Engineering erythropoietin-producing cells

工程化促红细胞生成素生成细胞

• 批准号: 9516535
• 负责人: Thomas Joseph Carroll
• 金额: $ 29.16万
• 依托单位: MAINEHEALTH
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-15 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9516535
• 关键词: Abdominal Cavity; Adult; Alpha Cell; Anemia; Biological Assay; Budgets; Cell Count; Cell Differentiation process; Cell Line; Cell Therapy; Cells; Cellular biology; Chronic Kidney Failure; Clinical; Depressed mood; Developmental Process; Encapsulated; Engineering; Enterobacteria phage P1 Cre recombinase; Erythrocytes; Erythropoietin; Event; Fibroblasts; Frequencies; Future; Genetic; Genetic Transcription; Goals; Growth; Heart Arrest; Hormones; Human; Hypoxia; Implant; Investigation; Kidney; Label; Location; Medicare; Microspheres; Molecular; Molecular Profiling; Morbidity - disease rate; Mouse Strains; Mus; Nephrectomy; Organoids; Patients; Polycythemia; Population; Production; Protocols documentation; Recombinant Erythropoietin; Reporter; Reporting; Source; Specific qualifier value; Stimulus; Stroke; Stromal Cells; Tamoxifen; Technology; Testing; Tissues; Variant; adult stem cell; cardiovascular risk factor; cell type; clinical translation; cost; economic incentive; experimental study; genetic analysis; induced pluripotent stem cell; interest; kidney cell; kidney medulla; nephrogenesis; precursor cell; tool; transcriptome; transcriptome sequencing; tumor; Abdominal Cavity; Adult; Alpha Cell; Anemia; Biological Assay; Budgets; Cell Count; Cell Differentiation process; Cell Line; Cell Therapy; Cells; Cellular biology; Chronic Kidney Failure; Clinical; Depressed mood; Developmental Process; Encapsulated; Engineering; Enterobacteria phage P1 Cre recombinase; Erythrocytes; Erythropoietin; Event; Fibroblasts; Frequencies; Future; Genetic; Genetic Transcription; Goals; Growth; Heart Arrest; Hormones; Human; Hypoxia; Implant; Investigation; Kidney; Label; Location; Medicare; Microspheres; Molecular; Molecular Profiling; Morbidity - disease rate; Mouse Strains; Mus; Nephrectomy; Organoids; Patients; Polycythemia; Population; Production; Protocols documentation; Recombinant Erythropoietin; Reporter; Reporting; Source; Specific qualifier value; Stimulus; Stroke; Stromal Cells; Tamoxifen; Technology; Testing; Tissues; Variant; adult stem cell; cardiovascular risk factor; cell type; clinical translation; cost; economic incentive; experimental study; genetic analysis; induced pluripotent stem cell; interest; kidney cell; kidney medulla; nephrogenesis; precursor cell; tool; transcriptome; transcriptome sequencing; tumor

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.

抽象的 肾脏是成人促红细胞生成素（EPO）的主要来源。丧失EPO产生 慢性肾脏疾病（CKD）患者的细胞会导致红细胞降低，并且是重要的 发病率的贡献者。尽管CKD患者可以通过全身性EPO治疗，但这导致 激素可用性的零星和过度峰值，这可能为某些肿瘤提供生长刺激。在 此外，对CKD患者的重组EPO或EPO刺激剂（ESA）的施用与 随着心血管事件（包括心脏骤停和中风）的风险增加。目前尚不清楚这些是否 病毒性是由EPO或靶向刺激的过度红细胞数（多余细胞）引起的 EPO本身的影响。最后，对CKD患者进行重组EPO的成本现在代表 医疗保险预算的很大比例。因此，有强烈的临床和经济激励措施 推导诱导多能干细胞（IPSC）衍生的EPO生产细胞，可用于治疗贫血 CKD患者。我们建议定义条件以通过定向分化生成EPO产生细胞 通过概括开发过程，该过程指定了发育中的肾脏中的这种细胞类型。 遗传分析表明，肾EPO产生细胞（REPC）是位于基质成纤维细胞 肾脏外髓质中的近端小管周围。 RETC源自表达FOXD1的基质 仅在主动肾脏发育期间存在的前体人群。 RETC人口 很难识别，因为EPO仅在缺氧方面表达，而其他标记是专门的 识别这些细胞缺乏。对RETC的分子身份有更深入的了解对于 定义针对IPSC分化的目标状态。因此，我们建议：1。定义分子 通过遗传标记和隔离的差异retc的曲线，以及2。使用Epo-Reporter IPSC测试 器官分化方案的能力产生缺氧反应性EPO产生细胞的能力。因为 EPO生产单元仅在鼠标中是空间定义的，我们看到了这种串联方法 小鼠遗传工具和类器官与基因修饰的人IPSC区分开来是必不可少的。这 实验在技术上是可行的，我们的小组在基质细胞生物学方面具有专业知识，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 将它们封装在微球中，并将其植入遭受的小鼠的腹腔 肾切除术。从长远来看，我们的目标是将缺氧反应性的EPO产生细胞纳入 致力于临床翻译的合成肾组织。