Inhibition Of P38 MAPK, MTORC1 And AHR Signals Increases The Number And Stemness Of Ex Vivo Expanded HUCB HSCs

Part IInhibition of P38 MAPK and mTORC1 signals during hUCB CD34+ cells expansion in vitro decreases the level of cell senescenceBackgroud:Hematopoietic stem cell transplantation (HSCT) is an effective treatment for various hematological disorders. However, a prominent problem is relatively insufficient number of hematopoietic stem cells (HSCs), which limits the success of HSCT in clinical therapy. Although the clinical study about human umbilical cord blood (hUCB) HSCT indicated that a 2-fold ex vivo expansion for HSCs would meet the clinical demand, past conventional methods for HSCs expansion, such as using cytokines or feeder layer cells, were difficult to obtain HSCs with long-term hematopoietic reconstitution ability. Hematopoietic reconstitution ability measures the ability of HSCs to differentiate into each lineage of blood cells in transplant recipient. Thus, sternness maintenance of HSCs during ex vivo expansion had been an important aim to us in the past.The developmental processes of HSCs include cell proliferation, differentiation, senescence and death, which can be regulated to promote HSCs expansion. β-Galactosidase is a biological marker for senescent cell, and increased activity of P-galactosidase represents cell senescence. Although senescent cells possess metabolic activity, the abilities of self-renewal and multilineage differentiation were lost. Our previous studies showed that inhibition of either activated P38 MAPK or mTORC1 signaling pathway could promote the expansion of HSCs and enhance the hematopoietic reconstitution ability of ex vivo expanded HSCs by inhibiting cell senescence. Thus, it is of a great interest to determine whether inhibition of these two pathways can further promote ex vivo expansion of HSCs and enhance the hematopoietic reconstitution ability by further inhibiting HSCs senescence.Methods:CD34+ cells derived from the normal puerpera’s umbilical cord blood were separated by magnetic activated cell sorting (MACS). Separated CD34+ cells were cultured in vitro with IL-6, TPO, SCF and Flt3-Ligand. The LY2228820 and (or) Rapamycin were utilized at the same time to inhibit the activated P38 MAPK and mTORC1 signaling pathways. We examined the expressions of intracellular p-P38, p-P70 S6, p-4E BP1 or p-mTOR after culture through western blot or flow cytometry. We also examined the expressions of CD34, CD38, CD90, and CD45RA on the surface of cells after culture by flow cytometry. CFSE, PI, and FDG staining were utilized to examine cell proliferation, cell cycle, and cell senescence by flow cytometry, respectively. Furthermore, colony forming unit (CFU) was determined through colony formation assay in vitro.Results:Our results indicated that LY2228820 and Rapamycin could separately inhibit the activated P38 MAPK and mTORC1 signaling pathways during the ex vivo culture of hUBC CD34+ cells. The percentage of CD34+, CD34+CD38-, and CD34+CD45RA-subpopulations was significantly higher in the progeny of hUBC CD34+ cells cultured with both LY2228820 and Rapamycin than those with LY2228820 or rapamycin alone in association of further inhibition of cellular senescence. However, combinated utilization of LY2228820 and Rapamycin did not increase the fold expansion of these cells compared with LY2228820 or Rapamycin treatment alone in part because Rapamycin inhibited cell proliferation through blocking more cultured cells in G0/G1 phases. The number of CFU-E, CFU-GM, CFU-M, CFU-GEMM and CFU-total was not significantly increased in the progeny of hUBC CD34+ cells cultured with both LY2228820 and rapamycin compared with cells cultured with LY2228820 or Rapamycin alone, which indicated the number of hematopoietic progenitor cells (HPCs) was not increased.Conclusion:Inhibiton of activated P38 MAPK and mTORC1 signaling pathways increases the percentage of hematopoietic stem/progenitor cells in the progeny of hUBC CD34+ cells cultured ex vivo through inhibiting cell senescence.Part IIInhibition of cell senescence and AHR signal promotes the expansion of hUCB HSCsBackgroud:We have demonstrated that co-inhibition of activated P38 MAPK and mTORCl signaling pathways during the ex vivo culture of hUCB CD34+ cells increases the percentage of HSCs/HPCs in the progeny of hUBC CD34+ cells cultured in vitro through inhibiting cell senescence, but can not promote those cells expansion. We analyzed this phenomenon and speculated that co-inhibition of activated P38 MAPK and mTORC1 signaling pathways might promote the stemness maintain of HSC. In addition, inhibition of the aryl hydrocarbon receptor (AHR) with SRI had been demonstrated with the ability of promoting hUCB HSCs expansion through inhibiting HSCs differentiation. Furthermore, AhR-KO mice showed an increased number of HSCs/HPCs in bone marrow, but also showed a significant decrease in self-renewal ability. We supposed that the inhibition of cell differentiation resulted by SRI treatment during ex vivo culture of HSCs was accompanied with increased cell senescence. Thus, it is of a great interest to us whether a combination of inhibition of cell senescence and differentiation can further promote the expansion of hUCB HSCs and increase the hematopoietic reconstitution ability of cultured cell. So, on the basis of our previous section, we utilized SRI to inhibit the activated AHR in our current experiments.Methods:We cultured the hUCB CD34+cells with additional agonist SRI following the foregoing methods. The current experiments include vehicle group, P38 MAPK and mTORC1 co-inhibition group, AHR inhibition group and combinated inhibition group. After culture, we examined the expressions of intracellular p-P38, p-mTOR and CYP1B1 by flow cytometry. We also examined the expressions of CD34, CD38, CD90 and CD45RA after culture by flow cytometry, as well as cell proliferation, cell cycle, and cell senescence by flow cytometry. Furthermore, colony forming unit (CFU) was determined through colony formation assay in vitro. We conducted the engraftment using NOD-Prkdcscid 1112rgnull mice which presented as severe combined immunodeficiency. Uncultured cells and cultured cells were transplanted into irradiated mice via tail vein injection. The chimerism of human cells in blood was determined post transplantation at week 1,4 and 8, and chimerism of human cells in bone morrow and spleen was determined at week 13 post transplantation. The chimerism of human T and B lymphoid cells, myeiod cells, red blood cells progenitors, megakaryocytes, NK cells and HSCs/HPCs in bone morrow was also determined at week 13 post transplantation.Results:Our results showed that LY2228820, Rapamycin and SRI could inhibit activated P38 MAPK, mTORC1 and AHR signaling pathways during the ex vivo culture of hUCB CD34+ cells, respectively. Co-inhibition of activated P38 MAPK and mTORC1, accompanied with SRI further increased the percentage of CD34+, CD34+CD38-, CD34+CD90+, CD34+CD45RA- subpopulations compared with P38 MAPK and mTORC1 co-inhibition group or SRI treatment separately. At the same time, co-inhibition of activated P38 MAPK, mTORC1 and AHR also increased the expansion fold of CD34+CD45RA-subpopulation which represented HSCs and multilineage progenitor cells compared with P38 MAPK and mTORC1 co-inhibition group or SRI treatment alone. Furthermore, co-inhibition of activated P38 MAPK, mTORC1 and AHR signals or inhibition of AHR signal alone increased the number of CFU-GM, CFU-GEMM and CFU-total, which indicated that the HPCs were expanded or the differentiation of HPC was inhibited. We explored the mechanism and found that co-inhibition of activated P38 MAPK, mTORC1 and AHR alleviated cell senescence, at the same time inhibited cell differentiation. The results of engraftment in vivo showed that co-inhibition of activated P38 MAPK and mTORC1 signals or inhibition of AHR alone increased the chimerism of human cells in blood and bone morrow of mice compared with uncultured group or vehicle group; co-inhibition of activated P38 MAPK, mTORC1 and AHR signals increased the chimerism of human cells in blood and bone morrow of mice compared with P38 MAPK and mTORC1 co-inhibition group or SRI treatment separately. The transplanted cells in mice possessed hematopoietic reconstitution ability.Conclusion:Co-inhibition of activated P38 MAPK, mTORC1 and AHR signals promotes the expansion of hUCB HSCs and increases the hematopoietic reconstitution ability of cultured cells through inhibiting cells senescence and differentiation.