Engineering erythropoietin-producing cells

摘要 肾脏是成年人产生促红细胞生成素(EPO)的主要来源。促红细胞生成素的损失 慢性肾脏疾病(CKD)患者的细胞导致红细胞产生抑制，是一种显著的 导致发病率的因素。虽然慢性肾脏病患者可以通过全身应用促红细胞生成素来治疗，但这会导致 零星和过度的激素供应高峰，这可能会为某些肿瘤提供生长刺激。在……里面 此外，对CKD患者给予重组EPO或EPO刺激剂(ESA)也是相关的 心血管事件的风险增加，包括心脏骤停和中风。目前还不清楚这些是否 红细胞增多症(红细胞增多症)是由促红细胞生成素或非靶向刺激引起的 促红细胞生成素本身的影响。最后，CKD患者使用重组促红细胞生成素的成本现在是 占医疗保险预算的很大比例。因此，有很强的临床和经济诱因 诱导多能干细胞(IPSC)来源的EPO产生细胞可用于治疗贫血 CKD患者。我们建议定义通过定向分化产生EPO产生细胞的条件 通过重述在发育中的肾脏中指定这种细胞类型的发育过程。 遗传学分析表明，肾脏EPO产生细胞(REPC)是一种间质成纤维细胞 在肾脏外髓的近端小管周围。RECs来源于Foxd1表达的基质 只有在肾脏发育活跃的时期才出现的前体群体。REPC人口 一直很难识别，因为EPO只在缺氧时表达，而其他标记物特异性地 确定这些细胞是否缺失。深入了解REPC的分子同一性对于 定义IPSC定向分化的目标状态。因此，我们建议：1.定义分子 通过基因标记和分离获得常氧内皮祖细胞的图谱，以及2.使用EPO报告基因芯片检测 类器官分化方案产生低氧反应的EPO产生细胞的能力。因为 产生EPO的细胞只在鼠标中空间定义，我们看到这种串联的方法使用 从转基因的人类ipscs分化而来的小鼠遗传工具和有机类化合物是必不可少的。这个 实验在技术上是可行的，我们小组拥有基质细胞生物学方面的专业知识，这将是必不可少的。 以发展分化条件。在短期内，我们的目标是通过以下方式对这些电池进行功能测试 将其包裹在微球中，并将其植入小鼠的腹腔内 肾切除术。从长远来看，我们的目标是将低氧反应、产生EPO的细胞整合到 用于临床移植的人造肾组织。

ABSTRACT The kidney is the major source of erythropoietin (EPO) production in adults. Loss of EPO-producing cells in chronic kidney disease (CKD) patients causes depressed red blood cell production and is a significant contributor to morbidity. Although CKD patients can be treated by systemic administration of EPO, this leads to sporadic and excessive peaks in hormone availability, which may provide growth stimuli to certain tumors. In addition, administration of recombinant EPO or EPO stimulating agents (ESAs) to CKD patients is associated with increased risk of cardiovascular events including cardiac arrest and stroke. It is not clear whether these morbidities are caused by excessive red blood cell number (polycythemia) stimulated by EPO or off-target effects of EPO itself. Finally, the cost of administering recombinant EPO to CKD patients now represents a significant proportion of the Medicare budget. Therefore, there are strong clinical and economic incentives for deriving an induced pluripotent stem cell (iPSC)-derived EPO-producing cell that could be used to treat anemic CKD patients. We propose to define conditions to generate EPO-producing cells by directed differentiation through recapitulating the developmental process that specifies this cell type within the developing kidney. Genetic analyses have revealed that the renal EPO-producing cell (REPC) is a stromal fibroblast located around proximal tubules in the outer medulla of the kidney. REPCs derive from the Foxd1-expressing stromal precursor population that is present only during the period of active kidney development. The REPC population has been difficult to identify because EPO is only expressed upon hypoxia, and other markers to specifically identify these cells are lacking. A deeper understanding of the molecular identity of the REPC is essential to define a target state for directed differentiation of iPSCs. We therefore propose to: 1. Define the molecular profile of normoxic REPCs through genetic labelling and isolation, and 2. Use EPO-reporter iPSCs to test the capacity of organoid differentiation protocols to generate hypoxia responsive EPO-producing cells. Because the EPO-producing cell has only been spatially defined in the mouse, we see this tandem approach using both mouse genetic tools and organoids differentiated from genetically modified human iPSCs as essential. The experiments are technically feasible, and our group has expertise in stromal cell biology, which will be essential to developing differentiation conditions. In the short term we would aim to functionally test these cells by encapsulating them in microspheres and implanting them into the abdominal cavities of mice subjected to nephrectomy. In the long term, we would aim to incorporate hypoxia-responsive, EPO-producing cells into synthetic kidney tissue destined for clinical translation.