Abstract
Umbilical cord blood (UCB) has been established as an alternative source for hematopoietic stem/progenitor cells (HSPC) for cell and gene therapies. Limited cell yields of UCB units have been tackled with the development of cytokine-based ex vivo expansion platforms. To improve the effectiveness of these platforms, namely targeting clinical approval, in this study, we optimized the cytokine cocktails in two clinically relevant expansion platforms for HSPC, a liquid suspension culture system (CS_HSPC) and a co-culture system with bone marrow derived mesenchymal stromal cells (BM MSC) (CS_HSPC/MSC). Using a methodology based on experimental design, three different cytokines [stem cell factor (SCF), fms-like tyrosine kinase 3 ligand (Flt-3L), and thrombopoietin (TPO)] were studied in both systems during a 7-day culture under serum-free conditions. Proliferation and colony-forming unit assays, as well as immunophenotypic analysis were performed. Five experimental outputs [fold increase (FI) of total nucleated cells (FI TNC), FI of CD34+ cells, FI of erythroid burst-forming unit (BFU-E), FI of colony-forming unit granulocyte-monocyte (CFU-GM), and FI of multilineage colony-forming unit (CFU-Mix)] were followed as target outputs of the optimization model. The novel optimized cocktails determined herein comprised concentrations of 64, 61, and 80 ng/mL (CS_HSPC) and 90, 82, and 77 ng/mL (CS_HSPC/MSC) for SCF, Flt-3L, and TPO, respectively. After cytokine optimization, CS_HSPC and CS_HSPC/MSC were directly compared as platforms. CS_HSPC/MSC outperformed the feeder-free system in 6 of 8 tested experimental measures, displaying superior capability toward increasing the number of hematopoietic cells while maintaining the expression of HSPC markers (i.e., CD34+ and CD34+CD90+) and multilineage differentiation potential. A tailored approach toward optimization has made it possible to individually maximize cytokine contribution in both studied platforms. Consequently, cocktail optimization has successfully led to an increase in the expansion platform performance, while allowing a rational side-by-side comparison among different platforms and enhancing our knowledge on the impact of cytokine supplementation on the HSPC expansion process.
Highlights
Hematopoietic cell transplantation (HCT) continues to be the leading cell therapy for malignant and non-malignant bloodbased disorders and advances in this field have expanded the options available for patients concerning graft source
Despite some expected variants between strategies, we identified the trio of cytokines stem cell factor (SCF), fms-like tyrosine kinase 3 ligand (Flt-3L) and TPO as the most used cytokine combination in the majority of expansion studies, including those which have progressed to Phase I/II clinical trials
Whether due to having been performed in non-human cells (Audet et al, 2002), used to study ex vivo hematopoietic differentiation rather than hematopoietic stem/progenitor cells (HSPC) expansion (Cortin et al, 2005; Lim et al, 2011) or to the lack of surface response models and optimization (Petzer et al, 1996; Tursky et al, 2012), previous attempts struggle in being transposed to present expansion protocols
Summary
Hematopoietic cell transplantation (HCT) continues to be the leading cell therapy for malignant and non-malignant bloodbased disorders and advances in this field have expanded the options available for patients concerning graft source. Umbilical cord blood (UCB) is an accepted and appealing alternative source of hematopoietic stem/progenitor cells (HSPC) for HCT (Hough et al, 2016; Woolfrey et al, 2016). Compared with bone marrow (BM) or mobilized peripheral blood, UCB transplants have shown similar survival outcomes with lower chances of developing graft vs host disease (GVHD) and lesser compatibility issues concerning human leukocyte antigen (HLA) matching (Rocha et al, 2001, 2004). Approaches have varied from promoting HSPC expansion with novel small molecules including StemRegenin-1 (Wagner et al, 2016), UM171 (Fares et al, 2014), and nicotinamide (Horwitz et al, 2014), co-culture with mesenchymal stromal cells (de Lima et al, 2012) and induction of Notch signaling pathways (Delaney et al, 2010)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
