Table_1_Tailored Cytokine Optimization for ex vivo Culture Platforms Targeting the Expansion of Human Hematopoietic Stem/Progenitor Cells.DOCX

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.

脐带血（UCB）已被确立为造血干细胞/祖细胞（HSPC）的替代来源，适用于细胞和基因治疗。针对脐带血单位中细胞产量有限的难题，已开发了基于细胞因子的原位外扩展平台。为提升这些平台的有效性，特别是针对临床批准，本研究中对两种临床相关的HSPC扩展平台进行了细胞因子混合物的优化，包括液体悬浮培养系统（CS_HSPC）和与骨髓来源的间充质干细胞（BM MSC）共培养系统（CS_HSPC/MSC）。通过基于实验设计的方案，在7天的无血清条件下，研究了两种系统中三种不同的细胞因子[干细胞因子（SCF）、fms样酪氨酸激酶3配体（Flt-3L）和血小板生成素（TPO）]。进行了增殖和集落形成单位检测，以及免疫表型分析。跟踪了五个实验输出[总核细胞（TNC）的倍增（FI TNC）、CD34+细胞的倍增、红系爆裂形成单位（BFU-E）的倍增、粒-单核细胞集落形成单位（CFU-GM）的倍增和多能性集落形成单位（CFU-Mix）的倍增]作为优化模型的目标输出。本研究确定的创新优化混合物分别包含SCF、Flt-3L和TPO的浓度为64、61和80 ng/mL（CS_HSPC）以及90、82和77 ng/mL（CS_HSPC/MSC）。经过细胞因子优化后，直接比较了CS_HSPC和CS_HSPC/MSC作为平台。在8项测试的实验指标中，CS_HSPC/MSC在6项中优于无饲养层系统，显示出在增加造血细胞数量的同时维持HSPC标志物（即CD34+和CD34+CD90+）的表达和多能性分化潜能的优越能力。针对优化的定制方法使得能够在两个研究平台中分别最大化细胞因子的贡献。因此，混合物优化成功提升了扩展平台的表现，同时允许在不同平台之间进行合理的平行比较，并加深了我们对于细胞因子补充对HSPC扩展过程影响的认知。