A small-molecule combination promotes ex vivo expansion of cord blood-derived hematopoietic stem cells

Abstract

Background & Aim Background Cord blood (CB) provides an excellent alternative source of hematopoietic stem cells (HSCs) for patients lacking human leukocyte antigen-matched peripheral blood or bone marrow graft for transplantation. However, the limited number of HSCs in CB graft restricts its widespread use. Although extensive efforts have led to multiple methods for ex vivo expansion of human HSCs by targeting single molecules or pathways, it remains unknown whether it is possible to simultaneously manipulate the large number of targets essential for HSCs expansion and self-renewal. And the effect of small molecules on the proliferation and differentiation of HSCs has not been reported at the single cell level. Here, we report a combination including the small molecules UM171, SR1, JNK-IN-8 (USK) and the cytokines TPO, IL-6, SCF, FLT-3L, which can efficiently promote ex vivo expansion of HSCs. Methods, Results & Conclusion Methods CB-derived CD34+ cells were cultured for 10 days with cytokines plus DMSO (Vehicle group) or with cytokines plus three small molecules (USK group). The fold of cell expansion and the immunophenotype of expanded cells were assessed by flow cytometry. Results USK treatment increased the proportion of CD34+ (92.29 ±2.54%), CD34+ CD38− cells (78.06 ± 5.52%) and CD34+CD38−CD45RA−CD90+ LT-HSCs (8.96 ± 2.83%) compared to vehicle treatment (52.57 ± 5.98%, 39.31 ± 4.62% and 0.80 ± 0.54%, respectively, n=5). USK treatment led to a 106.52-, 923.8-, and 603.04-fold increase in CD34+, CD34+ CD38− and LT-HSCs numbers relative to initial cells, respectively (n=5). Vehicle treatment led to a 72.82-, 508.81-, and 54.64-fold increase in CD34+, CD34+ CD38− and LT-HSCs numbers relative to initial cells, respectively (n=5). USK treatment led to an 11.04-fold increase in LT-HSC numbers as compared to vehicle cultures. Conclusions Our study shows that a small-molecule combination (USK) can efficiently promote ex vivo expansion of HSCs. Using this combination model combined with transcriptome analysis at the single cell level, the key pathways and molecules of hematopoietic stem cell self-renewal in vitro can be further studied, which is expected to provide new targets for in vitro expansion of hematopoietic stem cells.