Delineating Determinants of Renal Tubulogenesis in an ECM-based Co-culture Model

 描述(申请人提供)：慢性肾脏疾病在美国是一个严重且日益严重的健康问题。患有终末期肾病的患者需要以透析或肾移植的形式进行肾脏替代治疗。虽然肾移植带来了更好的患者结果，而且比透析更具成本效益，但可移植供体肾脏的严重短缺限制了临床医生将这种疗法推广到所有有需要的患者的能力。解决肾脏短缺的一种方法是将组织工程、再生医学和干细胞科学的最新进展结合起来，开发实验室培养的生物人造器官。被剥离细胞成分或脱细胞的不可移植肾脏可以重新植入患者特有的干细胞，以根据需要生成免疫兼容的肾移植。虽然肾脏脱细胞已被证明在技术上是可行的，但目前对肾细胞外基质(ECM)在调节实质细胞归巢到肾脏特定的壁龛以及随后它们组织成肾单位的成熟成分方面所起的作用知之甚少。此外，尚不清楚急性损伤后肾小管上皮细胞和其他常驻支持细胞之间的相互作用如何起作用。 无论是适当的组织再生还是肾脏纤维化的病理发展，都是导致肾损害导致肾功能衰竭的重要原因。因此，我们的目标是利用脱细胞大鼠肾脏作为一个三维的仿生全器官培养系统来研究体外小管再生的过程。在致力于设计具有功能的肾单位的长期目标的同时，我们试图通过将精选的上皮细胞和基质细胞接种于肾脏ECM来更多地了解控制适当的小管再生的生理因素。我们假设，随着脱细胞肾基质内小管的发育，体外诱导的缺血再灌注损伤将产生一种再生状态，在这种状态下，我们可以研究肾小管上皮细胞与人身体来源的肾脏修复细胞或基质成纤维细胞之间的相互作用。为了验证这一假设，我们将使用灌流生物反应器，通过限制含氧培养基短时间间隔的灌流，在再细胞肾支架中创造暂时的低氧状态，以造成缺血损伤。然后，我们将研究肾修复细胞或间质成纤维细胞在调节肾小管上皮细胞修复中的不同作用。通过在仿生培养环境中将脱细胞肾脏支架与人类来源的肾脏细胞相结合，我们将研究人类肾脏支持细胞在组织再生过程中如何决定肾脏重塑和上皮细胞分化。了解导致慢性肾脏疾病的正常肾脏再生和纤维化的过程将使我们能够开发更有效的模型，并最终改进治疗肾脏疾病的方法。

 DESCRIPTION (provided by applicant): Chronic kidney disease is a significant and growing health problem in the United States. Patients who suffer from end-stage renal disease require renal replacement therapy in the form of either dialysis or kidney transplantation. Although kidney transplantation leads to superior patient outcomes and is more cost effective than dialysis, the severe shortage of transplantable donor kidneys limits the ability of clinicians to extend this therapy to all patients in need. One approach to overcoming the kidney shortage would be to combine recent advances in tissue engineering, regenerative medicine, and stem cell science to develop laboratory-grown, bioartificial organs. Non-transplantable kidneys that have been stripped of their cellular component, or decellularized, could be repopulated with patient-specific stem cells to generate an immunologically compatible kidney graft on demand. While kidney decellularization has been demonstrated as technically feasible, at present there is a limited understanding of the role the renal extracellular matrix (ECM) plays in regulating homing of parenchymal cells to particular niches of the kidney, and their subsequent organization into mature components of the nephron. Additionally, it is unclear how interactions between tubular epithelial cells and other resident support cells following acute injury contribute to either proper tissue regeneration or pathological development of renal fibrosis, a significant cause of kidney damage leading to renal failure. It is therefore our aim to study the process of tubule regeneration ex vivo using decellularized rat kidneys as a three-dimensional, biomimetic whole-organ culture system. While working toward the long-term goal of engineering a functional nephron, we seek to learn more about the physiological factors that govern appropriate tubule regeneration by seeding the renal ECM with select populations of epithelial cells and stromal cells. We hypothesize that induction of ischemic reperfusion injury ex vivo following the development of tubules within decellularized renal matrices will create a regenerative state, by which we can study interactions between tubular epithelial cells and human cadaver-derived renal repair cells or stromal fibroblasts. To test this hypothesis, we will use perfusion bioreactors to create a temporary hypoxic state in recellularized kidney scaffolds, by limiting perfusion of oxygenated culture medium for a short time interval to create ischemic damage. We will then investigate the differential roles of renal repair cells or stromal fibroblast in modulating tubular epithelial cell repair. By combining decellularized kidney scaffolds with human-derived renal cells within a biomimetic culture environment, we will investigate how human renal support cells dictate both kidney remodeling and epithelial cell differentiation during tissue regeneration. Understanding the processes of normal kidney regeneration and fibrogenesis leading to chronic kidney disease will allow us to develop more effective models, and ultimately improve therapies for treating renal disease.