Abstract
Background & Aim Since numbers of harvested hematopoietic stem cells (HSCs) continues to limit not only laboratory research but also clinical HSC transplantation and gene therapies, development of ex vivo culture systems to expand harvested human hematopoietic stem-progenitor cells (HSPCs) remains a critical translational research quest. To address the barrier that HSCs generally die or differentiate in current ex vivo culture media, we have developed an xeno-free, serum-free medium - StemPro™ HSC Expansion Medium (Prototype) by extensive evaluation of iterative modification of medium constituents. Methods, Results & Conclusion Cultures of primary human CD34+ HSPCs immunopurified from cord blood, mobilized peripheral blood and bone marrow harvests generated greatly increased numbers of immunphenotyped-defined HSPC in StemPro™ HSC Expansion Medium (Prototype) supplemented with FLT3L, KITL (also known as SCF), TPO, IL-3, and IL-6 (FKT36), as compared to either uncultured day 0 cells or cells cultured in industry-standard culture media containing FKT36. For example, culture of primary human CD34+ cells from mobilized peripheral blood for 7 days in FKT36-containing StemPro™ HSC Expansion Medium (Prototype) resulted in ∼100-fold increased numbers of CD34+CD45+Lin− cells and ∼2000-fold increased numbers of CD34+Lin−CD90+CD45RA− cells (an early HSPC immunophenotype), as compared to uncultured day 0 cells. The ex vivo-cultured cells contained high frequencies of aldehyde dehydrogenase-containing cells and formed erythroid and non-erythroid hematopoietic colonies in vitro. In an ongoing in vivo hematopoietic chimera experiment, ex vivo-cultured mPB CD34+ HSPCs harbored robust in vivo-engrafting capacity at the 8-week post-transplant short-term HSC time point evaluated to date. Thus, it appears that StemPro™ HSC Expansion Medium (Prototype) supports superior HSPC expansion that includes self-renewal of short-term-HSCs. Evaluation of long-term-HSC capacity is in progress.
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