Optimized culture medium for enhanced ex vivo expansion of human hematopoietic stem cells

Abstract

Background & Aim Development of ex vivo culture systems to expand human hematopoietic stem-progenitor cells (HSPCs) remains a critical translational research goal that would enhance clinical hematopoietic stem cell (HSCs) transplantation and gene therapies. To address the problem that HSCs generally die or differentiate rapidly in current ex vivo culture media, we developed a xeno-free, serum-free medium – CTS™ StemPro™ HSC Expansion Medium. Methods, Results & Conclusion In a Design of Experiments approach, media constituents were systematically varied and iterations were evaluated with the goal to maximize ex vivo expansion of HSCP immunophenotypes. Culture of normal primary human CD34+ cells immunopurified from cord blood, mobilized peripheral blood and bone marrow in HSC Expansion Medium supplemented with FLT3L, SCF, TPO, IL3, and IL6 (FKT36), resulted in higher numbers of immunophenotype-defined HSPCs, 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 (mPB) for 7 days in FKT36-containing HSC Expansion Medium 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. CD34+ cells expanded in this medium maintained differentiation capacity in in-vitro colony forming assays, forming erythroid and non-erythroid cell colonies. In addition, using a directed differentiation method we showed that CD34+ cells were able to differentiate into erythroid lineage which generated red blood cells at > 80% efficiency as indicated by co-expression of CD71 and CD235a markers. Further, we demonstrated CTS™ StemPro™ HSC Expansion Medium enabled transduction of CD34+ cells with lentiviral vector generated using CTS™ LV-MAX System, and can be gene edited with CRISPR-Cas9 Gene editing tools that are suitable for cell and gene therapies CD34+ cells expanded with CTS™ StemPro™ HSC Expansion Medium exhibited long-term engraftment potential and multilineage chimerism at 6 months post-transplant. We demonstrated that ex vivo-cultured CD34+ HSPCs generate transplanted cell dose-dependent engraftment in NRG mouse bone marrows and spleens at 26 weeks post-transplant. Thus, CTS™ StemPro™ HSC Expansion Medium will be an impactful tool to enhance expansion of HSPCs that are suited for translational purposes such as transplantation, and gene therapies.