Single-cell longitudinal analysis of regeneration in human pancreatic slices

人胰腺切片再生的单细胞纵向分析

• 批准号: 10336196
• 负责人: Juan Dominguez-Bendala
• 金额: $ 38.38万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-20 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10336196
• 关键词: Adult; Antibodies; Architecture; B-Lymphocytes; BMPR1A gene; Beta Cell; Bioinformatics; Biological Process; Biomedical Engineering; Cells; Characteristics; Coupled; Development; Diabetes Mellitus; Dissection; Duct (organ) structure; Ductal Epithelial Cell; Endocrine; Event; Exposure to; Goals; Human; Immune; In Vitro; Inflammation; Insulin-Dependent Diabetes Mellitus; Leadership; Maps; Mediating; Mission; Modeling; Mus; National Institute of Diabetes and Digestive and Kidney Diseases; Natural regeneration; Nature; Non-Insulin-Dependent Diabetes Mellitus; Organ; Pancreas; Pancreatic Diseases; Pancreatic duct; Pathway interactions; Perinatal; Population; Process; Proliferating; Public Health; Regenerative capacity; Regenerative research; Research; Research Design; Research Institute; Resolution; Signal Transduction; Slice; Sorting - Cell Movement; Source; Stress; Structure of beta Cell of islet; Surface; System; Techniques; Testing; Therapeutic; Time; Tissues; Transgenic Organisms; Transplantation; Trees; Visualization; Withdrawal; Xenograft procedure; base; bone morphogenetic protein receptors; cell regeneration; cell type; design; developmental plasticity; diabetes mellitus therapy; diabetic; differential expression; experimental study; human model; in vivo; in vivo regeneration; islet; longitudinal analysis; method development; mouse model; movie; multidisciplinary; new technology; novel; novel therapeutic intervention; novel therapeutics; organ on a chip; progenitor; real time monitoring; regeneration potential; response; single cell analysis; stem cells; success; tool; transcriptomics; virtual

PROJECT SUMMARY We have described a subpopulation of human pancreatic ductal cells with progenitor-like characteristics. These cells can be sorted using antibodies against the BMP receptor 1A (ALK3bright+) and P2RY1, a surrogate surface marker for PDX1 identified by our team. ALK3bright+/P2RY1+ cells proliferate when exposed to BMP-7, and differentiate into all adult pancreatic cell types, including functional β-cells, upon BMP-7 withdrawal. scRNAseq of the ALK3bright+ fraction of human pancreatic donors confirmed the existence of clusters with progenitor-like features, with substantial evidence suggesting that such features may be acquired by de- differentiation. When transplanted into immune-deficient mice, sorted cell populations enriched in markers differentially expressed in these clusters self-organize into ‘micro-pancreata’ with native-like cytoarchitecture and functional endocrine cells. More recently, we have developed the means to study the dynamic processes of progenitor cell-dependent regeneration using human pancreatic slices (HPSs). We have achieved functional long-term (>10d) culture of HPSs, which allows for the longitudinal tracking of β-cell regeneration in a setting that is widely considered the closest approximation in vitro to a native pancreas. BMP signaling-dependent regeneration was established in slices from healthy and, more importantly, T1D and T2D donors. Numerous single-cell analyses of the pancreas, included our own, support the emerging view that specific lineages are in a state of flux between differentiation stages. However, all these analyses only offer a snapshot of the tissue at any given time point. Conclusions about potential developmental/regeneration paths are exclusively based on bioinformatics inferences. We hypothesize that the combination of long-term organotypic culture with lineage-tracing in vitro and sequential single-cell analyses will allow us, for the first time, to dynamically map cell fate changes –e.g., by conducting longitudinal scRNAseq of slices from the same donor across multiple time points after BMP-7 addition. Furthermore, we contend that this system will enable the real- time visualization and in-depth, single-cell resolution study of potential de-differentiation events, should they happen in response to different sources of stress in human slices. These approaches offer a wealth of new research possibilities that were downright unfeasible prior to the development of these methods. Our research design is expected to help us realize the full potential of single-cell transcriptomics to unveil dynamic biological processes, model human pancreatic disease, and, ultimately, enable the development of novel therapeutic approaches to induce regeneration. We will test the above hypotheses by pursuing the following specific aims: (1) Longitudinal scRNAseq of same-donor T1D/T2D HPSs following stimulation of the BMP pathway. (2) Study of the effect of stress, inflammation and de-differentiation on BMP-7-mediated induction of β-cell formation in HPSs; (3) Functional characterization of neogenic β-cells through “slice-on-a-chip” approaches; and (4) Determination of in vivo regeneration potential of progenitor cells sorted from healthy and T1D/T2D donors using a xenotransplantation model.

项目概要 我们描述了具有祖细胞样特征的人类胰腺导管细胞亚群。 可以使用针对 BMP 受体 1A (ALK3bright+) 和 P2RY1（替代品）的抗体对这些细胞进行分选 我们的团队鉴定出 PDX1 的表面标记。当暴露于 BMP-7 时，ALK3bright+/P2RY1+ 细胞会增殖， 并在 BMP-7 撤除后分化为所有成体胰腺细胞类型，包括功能性 β 细胞。 人类胰腺供体 ALK3bright+ 部分的 scRNAseq 证实存在以下簇： 祖样特征，有大量证据表明这些特征可能是通过去 差异化。当移植到免疫缺陷小鼠体内时，分选的细胞群富含标记物 这些簇中差异表达的细胞自组织成具有类似天然细胞结构的“微胰腺” 有功能的内分泌细胞。最近，我们开发了研究动态过程的方法 使用人胰腺切片（HPS）进行祖细胞依赖性再生。我们已经实现了功能化 HPS 的长期（>10 天）培养，允许在一定环境中纵向跟踪 β 细胞再生 这被广泛认为在体外最接近天然胰腺。 BMP 信号依赖 再生是在健康捐献者、更重要的是 T1D 和 T2D 捐献者的切片中建立的。 对胰腺的大量单细胞分析（包括我们自己的分析）支持新出现的观点，即特定的 谱系在分化阶段之间处于不断变化的状态。然而，所有这些分析都只提供了一个快照 任何给定时间点的组织。关于潜在发展/再生路径的结论是 完全基于生物信息学推论。我们假设长期器官型的组合 体外谱系追踪培养和连续单细胞分析将使我们第一次能够 动态绘制细胞命运变化图谱——例如，通过对来自同一供体的切片进行纵向 scRNAseq 添加 BMP-7 后的多个时间点。此外，我们认为该系统将使真正的 潜在去分化事件的时间可视化和深入的单细胞分辨率研究，如果它们 这是为了响应人体切片中不同的压力来源而发生的。这些方法提供了大量新的 在这些方法开发之前，研究可能性是完全不可行的。我们的研究 设计有望帮助我们充分发挥单细胞转录组学的潜力，揭示动态生物 过程，模拟人类胰腺疾病，并最终实现新型治疗方法的开发 诱导再生的方法。我们将通过追求以下具体目标来检验上述假设： (1) BMP 通路刺激后同供体 T1D/T2D HPS 的纵向 scRNAseq。 （二）学习 应激、炎症和去分化对 BMP-7 介导的 β 细胞形成诱导的影响 HPS； (3) 通过“芯片切片”方法表征新生β细胞的功能；和（4） 测定从健康和 T1D/T2D 供体中分选的祖细胞的体内再生潜力 异种移植模型。