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 成年人类胰腺的非内分泌室中祖细胞的存在 （尘土/腺泡）已经假设了数十年，但它们的特征被证明是难以捉摸的 假设BMP-7（具有双重TGF-β-β-β-β吸入/BMP激活能力的TGF-β配体）会诱导您 假定的胰腺祖先的扩散。 （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 多疗法胰组织。 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成熟胰腺标记呈阴性，包括碳赤霉素II （CAII，previosury认为是panductal标记）。 Purinergic受体P2Y1（P2RY1），PDX1+细胞的替代表面标记 细胞可以在定义的条件下培养。 祖细胞状菌落，并分化为BMP-7的多裂胰细胞类型（包括β样细胞） 提取。 与祖细胞泛滥相关的区分（ID）蛋白质以及上升 BMP-7撤回后所有成年胰腺谱系的标记。 PDX1+/Alk3bright+细胞在主要胰腺尘埃和胰腺灰尘中的人脉络中的位置 腺体。 我们的目标是剖析胰腺的角色和生物学 塑料/再生，无论是健康还是1型糖尿病（T1D）。 导管细胞的细节子集通过增殖和中断时对Alk3激动剂做出反应 刺激，沿多个成人内分泌和外分泌胰腺谱系进行区分 假设及其多重影响，我们将追求以下具体目的：（1）高分辨率 通过单细胞的胰腺导管亚洪流亚pop-In健康和T1D供体的表征 RNASEQ以及基于质谱的无标签蛋白质组学 在 在上皮祖细胞的殖民/迁移/差异上的作用。 使用Viral-Transdoded Reporter系统的再生现象中的现象中的活体胰腺切片培养物。 （3）在健康对照和T1D样品中对祖细胞的表征和定量代表 疾病的各个阶段。 P2RY1/ALK3BRIGHT细胞移植到免疫缺陷啮齿动物中。 我们的工作意志将阐明胰腺祖细胞的性质和能力的新灯，并可能建议 诱导β细胞再生的潜在干预措施与几个主题一致 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。