Mesenchymal stem cells derived from human limbal niche cells

Purpose. We investigated whether human limbal niche cells generate mesenchymal stem cells. Methods. Limbal niche cells were isolated from the limbal stroma by collagenase alone or following dispase removal of the limbal epithelium (D/C), and cultured on plastic in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS), or coated or three-dimensional Matrigel in embryonic stem cell medium with leukemia inhibitory factor and basic fibroblast growth factor. Expression of cell markers, colony-forming units-fibroblast, tri-lineage differentiation, and ability of supporting limbal epithelial stem/progenitor cells were compared to limbal residual stromal cells. Results. Stromal cells expressing angiogenesis markers were found perivascularly, subjacent to limbal basal epithelial cells, and in D/C and limbal residual stromal cells. When seeded in three-dimensional Matrigel, D/C but not limbal residual stromal cells yielded spheres of angiogenesis progenitors that stabilized vascular networks. Similar to collagenase-isolated cells, D/C cells could be expanded on coated Matrigel for more than 12 passages, yielding spindle cells expressing angiogenesis and mesenchymal stem cells markers, and possessing significantly higher colony-forming units-fibroblast and more efficient tri-lineage differentiation than D/C and limbal residual stromal cells expanded on plastic in DMEM with 10% FBS, of which both lost the pericyte phenotype while limbal residual stromal cells turned into myofibroblasts. Upon reunion with limbal epithelial stem/progenitor cells to form spheres, D/C cells expanded on coated Matrigel maintained higher expression of p63α and lower expression of cytokeratin 12 than those expanded on plastic in DMEM with 10% FBS, while spheres formed with human corneal fibroblasts expressed cytokeratin 12 without p63α. Conclusions. In the limbal stroma, cells subjacent to limbal basal epithelial cells serve as niche cells, and generate progenitors with angiogenesis and mesenchymal stem cells potentials. They might partake in angiogenesis and regeneration during corneal wound healing.     

Transplantation of human embryonic stem cells onto a partially wounded human cornea in vitro

Purpose: The aim of this study was to investigate whether cells originating from human embryonic stem cells (hESCs) could be successfully transplanted onto a partially wounded human cornea. A second aim was to study the ability of the transplanted cells to differentiate into corneal epithelial‐like cells. Methods: Spontaneously, differentiated hESCs were transplanted onto a human corneal button (without limbus) with the epithelial layer partially removed. The cells were cultured on Bowman’s membrane for up to 9 days, and the culture dynamics documented in a time‐lapse system. As the transplanted cells originated from a genetically engineered hESC line, they all expressed green fluorescent protein, which facilitated their identification during the culture experiments, tissue preparation and analysis. To detect any differentiation into human corneal epithelial‐like cells, we analysed the transplanted cells by immunohistochemistry using antibodies specific for CK3, CK15 and PAX6. Results: The transplanted cells established and expanded on Bowman’s membrane, forming a 1–4 cell layer surrounded by host corneal epithelial cells. Expression of the corneal marker PAX6 appeared 3 days after transplantation, and after 6 days, the cells were expressing both PAX6 and CK3. Conclusion: This shows that it is possible to transplant cells originating from hESCs onto Bowman’s membrane with the epithelial layer partially removed and to get these cells to establish, grow and differentiate into corneal epithelial‐like cells in vitro.     

Ocular surface reconstruction using autologous rabbit oral mucosal epithelial sheets fabricated ex vivo on a temperature-responsive culture surface

PURPOSE: Autologous stem cell transplantation for total limbal stem cell deficiency is immunologically preferable, to avoid allograft rejection. This study was undertaken to investigate the possibility of a novel tissue engineering approach for ocular surface reconstruction, using autologous oral mucosal epithelial stem cells expanded ex vivo on temperature-responsive cell culture surfaces. METHODS: Rabbit oral mucosal epithelial cells cultured on temperature-responsive culture surfaces with mitomycin-C-treated 3T3 feeder cells for 2 weeks produced confluent epithelial cell sheets. Putative progenitor cell populations were estimated by colony-forming assays. Autologous transplantation of these cell sheets to surgically manipulated eyes was performed, and ocular surface reconstruction and cell phenotypic modulation were examined. RESULTS: All cultured oral epithelial cells were nonenzymatically harvested as transplantable intact cell sheets by reducing culture temperature to 20 degrees C. Oral epithelial cells were stratified in three to five cell layers more similar to corneal epithelium than to oral mucosal epithelium. Colony-forming assays and immunofluorescence for p63, beta1-integrin, and connexin 43 indicated retention of viable stem and/or progenitor cell populations in cell sheets. Autologous transplantation to rabbit corneal surfaces successfully reconstructed the corneal surface, with restoration of transparency. Four weeks after transplantation, epithelial stratification was similar to that in the corneal epithelium, although the keratin expression profile retained characteristics of the oral mucosal epithelium. CONCLUSIONS: Cell sheet harvest technology enables fabrication of viable, transplantable, tissue-engineered epithelial cell sheets that retain putative progenitor cells from autologous oral mucosal epithelial cells. Promising clinical capabilities for autologous tissue-engineered epithelial cell sheets for ocular surface reconstruction are indicated.     

干细胞与角膜眼表重建研究进展

角膜是眼表的重要防护屏障和光学系统的重要组成部分,其完整性和透明性是实现有效视功能的重要前提。各种原因造成的眼表疾病未经及时有效治疗都有可能发展到终末阶段———角膜缘干细胞功能障碍,包括角膜缘干细胞数目的减少和微环境的病理改变。自体角膜移植无疑是治疗角膜缘干细胞功能障碍最有效的方法。但是供体角膜来源十分有限,且切除健眼组织行角膜移植可能会对健眼造成长期损害。随着干细胞研究的不断深入和组织工程技术的兴起,组织工程角膜应运而生,并且迅速发展。其基本原理为选用生物性能良好的支架材料,体外模拟角膜缘干细胞微环境,诱导各类种子干细胞分化为角膜类上皮,然后再行角膜眼表重建。常用种子细胞包括角膜缘干细胞、胚胎干细胞、诱导多能干细胞、骨髓间充质干细胞、皮肤干细胞、口腔黏膜干细胞。本文就上述干细胞在角膜眼表重建中的应用作一综述。     

A fibrin-based bioengineered ocular surface with human corneal epithelial stem cells

PURPOSE: The purpose of the investigation was to prepare a bioengineered ocular surface tissue replacement consisting of (presumed) human corneal epithelial stem cells in a cross-linked fibrin gel for potential transplant. METHODS: Presumed human epithelial stem cells were harvested, isolated, and cultivated as previously described from adult donor corneas obtained from a tissue and organ bank. The cultured corneal epithelial stem cells were suspended in a fibronectin/fibrin gel cross-linked by factor XIII. Plasma components were derived from a fibrinogen-rich cryoprecipitate of human plasma. Suspended cells proliferated in the fibrin gel, giving rise to colonies that eventually coalesced to near confluence over the 15 days of cultivation. The gels were sectioned and immunostained for keratin 3 (AE5) and keratin 19. RESULTS: The fibrin gel product with corneal stem cells was easily manageable and maneuverable. Addition of the protease inhibitor aprotinin to the incubation medium prevented gel degradation; once it was removed, gels disintegrated within 24 hours. All of the cells cultivated in the fibrin gel stained positively for keratin 3 (AE5), indicating differentiation along the corneal epithelium lineage. Cells located in the center of the colonies were keratin 19-positive, suggesting a more primitive cell type. Growth kinetics were documented. CONCLUSIONS: A bioengineered ocular surface with a combination of presumed corneal epithelial stem cells in a cross-linked fibrin gel represents a potential improvement in current attempts to create a transportable, pliable, and stable tissue replacement. Since both the cells and the plasma components of the fibrin gel are of human origin, this technique provides the potential for a totally autologous bioengineered replacement tissue.     

大鼠骨髓间充质干细胞体外可诱导分化为角膜上皮细胞

目的:探讨骨髓间充质干细胞 (mesenchymal stem cell,MSC) 分化为角膜上皮细胞的可塑性及其重建角膜上皮的可能性.方法:用密度梯度离心法结合贴壁培养法分离纯化大鼠骨髓MSC,经体外与角膜基质细胞共培养诱导分化,免疫荧光法检测角膜上皮细胞特异标志物K12的表达.结果:体外培养的大鼠骨髓MSC表现出很强的增殖潜能,原代培养的骨髓MSC CD29免疫荧光染色阳性,CD34和CD45为阴性,符合骨髓MSC的特征.MSC与角膜基质细胞共培养1wk后大部分细胞分化为间质细胞,少部分细胞形态上相对偏小,免疫荧光检测这部分细胞表达角膜上皮细胞特异性标志角蛋白K12.结论:体外培养的MSC在角膜基质细胞的诱导下可横向分化为角膜上皮细胞.     

干细胞移植修复眼表损伤的研究进展

角膜缘干细胞、口腔黏膜细胞、胚胎干细胞、骨髓问充质干细胞等多种来源干细胞在修复眼表损伤的研究中都取得了较大的进展.本文就角膜上皮的组织结构、功能特征及上皮细胞的生物学特点,对胚胎干细胞和多种成体干细胞向角膜上皮细胞的分化及其对眼表损伤修复过程的影响进行了阐述,并对其各自的临床应用前景和存在的问题进行了讨论.(中华眼科杂志,2009,45:658-662)     

角膜干细胞增殖与分化表达实验研究

目的 了解角膜缘干细胞体外生长的增殖分化特性,检测上皮细胞生长因子对干细胞增殖的促进作用。方法 采用ＤＭＥＭ和Ｆ１２培养基进行兔角膜上皮细胞培养,用克隆形成率（ＣＦＥ）、免疫组化染色和蛋白质免疫印迹等方法检测细胞的增殖力和分化表达状况。结果角膜缘干细胞（ＬＣ）在本培养条件下能够正常传代生长５代,ＣＦＥ为（５．０７±２．３５）％,而角膜中央上皮细胞（ＣＣ）仅第１次传代生长,ＣＦＥ为（１．１２±０．８     

An evaluation of cultivated corneal limbal epithelial cells,using cell-suspension culture

PURPOSE: A previous report has described an ocular surface reconstruction method involving the use of cultivated corneal epithelium derived from limbal explants. In the current study, a new culture system was developed involving the in vitro propagation on amniotic membrane (AM) of epithelial cells from enzymatically dissociated limbal epithelium. The purpose of this new method is to produce a cultivated epithelial cell layer that contains stem cells and that is superior to explanted cultivated epithelium. The new cell-suspension technique was compared with the existing explant method. METHODS: Limbal epithelial cells were dissociated from donor eyes by dispase and seeded on the denuded AM. Small pieces of limbal epithelium were also cultured on denuded AM as explant cultures. The cultivated epithelium was examined by electron microscopy and immunohistochemistry for cornea-specific keratins (K3 and K12). RESULTS: Both cell-suspension and explant culture methods produced a healthy epithelial cell layer. The cell-suspension culture had significantly (P < 0.001) more desmosomal junctions between the explant-cultured basal cells. In addition, the intercellular spaces between the cell-suspension's basal cells were significantly (P < 0.001) smaller than those between the explant-cultured basal cells. Both types of cultivated epithelium showed positive expression of K3 and K12 keratins. In the cell-suspension culture, expression of K3 and K12 keratins was more prominent in the superficial cells. CONCLUSIONS: Corneal epithelial cells were successfully regenerated in vitro by a cell-suspension culture system. The suspension-cultured epithelium must include some stem cells and morphologically is significantly superior to explant-cultured epithelium. Thus, this new technique is potentially more suitable for cultivated corneal limbal epithelial transplantation.     

骨髓间充质干细胞的视网膜保护研究进展

骨髓间充质干细胞( bone marrow mesenchymal stem cells, BMSCs)富集于骨髓,可跨胚层分化,是一种具有多向分化潜能的成体干细胞。 BMSC易于分离培养,可高效扩增和表达,具有组织修复能力。由于其具有免疫调节能力,能分泌神经营养因子,使BMSC可以作为移植治疗视网膜疾病的种子细胞。本文将对BMSCs的视网膜保护研究进展作一综述。     

结膜上皮干细胞的体外培养与分化的研究进展

结膜上皮干细胞可以分化为结膜上皮细胞和杯状细胞,在维持正常眼表与损伤修复中具有关键作用,具有较强的自我更新和增殖分化能力.因此,结膜上皮干细胞是结膜组织工程种子细胞的重要来源.现就结膜上皮干细胞的生物特性、在眼表的分布、常用的检测方法、体外培养情况、诱导分化为杯状细胞的研究进展进行分析.

Abstract：

Conjunctiva epithelial stem cells are bipotent cells giving rise to both conjunctival epithelial cells and goblet cells. They play a central role in ocular surface homeostasis and wound repair, and also have great capacities of self-renewal, proliferation and differentiation. As a result, conjunctival epithelial stem cells are main resources of seed cells for tissue engineering conjunctiva. To review the current research of conjunctival epithelia stem cells such as their features, distribution, ways to identify stem cells,in vitro culture and induced differentiation to goblet cells.     

人视网膜中神经干细胞样细胞的分离和体外培养

目的 分离培养发育基本成熟的人视网膜中神经干细胞样细胞。方法 取0～3岁猝死婴幼儿视网膜细胞，在干细胞选择性培养基中培养，观察原代和传代细胞的形态和增殖情况，并对其诱导分化。通过免疫细胞化学方法检测Nestin、MAP-2和GFAP的表达。结果 原代细胞可形成悬浮生长的致密球状细胞团，传代后能形成新的细胞球。部分细胞球表达Nestin，传代后阳性率增加。分化后细胞形态多样，每一团细胞相对一致，部分细胞表达MAP-2或GFAP。结论 0～3岁人视网膜中存在一种表现出神经干细胞特性的细胞。     

体外共培养诱导人羊膜间充质干细胞分化为眼表上皮细胞的研究

背景 人羊膜间充质干细胞（hAMSCs）在体外能诱导分化为多种体细胞,但目前有关hAMSCs分化为眼表细胞的研究鲜有报道. 目的 探讨体外共培养诱导hAMSCs向眼表细胞分化的可能性及其分化机制.方法 本研究经广州医科大学第二附属医院伦理委员会批准,在健康产妇的知情同意下从人胎盘中分离hAMSCs并进行培养,采用流式细胞术计数分析CD44、CD45、CD73、CD90在培养细胞中的表达以鉴定细胞,此外通过成骨诱导分化实验和成脂诱导分化实验对培养细胞进行体外分化鉴定.在患者知情同意下获取眼科手术中弃用的人眼球结膜组织,用组织块培养法分离和培养人球结膜成纤维细胞（hBCFs）,将hAMSCs与hBCFs在Transwell培养体系中进行共培养,分为hAMSCs培养组和hAMSCs与hBCFs共培养组,用含质量分数5％胎牛血清（FBS）的DMEM高糖/F12培养基培养7d,采用免疫荧光技术检测hAMSCs中对上皮细胞角蛋白19（CK19）的表达,评价hAMSCs向眼表细胞分化的程度,并检测hAMSCs中α-平滑肌肌动蛋白（α-SMA）的表达,评价其分化过程与间质上皮化转换（MET）过程的关系.结果 传代至3～7代的hAMSCs均为细长形,但随着传代增加,细胞体积增大,细胞中CD44、CD73、CD90表达呈强阳性,但不表达CD45.经成骨或成脂诱导分化后3～4周,细胞中茜素红S（ARS）染色或油红O染色阳性.hAMSCs与hBCFs共培养后1周,hAMSCs形态由细长形变为类上皮细胞型,共培养组的部分细胞中CK19表达阳性,所有细胞α-SMA表达呈微弱阳性,而hAMSCs细胞培养组可见呈绿色荧光的α-SMA阳性细胞,但不表达CK19. 结论 通过体外共培养可诱导hAMSCs向人眼表细胞分化,其分化机制可能与MET过程相关.     

经角膜基质细胞诱导的大鼠骨髓间充质干细胞在人羊膜上构建角膜上皮移植片

目的:研究将骨髓间充质干细胞(mesenchymal stem cells,MSC)诱导分化后在羊膜表面培养构建角膜上皮移植片的可行性。方法:取SD大鼠骨髓间充质干细胞和角膜基质细胞,分别培养并传2代后进行共培养,取共培养7d后的MSC覆载于新鲜人羊膜,再培养7d。对MSC及诱导后MSC进行免疫荧光染色和扫描电镜检查;对覆载细胞的羊膜进行HE染色及免疫荧光染色。结果:MSC在体外培养条件下贴壁生长,免疫荧光染色CD29和CD44阳性,CK12阴性。经角膜基质细胞诱导分化后,MSC细胞CK12染色转为阳性。诱导后MSC接种到羊膜表面,迅速贴壁生长,组织学特性无明显改变,CK12染色仍为阳性。结论:经角膜基质细胞诱导的MSC表现出角膜上皮细胞特征,在羊膜上生长后保持不变。     

Comparison of cell-suspension and explant culture of rabbit limbal epithelial cells

Currently, most investigators directly use limbal explants to culture corneal epithelial cells. However, it has not been identified that limbal stem cells do readily migrate from the limbal explants onto culture plate or amniotic membrane carrier. In this study a cell-suspension culture system for rabbit limbal stem cells was developed and compared with the direct explant method in the aspect of stem cells content in the culture system. Rabbit limbal epithelial cells were dissociated from rabbit eyes by dispase and single cell suspension was made for cell-suspension culture. DeltaNp63 expression of cultured rabbit limbal epithelial cells by cell-suspension technique and explant technique was detected. In cell-suspension culture, isolated cell-suspension was evaluated by flow cytometric analysis for vimentin expression and residual limbal tissue after dispase treatment was examined by scanning electron microscopy. In limbal epithelial cells suspension less than 5% cells were vimentin positive. Examination of residual limbal tissue confirmed that all the limbal epithelial cells had been removed. Histological examination revealed that with cell-suspension culture the cultured epithelial cells could differentiate better than with explant technique. In cells cultured with cell-suspension, there were much more cells expressing DeltaNp63 than in explant cultured cells. In cells cultured with explants, most of DeltaNp63 labelling cells mustered around the explants, and peripheral cells on the slides were DeltaNp63 negative. These results suggested that with pure limbal epithelial cells suspension including basal cells, which could directly enter into culture system, cell-suspension culture technique was significantly superior to explant culture technique in terms of stem cells content.     

Towards a defined,serum‐ and feeder‐free culture of stratified human oral mucosal epithelium for ocular surface reconstruction

Purpose: Ocular surface reconstruction with cultivated oral mucosal epithelial transplantation technique is a viable treatment option for severe ocular surface injuries and diseases with limbal stem cell deficiency. Currently, this technique is based on utilization of xenogenic, allogenic or undefined components such as murine 3T3 feeders, serum and amniotic membrane. In this study, we aimed to find a more defined culture method to generate stratified human oral mucosal epithelium. Methods: In this study, we have examined the formation of stratified cell sheets from human oral mucosal epithelial cells under serum‐free culture environment both in the absence and presence of fibroblast‐conditioned culture medium and elevated epidermal growth factor (EGF) concentration. Results: In all examined culture conditions, the cultivated oral epithelial cells formed a stratified tissue, which was positive for keratins K3/12, K4 and K13. The tissue‐engineered oral epithelia also expressed proliferation and progenitor markers Ki67 and p63 in the basal layer of the cell sheets, suggesting that the epithelia still had regenerative capacity. The cultures presented expression of tight junction proteins ZO‐1 and occludin and high transepithelial electrical resistance values. Conclusion: In this culture method, we have been able to produce stratified cell sheets successfully without serum, conditioning of the medium or increased EGF concentration. We provide a novel protocol to produce tight multi‐layered epithelium with proliferative potential, which can be easily adapted for cultivated oral mucosal epithelial transplantation.     

The Preliminary Experimental Study of Induced Differentiation of Embryonic Stem Cells into Corneal Epithelial Cells

Purpose: To study preliminarily induced differentiation of embryonic stem cells into corneal epithelial cells in vitro.Methods: Murine embryonic stem cells were co-cultured with Rabbit limbal corneal epithelial cells in Transwell system to induce differentiation. Mophological and immunohistochemical examination were implemented.Results: The induced cells from embryonic stem cells have an epithelial appearance. The cells formed a network and were confluent into film gradually after being co-cultured with rabbit limbal corneal epithelial cells for 24 ~ 96 hours. The cells ranged mosaic structure and localized together with clear rim. Most of the cells showed polygonal appearance. Transmission electron microscope showed lots of microvilli on the surface of induced cells and tight junctions between them. These epithelial-like cells expressed the corneal epithelial cell specific marker cytokeratin3/cytokeratin12. Conclusion: The potential mechanism of the differentiation of murine embryonic stem cel     

骨髓间充质干细胞及其在眼科应用的研究进展

骨髓间充质干细胞作为一类成体干细胞,由于具有易被分离、易被外源基因转染且稳定表达,分裂增殖能力强、以及体外操作简便等特点,成为组织工程、细胞移植及基因治疗理想的靶细胞。我们就骨髓间充质干细胞的概念、各项生理特征、向各组织的分化潜能及在眼科的应用等方面进行综述,并展望骨髓间充质干细胞的应用前景。