The insufficient number of hematopoietic stem cells (HSCs) poses a significant challenge for successful hematopoietic stem cell transplantation and gene-based therapies. To address this issue, ex vivo expansion of HSCs has been developed, improving engraftment and reducing morbidity risks in hematological disorders. Small molecules, known as stem cell agonists (SCAs), have been utilized to promote HSC expansion and have been implemented in clinical trials. While most HSC expansion protocols focus on the single use of SCAs, we describe a protocol using an optimized small molecule cocktail (SMC), X2A, to robustly enhance HSC yield. This protocol is applicable to human CD34+ hematopoietic stem and progenitor cells (HSPCs) derived from both umbilical cord blood and peripheral blood. In addition to the ex vivo HSC expansion protocol, we detail the CD34+ HSPC isolation technique and flow cytometry methods to characterize HSPC sub-populations from cell cultures. This culture protocol serves as a robust tool for pre-clinical studies in HSPCs and provides a foundation for further modifications to meet specific research needs.
Keywords: Cell expansion; Hematopoietic stem cells; Small molecules; Stem cell agonists.
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