HIGH-RESOLUTION CHARACTERIZATION OF HUMAN DUCTAL PROGENITOR CELLS AND THEIR REGENERATION POTENTIAL

人类导管祖细胞及其再生潜力的高分辨率表征

基本信息

批准号：
10252070
负责人：
Juan Dominguez-Bendala
金额：
$ 23.03万
依托单位：
UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-25 至 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10252070
关键词：
3-Dimensional Activins Adult Agonist Anatomy BMP7 gene BMPR1A gene Basic Science Beta Cell Bioinformatics Bromodeoxyuridine Carbonic Anhydrase II Cell Communication Cells Cellular biology Coupled Data Development Disease Duct (organ) structure Ductal Epithelial Cell Duodenum Endocrine Epithelial Equilibrium Exocrine pancreas Future Gland Goals Growth Health Homeobox Human Image Immunodeficient Mouse In Situ In Vitro Insulin Insulin-Dependent Diabetes Mellitus Interruption Intervention Islets of Langerhans Label Ligands Light Link Location Mass Spectrum Analysis Mission Molecular Mus National Institute of Diabetes and Digestive and Kidney Diseases Natural regeneration Nature Non-Insulin-Dependent Diabetes Mellitus Pancreas Pancreatic duct Patients Phosphotransferases Population Process Proliferating Proteins Proteomics Public Health Publishing Purinoceptor Quantitative Reverse Transcriptase PCR Rattus Regenerative capacity Reporter Reporting Research Research Support Resolution Rodent Role Sampling Signal Transduction Slice Stimulus Structure Surface System Testing Therapeutic Time Tissues Transcriptional Activation Transforming Growth Factor alpha Transforming Growth Factor beta Transgenic Organisms Translations Transplantation Up-Regulation Viral Withdrawal Work base body system bone morphogenetic protein receptors cell regeneration cell type cellular targeting design exposed human population illness length in vivo in vivo regeneration islet islet stem cells migration mouse model new technology novel perinatal period pre-clinical progenitor purinoceptor P2Y1 regeneration potential regenerative response single-cell RNA sequencing stem cell proliferation stem cells tool transcriptome sequencing

项目摘要

PROJECT SUMMARY The existence of progenitor cells in the non-endocrine compartments of the adult human pancreas (ductal/acinar) has been hypothesized for decades, but their characterization has proven elusive. We hypothesized that BMP-7 (a TGF-β ligand with dual TGF-β inhibition/BMP activation abilities) would induce the proliferation of putative resident pancreatic progenitors. Indeed, exposure of human non-endocrine tissue (hNEPT) to BMP-7 led to the formation and growth of colonies that, upon BMP-7 withdrawal, differentiated into multiple pancreatic tissues. In vitro lineage tracing showed that BMP-7-responsive cells in hNEPT express pancreatic duodenal homeobox (PDX1) and the BMP receptor 1A (also known as activin-like kinase-3, ALK3). However, they were negative for insulin and other mature pancreatic markers, including carbonic anhydrase II (CAII, previosuly considered a panductal marker). These cells can be sorted using ALK3 (bright fraction) and the purinergic receptor P2Y1 (P2RY1), a surrogate surface marker for PDX1+ cells. Sorted P2RY1+/ALK3bright+ cells can be cultured in defined conditions. They respond to BMP-7 by expanding as PDX1+/NKX6.1+ progenitor-like colonies, and differentiate into multiple pancreatic cell types (including β-like cells) upon BMP-7 withdrawal. qRT-PCR and RNAseq further confirmed the BMP-7-induced transcriptional activation of Inhibition of Differentiation (ID) proteins associated with progenitor cell proliferation, as well as the upregulation of markers of all adult pancreatic lineages following BMP-7 withdrawal. We have further identified the anatomic location of PDX1+/ALK3bright+ cells in the human pancreas within the major pancreatic ducts and pancreatic duct glands. Our goal is to dissect the role and biology of these cells in the context of pancreatic tissue plasticity/regeneration, both in health and type 1 diabetes (T1D). Based on our data, we hypothesize that only specific subsets of ductal cells respond to ALK3 agonism by proliferating and, upon interruption of this stimulation, differentiating along multiple adult endocrine and exocrine pancreatic lineages. To test this hypothesis and its multiple ramifications, we will pursue the following specific aims: (1) High-resolution characterization of pancreatic ductal sub-populations obtained from healthy and T1D donors by single-cell RNAseq as well as mass spectrometry-based label-free proteomics. In this context, we will also characterize other potentially supportive cells in the progenitor cell niche, including those at the ductal stroma that may have roles in proliferation/migration/differentiation of epithelial progenitors. (2) Dynamic imaging and characterization of regeneration phenomena using virally-transduced reporter systems in live human pancreatic slice cultures. (3) Characterization and quantification of progenitor cells in healthy controls and T1D samples representative of various stages of the disease. (4) Determination of the in vivo regeneration ability of sorted human P2RY1/ALK3bright cells upon transplantation into immunodeficient rodents. Our work will shed new light on the nature and capabilities of pancreatic progenitor cells, and may suggest potential interventions to induce β-cell regeneration in situ. These studies are aligned with several themes of this RFA and, if successful, will pave the way to the preclinical characterization of BMPR agonists as therapeutic leads.

项目概要成人胰腺非内分泌区室中祖细胞的存在（导管/腺泡）已经发展了数十年，但其特征却难以捉摸。刺激 BMP-7（一种具有双重 TGF-β 抑制/BMP 激活能力的 TGF-β 配体）会诱导假定的常驻胰腺祖细胞的增殖确实与人类非内分泌组织的暴露有关。 (hNEPT) 至 BMP-7 导致集落的形成和生长，在 BMP-7 撤除后，集落分化为体外谱系追踪显示 hNEPT 中 BMP-7 反应细胞表达。胰腺十二指肠同源盒 (PDX1) 和 BMP 受体 1A（也称为激活素样激酶 3，ALK3）。然而，它们对胰岛素和其他成熟胰腺标志物（包括碳酸酐酶 II）呈阴性（CAII，以前被认为是全导管标记）可以使用 ALK3（亮部分）和嘌呤能受体 P2Y1 (P2RY1)，PDX1+ 细胞的替代表面标记。细胞可以在规定的条件下培养，它们通过扩增为 PDX1+/NKX6.1+ 来响应 BMP-7。祖细胞样集落，并在 BMP-7 上分化为多种胰腺细胞类型（包括 β 样细胞） qRT-PCR 和 RNAseq 进一步证实了 BMP-7 诱导的转录激活抑制。与祖细胞增殖相关的分化（ID）蛋白，以及上调 BMP-7 停用后所有成人胰腺谱系的标记物我们进一步确定了解剖学。人胰腺中 PDX1+/ALK3bright+ 细胞在主胰管和胰管内的位置腺体。我们的目标是剖析这些细胞在胰腺组织中的作用和生物学健康和 1 型糖尿病 (T1D) 中的可塑性/再生根据我们的数据，我们仅捕获了这一点。特定的导管细胞亚群通过增殖来响应 ALK3 激动剂，并且在增殖中断后刺激，沿着多个成人内分泌和外分泌胰腺谱系进行分化来测试这一点。假设及其多重后果，我们将追求以下具体目标：（1）高分辨率通过单细胞对从健康和 T1D 供体获得的胰腺导管亚群进行表征在这种情况下，我们还将表征 RNAseq 以及基于质谱的无标记蛋白质组学。祖细胞生态位中的其他潜在支持细胞，包括那些可能具有导管基质的细胞 (2) 动态成像与表征使用病毒转导的报告系统在活人胰腺切片培养物中进行再生现象的研究。 (3) 健康对照和 T1D 代表样本中祖细胞的表征和定量 (4)分选人体内再生能力的测定 P2RY1/ALK3bright 细胞移植至免疫缺陷啮齿动物后。我们的工作将为胰腺祖细胞的性质和能力提供新的线索，并可能表明这些研究与诱导 β 细胞原位再生的潜在干预措施相一致。该 RFA，如果成功，将为 BMPR 激动剂的临床前表征铺平道路：治疗线索。