Differentiation of Cord Blood Mesenchymal Stem Cells to Corneal Endothelium

脐带血间充质干细胞向角膜内皮的分化

• 批准号: 7570902
• 负责人: NANCY C. JOYCE
• 金额: $ 30.24万
• 依托单位: SCHEPENS EYE RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7570902
• 关键词: Adipocytes; Aqueous Humor; Biomedical Engineering; Blindness; Cell Culture Techniques; Cell Size; Cells; Cellular Morphology; Characteristics; Chondrocytes; Coculture Techniques; Conditioned Culture Media; Contact Inhibition; Contralateral; Cornea; Corneal Diseases; Corneal Endothelium; Corneal Opacity; Cytoskeleton; Descemet' s membrane; Development; Disease; Endothelial Cells; Endothelium; Ensure; Epithelial Cells; Exhibits; Extracellular Matrix Proteins; Eye; Eye Development; Flow Cytometry; Functional disorder; Gene Expression; Gene Proteins; Genes; Goals; Growth; Human; Human Characteristics; Human Cloning; Inbred BALB C Mice; Incubated; Injury; Institutes; Keratoplasty; Laboratories; MSC gene; Maintenance; Medical Research; Mesenchymal; Mesenchymal Stem Cells; Mesenchyme; Methods; Microarray Analysis; Monitor; Morphology; Mus; Nature; Neural Crest; Neurons; Osteocytes; Patients; Phenotype; Philadelphia; Process; Proteins; Relative (related person); Research; Research Personnel; SCID Mice; Shapes; Source; Surface; Testing; Thick; Time; Tissues; Transmission Electron Microscopy; Transplantation; Umbilical Cord Blood; Undifferentiated; Visual Acuity; Western Blotting; Wound Healing; base; cell injury; cell type; in vivo; lens; monolayer; mouse model; novel strategies; protein expression; public health relevance

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.

描述（由申请人提供）：生物工程角膜内皮正在探索作为角膜移植的一种令人兴奋的、实用的替代方法，用于治疗因内皮损伤或疾病导致的视力丧失。目前，供体人角膜内皮细胞（HCEC）被用于开发生物工程构建体;然而，HCEC具有有限的分裂能力，因此限制了用于这些构建体的健康细胞的数量。我们的目标是开发一种新的方法，以确保生物工程角膜结构和组织修复的健康HCEC的可用供应。拟议的探索性研究是基于这样一个事实，即来自多种来源的间充质干细胞，包括人脐带血，具有分化成多种细胞类型的潜力。由于内皮细胞在眼部发育过程中从神经嵴来源的间充质细胞分化而来，因此有理由假设人脐带血间充质干细胞（UCB MSCs）可以通过重现有助于从神经嵴来源的间充质细胞发育为角膜内皮的微环境条件而分化为功能性内皮细胞。我们提出了三个具体目标来检验这一假设。目的1将建立一组MSC和HCEC的基线特征，可用于跟踪MSC分化为HCEC样细胞。微阵列分析将确定相对基因表达，流式细胞术将鉴定表面标志物表达，Western印迹将比较特异性细胞和基质蛋白的表达。Ki 67和ZO-1的免疫染色将测试增殖的接触抑制。透射电子显微镜检查细胞形态。目的2：确定脐血间充质干细胞向HCEC样细胞分化的培养条件。培养条件将试图重现在眼发育期间导致成熟HCEC形成的微环境。将UCB MSC接种在4种不同基质上，并依次在透镜上皮细胞条件培养基中培养或与透镜上皮细胞共培养;在HCEC条件培养基中培养或与HCEC共培养;然后在房水中培养。这些培养条件对MSC的影响将通过鉴定从未分化MSC表现出的特征到如目标1中的HCEC表现出的特征的变化来跟踪。目的3将使用体内小鼠模型测试分化的HCEC样MSC的功能。将分化的HCEC样MSC接种到已经去除内皮细胞的供体小鼠角膜上。将该角膜构建体移植到受体小鼠眼中。对侧眼、含有培养的HCEC的角膜移植物和具有正常小鼠内皮的供体角膜将作为对照。通过对角膜混浊和血管向内生长进行评分，在体内跟踪分化的MSC维持透明角膜的能力8周。还将每周取出角膜，以监测细胞大小、形状和数量，并检测接触抑制单层的维持情况。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，用于治疗由于这种生理上重要的组织功能障碍而丧失视力的患者。当角膜内皮细胞因损伤或疾病而死亡时，它们不会分裂以取代自身，导致角膜透明度和视力丧失。成功完成这些探索性研究，以测试人脐带血间充质干细胞是否可以分化形成角膜内皮细胞，通过提供一种新的健康的内皮细胞来源来治疗由于这种生理学上重要的组织功能障碍而失去角膜透明度和视力的患者，可能对角膜疾病的治疗产生重大影响。