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

• 批准号: 9788440
• 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
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-25 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9788440
• 关键词: Activins; Adult; Agonist; Anatomy; BMP7 gene; BMPR1A gene; Basic Science; Beta Cell; Bioinformatics; Bromodeoxyuridine; Carbonic Anhydrase II; Cell Communication; Cell Proliferation; Cells; Cellular biology; Coupled; Data; Development; Dimensions; Disease; Duct (organ) structure; Ductal; 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; Reporter; Reporting; Research; Research Support; Resolution; Rodent; Role; Sampling; Signal Transduction; Slice; Stem cells; 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 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; tool; transcriptome sequencing

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 成人胰腺非内分泌区存在祖细胞 （导管/腺泡）已经假设了几十年，但它们的特征已被证明是难以捉摸的。我们 假设BMP-7（一种具有双重TGF-β抑制/BMP激活能力的TGF-β配体）将诱导 假定的常驻胰腺祖细胞的增殖。事实上，人体非内分泌组织的暴露 （hNEPT）与BMP-7的结合导致集落的形成和生长，在BMP-7撤除后，集落分化为 多处胰腺组织体外谱系追踪显示hNEPT中BMP-7反应细胞表达 胰腺十二指肠同源框（PDX1）和BMP受体1A（也称为激活素样激酶-3，ALK 3）。 然而，他们的胰岛素和其他成熟的胰腺标志物，包括碳酸酐酶II阴性 (CAII以前认为是一种泛导管标记）。这些细胞可以使用ALK 3（亮级分）分选， 嘌呤能受体P2Y1（P2RY1），PDX1+细胞的替代表面标志物。已排序P2 RY 1 +/ALK 3bright + 细胞可以在确定的条件下培养。它们通过PDX1 +/NKX6.1+扩增对BMP-7作出反应 祖细胞样集落，并在BMP-7作用下分化为多种胰腺细胞类型（包括β样细胞） 戒断qRT-PCR和RNAseq进一步证实了BMP-7诱导的转录激活抑制作用 分化（ID）蛋白与祖细胞增殖，以及上调 BMP-7撤除后所有成年胰腺谱系的标志物。我们进一步确定了 人胰腺中PDX1 +/ALK 3bright+细胞在主胰管和胰管内的位置 腺体 我们的目标是在胰腺组织中解剖这些细胞的作用和生物学 可塑性/再生，无论是在健康和1型糖尿病（T1D）。根据我们的数据，我们假设只有 特定的导管细胞亚群通过增殖响应ALK 3激动，并且在这种增殖中断时， 刺激，沿沿着多种成体胰腺内分泌和外分泌谱系分化。为了验证这一 假设及其多重分支，我们将追求以下具体目标：（1）高分辨率 通过单细胞免疫分析表征从健康和T1D供体获得的胰腺导管亚群 RNAseq以及基于质谱的无标记蛋白质组学。在此背景下，我们还将 祖细胞龛中的其他潜在支持细胞，包括导管基质中可能具有 在上皮祖细胞增殖/迁移/分化中的作用。(2)动态成像和表征 的再生现象，使用病毒转导的报告系统在活的人胰腺切片培养。 (3)健康对照和T1D代表性样本中祖细胞的表征和定量 疾病的不同阶段。(4)测定分选的人骨髓基质细胞的体内再生能力 P2RY1/ALK 3bright细胞移植到免疫缺陷啮齿类动物中。 我们的工作将为胰腺祖细胞的性质和能力提供新的线索， 潜在的干预以诱导β细胞原位再生。这些研究与以下几个主题保持一致： 这种RFA，如果成功的话，将为BMPR激动剂的临床前表征铺平道路， 治疗引导。