Microporous scaffolds for enhancing efficiency of beta-cell progenitor maturation in vitro and in vivo

同种异体胰岛移植到肝脏已显示出治疗T1D的临床前景，但它们的供应 是有限的。人胚胎干细胞分化为胰岛素分泌细胞的研究进展 已经证明了它们作为细胞来源的潜力。体外培养人胚胎干细胞以产生胰岛素阳性 细胞，并被移植到β-细胞完成成熟。重要的是，这些出版物指出 并不是所有接受这些细胞的糖尿病患者血糖正常，这表明需要有这样的系统 持续有效地促进体外和体内分化，获得成熟的β-细胞，能够 恢复正常血糖。有机化合物3D培养的最新进展表明， 通过维持组织结构和细胞组织使组织变得复杂。我们建议 微孔支架可以提供促进分化的线索，支持细胞组织进入 胰岛状结构。Pi Shea博士已经开发出用于将原始胰岛移植到小鼠体内的支架 在临床上可翻译的部位，允许有效的植入和功能，并逆转 高血糖伴最小的胰岛肿块。斯宾塞博士是一位发育生物学家，擅长于 正在合作进行支架设计和体内成熟分析的有机培养。AIM 1将测试 HES来源的胰腺前体细胞在三维微孔支架上分化的假说 提高未成熟β-细胞的体外形成效率。脚手架将以受控方式创建 结构。目标2专注于促进从不成熟的β细胞向成熟的β细胞的转变，这一步骤可以 结构，并用ECM蛋白修饰以促进细胞组织和分化为胰岛样细胞 只有通过移植和体内环境，在临床上可翻译的部位才能实现。 重要的是，支架/细胞结构可以直接移植，这有望增强 通过避免在移植过程中细胞-基质和细胞-细胞接触的中断而植入和发挥功能 操作包括为移植准备2D培养细胞。脚手架将在 移植以传递血管内皮生长因子，从而招募能够提供营养和分配胰岛素的血管系统， 此外，还提供对体内内分泌分化至关重要的内皮细胞相互作用。传统型 包括聚合酶链式反应、免疫组化和流式细胞术的分析将被用来表征组织特性。 然而，我们应用转录因子(TF)报告系统来量化分化过程中的活性，并且 从而为生物材料设计的发展过程提供了前所未有的洞察力。 此外，我们将使用人胰岛素原启动子-C-肽-超级折叠绿色荧光蛋白 (hPro-CPepSfGFP)以评估个体细胞在体外和体内的胰岛素储存和分泌。 用于细胞分化为成熟的β细胞，具有逆转高血糖的潜力。 成功完成研究将确定棚架设计，以提高一致性和效率

Allogeneic islets transplanted into the liver have shown promise clinically for treatment of T1D, yet their supply is limited. Recent reports on the generation of insulin producing cells from human embryonic stem cells (hESC) has demonstrated their potential as a cell source. The hESC are cultured in vitro to generate insulin positive cells, and are transplanted to complete their maturation toward β-cells. Importantly, these publications note that not all diabetic recipients of these cells become normoglycemic, and indicates the need for systems that consistently and efficiently promote differentiation in vitro and in vivo to achieve mature β-cells capable of restoring normoglycemia. Recent advances in 3D culture of organoids have demonstrated the production of complex tissues through maintenance of tissue structure and cellular organization. We propose that microporous scaffolds can provide cues that promotes differentiation and supports the cellular organization into islet-like structures. PI Dr. Shea has developed the scaffolds for the transplantation of primary islets into mice at a clinically translatable site that allows for efficient engraftment and function, and the reversal of hyperglycemia with a minimal islet mass. co-PI Dr. Spence is a developmental biologist with expertise in organoid culture that is collaborating on the scaffold design and analysis of in vivo maturation. Aim 1 will test the hypothesis that the differentiation of hES-derived pancreatic progenitors on 3D microporous scaffolds can increase the efficiency for forming immature β-cells in vitro. Scaffolds will be created with controlled structures. Aim 2 focuses on facilitating the transition from immature β-cells to mature β-cells, a step that can architecture and modified with ECM proteins to facilitate organization and differentiation of cells into islet like only be achieved through transplantation and the in vivo environment, at a clinically translatable site. Importantly, the scaffold/cell constructs can be directly transplanted, which is anticipated to enhance engraftment and function by avoiding the disruption of cell-matrix and cell-cell contacts that occurs during the manipulation involved with preparing 2D cultured cells for transplant. The scaffolds will be augmented prior to transplantation to deliver VEGF and thereby recruit vasculature that can provide nutrients and distribute insulin, as well provide endothelial cells interactions that are critical for endocrine differentiation in vivo. Traditional assays involving PCR, IHC, and flow cytometry will be employed to characterize the tissue properties. However, we apply a transcription factor (TF) reporter system to quantify activity throughout differentiation, and thus provide unprecedented insight to the developmental processes as a function of the biomaterial design. Furthermore, we will employ human Pro-insulin promoter-C-peptide- Super Folded Green Florescent Protein (hPro-CpepSfGFP) to assess the insulin storage and secretion by individual cells in vitro and in vivo. for cell differentiation to mature β-cells, with the potential to reverse hyperglycemia. Successful completion of the studies would identify scaffold designs to enhance the consistency and efficiency