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

背景I型糖尿病是一种遗传性慢性自身免疫性疾病，与胰腺细胞功能缺陷和胰岛素分泌不足有关。胰腺中β细胞生态位的自我复制介导而非自我更新的维持，以及免疫系统对它们的靶向性，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自身免疫排斥反应的风险增加是成功进行胰岛细胞手术移植的主要障碍。通过绕过移植排斥反应的可能性，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。近年来，肽水凝胶的发展为再生医学组织培养开辟了新的可能性（Chawla et al., 2012）。这些凝胶的生物可降解性和生物相容性也表明，这种可注射材料（体内或原位）可以使患者避免手术移植手术（Castillo Diaz等，2016）。先前的研究已经证实MSCs能够分化为产生胰岛素的胰腺β细胞（Xin et al., 2016）。分化为胰岛素分泌细胞可以通过微环境操作和利用病毒载体的基因操作方法实现（Xie et al.， 2009和Allahverdi et al., 2015）。然而，这些研究都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方式较少的生理观点。因此，肽水凝胶将允许MSCs的3D培养及其分化为β细胞，为我们提供了对整个过程的更好的概述，并有可能将最终分化的细胞注射到患有I型糖尿病的生物体中进行临床试验。方法本课题将首先进行水凝胶的制备和间充质细胞的培养。我打算进行细胞活力测定（例如LIVE/DEAD测定），以确认凝胶是否适合3D MSC培养。为了使MSC分化为胰腺β细胞，我将在水凝胶中引入合适的培养基和分化刺激(例如：包括相关转录因子如Neurod1、Ngn3、Pdx1等的表达)。最后，重要的是我要用免疫荧光法确认细胞产生胰岛素和β细胞特异性标记物。在项目结束时，我将测试水凝胶的可降解性，以建议将其引入原位生物体的可能性和安全性（可能使用Castillo Diaz等人，2016年描述的评估水凝胶蛋白水解可降解性的方法）。

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).