人诱导多能干细胞诱导分化角膜内皮样细胞的研究

Corneal endothelial dysfunction is a thorny problem in the field of ophthalmology. Human corneal endothelial cells are terminally differentiated cells, once damaged could not renewable. Corneal transplantation is the only effective treatment. However, severe shortage of available donor corneas remains a global challenge. Inducing corneal endothelial cell regeneration will be the key to solving the problem. Up to now, it has been reported that CECs-like cells were obtained from stem cells, using the methods such as cell coculture, conditioned media and cytokine induction in vitro. Our group had successfully reprogrammed human peripheral blood mononuclear cells to iPSCs. Based on the previous theory, our group intends to conduct the following research: (1) Differentiation of human induced pluripotent stem cells into corneal endothelium-like cells by continuous induction with small molecule compounds; (2) Identification the function of corneal endothelium-like cells. The subject is based on the current research of corneal endothelial dysfunction diseases. It proposes a new method of inducing CEC-like cells，and offers new ideas for obtaining CECs for transplantation.

角膜内皮细胞（CECs）功能失代偿是眼科角膜病领域的棘手难题，因为成人CECs属终末分化细胞丧失增殖能力，此类疾病唯一治疗手段是角膜移植，而角膜移植的供体材料非常匮乏。诱导CECs再生将是解决问题的关键。目前已有研究报道了在体外由干细胞，经细胞共培养、条件培养基、细胞因子诱导等方法获得CECs样细胞。本课题组在成功将人外周血单个核细胞重编程为诱导多能干细胞（iPSCs）系的基础上，进行如下研究：（1）利用小分子化合物连续诱导的方法将iPSCs定向分化为CECs样细胞；（2）鉴定所得CECs样细胞性质、功能。本课题基于当前角膜内皮功能失代偿相关疾病的科研前沿提出，为诱导CECs样细胞提出新方法，为获得临床可用于移植的CECs提供新思路。

角膜内皮细胞（corneal endothelial cells ，CECs）通过屏障作用和泵功能维持角膜透明。衰老或疾病状态下CECs密度会逐渐降低引起角膜内皮失代偿，最终导致失明，角膜移植或细胞移植仍然是主要的治疗方法。我们通过双Smad抑制法利用化学成份明确的培养基诱导人诱导多能干细胞（human induced pluripotent stem cells ，hiPSCs）分化为神经嵴细胞（neural crest cells ，NCCs），通过小分子库筛选将NCCs诱导分化为CECs。实时荧光定量PCR和免疫荧光检测hiPSCs-CECs的标志物，蛋白质印迹法检测小分子调控的信号通路和关键因子。我们建立了一种联合小分子化合物和细胞因子高效诱导hiPSCs分化为CECs的方法，hiPSCs-CECs与人角膜内皮细胞相似，表达CECs的相关标志物ZO-1, AQP1, Vimentin和Na/K-ATPase。基于小分子筛选我们确定了A769662和AT13148两个小分子组合诱导效率最高，并且是通过上调PKA/AKT信号通路，FOXO1和PITX2促进NCCs分化为CECs。我们的研究结果为角膜内皮细胞再生提供一种新方法，为临床细胞移植治疗角膜疾病提供细胞来源。