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

项目摘要/摘要 目前的培养方法降低了造血干细胞成功植入的能力。 在一个宿主里。新兴的基因编辑技术，如CRISPR/Cas9，需要培养时间才能 用于纠正致病等位基因。因此，有必要开发新的方法来 培养造血干细胞。骨髓间充质干细胞等骨髓细胞与造血干细胞共培养的实验研究 (MSCs)是解决HSC培养问题的一种可能方案，因为这些细胞提供了 在体内支持造血干细胞。然而，MSCs不能在培养中长时间保持 并相当迅速地失去了生态位因子的表达。通过筛选候选转录因子， 我们的实验室确定了5个因子，当它们一起转导时，可以恢复利基因子的表达，并允许 以延长培养时间。这些因子是Kruppel样因子7(KLF7)，破骨细胞刺激因子 (Ostf1)、X盒结合蛋白(XBP1)、干扰素调节因子3(IRF3)和IRF7，我们发现 统称为KOXII因子。KOXII转导的MSCs能够扩增小鼠和 与模拟转导的骨髓间充质干细胞相比，人功能性造血干细胞的分化程度要大得多。因此，这些细胞 可能有助于体外扩增HSCs以进行基因校正。然而，监管方面存在障碍。 小鼠细胞在人类治疗中的应用。这里提出的工作将在一定程度上集中在 转导KOXII基因的人骨髓间充质干细胞的培养及特性在生成这些文件之后 细胞，我将确定KOXII因子是否影响人MSCs中利基因子的表达。我也会 使用流式细胞仪和干细胞异种移植来确定转导KOXII的MSCs是否 与未经修饰的MSCs相比，在推动HSC扩张方面更有效。最后，使用基于CRISPR/CAS9 以镰状细胞病患者的造血干细胞基因编辑为模型，我将确定是否 患者细胞与转导KOXII的MSCs共培养可提高基因编辑效率或 提高经过适当编辑的细胞的产量，而不是目前标准的HSC培养方法。同时，我 将使用小鼠KOXII转导的MSCs来更仔细地研究MSCs的利基信号。就像这些 细胞可以大量培养，是蛋白质组学研究的理想之选。在协作中 在Jeroen Krijgsveld的实验室里，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。使用体外和体内测试，我 将评估这些因素对HSC维持和增殖的影响，目的是 确定在MSC-HSC生态位相互作用中具有以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.