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: 10490330
• 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/10490330
• 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.

项目摘要 我们已经描述了具有祖细胞样特征的人类胰腺导管细胞的亚群。 可以使用针对BMP受体1a（ALK3BRIGHT+）和P2RY1的抗体对这些细胞进行分类 PDX1的表面标记是由我们的团队促进的。 并在BMP-7退出后分为所有成年胰腺细胞类型，包括功能性β细胞。 Alk3bright+人类胰腺供体的scrnaseq证实存在群集 祖细胞样特征，并有大量证据表明，这种特征可以通过DE-获得 分化时。 在这些群集中差异表达的自我组织形成“与天然的leaata”，具有本地类细胞结构和 功能性内分泌细胞。 祖细胞依赖性的使用人类胰腺切片（HPSS）。 HPSS的长期（> 10d）培养 这被广泛认为是与天然胰腺最接近的近似值。 在健康，更重要的是T1D和T2D供体的缝隙中建立了再生。 palcress的大量单细胞分析包括我们自己的，支持新兴观点 谱系处于分化阶段之间的通量。 在任何给定时间点的组织。 基于生物信息学推断，我们假设长期器官的结合 在体外和顺序单细胞分析中进行谱系追踪的培养将使我们首次进入。 动态的地图细胞命运变化 - 例如 在添加BMP-7之后的多个时间点。 时间可视化和深入的，单细胞的潜在脱不同事件的分辨率， 发生在人类切片中的不同压力来源的情况下发生。 在开发这些方法之前，研究可能性是彻头彻尾的 设计有望帮助我们实现单细胞转录组学揭示Dynabeil动态生物学的全部潜力 过程，建模人类胰腺疾病，并最终使新的治疗疗法的发展 诱导再生的方法。 （1）刺激BMP途径后，相同T1D/T2D HPS的纵向SCRNASEQ。 压力，炎症和脱节性对BMP-7介导的β细胞形成诱导的影响 HPSS;（3）通过“片段切片”方法对新生β细胞的功能表征； 使用健康和T2D供体排序的祖细胞的体内再生潜力的确定 异种解雇模型。