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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：
10186697
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 2022-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10186697
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 Epithelium 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 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 migration mouse model new technology novel perinatal period pre-clinical progenitor purinoceptor P2Y1 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.

项目总结