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带血造血干/祖细胞 细胞（HSPC），至可用于临床的市售量。这将允许 造血干细胞可商购获得，用于治疗癌症、自身免疫性 急性辐射综合征（ARS）。此外，众所周知，脐带 血液造血干细胞数量很少，不能用于成人移植，甚至不能用于成人移植。 同种异体移植，因为不到30%的患者可以找到MHC相容的供体。质量 Biological，Inc（QBI）一直在通过开发必要的离体技术来研究这种现象， 将有助于这些HSPC群体的总体细胞培养和分析。我们将这些 确定特异性细胞因子混合物（Flt-3、干细胞因子、促凝血酶生成素和IL-6; FST 6）在2D静态培养物中对人CD 34+细胞的作用。使用这种方法，我们注意到， 通过CD 34+细胞染色和流式细胞术测定HSPC在培养物中随时间的总体存在。到 为了进一步表征这些细胞，我们利用异种移植物连续移植模型， 胎羊或NOD/SCID小鼠人造血模型。人类的主要接受者 CD 34+细胞具有短期（祖细胞）和长期移植细胞（干细胞），而 第二受体仅具有长期移植细胞（干细胞）。这些体外和体内试验 使我们能够修改无血清培养条件，并系统地分析特定的 对干细胞/长期移植群体和/或祖细胞/短期移植群体的修饰 嫁接人口。在含有细胞因子的无血清培养基QBSF-60中培养的CD 34+细胞通常存活 仅需7天，同时保持其长期嫁接能力。相比之下， 细胞因子组合，FST 6，我们保持了CD 34+细胞的活力和它们的长期移植 培养能力长达14-21天，之后培养物仅由祖细胞组成。我们建议 在I期中评估单独的上述细胞因子组合以及在存在其他细胞因子的情况下， bFGF和TGF β在一个新的可扩展的，三维培养系统，模拟流体动力学的 造血系统在第二阶段的研究中，我们建议用最佳的方法来评估这些培养条件。 单独的细胞因子组合和在由CD 34+细胞和细胞类型组成的共培养物存在下 使用有限稀释和系列移植研究来确定 无论是长期移植的细胞已经扩增还是仅仅是更定型的祖细胞。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，这些细胞都已被证明是 在QBSF 60无血清培养基中增殖。这种方法对于体外扩增将是非常宝贵的。 和/或造血干细胞和祖细胞的分化以用于它们在临床模式中的用途。

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.