Induction of hematopoietic stem cell self-renewal and maintenance ex vivo through small molecule compounds

Many hematopoietic and nonhematopoietic diseases require chemotherapy and radiotherapy for treatment. Unfortunately, these regimens induce long-term damage, deleterious side effects and require multiple treatments for success. Hematopoietic stem cell (HSC) transplantation has proven effective in treatments but requires HLA-matching of these products to prevent complications, a process not easily achieved. Umbilical cord blood has become a standard in therapeutic transplantation primarily due to the ability to avoid more stringent HLA-matching. However, the low stem cell numbers in cord blood which limits use primarily in pediatric patients requires ex vivo expansion to increase patient availability. Clinical expansion is difficult to achieve and costly necessitating the need for enhanced protocols. The recent identification of inducing pluripotency by small molecule compounds has become an attractive option in HSC expansion. Our aim was to identify compounds that could induce self-renewal mechanisms of HSCs in vitro. To accomplish this, we developed a flow cytometry based small throughput screen to identify compounds. We identified four molecules that expanded HSCs and briefly validated their functional activity. Using various starting populations, we identified specific targeting of HSC populations by these compounds, a phenotype that was dramatically increased by adding compounds in combination. Our dual small molecule expanded cells also demonstrated long-term and multi-lineage engraftment validating our HSC expansion. Upon further study, we identified the compounds induced proliferation mechanisms while preventing differentiation, forcing the activation of HSC self-renewal genes. We also identified expansion of HSCs by inhibition of the p38 MAPK pathway, involved in differentiation and activated by ROS and normoxic conditions. This expansion also demonstrated long-term and multi-lineage engraftment. Moreover, addition of small molecule compounds with the p38 MAPK inhibitor promoted even greater expansion and replaced growth factors in cultures demonstrating the possibility of a cost-effective culture. Furthermore, IR-induced BM-injury could be rescued by in vivo injection of a p38 MAPK inhibitor. These findings suggest that inhibition of negative regulation of HSCs could force self-renewal and promote expansion, protecting the stem cells from negative regulation. More research is required to better understand small molecule activation of HSC self-renewal to develop an improved expansion culture for clinical therapeutics.