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，肾脏会产生数百万的肾单位，从而产生高度专业化细胞。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产物和溶质导致膀胱排泄。出生缺陷在肾脏，所有出生中的1/100均具有所谓的肾脏和尿路的先天异常（Cakut）。在卡库特最严重的一端，新生儿缺少肾脏功能，预期寿命小于一年。大多数异常没有目前的有效干预措施，遗传变化缺乏背景。有因此，迫切需要了解发育缺陷的出现并生成新的疗法恢复或更换肾功能。在我们的工作中，我们使用了单细胞磁术和分子表征人和小鼠肾脏提供蓝图，以形成肾单位和地图以在人类中复制它干细胞衍生的肾脏器官。在这样做的过程中，我们为基因提供了遗传和发育环境在Cakut患者中鉴定。在此提案中，我们将遵循这些潜在客户并解决三个杰出问题发育肾脏科。在AIM 1中，我们研究了远端肾小管小管片段的胚胎起源。我们将进行第一个连接发育和成人肾脏的单细胞磁分析。这提供了路线图用于细胞分化的方式。我们将使用新的遗传小鼠谱系追踪工具来测试远端早期的细胞肾单位与成熟肾脏中的功能细胞有关。这些实验将映射需要基因的位置肾脏发展。在AIM 2中，我们将研究开关基因的蛋白质如何控制远端肾单位的发展。我们将使用一种称为Cut＆Run的技术来分析基因经常突变在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式与Cakut相关的基因。这将使我们能够直接研究远端肾单元细胞如何提供因果关系在基因表达和调节之间。我们将使用新系统从人类干细胞中的 - 同步肾病。在此系统中，肾尖头同时发展和步伐，与体内的许多发育阶段彼此相近形成的肾单位不同。我们的系统提供在同一发育阶段研究，操纵和分离细胞的独特优势。这我们收集的数据将显示基因如何激活。在目标3中，我们解决了发展中的一个基本问题肾脏学 - 肾脏最初是如何形成的？为此，我们将使用合成细胞组织者分泌信号蛋白来对我们的同步肾病进行模型。我们将研究信号配体如何控制肾单位形成和图案。这也具有实际的应用，因为我们可以控制肾病图案。我们的系统将为我们努力建立大量平行型肾病以替换疗法和疾病建模。该提议的优势是与人类和人类相交的独特专业知识小鼠肾脏遗传学，这是一种新型的干细胞衍生肾脏器官和合成生物学系统。