Differentiation of Cord Blood Mesenchymal Stem Cells to Corneal Endothelium

描述(申请人提供)：生物工程角膜内皮细胞正在被探索作为角膜移植的一种令人兴奋的、实用的替代方案，以治疗由于内皮损伤或疾病而导致的视力丧失。目前，供体人角膜内皮细胞(HCEC)正被用于开发生物工程构建物；然而，HCEC具有有限的分裂能力，因此限制了用于这些构建物的健康细胞的数量。我们的目标是开发一种新的方法，以确保可用于生物工程角膜构建和组织修复的健康HCEC的供应。拟议的探索性研究是基于这样一个事实，即来自几个来源的间充质干细胞，包括人脐血，具有分化为几种细胞类型的潜力。由于人脐血间充质干细胞在眼发育过程中分化为神经脊来源的间充质细胞，因此，通过总结促进神经嵴来源的间充质形成角膜内皮的微环境条件，可以推测人脐血间充质干细胞可以分化为功能性的人脐血间充质干细胞。我们提出了三个具体目标来检验这一假设。AIM 1将建立一套MSCs和HCEC的基线特征，可用于跟踪MSC向HCEC样细胞的分化。微阵列分析将确定相关基因的表达，流式细胞仪将确定表面标志物的表达，Western blots将比较特定细胞和基质蛋白的表达。Ki67和ZO-1的免疫染色将检测接触性抑制增殖。透射电子显微镜将检查细胞的形态。目的2鉴定脐血间充质干细胞分化为人脐静脉内皮细胞样细胞的培养条件。培养条件将试图概括在眼睛发育过程中导致成熟HCEC形成的微环境。将脐血间充质干细胞接种于4种不同的基质上，依次在晶状体上皮细胞条件培养液或与晶状体上皮细胞共培养；在HCEC条件培养液或与HCEC共培养；然后在房水中培养。这些培养条件对MSCs的影响之后，将确定从未分化的MSCs表现出的特性变化到HCEC表现的变化，就像Aim 1中的那样。Aim 3将使用活体小鼠模型测试分化的HCEC样MSCs的功能。分化的HCEC样间充质干细胞将被种植到已经剥离内皮细胞的供体小鼠角膜上。这种角膜结构将被移植到受体小鼠的眼睛中。对侧眼、含有培养的人脐静脉内皮细胞的角膜移植和正常小鼠内皮细胞的供体角膜作为对照。分化后的MSCs保持透明角膜的能力将在体内进行8周的跟踪，并对角膜混浊和血管生长进行评分。每周还将取出角膜，以监测细胞的大小、形状和数量，并测试接触抑制单层的维护情况。这些探索性研究的成功完成可能会对角膜疾病的治疗产生重大影响，因为它提供了一种新的、健康的HCEC样细胞来源，用于治疗因这一生理重要组织功能障碍而失去视力的患者。公共卫生相关性当角膜内皮细胞因损伤或疾病死亡时，它们不会分裂以取代自己，导致角膜透明度和视力的丧失。成功完成这些探索性研究，以测试人脐血间充质干细胞是否可以分化为角膜内皮细胞，可能会对角膜疾病的治疗产生重大影响，因为它提供了一种新的、健康的内皮细胞来源，用于治疗因这一生理上重要的组织功能障碍而失去角膜透明度和视力的患者。

DESCRIPTION (provided by applicant): Bioengineered corneal endothelium is being explored as an exciting, practical alternative to corneal transplantation to treat vision loss due to endothelial injury or disease. Currently, donor human corneal endothelial cells (HCEC) are being used to develop bioengineered constructs; however, HCEC have a finite ability to divide, thus limiting the number of healthy cells for use in these constructs. Our goal is to develop a novel approach to ensure an available supply of healthy HCEC for bioengineered corneal constructs and tissue repair. The proposed exploratory studies are based on the fact that mesenchymal stem cells from several sources, including human umbilical cord blood, have the potential to differentiate into several cell types. Since HCEC differentiate from neural crest-derived mesenchymal cells during ocular development, it is reasonable to hypothesize that human umbilical cord blood mesenchymal stem cells (UCB MSCs) can be differentiated into functional HCEC by recapitulating microenvironmental conditions that contribute to the development of corneal endothelium from neural crest-derived mesenchyme. We propose three Specific Aims to test this hypothesis. Aim 1 will establish a set of baseline characteristics for MSCs and HCEC that can be used to follow MSC differentiation to HCEC-like cells. Microarray analysis will determine relative gene expression, flow cytometry will identify surface marker expression, Western blots will compare expression of specific cellular and matrix proteins. Immunostaining for Ki67 and ZO-1 will test for contact inhibition of proliferation. Transmission electron microscopy will examine cellular morphology. Aim 2 will identify culture conditions that differentiate UCB MSCs to HCEC-like cells. Culture conditions will try to recapitulate the microenvironment that leads to formation of mature HCEC during eye development. UCB MSCs will be seeded on 4 different matrices and successively cultured in lens epithelial cell-conditioned medium or co-cultured with lens epithelial cells; cultured in HCEC- conditioned medium or co-cultured with HCEC; and then cultured in aqueous humor. The effect of these culture conditions on MSCs will be followed by identifying changes in characteristics from those exhibited by undifferentiated MSCs to those exhibited by HCEC as in Aim 1. Aim 3 will test the function of differentiated HCEC-like MSCs using an in vivo mouse model. Differentiated, HCEC-like MSCs will be seeded onto donor mouse corneas that have been denuded of endothelial cells. This corneal construct will be transplanted into a recipient mouse eye. The contralateral eye, corneal transplants containing cultured HCEC, and donor corneas with normal mouse endothelium will act as controls. The ability of the differentiated MSCs to maintain a clear cornea will be followed in vivo over an 8-week period by scoring for corneal opacity and vessel in-growth. Corneas will also be removed at weekly intervals to monitor cell size, shape, and number, and to test for maintenance of a contact-inhibited monolayer. Successful completion of these exploratory studies could have a major impact on the treatment of corneal disease by providing a new, healthy source of HCEC-like cells to treat patients who have lost visual acuity due to dysfunction of this physiologically important tissue. PUBLIC HEALTH RELEVANCE When corneal endothelial cells die due to injury or disease, they do not divide to replace themselves, resulting in loss of corneal clarity and visual acuity. Successful completion of these exploratory studies to test whether human umbilical cord blood mesenchymal stem cells can be differentiated to form corneal endothelial cells could have a major impact on the treatment of corneal disease by providing a new, healthy source of endothelial cells to treat patients who have lost corneal clarity and visual acuity due to dysfunction of this physiologically important tissue.