Engineering erythropoietin-producing cells

摘要 肾脏是成人产生促红细胞生成素 (EPO) 的主要来源。 EPO生成能力丧失 慢性肾病 (CKD) 患者体内的细胞会导致红细胞生成减少，并且是一个重要的因素。 发病率的贡献者。虽然 CKD 患者可以通过全身施用 EPO 来治疗，但这会导致 激素可用性的零星和过度峰值可能会刺激某些肿瘤的生长。在 此外，给 CKD 患者施用重组 EPO 或 EPO 刺激剂 (ESA) 与 心血管事件的风险增加，包括心脏骤停和中风。目前尚不清楚这些是否 疾病是由 EPO 刺激的红细胞数量过多（红细胞增多症）或脱靶引起的 EPO 本身的影响。最后，现在向 CKD 患者施用重组 EPO 的费用相当于 医疗保险预算的很大一部分。因此，有强烈的临床和经济动机 衍生出诱导多能干细胞 (iPSC) 衍生的 EPO 生成细胞，可用于治疗贫血 慢性肾病患者。我们建议定义通过定向分化产生 EPO 生成细胞的条件 通过重述发育过程，确定发育中肾脏内的这种细胞类型。 遗传分析表明，肾 EPO 生成细胞 (REPC) 是一种基质成纤维细胞，位于 肾外髓质近端小管周围。 REPCs源自表达Foxd1的基质 仅在肾脏发育活跃期间存在的前体群体。 REPC 人口 很难识别，因为 EPO 仅在缺氧时表达，而其他标记物则专门针对 确定这些细胞是否缺乏。更深入地了解 REPC 的分子特性对于 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来分析含氧量正常的 REPC 的概况，以及 2. 使用 EPO 报告基因 iPSC 来测试 类器官分化方案产生缺氧反应性 EPO 生成细胞的能力。因为 EPO 生成细胞仅在小鼠体内进行了空间定义，我们看到这种串联方法同时使用了 小鼠遗传工具和类器官与转基因人类 iPSC 的区别至关重要。的 实验在技术上是可行的，我们的团队拥有基质细胞生物学方面的专业知识，这将是必不可少的 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细胞进行功能测试： 将它们封装在微球中并将其植入小鼠的腹腔中 肾切除术。从长远来看，我们的目标是将缺氧反应性、生成 EPO 的细胞纳入 用于临床转化的合成肾组织。

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.