all these pathways can further promote ex vivo expansion of HSCs by inhibiting HSCs senescence and differentiation.

To test this hypothesis, we first examined the effects of inhibition of both p38 and mTOR C1 with SB203580 and rapamycin on mouse BM HSCs expansion ex vivo. Our results showed that inhibition of p38 alone by SB203580 modestly promoted mouse BM HSCs expansion in association with a significant reduction in HSCs senescence. However, this also led increase in mTOR C1 activation in HSCs. Inhibition of mTOR C1 with rapamycin potentiated the effect of p38 inhibition on promotion of mouse BM HSCs expansion according to the results from CFC and CAFC assays in comparison with inhibition of p38 alone, which was confirmed by the competitive repopulating assay. Further mechanistic studies revealed that co-inhibition of p38 and mTOR C1 resulted in a great inhibition of HSCs senescence.

Next, we examined whether inhibition of both p38 and mTOR C1 can also potentiate the effect of inhibition of p38 or mTORC1 alone on promotion of human umbilical cord blood (hUCB) HSCs expansion ex vivo. We found that the percentage of CD34⁺, CD34⁺CD38^-, and CD34⁺CD45RA^- cells were significantly higher in the progeny of hUBC CD34⁺ cells cultured with both LY2228820 (a P38 MAPK inhibitor) and rapamycin than those with LY2228820 or rapamycin alone in association of inhibition of cellular senescence. However, the fold expansion of these cells was not significantly different between hUBC CD34⁺ cells cultured with both LY2228820 and rapamycin or LY2228820 or rapamycin alone in part because rapamycin inhibited cell proliferation through blocking more culture cells in G0/G1 phases.

However, co-inhibition of activated P38 and mTORC1, accompanied with SR1 further increased the percentages of CD34⁺/CD34⁺CD38^-/CD34⁺CD90⁺/CD34⁺CD45RA^- subpopulations by 42.7-130.9% compared with P38 and mTORC1 co-inhibition group or SR1 treatment separately. The combined treatment also increased expansion folds of CD34⁺/CD34⁺CD38^-/CD34⁺CD90⁺/CD34⁺CD45RA^- subpopulations by 75.9-155.4% compared with P38 and mTORC1 co-inhibition group. More importantly, the combined inhibition of activated mTORC1 and p38 along with SR1 maintained or increased the expansion folds of CD34⁺CD90⁺ and CD34⁺CD45RA^- subpopulation£¬which are enriched in HSCs and multilineage progenitor cells, compared to SR1 treatment group separately. The mechanism study further found that co-inhibition of P38 and mTORC1 alleviated cell apoptosis and activity of ¦Â-galactosidase induced by SR1, suggesting inhibition of senescnce. Finally, our combined culture system in 8 days led to a 16.2-fold increase for CD34⁺CD90⁺ subpopulation and a 6.1-fold increase for CD34⁺CD45RA^- subpopulation compared to input cells (Fig.1).

In conclusion, our results suggested that a combined inhibition of p38, mTOR C1 and AHR can inhibit HSCs senescence and differentiation in a HSCs expansion culture, which has the potential to be used to promote ex vivo expansion of HSCs.

Fig.1 Inhibition of both activated p38 MAPK and mTOR C1 potentiates the effect of SR1 on promotion of hematopoietic stem cell expansion ex vivo