Single-cell longitudinal analysis of regeneration in human pancreatic slices

项目总结 我们已经描述了一个具有祖细胞样特性的人胰腺导管细胞亚群。 这些细胞可以用抗骨形态发生蛋白受体1A(ALK3bright)和替代物P2RY1的抗体进行分离 我们的团队发现了PDX1的表面标记。ALK3bright/P2RY1细胞在BMP-7作用下增殖， 并在停用骨形态发生蛋白-7后分化为所有类型的成人胰腺细胞，包括功能正常的β细胞。 人胰腺供体ALK3组分的scRNAseq证实存在与 类似祖细胞的特征，有大量证据表明，这种特征可能是通过去分化获得的 差异化。当被移植到免疫缺陷小鼠体内时，分选的细胞群体富含标志物 在这些簇中差异表达的细胞自组织成具有天然细胞结构和 功能内分泌细胞。最近，我们开发了一种方法来研究 使用人胰腺切片进行祖细胞依赖的再生。我们已经实现了功能 长期(&gt；10d)培养β，允许在特定环境中纵向跟踪HPS细胞的再生 这被广泛认为是在体外最接近天然胰腺的方法。BMP信号依赖 再生是从健康的T1D和T2D捐赠者的切片中建立的，更重要的是，T1D和T2D捐赠者的切片再生。 许多对胰腺的单细胞分析，包括我们自己的，支持了新出现的观点，即特定的 谱系在分化阶段之间处于不断变化的状态。然而，所有这些分析只提供了一个快照 在任何给定的时间点。关于潜在的发展/再生途径的结论是 完全基于生物信息学的推论。我们假设，长期器官类型的组合 体外谱系追踪和连续单细胞分析的培养将使我们第一次能够 动态映射细胞命运的变化-例如，通过对来自同一供体的切片进行纵向scRNAseq 在添加BMP-7后的多个时间点。此外，我们认为这个系统将使真正的- 对潜在的去分化事件进行时间可视化和深入的单细胞分辨率研究，如果 会对人体切片中不同来源的压力做出反应。这些方法提供了大量新的 在开发这些方法之前，研究完全不可行的可能性。我们的研究 设计有望帮助我们实现单细胞转录的全部潜力，以揭示动态生物学 过程，模拟人类胰腺疾病，并最终使新的治疗方法的开发成为可能 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)同种供体T1D/T2D HPS在BMP途径刺激后的纵向scRNAseq。(2)研究 应激、炎症和去分化对骨形态发生蛋白-7介导的β细胞形成的影响 (3)通过“芯片上切片”方法对新生的β细胞进行功能表征；以及(4) 分离自健康和T1D/T2D供者的造血祖细胞体内再生潜能的测定 一种异种移植模型。

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.