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

项目总结 成人胰腺非内分泌区祖细胞的存在 (导管/腺泡)几十年来一直被假设，但它们的特征被证明是难以捉摸的。我们 假设骨形态发生蛋白-7(一种具有转化生长因子-β抑制/骨形态发生蛋白双重激活功能的转化生长因子-β配体)将诱导 假定的常驻胰脏祖细胞的增殖。事实上，人类非内分泌组织的暴露 (HNEPT)到BMP-7导致集落的形成和生长，当BMP-7退出时，集落分化为 多个胰腺组织。体外谱系追踪显示hNEPT中的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，他们的胰岛素和其他成熟的胰腺标志物，包括碳酸氢酶II，都是阴性的。 (CAII，以前被认为是胰管标记物)。这些细胞可以使用ALK3(亮度分数)和 嘌呤能受体P2Y1(P2RY1)，PDX1细胞的替代表面标志。排序的P2RY1/ALK3bright 细胞可以在特定的条件下培养。它们通过扩展为PDX1/Nkx6.1来响应BMP-7 在骨形态发生蛋白-7作用下，分化为多种类型的胰腺细胞(包括β样细胞) 戒烟。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血统的标记物。我们进一步确定了解剖结构 人胰腺中PDX1/ALK3亮细胞在主胰管和胰管内的定位 腺体。 我们的目标是在胰腺组织的背景下剖析这些细胞的作用和生物学。 可塑性/再生，在健康和1型糖尿病(T1D)中都有。根据我们的数据，我们假设只有 特定的导管细胞亚群对ALK3激动剂的反应是通过增殖，并在此过程中断时 刺激，沿多个成人内分泌和外分泌胰腺谱系分化。为了测试这一点 假设及其多重后果，我们将追求以下具体目标：(1)高分辨率 用单细胞技术鉴定健康供者和T1D供者的胰腺导管亚群 RNAseq以及基于质谱学的无标记蛋白质组学。在这方面，我们还将描述 祖细胞龛中其他潜在的支持性细胞，包括那些位于导管间质的细胞，这些细胞可能具有 上皮祖细胞增殖/迁移/分化中的作用。(2)动态成像与表征 使用病毒转导的报告系统在活体人胰腺切片培养中再生现象的研究。 (3)健康对照和有代表性的T1D患者外周血祖细胞的特征和定量 疾病的不同阶段。(4)分选人体内再生能力的测定 将P2RY1/ALK3细胞移植到免疫缺陷啮齿动物体内。 我们的工作将为胰腺前体细胞的性质和能力提供新的线索，并可能提示 诱导β细胞原位再生的潜在干预措施。这些研究与以下几个主题相一致 这种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。

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.