Single-cell longitudinal analysis of regeneration in human pancreatic slices

项目摘要 我们描述了一个具有祖细胞样特征的人胰腺导管细胞亚群。 这些细胞可以使用针对BMP受体1A（ALK 3bright+）和P2 RY 1（替代物）的抗体进行分选。 PDX 1的表面标记物。当暴露于BMP-7时，ALK 3bright +/P2 RY 1+细胞增殖， 并在BMP-7撤除后分化成所有成体胰腺细胞类型，包括功能性β细胞。 人胰腺供体的ALK 3bright+级分的scRNAseq证实了存在簇， 祖细胞样特征，有大量证据表明，这些特征可能是通过去- 分化当移植到免疫缺陷小鼠体内时， 在这些簇中差异表达的细胞自组织成具有天然样细胞结构的“微胰腺”， 功能性内分泌细胞最近，我们已经开发出了研究 使用人胰腺切片（HPS）的祖细胞依赖性再生。我们已经实现了功能性 HPS的长期（> 10天）培养，其允许在环境中纵向跟踪β细胞再生 这被广泛认为是体外最接近天然胰腺的。BMP信号依赖性 在来自健康供体，更重要的是T1 D和T2 D供体的切片中建立了再生。 包括我们自己在内的许多胰腺单细胞分析支持了一种新的观点， 谱系在分化阶段之间处于流动状态。然而，所有这些分析只提供了一个快照， 在任何给定的时间点。关于潜在发育/再生途径的结论是 完全基于生物信息学推论。我们假设长期的器官型 体外谱系追踪培养和连续的单细胞分析将使我们第一次， 动态映射细胞命运变化-例如，通过对来自同一供体的切片进行纵向scRNAseq， 在BMP-7添加后的多个时间点。此外，我们认为，这一制度将使真实的- 时间可视化和深入的，潜在的去分化事件的单细胞分辨率研究，他们应该 在人体切片中，不同的压力源会产生不同的反应。这些方法提供了丰富的新方法 在这些方法发展之前完全不可行的研究可能性。我们的研究 设计有望帮助我们实现单细胞转录组学的全部潜力， 过程，模拟人类胰腺疾病，并最终能够开发新的治疗方法， 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)刺激BMP途径后相同供体T1 D/T2 D HPS的纵向scRNAseq。(2)研究 研究了应激、炎症和去分化对BMP-7介导的β细胞形成的影响， （3）通过“芯片上切片”方法对新生β细胞进行功能表征;以及（4） 使用流式细胞仪测定从健康和T1 D/T2 D供体分选的祖细胞的体内再生潜力 异种移植模型。

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.