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.

项目摘要成年人类胰腺的非内分泌室中祖细胞的存在（导管/腺泡）已经假设了数十年，但它们的特征被证明难以捉摸。我们假设BMP-7（具有双重TGF-β的TGF-β配体/BMP激活能力）将诱导推定居民胰腺祖细胞的扩散。实际上，人类非内分泌组织的暴露（Hnept）到BMP-7导致菌落的形成和生长，而BMP-7退出后，将其分化为多个胰组织。体外谱系跟踪表明，Hnept Express中的BMP-7反应细胞胰腺十二指肠同型（PDX1）和BMP受体1a（也称为激活素样激酶-3，ALK3）。但是，它们对胰岛素和其他成熟的胰腺标志物（包括碳酸酐酶II）呈阴性（CAII，被认为是panducal标记）。可以使用ALK3（明亮的分数）对这些单元进行分类，并且嘌呤能受体P2Y1（P2RY1），PDX1+细胞的替代表面标记。排序的p2ry1+/alk3bright+ 细胞可以在定义的条件下进行培养。他们通过扩展为PDX1+/NKX6.1+来响应BMP-7 祖细胞状菌落，并分化为BMP-7的多种胰腺细胞类型（包括β样细胞）提取。 QRT-PCR和RNASEQ进一步证实了BMP-7诱导的抑制转录激活与祖细胞增殖相关的分化（ID）蛋白质，以及 BMP-7退出后所有成年胰腺谱系的标记。我们进一步确定了解剖学 PDX1+/Alk3bright+细胞在主要胰管和胰管内的人胰腺中的位置腺体。我们的目标是在胰组织中剖析这些细胞的作用和生物学可塑性/再生，无论是健康还是1型糖尿病（T1D）。根据我们的数据，我们假设只有导管细胞的特定子集通过增殖和中断时对ALK3激动剂作出反应刺激，沿多个成人内分泌和外分泌胰腺谱系区分。测试这个假设及其多重影响，我们将追求以下具体目的：（1）高分辨率通过单细胞从健康和T1D供体获得的胰腺导管亚种群的表征 RNASEQ以及基于质谱的无标签蛋白质组学。在这种情况下，我们还将表征祖细胞细胞生态位中的其他潜在受支持的细胞，包括可能具有的导管基质的细胞上皮祖细胞增殖/迁移/分化中的作用。（2）动态成像和表征在人类胰腺切片培养物中使用虚拟转导的记者系统的再生现象。（3）健康对照和T1D样品中祖细胞的表征和定量代表疾病的各个阶段。（4）确定分类人的体内再生能力 P2RY1/ALK3BRIGHT细胞移植到免疫缺陷啮齿动物中。我们的工作将为胰腺祖细胞的性质和能力提供新的启示，并可能建议潜在的干预措施以诱导原位β细胞再生。这些研究与几个主题保持一致这种RFA，如果成功的话，将为BMPR激动剂的临床前描述为治疗铅。