Hybrid microenvironments for the ex-vivo expansion of Hematopoietic Stem Cells

用于造血干细胞离体扩增的混合微环境

• 批准号: 1944819
• 金额: --
• 依托单位: University of Glasgow
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2017
• 资助国家: 英国
• 起止时间: 2017 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-1944819
• 关键词: Hybrid; microenvironments; ex; vivo; expansion

BackgroundType I diabetes is a genetically based chronic autoimmune disease that is associated with deficient pancreatic beta cell function and insufficient insulin production. The self-duplication mediated rather than self-renewal maintenance of the beta cell niche in the pancreas and their targeting by the immune system creates the need for external insulin intake or pancreatic islet cell transplantation in patients suffering from the disease (Murtaugh, 2007). However, the inadequate supply of functional insulin producing cells as well as the increased risk for autoimmune rejection of the transplanted tissue pose major obstacles to a successful surgical transplantation of islet cells. By bypassing the possibility of transplant rejection, the differentiation of mesenchymal stem cells to insulin producing beta cells for autologous transplantation may provide an alternative treatment of type I diabetes. Aims of the projectIn recent years, the development of peptide hydrogels has opened up new possibilities for tissue culture in regenerative medicine (Chawla et al., 2012). The biodegradability and biocompatibility of these gels have also suggested the possibility of this injectable material (in vivo or in situ) to enable patients to avoid surgical transplantation procedures (Castillo Diaz et al., 2016). Previous studies have confirmed the ability of MSCs to differentiate into insulin producing pancreatic beta cells (Xin et al., 2016). The differentiation into insulin secreting cells has been achieved both by microenvironmental manipulation and by gene manipulation methods using viral vectors (Xie et al., 2009 and Allahverdi et al., 2015). However, these studies have been performed in tissue culture plates and therefore represent a less physiological view of the whole process in the way it occurs at an organism level. Consequently, a peptide hydrogel would allow the 3D culture of MSCs and their differentiation into beta cells, providing us with both a better overview of the whole process and the possibility to inject the terminally differentiated cells in organisms suffering from type I diabetes for clinical trials. MethodsThe project will initially involve the preparation of the hydrogel and the initiation of the mesenchymal cell culture. I intend to perform a cell viability assay (e.g. LIVE/DEAD assay) to confirm the suitability of the gel for a 3D MSC culture. In order to achieve MSC differentiation into pancreatic beta cells, I will introduce the appropriate culturing media and differentiation stimuli into the hydrogel (eg. to include the expression of relevant transcription factors such as Neurod1, Ngn3, Pdx1 etc). Finally, it is important that I confirm the production of insulin and beta cell specific markers by the cells by immunofluorescence. At the end of the project, I will test the degradability of the hydrogel to suggest the possibility and safety of its potential introduction to an organism in situ (possibly using the method for evaluating the hydrogel proteolytic degradability described by Castillo Diaz et al., 2016).

背景I型糖尿病是一种以遗传为基础的慢性自身免疫性疾病，与胰腺β细胞功能缺陷和胰岛素分泌不足有关。胰腺中β细胞小生境的自我复制介导而非自我更新维持及其被免疫系统靶向，使得患有该疾病的患者需要外部胰岛素摄入或胰岛细胞移植（Murtaugh，2007）。然而，功能性胰岛素产生细胞的供应不足以及移植组织的自身免疫排斥的风险增加对胰岛细胞的成功手术移植构成了主要障碍。通过绕过移植排斥的可能性，间充质干细胞分化为用于自体移植的胰岛素产生β细胞可以提供I型糖尿病的替代治疗。项目目标近年来，肽水凝胶的发展为再生医学中的组织培养开辟了新的可能性（Chawla等人，2012年）。这些凝胶的生物降解性和生物相容性也表明了这种可注射材料（体内或原位）使患者能够避免外科移植手术的可能性（Castillo迪亚兹et al.，2016年）。先前的研究已经证实了MSC分化成产生胰岛素的胰腺β细胞的能力（Xin等人，2016年）。通过微环境操作和使用病毒载体的基因操作方法已经实现了向胰岛素分泌细胞的分化（Xie et al.，2009和Allahverdi等人，2015年）的报告。然而，这些研究是在组织培养板中进行的，因此代表了整个过程在生物体水平上发生的方式的较少生理学观点。因此，肽水凝胶将允许MSC的3D培养及其分化为β细胞，为我们提供了整个过程的更好概述以及将终末分化细胞注射到患有I型糖尿病的生物体中进行临床试验的可能性。方法该项目将首先涉及水凝胶的制备和间充质细胞培养的启动。我打算进行细胞活力试验（例如活/死试验），以确认凝胶对3D MSC培养的适用性。为了实现MSC分化为胰腺β细胞，我将在水凝胶中引入适当的培养基和分化刺激物（例如，包括相关转录因子如Neurod 1、Ngn 3、Pdx 1等的表达）。最后，重要的是我通过免疫荧光证实细胞产生胰岛素和β细胞特异性标志物。在项目结束时，我将测试水凝胶的降解性，以表明其潜在引入原位生物体的可能性和安全性（可能使用Castillo迪亚兹等人描述的评估水凝胶蛋白水解降解性的方法，2016年）。