Inhibition of Both Activated p38 Mapkα and mTORC1 Potentiates the Effect of SR1 on Promoting Hematopoietic Reconstitution of Ex Vivo Expanded Human Umbilical Cord Blood Hematopoietic Stem Cells

Hematopoietic stem cells (HSCs) transplantation is a curative treatment for various hematological disorders. However, its use has been limited in part by the difficulty to obtain sufficient numbers of transplantable HSCs, especially from human umbilical cord bloods (hUCBs). Ex vivo expansion of cord blood HSCs can potentially mitigate the shortfall if the expanded HSCs can maintain their stemness for long-term hematopoietic reconstitution.

Our previous studies have shown that some hUCB HSCs lose their "stemness" during ex vivo expansion in part due to induction of cellular senescence via p38MAPKα(p38) or mTORC1 activation and inhibition of p38 and mTOR C1 promotes HSCs expansion (Ann Hematol. 2012; 91:813-823; Transplantation. 2014; 97:20-29). Recently, it was shown that StemRegenin 1 (SR1), an antagonist of the aryl hydrocarbon receptor (AHR), could significantly promote ex vivo expansion of HSCs, primarily via inhibition of HSCs differentiation. Therefore, we investigated whether the combination of SR1 with LY2228820 (LY) and rapamycin (Rapa) to inhibit p38 and mTOR C1, respectively, can enhance ex vivo expansion of hUBC HSCs by inhibiting HSCs differentiation and senescence.

Next, we confirmed that co-inhibition of activated p38 and mTORC1 promotes SR1 induced HSCs expansion ex vivo through inhibition of senescence.Our combined culture system in 7 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 (Blood,126(23):381-381) .To determine the hematopoietic reconstitution ability of the progeny of hUCB CD34⁺ cells expanded with inhibitors of p38, mTORC1, and AHR, we transplanted 1000 to 30000 uncultured hUCB CD34⁺ cells or a fraction of the final culture equivalent to 1000 to 10000 starting hUCB CD34⁺ cells into sub-lethally irradiated 6 to 10-week-old NOD-Prkdc^scid Il2rg^null (NOG) mice. Donor cell engraftment was analyzed at 8 and 13 weeks after the transplantation. The results showed that the progeny of 10,000 hUCB CD34⁺ cells cultured with LY, Rapa and SR1 produced significant greater donor cell engraftment in the recipients' peripheral blood compared with the same number of un-cultured hUCB CD34⁺ cells (2.19-fold, p<0.01), the progeny of 10,000 hUCB CD34⁺ cells cultured with vehicle (2.76-fold, p<0.01), or the progeny of 10,000 hUCB CD34⁺ cells cultured with SR1 alone (1.72-fold, p<0.05). The similar results were also found in the recipients' bone marrow and spleen. In addition, the progeny of 3,000 hUCB CD34⁺ cells cultured with LY, Rapa and SR1 generated the levels of peripheral blood donor cell engraftment that similar to these produced by 30,000 un-cultured hUCB CD34⁺ cells and the progeny of 10,000 hUCB CD34⁺ cells cultured with SR1 alone. More importantly, the hUCB CD34⁺ cells cultured with LY, Rapa and SR1 retained multi-lineage differentiation potential as they were able to produce human B cells, myeloid cells, red blood cell progenitors, megakaryocytes, and NK cells in the recipients' bone marrow. Furthermore, hUCB CD34⁺ cells cultured with LY, Rapa and SR1 produced the highest levels of human HSCs (CD34⁺CD38^-/CD34⁺CD90⁺/CD34⁺CD45RA^- cells) engraftment compared to all other cells.

Collectively, these findings suggest that the combined inhibition of p38, mTORC1 and AHR is more effective in promoting the expansion and hematopoietic reconstitution of hUBC HSCs than inhibition of p38, mTORC1 or ARH alone probably via inhibition of HSCs senescence and differentiation. This new strategy may alleviate the limitation of hUBC for transplantation in adult patients.