Optimization of the Ex Vivo 3-Dimensional Culture Conditions that Controls the Formation and Development of Human Hematopoietic Stem and Progenitor Cell Populations

摘要：本项目的总体目标是扩增人脐血造血干/祖细胞。 细胞(HSPC)，到可以在临床上使用的商业可用数量。这将允许 造血干细胞将在商业上用于治疗癌症、自身免疫 疾病，以及急性辐射综合征(ARS)的病例。此外，众所周知，脐带 血液中的造血干细胞数量很少，不能用于成人移植，甚至不能用于 异基因移植，因为只有不到30%的患者能找到MHC相合的供者。质量 生物公司(QBI)一直在通过开发必要的体外技术来研究这种现象 将有助于这些HSPC群体的整体细胞培养和分析。我们已经延长了这些 研究确定特定的细胞因子鸡尾酒(Flt-3、干细胞因子、血小板生成素和IL-6； FST6)在2D静态培养中对人CD34+细胞的影响。使用这种方法，我们注意到 CD34+细胞染色和流式细胞仪检测HSPC的总存在和培养时间。至 为了进一步鉴定这些细胞，我们利用了异种移植系列模型 胎羊或NOD/SCID小鼠模型用于人类造血。人类的主要接受者 CD34+细胞同时存在短期(祖细胞)和长期移植细胞(干细胞)，而 二次受者仅有长期移植细胞(干细胞)。这些体外和体内检测 使我们能够修改无血清培养条件，并系统地分析特定的 修饰对干细胞/长期移植群体和/或祖细胞/短期 嫁接种群。在含有细胞因子的QBSF-60无血清培养液中培养的CD34+细胞通常存活 仅需7天，同时保持其长期的嫁接能力。相比之下，在有 细胞因子组合FST6，我们维持了CD34+细胞的活性及其长期移植 培养长达14-21天，之后的培养仅由祖细胞组成。我们建议 在阶段1中，单独评估上述细胞因子组合以及在其他细胞因子存在的情况下 碱性成纤维细胞生长因子和转化生长因子β在一种新的可扩展的三维培养系统中，该系统模拟了 造血系统。在第二阶段的研究中，我们建议用最优的 细胞因子单独结合以及在由CD34+细胞和细胞类型组成的共培养情况下 使用有限稀释和系列移植研究来确定与造血相关的 长期移植的细胞是否已经扩大，还是只有更坚定的祖细胞。这些细胞 类型将包括人类内皮细胞、间充质干细胞和成纤维细胞，所有这些都已经显示出来。 在QBSF60无血清培养液中增殖。这种方法对于体外扩增将是无价的。 和/或造血干细胞和造血祖细胞的分化，以供临床使用。

Abstract: The overall goal of this project is to expand human cord blood hematopoietic stem and progenitor cells (HSPC), to commercially available quantities that can be used in the clinic. This would allow the hematopoietic stem cells to be commercially available for use in either the treatment of cancers, autoimmune diseases, and in cases of Acute Radiation Syndrome (ARS). Furthermore, it is well known that umbilical cord blood hematopoietic stem cells are few in number, and cannot be used for an adult transplant or even in allogenic transplants because less than 30% of the patients can find an MHC compatible donor. Quality Biological, Inc (QBI) has been studying this phenomena by developing the necessary ex-vivo technologies that will contribute to the overall cell culture and analysis of these HSPC populations. We have extended these studies to determine the effect of specific cytokine cocktails (Flt-3, Stem Cell Factor, Thrombopotin, and IL-6; FST6) have on human CD34+ cells in 2D static cultures. Using this approach we noted an increase in the overall presence of HSPCs with time in culture as determined by CD34+ cell staining and flow cytometry. To further characterize these cells we took advantage of the xenograft serial transplantation models using either the fetal sheep or the NOD/SCID mice models for human hematopoiesis. The primary recipients of the human CD 34+cell had both short-term (progenitor cells) and long-term engrafting cells (stem cells) present, whereas, the secondary recipients only had the long-term engrafting cells (stem cells). These in vitro and in vivo assays allowed us to modify the serum-free culture conditions and systematically analyze the effect a specific modification had on the stem cell/long-term engrafting population and/or the progenitor cell/short-term engrafting population. CD34+ cells cultured in serum-free medium QBSF-60 with cytokines typically survive for only 7 days while maintaining their long-term engrafting capacity. In comparison, in the presence of the cytokine combination, FST6, we have maintained the viability of the CD34+ cells and their long-term engrafting capacity for up to 14-21 days, after which the cultures consisted of only progenitor cells. We propose to evaluate in the Phase 1 the above cytokine combinations alone and in the presence of other cytokines as bFGF and TGF β in a novel scalable, 3-Dimensional culture system that mimics the fluidic dynamics of the hematopoietic system. In the Phase II study we propose to evaluate these culture conditions with the optimal cytokine combination alone and in the presence of co-cultures comprised of CD34+ cells and cell types associated with the hematopoiesis using a limiting dilution and serial transplantation studies to determine whether the long-term engrafting cells have expanded or only the more committed progenitors. These cell types will include human endothelial cells, mesenchymal stem cells, and fibroblasts that have all been shown to proliferate in QBSF 60 serum-free medium. Such an approach will be invaluable for the ex-vivo expansion and/or differentiation of hematopoietic stem and progenitor cells for their use in clinical modalities.