Developing a novel ex vivo platform to support hematopoietic cells and characterize the stem cell niche

项目总结/摘要 目前的培养方法降低了造血干细胞（HSC）成功植入的能力 在一个主机。新兴的基因编辑技术，如CRISPR/Cas9，需要培养时间， 用于纠正致病等位基因。因此，需要开发新的方法， 培养HSC。HSC与骨髓间充质干细胞等骨髓小生境细胞的共培养 骨髓间充质干细胞（MSC）是HSC培养中问题的一种可能的解决方案，因为这些细胞提供了 在体内支持HSC。然而，MSC不能在培养物中维持延长的时间段 并且相当迅速地失去生态位因子的表达。通过筛选候选转录因子， 我们的实验室鉴定了5种因子，当它们一起转导时，可以恢复小生境因子的表达， 长期培养。这些因子是Kruppel样因子7（Klf 7）、破骨细胞刺激因子 Ostf 1、X-box结合蛋白（XBP 1）、干扰素调节因子3（Irf 3）和Irf 7， 统称为KOXII因子。KOXII转导的MSC能够扩增小鼠和小鼠骨髓细胞。 在更大程度上比模拟转导的MSC诱导人功能性HSC。因此，这些细胞 可用于离体扩增HSC以进行基因校正。然而，存在监管障碍 涉及鼠细胞在人类治疗中的应用。这里提出的工作将部分侧重于 KOXII转导的人MSC的开发和表征。在生成这些 细胞，我将确定KOXII因子是否影响人MSC中的小生境因子的表达。我也会 使用流式细胞术和干细胞异种移植来确定KOXII转导的MSC是否 在驱动HSC扩增方面比未修饰的MSC更有效。最后，使用基于CRISPR/Cas9 作为来自镰状细胞病患者的HSC的基因编辑模型，我将确定是否 患者细胞与KOXII转导的MSC的共培养可以提高基因编辑的效率，或 相对于目前的标准HSC培养方法，增加适当编辑的细胞的产量。同时，我 将使用鼠KOXII转导的MSC来更仔细地检查MSC的小生境信号传导。因为这些 细胞可以相对大量地培养，它们是蛋白质组学研究的理想选择。合作 与Jeroen Krijgsveld的实验室合作，我将检查这些细胞的分泌组，以确定 其分泌被KOXII因子上调的蛋白质。使用体外和体内试验， 将评估这些因素对HSC维持和增殖的影响，目的是 鉴定在MSC-HSC生态位相互作用中具有先前未被认识的作用的分泌蛋白。

PROJECT SUMMARY / ABSTRACT Current culture methods reduce the ability of hematopoietic stem cells (HSCs) to successfully engraft in a host. Emerging gene editing technologies such as CRISPR/Cas9 require time in culture to allow for the correction of disease-causing alleles. There is therefore a need to develop new methods of culturing HSCs. Coculture of HSCs with bone marrow niche cells such as mesenchymal stem cells (MSCs) is one possible solution to problems in HSC culture, as these cells provide factors that support HSCs in vivo. However, MSCs cannot be maintained in culture for extended periods of time and fairly rapidly lose expression of niche factors. Through a screen of candidate transcription factors, our lab identified 5 factors that when transduced together restore niche factor expression and allow for prolonged culture. These factors are Kruppel-like factor 7 (Klf7), Osteoclast stimulating factor (Ostf1), X-box binding protein (XBP1), Interferon regulatory factor 3 (Irf3), and Irf7, which we collectively dubbed the KOXII factors. KOXII-transduced MSCs were able to expand both murine and human funtional HSCs to a much greater extent than mock-transduced MSCs. These cells therefore may be useful in expanding HSCs ex vivo for gene correction. However, there are regulatory barriers to the application of murine cells in human therapeutics. The work proposed here will in part focus on the development and characterization of KOXII-transduced human MSCs. After generating these cells, I will determine if the KOXII factors affect expression of niche factors in human MSCs. I will also use flow cytometry and stem cell xenotransplantation to determine if KOXII-transduced MSCs are more effective at driving HSC expansion than unmodified MSCs. Finally, using CRISPR/Cas9-based gene editing of HSCs derived from patients with sickle cell disease as a model, I will determine if coculture of patient cells with KOXII-transduced MSCs can improve the efficiency of gene editing or increase the yield of properly edited cells over current standard HSC culture methods. In parallel, I will use murine KOXII-transduced MSCs to more closely examine niche signalling by MSCs. As these cells can be cultured in relatively large numbers, they are ideal for proteomic studies. In collaboration with the lab of Jeroen Krijgsveld, I will examine the secretome of these cells in order to identify proteins whose secretion is upregulated by the KOXII factors. Using both in vitro and in vivo assays, I will evaluate the effect of these factors on HSC maintenance and proliferation, with the aim of identifying secreted proteins with previously unappreciated roles in MSC-HSC niche interactions.