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

I型糖尿病是一种以遗传为基础的慢性自身免疫性疾病，与胰岛β细胞功能低下和胰岛素分泌不足有关。自我复制调节而不是自我更新维持了胰腺中的β细胞生态位，免疫系统以它们为靶标，导致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自身免疫排斥反应的风险增加是成功进行胰岛细胞外科移植的主要障碍。通过绕过移植排斥的可能性，将间充质干细胞分化为产生胰岛素的β细胞用于自体移植可能为I型糖尿病提供另一种治疗方法。该项目的目标近年来，多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兼容性也表明这种可注射材料(体内或原位)使患者能够避免外科移植程序(Castillo Diaz等人，2016)。以前的研究已经证实了MSCs分化为产生胰岛素的胰岛β细胞的能力(Xin等人，2016)。分化为胰岛素分泌细胞是通过微环境操作和使用病毒载体的基因操作方法实现的(谢等人，2009和Allahverdi等人，2015)。然而，这些研究是在组织培养板上进行的，因此代表了整个过程在生物体水平上发生的较少的生理学观点。因此，多肽水凝胶将允许MSCs的3D培养和向β细胞的分化，为我们提供了更好的整个过程的概述，并使我们有可能将终末分化的细胞注射到患有I型糖尿病的生物体内进行临床试验。方法该项目最初将包括水凝胶的制备和间充质细胞培养的启动。我打算进行细胞活性测试(例如，活/死实验)，以确认该凝胶是否适合3D MSC培养。为了实现MSC向胰岛β细胞的分化，我将在水凝胶中引入合适的培养液和分化刺激物(例如。包括Neurod1、Ngn3、Pdx1等相关转录因子的表达)。最后，重要的是，我要确认细胞通过免疫荧光产生胰岛素和β细胞特异性标记物。在项目结束时，我将测试水凝胶的降解性，以建议其潜在地引入生物体的可能性和安全性(可能使用Castillo Diaz等人描述的评估水凝胶蛋白分解降解性的方法，2016)。

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).