Establishing and Mimicking Patterning Mechanisms in the Distal Nephron Tubule and Kidney Organoid

建立和模拟远端肾小管和肾类器官的模式机制

基本信息

批准号：
10719178
负责人：
Nils Olof Lindstrom
金额：
$ 65.02万
依托单位：
UNIVERSITY OF SOUTHERN CALIFORNIA
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-08-01 至 2028-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10719178
关键词：
Address Adopted Adult Automobile Driving Birth Bladder Blood Pressure Blood flow Body Fluids Cell Differentiation process Cell Lineage Cell Separation Cells Childhood Chromosome Mapping Clinical Confusion Congenital Abnormality Cues DNA Data Defect Development Developmental Anatomy Dialysis procedure Disease model Distal Dose Drainage procedure Embryo End stage renal failure Ensure Etiology Excretory function Fluid Balance Gene Expression Gene Expression Regulation Genes Genetic Genetic Markers Genetic Transcription Goals Human In Vitro Infant Kidney Kidney Diseases Life Life Expectancy Ligands Link Maps Mediating Molecular Mouse Strains Mus Mutate Mutation Nephrology Nephrons Newborn Infant Organ Transplantation Organoids Patients Pattern Physiological Physiology Population Production Proteins Protocols documentation Publishing Regulation Regulator Genes Renal function Replacement Therapy Running Series Signal Pathway Signal Transduction Signal Transduction Pathway Signaling Protein Sodium Chloride Specific qualifier value System Techniques Testing Therapeutic Time Transcriptional Regulation Urinary tract Urine Validation Variant Water Work absorption beta catenin blood filtration blood pressure control cell replacement therapy cell type congenital anomalies of the kidney diagnostic tool effective intervention experimental study fetal human stem cells improved in vitro Model in vivo induced pluripotent stem cell insight molecular modeling mortality nephrogenesis novel novel therapeutics practical application programs regenerative therapy solute stem cell model stem cells synthetic biology tool transcription factor

项目摘要

During embryonic and fetal stages, the kidneys develop millions of nephrons that generate highly specialized cells. These cells ensure that blood flowing into the kidney is filtered and required substances are reabsorbed while unwanted metabolites and solutes are led to the bladder for excretion. Birth defects are common in the kidney, ~ 1/100 of all births have a so-called Congenital Anomaly of the Kidney and Urinary Tract (CAKUT). At the most severe end of CAKUT, newborns are missing kidney functionality, and their life expectancy is less than one year. Most abnormalities have no current effective interventions and genetic changes lack context. There is thus a critical need to understand where developmental defects arise and to generate new therapies restoring or replacing kidney function. In our work we have used single cell omics and molecular characterizations of human and mouse kidneys to provide a blueprint for how nephrons form and maps for to replicate this in human stem cell-derived kidney organoids. In doing so we provide a genetic and developmental context to genes identified in CAKUT patients. In this proposal we will follow these leads and address three outstanding questions in developmental nephrology. In Aim 1, we investigate the embryonic origins of distal nephron tubule segments. We will perform the first single cell omic analysis linking developing and adult kidneys. This provides a roadmap for how cells differentiate. We will use new genetic mouse lineage-tracing tools to test how cells in the early distal nephron relate to functional cells in mature kidneys. These experiments will map where genes are required as the nephron develops. In Aim 2, we will investigate how proteins that turn genes on and off control the development of the distal nephron. We will use a technique called Cut&Run to analyze how genes often mutated in CAKUT, control DNA and gene expression. We will also activate signaling pathways and alter the expression of genes linked to CAKUT. This will allow us to directly study how distal nephron cells form provide causality between gene expression and regulation. We will use our new system to generate hundreds of nephrons from human stem cells in - synchronized nephroids. In this system, nephrons develop at the same time and pace, unlike in the body where nephrons from many developmental stages form near each other. Our system provides a unique advantage to study, manipulate, and isolate cells from nephrons at the same developmental stage. The data we collect will show how genes are activated. In Aim 3 we address a fundamental question in developmental nephrology - how is the nephron initially patterned? To do this we will use synthetic cellular organizers that secrete signaling proteins to pattern our synchronized nephroids. We will study how signal ligands control nephron formation and patterning. This also has a practical application as we can gain control over nephroid patterning. Our system will inform our efforts to build massive parallel arrays of nephroids for replacement therapies and disease modeling. A strength of the proposal is the unique expertise intersecting human and mouse kidney genetics, a novel system of stem cell derived kidney organoids, and synthetic biology.

在胚胎和胎儿阶段，肾脏发育出数百万个肾单位，产生高度专业化的肾单位细胞。这些细胞确保流入肾脏的血液被过滤并重新吸收所需的物质而不需要的代谢物和溶质则被引导至膀胱排泄。出生缺陷常见于肾脏，约 1/100 的新生儿患有所谓的肾脏和尿路先天性异常 (CAKUT)。在 CAKUT 最严重的结局是，新生儿丧失肾功能，并且他们的预期寿命低于一年。大多数异常目前没有有效的干预措施，并且基因变化缺乏背景。有因此，迫切需要了解发育缺陷出现的位置，并制定新的疗法来恢复发育缺陷。或替代肾功能。在我们的工作中，我们使用了单细胞组学和分子特征人类和小鼠肾脏，为肾单位如何形成提供蓝图，并绘制在人类中复制这一点的蓝图干细胞衍生的肾脏类器官。在此过程中，我们为基因提供了遗传和发育背景在 CAKUT 患者中发现。在本提案中，我们将遵循这些线索并解决三个悬而未决的问题在发育肾病学中。在目标 1 中，我们研究远端肾单位小管段的胚胎起源。我们将进行首次连接发育中肾脏和成年肾脏的单细胞组学分析。这提供了一个路线图了解细胞如何分化。我们将使用新的小鼠遗传谱系追踪工具来测试早期远端细胞如何肾单位与成熟肾脏中的功能细胞有关。这些实验将绘制出需要基因的位置：肾单位发育。在目标 2 中，我们将研究开启和关闭基因的蛋白质如何控制远端肾单位的发育。我们将使用一种称为 Cut&Run 的技术来分析基因如何经常突变在 CAKUT 中，控制 DNA 和基因表达。我们还将激活信号通路并改变表达与 CAKUT 相关的基因。这将使我们能够直接研究远端肾单位细胞的形成方式并提供因果关系基因表达和调控之间。我们将使用我们的新系统生成数百个肾单位同步肾病中的人类干细胞。在这个系统中，肾单位同时和同步发育，与体内不同，来自多个发育阶段的肾单位彼此靠近形成。我们的系统提供具有在同一发育阶段研究、操作和分离肾单位细胞的独特优势。这我们收集的数据将显示基因是如何被激活的。在目标 3 中，我们解决了发展中的一个基本问题肾脏病学 - 肾单位最初是如何形成的？为此，我们将使用合成细胞组织者分泌信号蛋白来模拟我们的同步肾病。我们将研究信号配体如何控制肾单位的形成和模式。这也有实际应用，因为我们可以控制肾病图案化。我们的系统将为我们构建大规模并行肾细胞阵列以进行替换的努力提供信息疗法和疾病建模。该提案的一个优势是独特的专业知识交叉人类和小鼠肾脏遗传学、干细胞衍生的肾脏类器官的新系统以及合成生物学。