小鼠眼部碱烧伤后骨髓间充质干细胞归巢到角膜组织的检测及影响因素分析
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摘要
第一章小鼠碱烧伤完全性角膜缘干细胞功能失代偿模型的建立及评价
目的
建立模拟临床的小鼠碱烧伤完全性角膜缘干细胞功能失代偿模型,为研究碱烧伤引发的完全性角膜缘干细胞功能失代偿提供简单、稳定的动物模型。
方法
选取6-8周龄C57小鼠30只,全身麻醉后将浸有0.5mol/L氢氧化钠溶液的环形滤纸片(内径3mm,外径5mm)置于角膜缘30秒后取下,然后应用生理盐水冲洗结膜囊30秒；术后1天、3天、5天、7天、10天、14天、21天及30天,应用裂隙灯显微镜观察角膜上皮完整性、基质透明性及新生血管化情况；每个时间点随机抽取3只小鼠取材,进行组织病理学检查；术后21天进行角膜免疫印迹细胞学检查。
结果
①裂隙灯检查：术后1天：角膜缘缺血,可见边界清楚的灰白色环状混浊区,大小与滤纸片直径一致,全角膜水肿、上皮缺损；术后3-5天：角膜灰白色混浊,水肿加重；术后7-10天：角膜水肿逐渐减轻,上皮恢复完整；术后14天：角膜缘可见毛刷状新生血管生长；术后21天：角膜缘较多新生血管长入角膜基质；术后30天：角膜混浊、新生血管化,部分模型发生睑球粘连；
②HE染色：术后1-5天：角膜上皮层、基质层内可见大量炎症细胞浸润,角膜基质水肿、明显增厚、胶原纤维排列不规则、较疏松；术后7-10天：角膜表面单层上皮覆盖,基质水肿逐渐减轻,炎症细胞浸润减少；术后14天：角膜表面覆盖2-3层上皮细胞,基质无明显水肿,胶原纤维排列相对整齐；术后21天：角膜基质内较多新生血管增生,管腔内有成熟红细胞；术后30天：角膜上皮层内可见较多杯状细胞；
③免疫印迹细胞学检查：角膜表面可见较多核大、核质比例约1/2、嗜伊红染色的杯状细胞,PAS染色阳性,为结膜细胞表型。
结论
①应用环形滤纸片碱烧伤的方法制作小鼠完全性角膜缘干细胞功能失代偿模型,角膜表面可见较多杯状细胞,裂隙灯检查见小鼠角膜的体征符合临床上“完全性角膜缘干细胞功能失代偿”的诊断标准。
②此方法操作简单,易于重复,为下一步的在体研究提供了良好的动物模型。
第二章小鼠眼部碱烧伤急性期骨髓间充质干细胞归巢到角膜组织的检测
目的
观察小鼠眼部碱烧伤急性期骨髓间充质干细胞归巢到角膜组织的时间窗、定植位置、以及影响因素。
方法
选取6-8周龄C57小鼠80只,随机分为骨髓功能正常组、骨髓功能动员组(皮下注射重组人粒细胞集落刺激因子)及球结膜下注射组(球结膜下注射GFP转染的小鼠骨髓间充质干细胞),建立完全性角膜缘干细胞功能失代偿模型,分别于碱烧伤后1天、3天、5天、7天、10天、14d天、21天及30天取材：
①冰冻切片行CD29、CD44及CD45免疫荧光染色；球结膜下注射组应用荧光显微镜观察GFP阳性细胞在眼表的位置；
②实时定量PCR检测CD29、CD44、CD45、CD90、CD105及基质衍生因子1(SDF-1)的表达；
③包埋切片行SDF-1免疫组织化学检测。
结果
①免疫荧光染色：两组呈现类似的表达规律：碱烧伤术后1-10天,角膜中央、周边及角膜缘均未见CD29、CD44及CD45表达；术后14d、21d、30d,角膜缘可见少量CD29、CD44阳性表达,阳性反应物主要位于胞膜,但CD45均为阴性。
②荧光显微镜：术后1天、3天、5天及7天,球结膜下、角膜缘及角膜中央均未见GFP阳性细胞；
③实时定量PCR：两组呈现类似的表达规律：CD29、CD44、CD90及CD105：碱烧伤术后1-7天均未见表达,术后10天、14天、21天、30天呈现低水平表达；CD45：各个时间点均为阴性。
④免疫组织化学检测：两组呈现类似的表达规律：术后7天：SDF-1呈现强阳性表达,阳性反应物主要位于角膜上皮细胞胞浆内；术后10-14天：阳性反应物逐渐减少；术后21天：SDF-1表达呈现阴性。
结论
①小鼠眼部碱烧伤后14-30天,仅有少量骨髓间充质干细胞归巢到角膜组织并聚集在角膜缘部位。
②小鼠眼部碱烧伤后SDF-1表达增加,时间-剂量关系与心肌等组织相似,不是抑制骨髓间充质干细胞归巢的关键因素。
第三章眼表炎症因子及高渗透压环境对小鼠骨髓间充质干细胞生物学性状的影响
目的
检测眼表主要炎症因子(白介素-lβ)及高渗透压环境(406Osm)对小鼠骨髓间充质干细胞形态、活细胞率、增殖及迁移能力等生物学性状的影响。
方法
体外分离小鼠骨髓间充质干细胞进行培养,设置正常对照组、5ng/ml IL-1β组、10ng/ml IL-1β组、20ng/ml IL-1β组及高渗组,分别应用MTT法、细胞克隆形成试验、划痕法进行检测。
结果
①细胞形态：原代培养的细胞呈梭形,排列具有方向性,胞浆丰富,细胞核较大,呈圆形,位于细胞中央；5ng/ml IL-1β组细胞形态无明显差异；随着培养基中IL-1β浓度升高,出现较多漂浮细胞,细胞变小,多呈圆形；高渗组培养基中出现大量漂浮细胞,贴壁细胞排列松散,形态不规则,失去梭形外观,细胞膜皱缩,反光增强。
②MTT法：正常对照组吸光度(0D值)为0.498±0.043(n=5)；5ng/m1IL-1β组0D值为0.499±0.038(n=5),与正常对照组相比,t=-0.035,P=-0.974,差异无统计学意义；10ng/ml IL-1β组、20ng/ml IL-1β组及高渗组0D值明显减少,以高渗组为著,与正常对照组相比,tr,=6.209,Pt=0.003,t2=8.881,P2=0.001,tr=10.146,P.0.001,差异均具有统计学意义；
③细胞克隆形成试验：正常对照组细胞能形成较多克隆,克隆形成率为18.200±1.923％(n=3)；5ng/ml IL-1β组克隆形成率下降至17.400±2.074%(n=3),与正常对照组相比,t=0.667,P=0.541,差异无统计学意义；10ng/ml IL-1β组、20ng/m1IL-1β组及高渗组克隆形成率进一步减少,以高渗组为著,与正常对照组相比,t1,=12.348,P1=0.000,t2=17.250,P2=0.000,t3=11.943,P3=0.000,差异均具有统计学意义；
④划痕法：正常对照组细胞迁移能力旺盛,72小时全部越过划痕；5ng/ml IL-1β组细胞迁移能力未受明显影响,72小时全部越过划痕；10ng/ml IL-1β组、20nng/mlIL-1β组及高渗组24小时、48小时及72小时细胞迁移能力均明显下降,以高渗组为著,与正常对照组相比,差异均具有统计学意义。
结论
①眼表高渗透压环境能够显著地抑制骨髓间充质干细胞的迁移、增殖能力,较多细胞出现死亡现象。
②低浓度IL-1β(5ng/ml)不具有细胞毒性,并且对骨髓间充质干细胞的迁移、增殖能力无明显影响。
③随着培养基中IL-18浓度的升高(lOng/ml,20ng/ml),细胞毒性逐渐显现,不同程度地抑制了骨髓间充质干细胞的迁移、增殖能力。
第四章小鼠眼部碱烧伤稳定期骨髓间充质干细胞归巢到角膜组织的检测
目的
观察小鼠眼部碱烧伤稳定期骨髓间充质干细胞归巢到角膜组织的时间窗及定植位置。
方法
选取6-8周龄C57小鼠80只,建立完全性角膜缘干细胞功能失代偿模型,眼局部应用抗炎药物一个月后进行后续实验。随机分为骨髓功能正常组(30只)、骨髓功能动员组(30只)及球结膜下注射组(20只)。分别于碱烧伤后1天、3天、5天、7天、10天、14天、21天及30天取材,进行裂隙灯照相、免疫荧光染色以及实时定量PCR检测。
结果
①裂隙灯照相：骨髓功能动员组及球结膜下注射组：小鼠角膜新生血管化程度降低,两组之间无明显差异。
②免疫荧光染色：骨髓功能正常组：各个时间点,角膜缘可见极少量CD29、CD44表达,CD45均为阴性。骨髓功能动员组：术后1-3天,角膜缘可见少量CD29、CD44表达；术后5-10天,CD29、CD44阳性表达增加,CD45表达均为阴性；术后14天,CD29、CD44阳性表达开始减少；术后21天,CD29、CD44及CD45表达均为阴性；
③实时定量PCR：
CD29、CD44、CD90及CD105的表达规律：骨髓功能正常组：各个时间点表达量较低；骨髓功能动员组：术后1-3天,表达量较低；5天开始升高,7-10天维持较高水平,14天时开始下降,21天少量表达,30天呈现阴性；球结膜下注射组：术后ld呈现高表达,以后逐渐降低,10d表达呈现阴性。骨髓功能动员组及球结膜下注射组角膜缘CD29、CD44、CD90及CD105的表达量明显高于中央角膜。
ABCG2、P63、CK3/12及CK19的表达规律：骨髓功能动员组：ABCG2、P63及CK3/12表达量较高,CK19表达量较低,与骨髓功能正常组相比,差异均具有统计学意义；球结膜下注射组：ABCG2、P63及CK3/12表达量低于骨髓功能动员组,CK19表达量高于骨髓功能动员组,但二者相比,差异均不具有统计学意义。
结论
①眼表微环境改善后,归巢到角膜组织的骨髓间充质干细胞明显增多,并有利于眼表向角膜上皮表型分化；应用粒细胞集落刺激因子进行骨髓动员可以提高归巢效率。
②稳定期球结膜下注射骨髓间充质干细胞的眼表重建效果略优于骨髓动员,但持续时间较短。
③经骨髓动员或结膜下注射归巢到角膜的骨髓间充质干细胞均聚集在角膜缘处,不向角膜中央发生移动。
Part I The Establishment and Evaluation of Alkali Burn Related Total Limbal Stem Cell Deficiency Mouse Model
Objective
To establish the mouse model of alkali burn related total limbal stem cell deficiency (LSCD), and to provide a simple and effective model for further research work.
Method
First, put an annular filter paper infiltrating with0.5mol/L sodium hydroxide with3mm inner diameter and5mm external diameter on the corneal limbus of a6-8weeks old mouse for30seconds, and then remove. Complete ocular examinations were performed, including slit-lamp examination, fluorescein staining and impression cytology after1d,3d,5d,7d,10d,14d,21d and30d. In addition, hematoxylin-eosin staining was made after the eyeballs were removed and embed in the paraffin.
Results
①Slit-lamp examination:The limbus became ischemic, a gray ring, the size of which was consistent with the diameter of the annular filter paper, was observed on Day1. The cornea became edematous and opacified on Day3-5. The corneal opacification and edematous were relieved on Day7-10. The circumlimbal hyperemia occurred with hyperplastic vessels, and extended up to the corneal stroma on Day14. Much more hyperplastic vessels were observed around the corneal limbus on Day21. The cornea became conjunctivalization and neovascularization on Day30.
②Hematoxylin-eosin staining:Large number of inflammatory cells were infiltrated in corneal epithelium and stroma on Day1-5. The edematous of corneal stroma was relieved on Day7-10.2-3layers of corneal epithelium was observed on Day14. On Day21, goblet cells were observed on corneal epithelium. Hyperplastic vessels with mature red blood cells inside were observed on corneal stroma on Day30.
③Impression cytology:A lot of goblet cells with positive staining of PAS were observed on corneal epitheliu.
Conclusion
The method of annular filter paper infiltrating with sodium hydroxide is effective in establishing total limbal stem cell deficiency mouse model, which can provide a simple and stable model for further research work. Part II The Observation of Mouse Bone Marrow Mesenchymal Stem Cells Homing to cornea after ocular alkali burns
Objective
To observe the timing and location of mouse bone marrow mesenchymal stem cells homing to cornea after ocular alkali burns.
Method
80mouse models were randomly divided into three group:group of normal bone marrow function, mobilized bone marrow function with G-CSF and subconjunctival injection with GFP transfected mouse bone marrow mesenchymal stem cells. The operated and contralateral control eyeballs were removed at1d,3d,5d,7d,10d,14d,21d and30d. The immunofluorescence staining of CD29, CD44and CD45and immunohistochemical staining of SDF-1were performed. Laser scanning confocal microscope was used to detect GFP transfected mouse bone marrow mesenchymal stem cells. Real-time quantitative polymerase chain reaction was performed to analyse the expression of CD44, CD45, CD90, CD105and SDF-1.
Results
①Immunofluorescence staining:No positive staining of CD29, CD44and CD45was found on Day1-10. A few positive staining of CD29and CD44in corneal limbus were detected on Day14-30.
②Laser scanning confocal microscope:No GFP transfected mouse bone marrow mesenchymal stem cells was detected in cornea and subconjunctiva.
③Real-time quantitative polymerase chain reaction:No expression of CD29, CD44, CD45, CD90and CD105was detected on Day1-10, a low level expression was detected on Day14-30.
④Immunohistochemical staining:The positive staining of SDF-1was observed on Day7, and became less gradually. No positive staining was observed on Day21.
Conclusion
①A few mouse bone marrow mesenchymal stem cells were detected homing to corneal limbus after14-30days of ocular alkali burns.
②The expression of SDF-1, which was consistent with myocardial tissue, was not the key factor affecting the homing of mouse bone marrow mesenchymal stem cells.
Part III The influence of inflammatory cytokine and hyperosmosis after ocular alkali burns on mouse bone marrow mesenchymal stem cells
Objective
To observe the influence of inflammatory cytokine (IL-1β) and hyperosmosis (406Osm) after ocular alkali burns on mouse bone marrow mesenchymal stem cells.
Method
Mouse bone marrow mesenchymal stem cells were isolated and incubated in vitro, and then divided into five groups:group of control,5ng/ml IL-1β, lOng/ml IL-1β,20ng/ml IL-1β and hyperosmosis. MTT assay, cell clone forming test and scratch assay were performed.
Results
①Cell morphology:The original generation of cultured mouse bone marrow mesenchymal stem cells were fusiform with abundant cytoplasm and circular large nuclei. The group of5ng/ml IL-1β:There was no difference in cell morphology between the control and5ng/ml IL-1β group. With the increase of concentration of IL-1β in medium, more floating cells appeared. The group of hyperosmosis:The cultured cells present as irregular shape with shrinking and reflective cell membrane.
②MTT assay:The value of OD in control group was0.498±0.043(n=5) and0.499±0.038(n=5) in5ng/ml IL-1group. There was no statistical significance between the two groups. But The difference was of significance in statistics among the control and10ng/ml IL-1β,20ng/ml IL-1P and hyperosmosis group.
③Cell clone forming test:The cell clone forming rate in control group was18.200±1.923%(n=3), and decreased to17.400±2.074%(n=3)in5ng/ml IL-1group. But there was no statistical significance between the two groups. The cell clone forming rate was further decreased in groups of10ng/ml IL-1β,20ng/ml IL-1β and hyperosmosis, the difference was of significance in statistics among them and the control.
④Seratch assay:The cell migration ability was not affected in5ng/ml IL-1group, but apparently decreased in groups of10ng/ml IL-1β,20ng/ml IL-1β and hyperosmosis in24hours,48hours and72hours. The difference was of significance in statistics among them and the control.
Conclusion
①The hyperosmosis in ocular surface apparently affected clone forming rate and migration ability of mouse bone marrow mesenchymal stem cells.
②Low concentration of IL-1β was proved to be non-cytotoxicity, and had no influence on cell clone forming rate and migration ability.
③With the increase of concentration of IL-1β in medium, cell toxicity gradually appeared, and clone forming rate and migration ability was inevitably affected. Part IV The Observation of Mouse Bone Marrow Mesenchymal Stem Cells Homing to cornea in the stable stage after ocular alkali burns
Objective
To observe the timing and location of mouse bone marrow mesenchymal stem cells homing to cornea in the stable stage after ocular alkali burns.
Method
80mouse models in the stable stage of ocular alkali burns were randomly divided into three group:normal bone marrow function group, mobilized bone marrow function group and subconjunctival injection group. The operated and contralateral control eyeballs were removed at Id,3d,5d,7d,10d,14d,21d and30d. The immunofluorescence staining of CD29, CD44and CD45were performed. Laser scanning confocal microscope was used to detect GFP transfected mouse bone marrow mesenchymal stem cells. Real-time quantitative polymerase chain reaction was performed to analyse the expression of CD44, CD45, CD90, CD105, ABCG2, P63, CK3/12and CK19.
Results
②Slit-lamp examination:The corneal clarity was partly restored, and corneal neovascularization decreased in mobilized bone marrow function group and subconjunctival injection group. There was no obvious change in normal bone marrow function group.
②Immunofluorescence staining:A few positive staining of CD29and CD44in corneal limbus were detected in normal bone marrow function group. Similarly, a few positive staining of CD29and CD44in corneal limbus were detected on Day1-3, and sharply increased on Day5-10, gradually decreased on Day14in mobilized bone marrow function group. No positive staining was found on Day21-30.
③Laser scanning confocal microscope:A lot of GFP positive cells in corneal limbus were detected on Day1-3, but gradually decreased as time passed. No positive staining was found on Day10.
④Real-time quantitative polymerase chain reaction:A low level expression of CD29, CD44, CD90and CD105was detected in normal bone marrow function group. In mobilized bone marrow function group, the expression of CD29, CD44, CD90and CD105obviously increased on Day5, maintained a high level on Day7-10, and gradually decreased after Day14. A higher level expression of CD29, CD44, CD90and CD105was detected in subconjunctival injection group, but gradually decreased as time passed. No positive staining was found on Day10.
When compared to normal bone marrow function group, a higher level expression of ABCG2, P63and CK3/12, and lower level of CK19were found in mobilized bone marrow function group. The difference was of significance in statistics between the two group. When compared to mobilized bone marrow function group, a higher level expression of ABCG2, P63and CK3/12, and lower level of CK19were found in subconjunctival injection group. But there was no statistical significance between the two groups.
Conclusion
①Much more mouse bone marrow mesenchymal stem cells were detected homing to corneal limbus in the stable stage after ocular alkali burns. Mobilizing bone marrow function with G-CSF benefited in improving homing efficience.
②The effect of ocular surface reconstruction in subconjunctival injection group was a litter better than that in mobilized bone marrow function group.
③The homing mouse bone marrow mesenchymal stem cells were located in the corneal limbus.
目的
建立模拟临床的小鼠碱烧伤完全性角膜缘干细胞功能失代偿模型,为研究碱烧伤引发的完全性角膜缘干细胞功能失代偿提供简单、稳定的动物模型。
方法
选取6-8周龄C57小鼠30只,全身麻醉后将浸有0.5mol/L氢氧化钠溶液的环形滤纸片(内径3mm,外径5mm)置于角膜缘30秒后取下,然后应用生理盐水冲洗结膜囊30秒；术后1天、3天、5天、7天、10天、14天、21天及30天,应用裂隙灯显微镜观察角膜上皮完整性、基质透明性及新生血管化情况；每个时间点随机抽取3只小鼠取材,进行组织病理学检查；术后21天进行角膜免疫印迹细胞学检查。
结果
①裂隙灯检查：术后1天：角膜缘缺血,可见边界清楚的灰白色环状混浊区,大小与滤纸片直径一致,全角膜水肿、上皮缺损；术后3-5天：角膜灰白色混浊,水肿加重；术后7-10天：角膜水肿逐渐减轻,上皮恢复完整；术后14天：角膜缘可见毛刷状新生血管生长；术后21天：角膜缘较多新生血管长入角膜基质；术后30天：角膜混浊、新生血管化,部分模型发生睑球粘连；
②HE染色：术后1-5天：角膜上皮层、基质层内可见大量炎症细胞浸润,角膜基质水肿、明显增厚、胶原纤维排列不规则、较疏松；术后7-10天：角膜表面单层上皮覆盖,基质水肿逐渐减轻,炎症细胞浸润减少；术后14天：角膜表面覆盖2-3层上皮细胞,基质无明显水肿,胶原纤维排列相对整齐；术后21天：角膜基质内较多新生血管增生,管腔内有成熟红细胞；术后30天：角膜上皮层内可见较多杯状细胞；
③免疫印迹细胞学检查：角膜表面可见较多核大、核质比例约1/2、嗜伊红染色的杯状细胞,PAS染色阳性,为结膜细胞表型。
结论
①应用环形滤纸片碱烧伤的方法制作小鼠完全性角膜缘干细胞功能失代偿模型,角膜表面可见较多杯状细胞,裂隙灯检查见小鼠角膜的体征符合临床上“完全性角膜缘干细胞功能失代偿”的诊断标准。
②此方法操作简单,易于重复,为下一步的在体研究提供了良好的动物模型。
第二章小鼠眼部碱烧伤急性期骨髓间充质干细胞归巢到角膜组织的检测
目的
观察小鼠眼部碱烧伤急性期骨髓间充质干细胞归巢到角膜组织的时间窗、定植位置、以及影响因素。
方法
选取6-8周龄C57小鼠80只,随机分为骨髓功能正常组、骨髓功能动员组(皮下注射重组人粒细胞集落刺激因子)及球结膜下注射组(球结膜下注射GFP转染的小鼠骨髓间充质干细胞),建立完全性角膜缘干细胞功能失代偿模型,分别于碱烧伤后1天、3天、5天、7天、10天、14d天、21天及30天取材：
①冰冻切片行CD29、CD44及CD45免疫荧光染色；球结膜下注射组应用荧光显微镜观察GFP阳性细胞在眼表的位置；
②实时定量PCR检测CD29、CD44、CD45、CD90、CD105及基质衍生因子1(SDF-1)的表达；
③包埋切片行SDF-1免疫组织化学检测。
结果
①免疫荧光染色：两组呈现类似的表达规律：碱烧伤术后1-10天,角膜中央、周边及角膜缘均未见CD29、CD44及CD45表达；术后14d、21d、30d,角膜缘可见少量CD29、CD44阳性表达,阳性反应物主要位于胞膜,但CD45均为阴性。
②荧光显微镜：术后1天、3天、5天及7天,球结膜下、角膜缘及角膜中央均未见GFP阳性细胞；
③实时定量PCR：两组呈现类似的表达规律：CD29、CD44、CD90及CD105：碱烧伤术后1-7天均未见表达,术后10天、14天、21天、30天呈现低水平表达；CD45：各个时间点均为阴性。
④免疫组织化学检测：两组呈现类似的表达规律：术后7天：SDF-1呈现强阳性表达,阳性反应物主要位于角膜上皮细胞胞浆内；术后10-14天：阳性反应物逐渐减少；术后21天：SDF-1表达呈现阴性。
结论
①小鼠眼部碱烧伤后14-30天,仅有少量骨髓间充质干细胞归巢到角膜组织并聚集在角膜缘部位。
②小鼠眼部碱烧伤后SDF-1表达增加,时间-剂量关系与心肌等组织相似,不是抑制骨髓间充质干细胞归巢的关键因素。
第三章眼表炎症因子及高渗透压环境对小鼠骨髓间充质干细胞生物学性状的影响
目的
检测眼表主要炎症因子(白介素-lβ)及高渗透压环境(406Osm)对小鼠骨髓间充质干细胞形态、活细胞率、增殖及迁移能力等生物学性状的影响。
方法
体外分离小鼠骨髓间充质干细胞进行培养,设置正常对照组、5ng/ml IL-1β组、10ng/ml IL-1β组、20ng/ml IL-1β组及高渗组,分别应用MTT法、细胞克隆形成试验、划痕法进行检测。
结果
①细胞形态：原代培养的细胞呈梭形,排列具有方向性,胞浆丰富,细胞核较大,呈圆形,位于细胞中央；5ng/ml IL-1β组细胞形态无明显差异；随着培养基中IL-1β浓度升高,出现较多漂浮细胞,细胞变小,多呈圆形；高渗组培养基中出现大量漂浮细胞,贴壁细胞排列松散,形态不规则,失去梭形外观,细胞膜皱缩,反光增强。
②MTT法：正常对照组吸光度(0D值)为0.498±0.043(n=5)；5ng/m1IL-1β组0D值为0.499±0.038(n=5),与正常对照组相比,t=-0.035,P=-0.974,差异无统计学意义；10ng/ml IL-1β组、20ng/ml IL-1β组及高渗组0D值明显减少,以高渗组为著,与正常对照组相比,tr,=6.209,Pt=0.003,t2=8.881,P2=0.001,tr=10.146,P.0.001,差异均具有统计学意义；
③细胞克隆形成试验：正常对照组细胞能形成较多克隆,克隆形成率为18.200±1.923％(n=3)；5ng/ml IL-1β组克隆形成率下降至17.400±2.074%(n=3),与正常对照组相比,t=0.667,P=0.541,差异无统计学意义；10ng/ml IL-1β组、20ng/m1IL-1β组及高渗组克隆形成率进一步减少,以高渗组为著,与正常对照组相比,t1,=12.348,P1=0.000,t2=17.250,P2=0.000,t3=11.943,P3=0.000,差异均具有统计学意义；
④划痕法：正常对照组细胞迁移能力旺盛,72小时全部越过划痕；5ng/ml IL-1β组细胞迁移能力未受明显影响,72小时全部越过划痕；10ng/ml IL-1β组、20nng/mlIL-1β组及高渗组24小时、48小时及72小时细胞迁移能力均明显下降,以高渗组为著,与正常对照组相比,差异均具有统计学意义。
结论
①眼表高渗透压环境能够显著地抑制骨髓间充质干细胞的迁移、增殖能力,较多细胞出现死亡现象。
②低浓度IL-1β(5ng/ml)不具有细胞毒性,并且对骨髓间充质干细胞的迁移、增殖能力无明显影响。
③随着培养基中IL-18浓度的升高(lOng/ml,20ng/ml),细胞毒性逐渐显现,不同程度地抑制了骨髓间充质干细胞的迁移、增殖能力。
第四章小鼠眼部碱烧伤稳定期骨髓间充质干细胞归巢到角膜组织的检测
目的
观察小鼠眼部碱烧伤稳定期骨髓间充质干细胞归巢到角膜组织的时间窗及定植位置。
方法
选取6-8周龄C57小鼠80只,建立完全性角膜缘干细胞功能失代偿模型,眼局部应用抗炎药物一个月后进行后续实验。随机分为骨髓功能正常组(30只)、骨髓功能动员组(30只)及球结膜下注射组(20只)。分别于碱烧伤后1天、3天、5天、7天、10天、14天、21天及30天取材,进行裂隙灯照相、免疫荧光染色以及实时定量PCR检测。
结果
①裂隙灯照相：骨髓功能动员组及球结膜下注射组：小鼠角膜新生血管化程度降低,两组之间无明显差异。
②免疫荧光染色：骨髓功能正常组：各个时间点,角膜缘可见极少量CD29、CD44表达,CD45均为阴性。骨髓功能动员组：术后1-3天,角膜缘可见少量CD29、CD44表达；术后5-10天,CD29、CD44阳性表达增加,CD45表达均为阴性；术后14天,CD29、CD44阳性表达开始减少；术后21天,CD29、CD44及CD45表达均为阴性；
③实时定量PCR：
CD29、CD44、CD90及CD105的表达规律：骨髓功能正常组：各个时间点表达量较低；骨髓功能动员组：术后1-3天,表达量较低；5天开始升高,7-10天维持较高水平,14天时开始下降,21天少量表达,30天呈现阴性；球结膜下注射组：术后ld呈现高表达,以后逐渐降低,10d表达呈现阴性。骨髓功能动员组及球结膜下注射组角膜缘CD29、CD44、CD90及CD105的表达量明显高于中央角膜。
ABCG2、P63、CK3/12及CK19的表达规律：骨髓功能动员组：ABCG2、P63及CK3/12表达量较高,CK19表达量较低,与骨髓功能正常组相比,差异均具有统计学意义；球结膜下注射组：ABCG2、P63及CK3/12表达量低于骨髓功能动员组,CK19表达量高于骨髓功能动员组,但二者相比,差异均不具有统计学意义。
结论
①眼表微环境改善后,归巢到角膜组织的骨髓间充质干细胞明显增多,并有利于眼表向角膜上皮表型分化；应用粒细胞集落刺激因子进行骨髓动员可以提高归巢效率。
②稳定期球结膜下注射骨髓间充质干细胞的眼表重建效果略优于骨髓动员,但持续时间较短。
③经骨髓动员或结膜下注射归巢到角膜的骨髓间充质干细胞均聚集在角膜缘处,不向角膜中央发生移动。
Part I The Establishment and Evaluation of Alkali Burn Related Total Limbal Stem Cell Deficiency Mouse Model
Objective
To establish the mouse model of alkali burn related total limbal stem cell deficiency (LSCD), and to provide a simple and effective model for further research work.
Method
First, put an annular filter paper infiltrating with0.5mol/L sodium hydroxide with3mm inner diameter and5mm external diameter on the corneal limbus of a6-8weeks old mouse for30seconds, and then remove. Complete ocular examinations were performed, including slit-lamp examination, fluorescein staining and impression cytology after1d,3d,5d,7d,10d,14d,21d and30d. In addition, hematoxylin-eosin staining was made after the eyeballs were removed and embed in the paraffin.
Results
①Slit-lamp examination:The limbus became ischemic, a gray ring, the size of which was consistent with the diameter of the annular filter paper, was observed on Day1. The cornea became edematous and opacified on Day3-5. The corneal opacification and edematous were relieved on Day7-10. The circumlimbal hyperemia occurred with hyperplastic vessels, and extended up to the corneal stroma on Day14. Much more hyperplastic vessels were observed around the corneal limbus on Day21. The cornea became conjunctivalization and neovascularization on Day30.
②Hematoxylin-eosin staining:Large number of inflammatory cells were infiltrated in corneal epithelium and stroma on Day1-5. The edematous of corneal stroma was relieved on Day7-10.2-3layers of corneal epithelium was observed on Day14. On Day21, goblet cells were observed on corneal epithelium. Hyperplastic vessels with mature red blood cells inside were observed on corneal stroma on Day30.
③Impression cytology:A lot of goblet cells with positive staining of PAS were observed on corneal epitheliu.
Conclusion
The method of annular filter paper infiltrating with sodium hydroxide is effective in establishing total limbal stem cell deficiency mouse model, which can provide a simple and stable model for further research work. Part II The Observation of Mouse Bone Marrow Mesenchymal Stem Cells Homing to cornea after ocular alkali burns
Objective
To observe the timing and location of mouse bone marrow mesenchymal stem cells homing to cornea after ocular alkali burns.
Method
80mouse models were randomly divided into three group:group of normal bone marrow function, mobilized bone marrow function with G-CSF and subconjunctival injection with GFP transfected mouse bone marrow mesenchymal stem cells. The operated and contralateral control eyeballs were removed at1d,3d,5d,7d,10d,14d,21d and30d. The immunofluorescence staining of CD29, CD44and CD45and immunohistochemical staining of SDF-1were performed. Laser scanning confocal microscope was used to detect GFP transfected mouse bone marrow mesenchymal stem cells. Real-time quantitative polymerase chain reaction was performed to analyse the expression of CD44, CD45, CD90, CD105and SDF-1.
Results
①Immunofluorescence staining:No positive staining of CD29, CD44and CD45was found on Day1-10. A few positive staining of CD29and CD44in corneal limbus were detected on Day14-30.
②Laser scanning confocal microscope:No GFP transfected mouse bone marrow mesenchymal stem cells was detected in cornea and subconjunctiva.
③Real-time quantitative polymerase chain reaction:No expression of CD29, CD44, CD45, CD90and CD105was detected on Day1-10, a low level expression was detected on Day14-30.
④Immunohistochemical staining:The positive staining of SDF-1was observed on Day7, and became less gradually. No positive staining was observed on Day21.
Conclusion
①A few mouse bone marrow mesenchymal stem cells were detected homing to corneal limbus after14-30days of ocular alkali burns.
②The expression of SDF-1, which was consistent with myocardial tissue, was not the key factor affecting the homing of mouse bone marrow mesenchymal stem cells.
Part III The influence of inflammatory cytokine and hyperosmosis after ocular alkali burns on mouse bone marrow mesenchymal stem cells
Objective
To observe the influence of inflammatory cytokine (IL-1β) and hyperosmosis (406Osm) after ocular alkali burns on mouse bone marrow mesenchymal stem cells.
Method
Mouse bone marrow mesenchymal stem cells were isolated and incubated in vitro, and then divided into five groups:group of control,5ng/ml IL-1β, lOng/ml IL-1β,20ng/ml IL-1β and hyperosmosis. MTT assay, cell clone forming test and scratch assay were performed.
Results
①Cell morphology:The original generation of cultured mouse bone marrow mesenchymal stem cells were fusiform with abundant cytoplasm and circular large nuclei. The group of5ng/ml IL-1β:There was no difference in cell morphology between the control and5ng/ml IL-1β group. With the increase of concentration of IL-1β in medium, more floating cells appeared. The group of hyperosmosis:The cultured cells present as irregular shape with shrinking and reflective cell membrane.
②MTT assay:The value of OD in control group was0.498±0.043(n=5) and0.499±0.038(n=5) in5ng/ml IL-1group. There was no statistical significance between the two groups. But The difference was of significance in statistics among the control and10ng/ml IL-1β,20ng/ml IL-1P and hyperosmosis group.
③Cell clone forming test:The cell clone forming rate in control group was18.200±1.923%(n=3), and decreased to17.400±2.074%(n=3)in5ng/ml IL-1group. But there was no statistical significance between the two groups. The cell clone forming rate was further decreased in groups of10ng/ml IL-1β,20ng/ml IL-1β and hyperosmosis, the difference was of significance in statistics among them and the control.
④Seratch assay:The cell migration ability was not affected in5ng/ml IL-1group, but apparently decreased in groups of10ng/ml IL-1β,20ng/ml IL-1β and hyperosmosis in24hours,48hours and72hours. The difference was of significance in statistics among them and the control.
Conclusion
①The hyperosmosis in ocular surface apparently affected clone forming rate and migration ability of mouse bone marrow mesenchymal stem cells.
②Low concentration of IL-1β was proved to be non-cytotoxicity, and had no influence on cell clone forming rate and migration ability.
③With the increase of concentration of IL-1β in medium, cell toxicity gradually appeared, and clone forming rate and migration ability was inevitably affected. Part IV The Observation of Mouse Bone Marrow Mesenchymal Stem Cells Homing to cornea in the stable stage after ocular alkali burns
Objective
To observe the timing and location of mouse bone marrow mesenchymal stem cells homing to cornea in the stable stage after ocular alkali burns.
Method
80mouse models in the stable stage of ocular alkali burns were randomly divided into three group:normal bone marrow function group, mobilized bone marrow function group and subconjunctival injection group. The operated and contralateral control eyeballs were removed at Id,3d,5d,7d,10d,14d,21d and30d. The immunofluorescence staining of CD29, CD44and CD45were performed. Laser scanning confocal microscope was used to detect GFP transfected mouse bone marrow mesenchymal stem cells. Real-time quantitative polymerase chain reaction was performed to analyse the expression of CD44, CD45, CD90, CD105, ABCG2, P63, CK3/12and CK19.
Results
②Slit-lamp examination:The corneal clarity was partly restored, and corneal neovascularization decreased in mobilized bone marrow function group and subconjunctival injection group. There was no obvious change in normal bone marrow function group.
②Immunofluorescence staining:A few positive staining of CD29and CD44in corneal limbus were detected in normal bone marrow function group. Similarly, a few positive staining of CD29and CD44in corneal limbus were detected on Day1-3, and sharply increased on Day5-10, gradually decreased on Day14in mobilized bone marrow function group. No positive staining was found on Day21-30.
③Laser scanning confocal microscope:A lot of GFP positive cells in corneal limbus were detected on Day1-3, but gradually decreased as time passed. No positive staining was found on Day10.
④Real-time quantitative polymerase chain reaction:A low level expression of CD29, CD44, CD90and CD105was detected in normal bone marrow function group. In mobilized bone marrow function group, the expression of CD29, CD44, CD90and CD105obviously increased on Day5, maintained a high level on Day7-10, and gradually decreased after Day14. A higher level expression of CD29, CD44, CD90and CD105was detected in subconjunctival injection group, but gradually decreased as time passed. No positive staining was found on Day10.
When compared to normal bone marrow function group, a higher level expression of ABCG2, P63and CK3/12, and lower level of CK19were found in mobilized bone marrow function group. The difference was of significance in statistics between the two group. When compared to mobilized bone marrow function group, a higher level expression of ABCG2, P63and CK3/12, and lower level of CK19were found in subconjunctival injection group. But there was no statistical significance between the two groups.
Conclusion
①Much more mouse bone marrow mesenchymal stem cells were detected homing to corneal limbus in the stable stage after ocular alkali burns. Mobilizing bone marrow function with G-CSF benefited in improving homing efficience.
②The effect of ocular surface reconstruction in subconjunctival injection group was a litter better than that in mobilized bone marrow function group.
③The homing mouse bone marrow mesenchymal stem cells were located in the corneal limbus.
引文
1. Li F. System of ophthalmology. Beijing:People's Medical Publishing House;1996:3159.
2. Hong J, Qiu T, Wei A, Sun X, Xu J. Clinical characteristics and visual outcome of severe ocular chemical injuries in Shanghai. Ophthalmology 2010;117:2268-2272.
3. Fish R, Davidson RS. Management of ocular thermal and chemical injuries, including amniotic membrane therapy. Curr Opin Ophthalmol 2010;21:317-321.
4. Cauchi PA, Ang GS, Azuara-Blanco A, Burr JM. A systematic literature review of surgical interventions for limbal stem cell deficiency in humans. Am J Ophthalmol 2008:146:251-259.
5. Avunduk AM, Tekelioglu Y. Therapeutic use of limbal stem cells. Curr Stem Cell Res Ther 2006;1:231-238.
6. Koizumi N, Inatomi T, Suzuki T, et al. Cultivated corneal epithelial stem cell transplantation in ocular surface disorders. Ophthalmology 2001;108:1569-1574.
7. Koizumi N, Inatomi T, Suzuki T, et al. Cultivated corneal epithelial transplantation for ocular surface reconstruction in acute phase of Stevens-Johnson syndrome. Arch Ophthalmol 2001;119:298-300.
8. Nakamura T, Sotozono C, Bentley AJ, et al. Long-term phenotypic study after allogeneic cultivated corneal limbal epithelial transplantation for severe ocular surface diseases. Ophthalmology 2010;117:2247-2254.
9. Tsai RJ, Li LM, Chen JK. Reconstruction of damaged corneas by transplantation of autologous limbal epithelial cells. N Engl J Med 2000:343:86-93.
10. Nishida K, Yamato M, Hayashida Y, et al. Corneal reconstruction with tissue-engineered cell sheets composed of autologous oral mucosal epithelium. N Engl J Med 2004;351:1187-1196.
11. Rama P, Matuska S, Paganoni G, et al. Limbal stem-cell therapy and long-term corneal regeneration. N Engl J Med 2010;363:147-155.
12. Burman S, Sangwan V. Cultivated limbal stem cell transplantation for ocular surface reconstruction. Clin Ophthalmol 2008;2(3):489-502.
13. Khaldoyanidi S. Directing stem cell homing. Cell Stem Cell 2008;2(3):198-200.
14. Karp JM, Leng Teo GS. Mesenchymal stem cell homing:the devil is in the details. Cell Stem Cell 2009;4(3):206-216.
15. Keating A. Mesenchymal stromal cells:new directions. Cell Stem Cell 2012;10(6):709-716.
16. Sitalakshmi G, Sudha B, Madhavan HN, et al. Ex vivo cultivation of corneal limbal epithelial cells in a thermoreversible polymer (Mebiol Gel) and their transplantation in rabbits:an animal model. Tissue Eng Part A 2009; 15(2):407-415.
17. Choi SH, Jung SY, Kwon SM, Baek SH. Perspectives on stem cell therapy for cardiac regeneration. Advances andchallenges. Circ J 2012;76(6):1307-1312.
18. Cucchiarini M, Venkatesan JK, Ekici M, Schmitt G, Madry H. Human mesenchymal stem cells overexpressing therapeutic genes:from basicscience to clinical applications for articular cartilage repair.Biomed Mater Eng 2012;22(4):197-208.
19. Ricart E. Current status of mesenchymal stem cell therapy and bone marrow transplantationin IBD. Dig Dis 2012;30 (4):387-391.
20. Nelson ER, Wong VW, Krebsbach PH, Wang SC, Levi B. Heterotopic ossification following burn injury:the role of stem cells. J Burn Care Res 2012;33(4):463-470.
21. Xie L, Shi W. Cornea. Beijing:People's Medical Publishing House;2007:508-509.
22. Shortt AJ, Seeker GA, Notara MD, et al. Transplantation of ex vivo cultured limbal epithelial stem cells:a review of techniques and clinical results. Surv Ophthalmol 2007:52:483-502.
23. Espana EM, Di Pascuale MA, et al. Characterization of corneal pannus removed from patients with total limbal stem cell deficiency. Invest Ophthalmol Vis Sci 2004:45(9):2961-2966.
24. Askari AT, Unzek S, Popovic ZB, et al. Effect of stromal-cell-derived factor 1 on stem-cell homing and tissue regeneration inischaemic cardiomyopathy. Lancet 2003:362(9385):697-703.
25. Penn MS, Pastore J, Miller T, Aras R. SDF-1 in myocardial repair. Gene Ther 2012;19 (6):583-587.
26. Zhang Y, Shen W, Hua J, et al. Pancreatic islet-like clusters from bone marrow mesenchymal stem cells of human first-trimester abortus can cure streptozocin-induced mouse diabetes. Rejuvenation Res 2010;13(6):695-706.
27. Joe AW, Gregory-Evans K. Mesenchymal stem cells and potential applications in treating ocular disease. Curr Eye Res 2010；35(11):941-952.
28. Ren G, Chen X, Dong F, et al. Concise review:mesenchymal stem cells and translational medicine:emergingissues. Stem Cells Transl Med 2012;1(1):51-58.
29. Yu J, Vodyanik MA, Smuga-Otto K et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 2007；318:1917-1920.
30. Miura K, Okada Y, Aoi T et al. Variation in the safety of induced pluripotent stem cell lines. Nat Biotechnol 2009;27:743-745.
31. Gutierrez-Arandal, Ramos-MejiaV, BuenoC, et al. Human induced pluripotent stem cells develop teratoma more efficiently and faster than human embryonic stem cells regardless the site of injection. Stem Cells 2010;28:1568-1570.
32. Wong DJ, Liu H, Ridky TW et al. Module map of stem cell genes guides creation of epithelial cancer stem cells. Cell Stem Cell 2008;2:333-344.
33. Fink DW Jr. FDA regulation of stem cell-based products. Science 2009;324:1662-1663.
34. Chute JP. Stem cell homing. Curr Opin Hematol 2006;13(6):399-406.
35. Shiozawa Y, Havens AM, Pienta KJ, Taichman RS. The bone marrow niche:habitat to hematopoietic and mesenchymal stem cells, and unwitting host to molecular parasites. Leukemia 2008;22(5):941-950.
36. Saito T, Kuang JQ, Bittira B, Al-Khaldi A, Chiu RC. Xenotransplant cardiac chimera: immune tolerance of adult stem cells. Ann Thorac Surg 2002;74(1):19-24; discussion 24.
37. Belema-Bedada F, Uchida S, Martire A, Kostin S, Braun T. Efficient homing of multipotent adult mesenchymal stem cells depends on FROUNT-mediated clustering of CCR2. Cell Stem Cell 2008;2(6):566-575.
38. Tomita M, Adachi Y, Yamada H, et al. Bone marrow-de -rived stem cells can differentiate into retinal cells in injured rat retina. Stem Cells 2002;20:279-283.
39. Kicic A, Shen WY, Wilson AS, et al. Differentiation of marrow stromal cells into photoreceptors in the rat eye. J Neurosci 2003;23:7742-7749.
40. Tomita M, Mori T, Maruyama K, et al. A comparison of neural differentiation and retinal transplantation with bone marrow-derived cells and retinal progenitor cells. Stem Cells 2006;24:2270-2278.
41. Gong L, Wu Q, Song B, et al. Differentiation of rat mesenchymal stem cells transplanted into the subretinal space of sodium iodate-injected rats. Clin Experiment Ophthalmol 2008;36:666-671.
42. Arnhold S, Heiduschka P, Klein H, et al. Adenovirally transduced bone marrow stromal cells differentiate into pigment epithelial cells and induce rescue effects in RCS rats. Invest Ophthalmol Vis Sci 2006:47:4121-4129.
43. Ma Y, Xu Y, Xiao Z, et al. Reconstruction of chemically burned rat corneal surface by bone marrow-derived human mesenchymal stem cells. Stem Cells 2006;24:315-321.
44. Oh JY, Kim MK, Shin MS, et al. The anti-inflammatory and anti-angiogenic role of mesenchymal stem cells in corneal wound healing following chemical injury. Stem Cells 2008:26:1047-1055.
45. Oh JY, Kim MK, Shin MS, et al. Cytokine secretion by human mesenchymal stem cells co-cultured with damaged corneal epithelial cells. Cytokine 2009:46:100-103.
46. McIntosh Ambrose W, Schein 0, Elisseeff J. A tale of two tissues:Stem cells in cartilage and corneal tissue engineering. Curr Stem Cell Res Ther 2010;5:37-48.
47. De Miguel MP, Alio JL, Arnalich-Montiel F, et al. Cornea and ocular surface treatment. Curr Stem Cell Res Ther 2010:5:195-204.
48. Oh JY, Kim MK, Ko JH, et al. Rat allogeneic mesenchymal stem cells did not prolong the survival of corneal xenograft in a pig-to-rat model. Vet Ophthalmol 2009:12:35-40.
49. Zoukhri D. Mechanisms involved in injury and repair of the murine lacrimal gland: Role of programmed cell death and mesenchymal stem cells. Ocul Surf 2010;8:60-69.
50. Zhang JJ, Yi ZW, Dang XQ, He XJ, Wu XC. Mobilization effects of SCF along with G-CSF on bone marrow stem cells and endothelial progenitor cells in rats with unilateral ureteral obstruction. Zhongguo Dang Dai Er Ke Za Zhi 2007;9(2):144-148.
51. Cashen AF, Lazarus HM, Devine SM. Mobilizing stem cells from normal donors:is it possible to improve upon G-CSF? Bone Marrow Transplant 2007:39(10):577-588.
52. Moog R. Management strategies for poor peripheral blood stem cell mobilization. Transfus Apher Sci 2008:38(3):229-236.
53. Ince H, Petzsch M, Rehders TC, Dunkelmann S, Nienaber CA. G-CSF in acute myocardial infarction-experimental and clinical findings. Anadolu Kardiyol Derg 2006:6(3):261-263.
54. Korbling M. In vivo expansion of the circulating stem cell pool. Stem Cells 1998; 16 Suppl 1:131-138.
55. Xie HT, Chen SY, Li GG, Tseng SC. Limbal epithelial stem/progenitor cells attract stromal niche cells by SDF-1/CXCR4signaling to prevent differentiation. Stem Cells 2011:29(11):1874-1885.
56. Wynn RF, Hart CA, Corradi-Perini C et al. A small proportion of mesenchymal stem cells strongly expresses functionally active CXCR4 receptor capable of promoting migration to bone marrow. Blood 2004:104:2643-2645.
57. Belema-Bedada F, Uchida S, Martire A et al. Efficient homing of multipotent adult mesenchymal stem cells depends on FROUNT-mediated clustering of CCR2. Cell Stem Cell 2008;2:566-575.
58. Cheng Z, Ou L, Zhou X et al. Targeted migration of mesenchymal stem cells modified with CXCR4 gene to infarcted myocardium improves cardiac performance. Mol Ther 2008:16:571-579.
59. Hatch HM, Zheng D, Jorgensen ML, et al. SDF-lalpha/CXCR4:A mechanism for hepatic oval cell activation and bone marrow stem cell recruitment to the injured liver of rats. Cloning Stem Cells 2002:4:339-351.
60. Sordi V, Malosio ML, Marchesi F, et al. Bone marrow mesenchymal stem cells express a restricted set of functionally active chemokine receptors capable of promoting migration to pancreatic islets. Blood 2005:106:419-427.
61. Ma J, Shi M, Li J, Liao L, et al. Regulation of CXCR4 expression in human mesenchymal stem cells by cytokine treat-ment:Role in homing efficiency in NOD/SCID mice. Haematologica 2007:92:897-904.
62. Ge J, Zhang S, et al. Time course of myocardial stromal cell-derived factor 1 expression and beneficial effects of intravenously administered bone marrow stem cells in rats with experimental myocardial infarction. Basic Res Cardiol 2005:100(3):217-223.
63. Ponte AL, Marais E, Gallay N, et al. The in vitro migration capacity of human bone marrow mesenchymal stem cells:comparison of chemokine and growth factor chemotactic activities. Stem Cells 2007:25(7):1737-1745.
64. Ren G, Zhang L, Zhao X, et al. Mesenchymal stem cell-mediated immunosuppression occurs via concerted actionof chemokines and nitric oxide. Cell Stem Cell 2008:2(2):141-150.
65. Hemeda H, Jakob M, Ludwig AK, et al. Interferon-gamma and tumor necrosis factor-alpha differentially affect cytokineexpression and migration properties of mesenchymal stem cells. Stem Cells Dev 2010;19(5):693-706.
66. Jackson WM, Nesti LJ, Tuan RS. Concise review:clinical translation of wound healing therapies based onmesenchymal stem cells. Stem Cells Transl Med 2012; 1(1):44-50.
67. Gipson IK. Distribution of mucins at the ocular surface. Exp Eye Res. 2004:78(3):379-388.
68. Ramaesh K, Dhillon B. Ex vivo expansion of corneal limbal epithelial/stem cells for corneal surface reconstruction. Eur J Ophthalmol 2003:13:515-524.
69. Gnecchi M, Melo LG. Bone marrow-derived mesenchymal stem cells:isolation, expansion, characterization, viral transduction, and production of conditioned medium. Methods Mol Biol 2009:482:281-294.
70. Xu S, De Becker A, Van Camp B, Vanderkerken K, Van Riet I. An improved harvest and in vitro expansion protocol for murine bone marrow-derivedmesenchymal stem cells. J Biomed Biotechnol 2010:2010:105940.
71. Yuan Y, Kallos MS, Hunter C, Sen A. Improved expansion of human bone marrow-derived mesenchymal stem cells in microcarrier-based suspension culture. J Tissue Eng Regen Med 2012 Jun 11.
72. Yew TL, Chang MC, Hsu YT, et al. Efficient expansion of mesenchymal stem cells from mouse bone marrow under hypoxic conditions. J Tissue Eng Regen Med 2012 May 24. [Epub ahead of print]
73. Chang Y, Hsieh PH, Chao CC. The efficiency of Percoll and Ficoll density gradient media in the isolation of marrow derived human mesenchymal stem cells with osteogenic potential. Chang Gung Med J 2009 May-Jun;32(3):264-275.
74. Crisan M, Yap S, Casteilla L, et al. A perivascular origin for mesenchymal stem cells in multiple human organs. Cell Stem Cell 2008:3:301-313.
75. Dellavalle A, Sampaolesi M, Tonlorenzi R, et al. Pericytes of human skeletal muscle are myogenic precursors distinct from satellite cells. Nat Cell Biol 2007:9:255-267.
76. Farrington-Rock C, Crofts NJ, Doherty MJ, et al. Chondrogenic and adipogenic potential of microvascular pericytes. Circulation 2004:110:2226-2232.
77. Collett GD, Canfield AE. Angiogenesis and pericytes in the initiation of ectopic calcification. Circ Res 2005;96:930-938.
78. Ren G, Zhao X, Zhang L et al. Inflammatory cytokine-induced intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in mesenchymal stem cells are critical for immunosuppression. J Immunol 2010:184:2321-2328.
79. Yilmaz Z, Dogan AL, Ozdemir 0, Serper A. Evaluation of the cytotoxicity of different root canal sealers on L929 cell line by MTT assay. Dent Mater J 2012;31 (6):1028-1032.
80. Wang X, Ge J, Wang K, Qian J, Zou Y. Evaluation of MTT assay for measurement of emodin-induced cytotoxicity. Assay Drug Dev Technol 2006;4(2):203-207.
81. Wang X, Xia Y, Liu L, et al. Comparison of MTT assay, flow cytometry, and RT-PCR in the evaluation of cytotoxicity of five prosthodontic materials. J Biomed Mater Res B Appl Biomater 2010:95(2):357-364.
82. Plaisant M, Giorgetti-Peraldi S, Gabrielson M, et al. Inhibition of hedgehog signaling decreases proliferation and clonogenicity of human mesenchymal stem cells. PLoS One 2011;6(2):e16798.
83. Morikawa S, Mabuchi Y, Kubota Y, et al. Prospective identification, isolation, and systemic transplantation of multipotent mesenchymal stem cells in murine bone marrow. J Exp Med 2009:206(11):2483-2496.
84. Kastrinaki MC, Andreakou I, Charbord P, Papadaki HA. Isolation of human bone marrow mesenchymal stem cells using different membrane markers:comparison of colony/cloning efficiency, differentiation potential, and molecular profile. Tissue Eng Part C Methods 2008;14(4):333-339.
85. Lin JR, Guo KY, Li JQ, Yan DA. In vitro culture of human bone marrow mesenchymal stem cell clonies and induced differentiation into neuron-like cells. Di Yi Jun Yi Da Xue Xue Bao 2003:23(3):251-253,264.
86. Walter MN, Wright KT, Fuller HR, MacNeil S, Johnson WE. Mesenchymal stem cell-conditioned medium accelerates skin wound healing:an in vitro study of fibroblast and keratinocyte scratch assays. Exp Cell Res 2010:316(7):1271-1281.
87. Hu XY, Wang WX, Yu MJ, et al. Tongxinluo promotes mesenchymal stem cell tube formation in vitro. J Zhejiang Univ Sci B 2011;12(8):644-651.
88. Zhang Z, Chen J, Guo F, et al. A high-temporal resolution technology for dynamic proteomic analysis based on 35S labeling. PLoS One 2008;3(8):e2991.
89. Calkins MJ, Reddy PH. Assessment of newly synthesized mitochondrial DNA using BrdU labeling in primary neurons from Alzheimer's disease mice:Implications for impaired mitochondrial biogenesis and synaptic damage. Biochim Biophys Acta 2011:1812(9):1182-1189.
90. Zhao W, Li JJ, Cao DY, et al. Intravenous injection of mesenchymal stem cells is effective in treating liver fibrosis. World J Gastroenterol 2012:18(10):1048-1058.
91. Kapuscinski J. DAPI:a DNA-specific fluorescent probe. Biotech Histochem 1995:70(5):220-233.
92. Li SL, Liu Y, Hui L. Construction of engineering adipose-like tissue in vivo utilizing human insulin gene-modified umbilical cord mesenchymal stromal cells with silk fibroin 3D scaffolds. J Tissue Eng Regen Med 2013 Mar 19. [Epub ahead of print]
93. Kruyt MC, DeBruijn J, Veenhof M, et al. Application and limitations of chloromethyl-benzamidodialkylcarbocyanine for tracing cells used in bone Tissue engineering. Tissue Eng 2003;9(1):105-115.
94. Yang S, Gao Q, Bao L, et al. Striatal extracts promote the dopaminergic differentiation of GFP-bone mesenchymal stem cells. Neurosci Lett 2012:530(2):115-120.
95. Hodgetts SI, Simmons PJ, Plant GW. Human mesenchymal precursor cells (stro-1(+)) from spinal cord injury patients improve functional recovery and tissue sparing in an acute spinal cord injury rat model. Cell Transplant 2013;22(3):393-412.
96. Lan Y, Kodati S, Lee HS, et al. Kinetics and function of mesenchymal stem cells in corneal injury. Invest Ophthalmol Vis Sci 2012;53(7):3638-3644.
97. Lyngholm M, Vorum H, Nielsen K, et al. Differences in the protein expression in limbal versus central human corneal epithelium--a search for stem cell markers. Exp Eye Res 2008;87(2):96-105.
98. Majo F, Rochat A, Nicolas M, Jaoude GA, Barrandon Y. Oligopotent stem cells are distributed throughout the mammalian ocular surface. Nature 2008:456(7219):250-254.
99. Yao L, Li ZR, Su WR, et al. Role of mesenchymal stem cells on cornea wound healing induced by acute alkali burn. PLoS One 2012;7(2):e30842.
100. Labouyrie E, Dubus P, Groppi A, et al. Expression of neurotrophins and their receptors in human bone marrow. Am J Pathol 1999:154:405-415.
101. Lin N, Hu K, Chen S, et al. Nerve growth factor-mediated paracrine regulation of hepatic stellate cells by multipotent mesenchymal stromal cells. Life Sci 2009:85:291-295.
102.Parr AM, Tator CH, Keating A. Bone marrow-derived mesenchymal stromal cells for the repair of central nervous system injury. Bone Marrow Transpl 2007:40:609-619.
103. Karussis D, Kassis I, Kurkalli BG, et al. Immunomodulation and neuroprotection with mesenchymal bone marrow stem cells (MSCs):A proposed treatment for multiple sclerosis and other neuroimmunological/neurodegenerative diseases. J Neurol Sci 2008;265:131-135.
104. Torrente Y, Polli E. Mesenchymal stem cell transplantation for neurodegenerative diseases. Cell Transplant 2008:17:1103-1113.
105. Caplan AI, Dennis JE. Mesenchymal stem cells as trophic mediators. J Cell Biochem 2006:98(5):1076-1084.
106. Alexanian AR. Epigenetic modifiers promote efficient generation of neural-like cells from bone marrow-derived mesenchymal cells grown in neural environment. J Cell Biochem 2007;100(2):362-371.
107. Ueda H, Fujita R, Yoshida A, Matsunaga H, Ueda M. Identification of prothymosin-alphal, the necrosis-apoptosis switch molecule in cortical neuronal cultures,J Cell Biol 2007;176(6):853-862.
108.Li L, Jiang J. Regulatory factors of mesenchymal stem cell migration into injured tissues and their signal transduction mechanisms. Front Med 2011;5(1):33-39.
109. Li GG, Chen SY, Xie HT, Zhu YT, Tseng SC. Angiogenesis potential of human limbal stromal niche cells. Invest Ophthalmol Vis Sci 2012;53(7):3357-3367.
1. Ren G, Chen X, Dong F, et al. Concise review:mesenchymal stem cells and translational medicine:emergingissues. Stem Cells Transl Med 2012;1(1):51-58.
2. Yu J, Vodyanik MA, Smuga-Otto K, et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 2007;318:1917-1920.
3. Miura K, Okada Y, Aoi T, et al. Variation in the safety of induced pluripotent stem cell lines. Nat Biotechnol 2009:27:743-745.
4. Gutierrez-Arandal, Ramos-MejiaV, BuenoC, et al. Human induced pluripotent stem cells develop teratoma more efficiently and faster than human embryonic stem cells regardless the site of injection. Stem Cells 2010;28:1568-1570.
5. Wong DJ, Liu H, Ridky TW, et al. Module map of stem cell genes guides creation of epithelial cancer stem cells. Cell Stem Cell 2008;2:333-344.
6. Fink DW Jr. FDA regulation of stem cell-based products. Science 2009; 324:1662-1663.
7. Zhang Y, Shen W, Hua J, et al. Pancreatic islet-like clusters from bone marrow mesenchymal stem cells of human first-trimester abortus can cure streptozocin-induced mouse diabetes. Rejuvenation Res 2010;13(6):695-706.
8. Crisan M, Yap S, Casteilla L, et al. A perivascular origin for mesenchymal stem cells in multiple human organs. Cell Stem Cell 2008;3:301-313.
9. Dellavalle A, Sampaolesi M, Tonlorenzi R, et al. Pericytes of human skeletal muscle are myogenic precursors distinct from satellite cells. Nat Cell Biol 2007:9:255-267.
10. Farrington-Rock C, Crofts NJ, Doherty MJ, et al. Chondrogenic and adipogenic potential of microvascular pericytes. Circulation 2004;110:2226-2232.
11. Collett GD, Canfield AE. Angiogenesis and pericytes in the initiation of ectopic calcification. Circ Res 2005;96:930-938.
12. Horwitz EM, Gordon PL, Koo WK, et al. Isolated allogeneic bone marrow-derived mesenchymal cells engraft and stimulate growth in children with osteogenesis imperfecta:Implications for cell therapy of bone. Proc Natl Acad SciUSA 2002;99:8932-8937.
13. Liechty KW, MacKenzie TC, Shaaban AF, et al. Human mesenchymal stem cells engraft and demonstrate site-specific differentiation after in utero transplantation in sheep. Nat Med 2000;6:1282-1286.
14. Kawada H, Fujita J, Kinjo K, et al. Nonhematopoietic mesenchymal stem cells can be mobilized and differentiate into cardiomyocytes after myocardial infarction. Blood 2004:104:3581-3587.
15. Dennis JE, Konstantakos EK, Arm D, et al. In vivo osteogenesis assay:A rapid method for quantitative analysis. Biomaterials 1998;19:1323-1328.
16. Ohgushi H, Kotobuki N, Funaoka H, et al. Tissue engineered ceramic artificial joint-ex vivo osteogenic differentiation of patient mesenchymal cells on total ankle joints for treat-ment of osteoarthritis. Biomaterials 2005:26:4654-4661.
17. Kon E, Muraglia A, Corsi A, et al. Autologous bone marrow stromal cells loaded onto porous hydroxyapatite ceramic accelerate bone repair in critical-size defects of sheep long bones. J Biomed Mater Res 2000:49:328-337.
18. Solchaga LA, Temenoff JS, Gao J, et al. Repair of osteochondral defects with hyaluronan-and polyester-based scaffolds. Osteoarthritis Cartilage 2005:13:297-309.
19. Horwitz EM, Gordon PL, Koo WK, et al. Isolated allogeneic bone marrow-derived mesenchymal cells engraft and stimulate growth in children with osteogenesis imperfecta:Implications for cell therapy of bone. Proc Natl Acad SciUSA 2002:99:8932-8937.
20. Mahmood A, Lu D, Lu M, et al. Treatment of traumatic brain injury in adult rats with intravenous administration of human bone marrow stromal cells. Neurosurgery 2003:53:697-702, discussion 702-693.
21. Ueda H, Fujita R, Yoshida A, Matsunaga H, Ueda M. Identification of prothymosin-alphal, the necrosis-apoptosis switch molecule in cortical neuronal cultures. J Cell Biol 2007;176(6):853-862.
22. Xie HT, Chen SY, Li GG, Tseng SC. Limbal epithelial stem/progenitor cells attract stromal niche cells by SDF-1/CXCR4signaling to prevent differentiation. Stem Cells 2011:29(11):1874-1885.
23. Wynn RF, Hart CA, Corradi-Perini C, et al. A small proportion of mesenchymal stem cells strongly expresses functionally active CXCR4 receptor capable of promoting migration to bone marrow. Blood 2004;104:2643-2645.
24. Belema-Bedada F, Uchida S, Martire A, et al. Efficient homing of multipotent adult mesenchymal stem cells depends on FROUNT-mediated clustering of CCR2. Cell Stem Cell 2008:2:566-575.
25. Cheng Z, Ou L, Zhou X, et al. Targeted migration of mesenchymal stem cells modified with CXCR4 gene to infarcted myocardium improves cardiac performance. Mol Ther 2008:16:571-579.
26. Hatch HM, Zheng D, Jorgensen ML, et al. SDF-lalpha/CXCR4:A mechanism for hepatic oval cell activation and bone marrow stem cell recruitment to the injured liver of rats. Cloning Stem Cells 2002:4:339-351.
27. Sordi V, Malosio ML, Marchesi F, et al. Bone marrow mesenchymal stem cells express a restricted set of functionally active chernokine receptors capable of promoting migration to pancreatic islets. Blood 2005;106:419-427.
28. Ma J, Shi M, Li J, Liao L, et al. Regulation of CXCR4 expression in human mesenchymal stem cells by cytokine treat-ment:Role in homing efficiency in NOD/SCID mice. Haematologica 2007;92:897-904.
29. Ruster B, Gottig S, Ludwig RJ, et al. Mesenchymal stem cells display coordinated rolling and adhesion behavior on endothelial cells. Blood 2006:108:3938-3944.
30. Ko IK, Kim BG, Awadallah A, et al. Targeting improves MSC treatment of inflammatory bowel disease. Mol Ther 2010;18:1365-1372.
31. Shi M, Li J, Liao L, et al. Regulation of CXCR4 expression in human mesenchymal stem cells by cytokine treatment:Role in homing efficiency in NOD/SCID mice. Haematologica 2007;92:897-904.
32. Ren G, Zhao X, Zhang L, et al. Inflammatory cytokine-induced intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in mesenchymal stem cells are critical for immunosuppression. J Immunol 2010;184:2321-2328.
33. Crisostomo PR, Wang Y, Markel TA, et al. Human mesenchymal stem cells stimulated by TNF-a, LPS, or hypoxia produce growth factors by an NF κB-but not JNK-dependent mechanism. Am J Physiol Cell Physiol 2008;294:C675-C682.
34. Caplan AI, Dennis JE. Mesenchymal stem cells as trophic mediators. J Cell Biochem 2006:98:1076-1084.
35. Xu G, Zhang Y, Zhang L, et al. The role of IL-6 in inhibition of lymphocyte apoptosis by mesenchymal stem cells. Biochem Biophys Res Commun 2007:361:745-750.
36. Cao W, Yang Y, Wang Z, et al. Leukemia inhibitory factor inhibits T helper 17 cell differentiation and confers treatment effects of neural progenitor cell therapy in autoimmune disease. Immunity 2011:35:273-284.
37. Hahn JY, Cho HJ, Kang HJ, et al. Pre-treatment of mesenchymal stem cells with a combination of growth factors enhances gap junction formation, cytoprotective effect on cardiomyocytes, and therapeutic efficacy for myocardial infarction. J Am Coll Cardiol 2008:51:933-943.
38. Schnabel LV, Lynch ME, van der Meulen MC, et al. Mesenchymal stem cells and insulin-like growth factor-I gene-enhanced mesenchymal stem cells improve structural aspects of healing in equine flexor digitorum superficialis tendons. J Orthop Res 2009:27:1392-1398.
39. Uccelli A, Moretta L, Pistoia V. Mesenchymal stem cells in health and disease. Nat Rev Immunol 2008:8:726-736.
40. Barry FP, Murphy JM, English K, et al. Immunogenicity of adult mesenchymal stem cells:Lessons from the fetal allograft. Stem Cells Dev 2005:14:252-265.
41. Toubai T, Paczesny S, Shono Y, et al. Mesenchymal stem cells for treatment and prevention of graft-versus-host disease after allogeneic hematopoietic cell transplantation. Curr Stem Cell Res Ther 2009;4:252-259.
42. Osawah H, Maruyama K, Streilein JW. CD95 ligand expression on corneal epithelium and endothelium influences the fates of orthotopic and heterotopic corneal allografts in mice. Invest Ophthalmol Vis Sci 2004:45:1908-1915.
43. Kwidzinskie E, Bunse J, Kovac AD, et al. Indolamine 2,3-dioxygenase is expressed in the CNS and down-regulates autoimmune inflammation. FASEB J 2005:19:1347-1349.
44. Waterman RS, Tomchuck SL, Henkle SL, et al. A new mesenchymal stem cell (MSC) paradigm:Polarization into a pro-inflammatory MSC1 or an immunosuppressive MSC2 phenotype. PLoS One.2010;5:e10088.
45. Ren G, Su J, Zhang L, et al. Species variation in the mechanisms of mesenchymal stem cell-mediated immunosuppression. Stem Cells.2009:27:1954-1962.
46. Allison M. Genzyme backs Osiris, despite Prochymal flop. Nat Biotechnol. 2009:27:966-967.
47. Tomita M, Adachi Y, Yamada H, et al. Bone marrow-derived stem cells can differentiate into retinal cells in injured rat retina. Stem Cells 2002;20:279-283.
48. Kicic A, Shen WY, Wilson AS, et al. Differentiation of marrow stromal cells into photoreceptors in the rat eye. J Neurosci 2003;23:7742-7749.
49. Tomita M, Mori T, Maruyama K, et al. A comparison of neural differentiation and retinal transplantation with bone marrow-derived cells and retinal progenitor cells. Stem Cells 2006:24:2270-2278.
50. Gong L, Wu Q, Song B, et al. Differentiation of rat mesenchymal stem cells transplanted into the subretinal space of sodium iodate-injected rats. Clin Experiment Ophthalmol 2008;36:666-671.
51. Castanheira P, Torquetti L, Nehemy MB, et al. Retinal incorporation and differentiation of mesenchymal stem cells intravitreally injected in the injured retina of rats. Arq Bras Oftalmol 2008:71:644-650.
52. Zhang ZQ, Dong FT. In vitro differentiation of rat mesenchymal stem cells into photoreceptors. Zhonghua Yan Ke Za Zhi 2008;44:540-544.
53. Vossmerbaeumer U, Ohnesorge S, Kuehl S, et al. Retinal pigment epithelial phenotype induced in human adipose tissue-derived mesenchymal stromal cells. Cytotherapy 2009:11:177-188.
54. Arnhold S, Heiduschka P, Klein H, et al. Adenovirally transduced bone marrow stromal cells differentiate into pigment epithelial cells and induce rescue effects in RCS rats. Invest Ophthalmol Vis Sci 2006;47:4121-4129.
55. Yu S, Tanabe T, Dezawa M, et al. Effects of bone marrow stromal cell injection in an experimental glaucoma model. Biochem Biophys Res Commun 2006:344:1071-1079.
56. Azizi SA, Stokes D, Augelli BJ, et al. Engraftment and migration of human bone marrow stromal cells implanted in the brains of albino rats—Similarities to astrocyte grafts. Proc Natl Acad Sci U S A 1998;95:3908-3913.
57. Li N, Li XR, Yuan JQ. Effects of bone-marrow mesenchymal stem cells transplanted into vitreous cavity of rat injured by ischemia/reperfusion. Graefes Arch Clin Exp Ophthalmol 2009;247:503-514.
58. Inoue Y, Iriyama A, Ueno S, et al. Subretinal transplantation of bone marrow mesenchymal stem cells delays retinal degeneration in the RCS rat model of retinal degeneration. Exp Eye Res 2007;85:234-241.
59. Wang S, Lu B, Girman S, et al. Non-invasive stem cell therapy in a rat model for retinal degeneration and vascular pathology. PLoS One 2010;5:e9200.
60. McIntosh Ambrose W, Schein O, Elisseeff J. A tale of two tissues:Stem cells in cartilage and corneal tissue engineering. Curr Stem Cell Res Ther 2010;5:37-48.
61. De Miguel MP, Alio JL, Arnalich-Montiel F, et al. cornea and ocular surface treatment. Curr Stem Cell Res Ther 2010;5:195-204.
62. Ma Y, Xu Y, Xiao Z, et al. Reconstruction of chemically burned rat corneal surface by bone marrow-derived human mesenchymal stem cells. Stem Cells 2006;24:315-321.
63. Oh JY, Kim MK, Shin MS, et al. The anti-inflammatory and anti-angiogenic role of mesenchymal stem cells in corneal wound healing following chemical injury. Stem Cells 2008;26:1047-1055.
64. Oh JY, Kim MK, Shin MS, et al. Cytokine secretion by human mesenchymal stem cells cocultured with damaged corneal epithelial cells. Cytokine 2009:46:100-103.
65. Oh JY, Kim MK, Ko JH, et al. Rat allogeneic mesenchymal stem cells did not prolong the survival of corneal xenograft in a pig-to-rat model. Vet Ophthalmol 2009:12:35-40.
66. Zoukhri D. Mechanisms involved in injury and repair of the murine lacrimal gland: Role of programmed cell death and mesenchymal stem cells. Ocul Surf 2010:8:60-69.
67. Zoukhri D, Fix A, Alroy J, et al. Mechanisms of murine lacrimal gland repair after experimentally induced inflammation. Invest Ophthalmol Vis Sci 2008:49:4399-4406.
68. Pease ME, McKinnon SJ, Quigley HA, et al. Obstructe axonal transport of BDNF and its receptor TrkB in experimental glaucoma. Invest Ophthalmol Vis Sci 2000;41:764-774.
69. Murphy JA, Clarke DB. Target-derived neurotrophin may influence the survival of adult retinal ganglio cells when local neurotrophic support is disrupted: Implications for glaucoma. Med Hypotheses 2006:67:1208-1212.
70. Yu S, Tanabe T, Dezawa M, et al. Effects of bone marrow stromal cell injection in an experimental glaucoma model. Biochem Biophys Res Commun 2006:344:1071-1079.
71. Osborne NN. Recent clinical findings with memantine should not mean that the idea of neuroprotection in glaucoma is abandoned. Acta Ophthalmol 2009:87:450-454.
72. Caspi RR. Th1 and Th2 responses in pathogenesis and regulation of experimental Autoimmune uveoretinitis. Int Rev Immunol 2002,21(2-3):197-208.
73. Gonzalez MA, Gonzalez-Rey E, Rico L, et al. Treatment of experimental arthritis by inducing immune tolerance with human adipose-derived mesenchymal stem cells. Arthritis Rheum 2009:60:1006-1019.
74. Parekkadan B, Tilles AW, Yarmush ML. Bone marrow-derived mesenchymal stem cells ameliorate autoimmune enteropathy independently of regulatory T cells. Stem Cells 2008; 26:1913.
75. Fiorina P, Jurewicz M, Augello A, et al. Immunomodulatory function of bone marrow-derived mesenchymal stem cells in experimental autoimmune type 1 diabetes. J Immunol 2009;183:993.
76. Marmont AM, Gualandi F, Piaggio G, et al. Allogeneic bone marrow transplantation (BMT) for refractory Behcet's disease with severe CNS involvement. Bone Marrow Transplant 2006;37:1061-1063.
77. Erokhin VV, Vasil'eva IA, Konopliannikov AG, et al. Systemic transplantation of autologous mesenchymal stem cells of the bone marrow in the treatment of patients with multidrug-resistant pulmonary tuberculosis. Probl Tuberk Bolezn Legk 2008;10:3-6.
78. Sacchetti B, Funari A, Michienzi S, et al. Self-renewing osteoprogenitors in bone marrow sinusoids can organize a hematopoietic microenvironment. Cell 2007:131:324-336.
79. Kuo CK, Tuan RS. Tissue engineering with mesenchymal stem cells. IEEE Eng Med Biol Mag 2003:22:51-56.
80. Chen F, Tuan R. Adult stem cells for cartilage tissue engineering and regeneration. Curr Rhematol Rev 2008;4:149-154.
81. Patel M, Betz MW, Geibel E, et al. Cyclic acetal hydroxyapatite nanocomposites for orbital bone regeneration. Tissue Eng Part A 2010;16:55-65.
82.82. Wright V, Peng H, Usas A, et al. BMP4-expressing muscle-derived stem cells differentiate into osteogenic lineage and improve bone healing in immunocompetent mice. Mol Ther 2002;6:169-178.
83. Peng H, Usas A, Olshanski A, et al. VEGF improves, whereas sFltl inhibits, BMP2-induced bone formation and bone healing through modulation of angiogenesis. J Bone Miner Res 2005;20:2017-2027.
84. Montarras D, Morgan J, Collins C, et al. Direct isolation of satellite cells for skeletal muscle regeneration. Science 2005;309:2064-2067.
85. Kuang S, Kuroda K, Le Grand F, et al. Asymmetric self-renewal and commitment of satellite stem cells in muscle. Cell 2007;129:999-1010.
86. Scheef EA, Sorenson CM, Sheibani N. Attenuation of proliferation and migration of retinal pericytes in the absence of thrombospondin-1. Am J Physiol Cell Physiol 2009;296:C724-C734.
87. Li Y, Huard J. Differentiation of muscle-derived cells into myofibroblasts in injured skeletal muscle. Am J Pathol 2002;161:895-907.
88. Quintero AJ, Wright VJ, Fu FH, et al. Stem cells for the treatment of skeletal muscle injury. Clin Sports Med 2009;28:1-11.
89. Li Y, Foster W, Deasy BM, et al. Transforming growth factor-betal induces the differentiation of myogenic cells into fibrotic cells in injured skeletal muscle: A key event in muscle fibrogenesis. Am J Pathol 2004;164:1007-1019.
90. Lu L, Saulis AS, Liu WR, et al. The temporal effects of anti-TGF-betal,2, and 3 monoclonal antibody on wound healing and hypertrophic scar formation. J Am Coll Surg 2005:201:391-397.
91. Rodeheffer MS, Birsoy K, Friedman JM. Identification of white adipocyte progenitor cells in vivo. Cell 2008;135:240-249.
92. Tang W, Zeve D, Suh JM, et al. White fat progenitor cells reside in the adipose vasculature. Science 2008;322:583-586.
93. Perros P, Krassas GE. Graves orbitopathy:A perspective. Nat Rev Endocrinol 2009:5:312-318.
94. Stenn K, Parimoo S, Zheng Y, et al. Bioengineering the hair follicle. Organogenesis 2007;3:6-13.
95. Nakagawa H, Akita S, Fukui M, et al. Human mesenchymal stem cells successfully improve skin-substitute wound healing. Br J Dermatol 2005:153:29-36.
96. Borue X, Lee S, Grove J, et al. Bone marrow-derived cells contribute to epithelial engraftment during wound healing. Am J Pathol 2004:165:1767-1772.
2. Hong J, Qiu T, Wei A, Sun X, Xu J. Clinical characteristics and visual outcome of severe ocular chemical injuries in Shanghai. Ophthalmology 2010;117:2268-2272.
3. Fish R, Davidson RS. Management of ocular thermal and chemical injuries, including amniotic membrane therapy. Curr Opin Ophthalmol 2010;21:317-321.
4. Cauchi PA, Ang GS, Azuara-Blanco A, Burr JM. A systematic literature review of surgical interventions for limbal stem cell deficiency in humans. Am J Ophthalmol 2008:146:251-259.
5. Avunduk AM, Tekelioglu Y. Therapeutic use of limbal stem cells. Curr Stem Cell Res Ther 2006;1:231-238.
6. Koizumi N, Inatomi T, Suzuki T, et al. Cultivated corneal epithelial stem cell transplantation in ocular surface disorders. Ophthalmology 2001;108:1569-1574.
7. Koizumi N, Inatomi T, Suzuki T, et al. Cultivated corneal epithelial transplantation for ocular surface reconstruction in acute phase of Stevens-Johnson syndrome. Arch Ophthalmol 2001;119:298-300.
8. Nakamura T, Sotozono C, Bentley AJ, et al. Long-term phenotypic study after allogeneic cultivated corneal limbal epithelial transplantation for severe ocular surface diseases. Ophthalmology 2010;117:2247-2254.
9. Tsai RJ, Li LM, Chen JK. Reconstruction of damaged corneas by transplantation of autologous limbal epithelial cells. N Engl J Med 2000:343:86-93.
10. Nishida K, Yamato M, Hayashida Y, et al. Corneal reconstruction with tissue-engineered cell sheets composed of autologous oral mucosal epithelium. N Engl J Med 2004;351:1187-1196.
11. Rama P, Matuska S, Paganoni G, et al. Limbal stem-cell therapy and long-term corneal regeneration. N Engl J Med 2010;363:147-155.
12. Burman S, Sangwan V. Cultivated limbal stem cell transplantation for ocular surface reconstruction. Clin Ophthalmol 2008;2(3):489-502.
13. Khaldoyanidi S. Directing stem cell homing. Cell Stem Cell 2008;2(3):198-200.
14. Karp JM, Leng Teo GS. Mesenchymal stem cell homing:the devil is in the details. Cell Stem Cell 2009;4(3):206-216.
15. Keating A. Mesenchymal stromal cells:new directions. Cell Stem Cell 2012;10(6):709-716.
16. Sitalakshmi G, Sudha B, Madhavan HN, et al. Ex vivo cultivation of corneal limbal epithelial cells in a thermoreversible polymer (Mebiol Gel) and their transplantation in rabbits:an animal model. Tissue Eng Part A 2009; 15(2):407-415.
17. Choi SH, Jung SY, Kwon SM, Baek SH. Perspectives on stem cell therapy for cardiac regeneration. Advances andchallenges. Circ J 2012;76(6):1307-1312.
18. Cucchiarini M, Venkatesan JK, Ekici M, Schmitt G, Madry H. Human mesenchymal stem cells overexpressing therapeutic genes:from basicscience to clinical applications for articular cartilage repair.Biomed Mater Eng 2012;22(4):197-208.
19. Ricart E. Current status of mesenchymal stem cell therapy and bone marrow transplantationin IBD. Dig Dis 2012;30 (4):387-391.
20. Nelson ER, Wong VW, Krebsbach PH, Wang SC, Levi B. Heterotopic ossification following burn injury:the role of stem cells. J Burn Care Res 2012;33(4):463-470.
21. Xie L, Shi W. Cornea. Beijing:People's Medical Publishing House;2007:508-509.
22. Shortt AJ, Seeker GA, Notara MD, et al. Transplantation of ex vivo cultured limbal epithelial stem cells:a review of techniques and clinical results. Surv Ophthalmol 2007:52:483-502.
23. Espana EM, Di Pascuale MA, et al. Characterization of corneal pannus removed from patients with total limbal stem cell deficiency. Invest Ophthalmol Vis Sci 2004:45(9):2961-2966.
24. Askari AT, Unzek S, Popovic ZB, et al. Effect of stromal-cell-derived factor 1 on stem-cell homing and tissue regeneration inischaemic cardiomyopathy. Lancet 2003:362(9385):697-703.
25. Penn MS, Pastore J, Miller T, Aras R. SDF-1 in myocardial repair. Gene Ther 2012;19 (6):583-587.
26. Zhang Y, Shen W, Hua J, et al. Pancreatic islet-like clusters from bone marrow mesenchymal stem cells of human first-trimester abortus can cure streptozocin-induced mouse diabetes. Rejuvenation Res 2010;13(6):695-706.
27. Joe AW, Gregory-Evans K. Mesenchymal stem cells and potential applications in treating ocular disease. Curr Eye Res 2010；35(11):941-952.
28. Ren G, Chen X, Dong F, et al. Concise review:mesenchymal stem cells and translational medicine:emergingissues. Stem Cells Transl Med 2012;1(1):51-58.
29. Yu J, Vodyanik MA, Smuga-Otto K et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 2007；318:1917-1920.
30. Miura K, Okada Y, Aoi T et al. Variation in the safety of induced pluripotent stem cell lines. Nat Biotechnol 2009;27:743-745.
31. Gutierrez-Arandal, Ramos-MejiaV, BuenoC, et al. Human induced pluripotent stem cells develop teratoma more efficiently and faster than human embryonic stem cells regardless the site of injection. Stem Cells 2010;28:1568-1570.
32. Wong DJ, Liu H, Ridky TW et al. Module map of stem cell genes guides creation of epithelial cancer stem cells. Cell Stem Cell 2008;2:333-344.
33. Fink DW Jr. FDA regulation of stem cell-based products. Science 2009;324:1662-1663.
34. Chute JP. Stem cell homing. Curr Opin Hematol 2006;13(6):399-406.
35. Shiozawa Y, Havens AM, Pienta KJ, Taichman RS. The bone marrow niche:habitat to hematopoietic and mesenchymal stem cells, and unwitting host to molecular parasites. Leukemia 2008;22(5):941-950.
36. Saito T, Kuang JQ, Bittira B, Al-Khaldi A, Chiu RC. Xenotransplant cardiac chimera: immune tolerance of adult stem cells. Ann Thorac Surg 2002;74(1):19-24; discussion 24.
37. Belema-Bedada F, Uchida S, Martire A, Kostin S, Braun T. Efficient homing of multipotent adult mesenchymal stem cells depends on FROUNT-mediated clustering of CCR2. Cell Stem Cell 2008;2(6):566-575.
38. Tomita M, Adachi Y, Yamada H, et al. Bone marrow-de -rived stem cells can differentiate into retinal cells in injured rat retina. Stem Cells 2002;20:279-283.
39. Kicic A, Shen WY, Wilson AS, et al. Differentiation of marrow stromal cells into photoreceptors in the rat eye. J Neurosci 2003;23:7742-7749.
40. Tomita M, Mori T, Maruyama K, et al. A comparison of neural differentiation and retinal transplantation with bone marrow-derived cells and retinal progenitor cells. Stem Cells 2006;24:2270-2278.
41. Gong L, Wu Q, Song B, et al. Differentiation of rat mesenchymal stem cells transplanted into the subretinal space of sodium iodate-injected rats. Clin Experiment Ophthalmol 2008;36:666-671.
42. Arnhold S, Heiduschka P, Klein H, et al. Adenovirally transduced bone marrow stromal cells differentiate into pigment epithelial cells and induce rescue effects in RCS rats. Invest Ophthalmol Vis Sci 2006:47:4121-4129.
43. Ma Y, Xu Y, Xiao Z, et al. Reconstruction of chemically burned rat corneal surface by bone marrow-derived human mesenchymal stem cells. Stem Cells 2006;24:315-321.
44. Oh JY, Kim MK, Shin MS, et al. The anti-inflammatory and anti-angiogenic role of mesenchymal stem cells in corneal wound healing following chemical injury. Stem Cells 2008:26:1047-1055.
45. Oh JY, Kim MK, Shin MS, et al. Cytokine secretion by human mesenchymal stem cells co-cultured with damaged corneal epithelial cells. Cytokine 2009:46:100-103.
46. McIntosh Ambrose W, Schein 0, Elisseeff J. A tale of two tissues:Stem cells in cartilage and corneal tissue engineering. Curr Stem Cell Res Ther 2010;5:37-48.
47. De Miguel MP, Alio JL, Arnalich-Montiel F, et al. Cornea and ocular surface treatment. Curr Stem Cell Res Ther 2010:5:195-204.
48. Oh JY, Kim MK, Ko JH, et al. Rat allogeneic mesenchymal stem cells did not prolong the survival of corneal xenograft in a pig-to-rat model. Vet Ophthalmol 2009:12:35-40.
49. Zoukhri D. Mechanisms involved in injury and repair of the murine lacrimal gland: Role of programmed cell death and mesenchymal stem cells. Ocul Surf 2010;8:60-69.
50. Zhang JJ, Yi ZW, Dang XQ, He XJ, Wu XC. Mobilization effects of SCF along with G-CSF on bone marrow stem cells and endothelial progenitor cells in rats with unilateral ureteral obstruction. Zhongguo Dang Dai Er Ke Za Zhi 2007;9(2):144-148.
51. Cashen AF, Lazarus HM, Devine SM. Mobilizing stem cells from normal donors:is it possible to improve upon G-CSF? Bone Marrow Transplant 2007:39(10):577-588.
52. Moog R. Management strategies for poor peripheral blood stem cell mobilization. Transfus Apher Sci 2008:38(3):229-236.
53. Ince H, Petzsch M, Rehders TC, Dunkelmann S, Nienaber CA. G-CSF in acute myocardial infarction-experimental and clinical findings. Anadolu Kardiyol Derg 2006:6(3):261-263.
54. Korbling M. In vivo expansion of the circulating stem cell pool. Stem Cells 1998; 16 Suppl 1:131-138.
55. Xie HT, Chen SY, Li GG, Tseng SC. Limbal epithelial stem/progenitor cells attract stromal niche cells by SDF-1/CXCR4signaling to prevent differentiation. Stem Cells 2011:29(11):1874-1885.
56. Wynn RF, Hart CA, Corradi-Perini C et al. A small proportion of mesenchymal stem cells strongly expresses functionally active CXCR4 receptor capable of promoting migration to bone marrow. Blood 2004:104:2643-2645.
57. Belema-Bedada F, Uchida S, Martire A et al. Efficient homing of multipotent adult mesenchymal stem cells depends on FROUNT-mediated clustering of CCR2. Cell Stem Cell 2008;2:566-575.
58. Cheng Z, Ou L, Zhou X et al. Targeted migration of mesenchymal stem cells modified with CXCR4 gene to infarcted myocardium improves cardiac performance. Mol Ther 2008:16:571-579.
59. Hatch HM, Zheng D, Jorgensen ML, et al. SDF-lalpha/CXCR4:A mechanism for hepatic oval cell activation and bone marrow stem cell recruitment to the injured liver of rats. Cloning Stem Cells 2002:4:339-351.
60. Sordi V, Malosio ML, Marchesi F, et al. Bone marrow mesenchymal stem cells express a restricted set of functionally active chemokine receptors capable of promoting migration to pancreatic islets. Blood 2005:106:419-427.
61. Ma J, Shi M, Li J, Liao L, et al. Regulation of CXCR4 expression in human mesenchymal stem cells by cytokine treat-ment:Role in homing efficiency in NOD/SCID mice. Haematologica 2007:92:897-904.
62. Ge J, Zhang S, et al. Time course of myocardial stromal cell-derived factor 1 expression and beneficial effects of intravenously administered bone marrow stem cells in rats with experimental myocardial infarction. Basic Res Cardiol 2005:100(3):217-223.
63. Ponte AL, Marais E, Gallay N, et al. The in vitro migration capacity of human bone marrow mesenchymal stem cells:comparison of chemokine and growth factor chemotactic activities. Stem Cells 2007:25(7):1737-1745.
64. Ren G, Zhang L, Zhao X, et al. Mesenchymal stem cell-mediated immunosuppression occurs via concerted actionof chemokines and nitric oxide. Cell Stem Cell 2008:2(2):141-150.
65. Hemeda H, Jakob M, Ludwig AK, et al. Interferon-gamma and tumor necrosis factor-alpha differentially affect cytokineexpression and migration properties of mesenchymal stem cells. Stem Cells Dev 2010;19(5):693-706.
66. Jackson WM, Nesti LJ, Tuan RS. Concise review:clinical translation of wound healing therapies based onmesenchymal stem cells. Stem Cells Transl Med 2012; 1(1):44-50.
67. Gipson IK. Distribution of mucins at the ocular surface. Exp Eye Res. 2004:78(3):379-388.
68. Ramaesh K, Dhillon B. Ex vivo expansion of corneal limbal epithelial/stem cells for corneal surface reconstruction. Eur J Ophthalmol 2003:13:515-524.
69. Gnecchi M, Melo LG. Bone marrow-derived mesenchymal stem cells:isolation, expansion, characterization, viral transduction, and production of conditioned medium. Methods Mol Biol 2009:482:281-294.
70. Xu S, De Becker A, Van Camp B, Vanderkerken K, Van Riet I. An improved harvest and in vitro expansion protocol for murine bone marrow-derivedmesenchymal stem cells. J Biomed Biotechnol 2010:2010:105940.
71. Yuan Y, Kallos MS, Hunter C, Sen A. Improved expansion of human bone marrow-derived mesenchymal stem cells in microcarrier-based suspension culture. J Tissue Eng Regen Med 2012 Jun 11.
72. Yew TL, Chang MC, Hsu YT, et al. Efficient expansion of mesenchymal stem cells from mouse bone marrow under hypoxic conditions. J Tissue Eng Regen Med 2012 May 24. [Epub ahead of print]
73. Chang Y, Hsieh PH, Chao CC. The efficiency of Percoll and Ficoll density gradient media in the isolation of marrow derived human mesenchymal stem cells with osteogenic potential. Chang Gung Med J 2009 May-Jun;32(3):264-275.
74. Crisan M, Yap S, Casteilla L, et al. A perivascular origin for mesenchymal stem cells in multiple human organs. Cell Stem Cell 2008:3:301-313.
75. Dellavalle A, Sampaolesi M, Tonlorenzi R, et al. Pericytes of human skeletal muscle are myogenic precursors distinct from satellite cells. Nat Cell Biol 2007:9:255-267.
76. Farrington-Rock C, Crofts NJ, Doherty MJ, et al. Chondrogenic and adipogenic potential of microvascular pericytes. Circulation 2004:110:2226-2232.
77. Collett GD, Canfield AE. Angiogenesis and pericytes in the initiation of ectopic calcification. Circ Res 2005;96:930-938.
78. Ren G, Zhao X, Zhang L et al. Inflammatory cytokine-induced intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in mesenchymal stem cells are critical for immunosuppression. J Immunol 2010:184:2321-2328.
79. Yilmaz Z, Dogan AL, Ozdemir 0, Serper A. Evaluation of the cytotoxicity of different root canal sealers on L929 cell line by MTT assay. Dent Mater J 2012;31 (6):1028-1032.
80. Wang X, Ge J, Wang K, Qian J, Zou Y. Evaluation of MTT assay for measurement of emodin-induced cytotoxicity. Assay Drug Dev Technol 2006;4(2):203-207.
81. Wang X, Xia Y, Liu L, et al. Comparison of MTT assay, flow cytometry, and RT-PCR in the evaluation of cytotoxicity of five prosthodontic materials. J Biomed Mater Res B Appl Biomater 2010:95(2):357-364.
82. Plaisant M, Giorgetti-Peraldi S, Gabrielson M, et al. Inhibition of hedgehog signaling decreases proliferation and clonogenicity of human mesenchymal stem cells. PLoS One 2011;6(2):e16798.
83. Morikawa S, Mabuchi Y, Kubota Y, et al. Prospective identification, isolation, and systemic transplantation of multipotent mesenchymal stem cells in murine bone marrow. J Exp Med 2009:206(11):2483-2496.
84. Kastrinaki MC, Andreakou I, Charbord P, Papadaki HA. Isolation of human bone marrow mesenchymal stem cells using different membrane markers:comparison of colony/cloning efficiency, differentiation potential, and molecular profile. Tissue Eng Part C Methods 2008;14(4):333-339.
85. Lin JR, Guo KY, Li JQ, Yan DA. In vitro culture of human bone marrow mesenchymal stem cell clonies and induced differentiation into neuron-like cells. Di Yi Jun Yi Da Xue Xue Bao 2003:23(3):251-253,264.
86. Walter MN, Wright KT, Fuller HR, MacNeil S, Johnson WE. Mesenchymal stem cell-conditioned medium accelerates skin wound healing:an in vitro study of fibroblast and keratinocyte scratch assays. Exp Cell Res 2010:316(7):1271-1281.
87. Hu XY, Wang WX, Yu MJ, et al. Tongxinluo promotes mesenchymal stem cell tube formation in vitro. J Zhejiang Univ Sci B 2011;12(8):644-651.
88. Zhang Z, Chen J, Guo F, et al. A high-temporal resolution technology for dynamic proteomic analysis based on 35S labeling. PLoS One 2008;3(8):e2991.
89. Calkins MJ, Reddy PH. Assessment of newly synthesized mitochondrial DNA using BrdU labeling in primary neurons from Alzheimer's disease mice:Implications for impaired mitochondrial biogenesis and synaptic damage. Biochim Biophys Acta 2011:1812(9):1182-1189.
90. Zhao W, Li JJ, Cao DY, et al. Intravenous injection of mesenchymal stem cells is effective in treating liver fibrosis. World J Gastroenterol 2012:18(10):1048-1058.
91. Kapuscinski J. DAPI:a DNA-specific fluorescent probe. Biotech Histochem 1995:70(5):220-233.
92. Li SL, Liu Y, Hui L. Construction of engineering adipose-like tissue in vivo utilizing human insulin gene-modified umbilical cord mesenchymal stromal cells with silk fibroin 3D scaffolds. J Tissue Eng Regen Med 2013 Mar 19. [Epub ahead of print]
93. Kruyt MC, DeBruijn J, Veenhof M, et al. Application and limitations of chloromethyl-benzamidodialkylcarbocyanine for tracing cells used in bone Tissue engineering. Tissue Eng 2003;9(1):105-115.
94. Yang S, Gao Q, Bao L, et al. Striatal extracts promote the dopaminergic differentiation of GFP-bone mesenchymal stem cells. Neurosci Lett 2012:530(2):115-120.
95. Hodgetts SI, Simmons PJ, Plant GW. Human mesenchymal precursor cells (stro-1(+)) from spinal cord injury patients improve functional recovery and tissue sparing in an acute spinal cord injury rat model. Cell Transplant 2013;22(3):393-412.
96. Lan Y, Kodati S, Lee HS, et al. Kinetics and function of mesenchymal stem cells in corneal injury. Invest Ophthalmol Vis Sci 2012;53(7):3638-3644.
97. Lyngholm M, Vorum H, Nielsen K, et al. Differences in the protein expression in limbal versus central human corneal epithelium--a search for stem cell markers. Exp Eye Res 2008;87(2):96-105.
98. Majo F, Rochat A, Nicolas M, Jaoude GA, Barrandon Y. Oligopotent stem cells are distributed throughout the mammalian ocular surface. Nature 2008:456(7219):250-254.
99. Yao L, Li ZR, Su WR, et al. Role of mesenchymal stem cells on cornea wound healing induced by acute alkali burn. PLoS One 2012;7(2):e30842.
100. Labouyrie E, Dubus P, Groppi A, et al. Expression of neurotrophins and their receptors in human bone marrow. Am J Pathol 1999:154:405-415.
101. Lin N, Hu K, Chen S, et al. Nerve growth factor-mediated paracrine regulation of hepatic stellate cells by multipotent mesenchymal stromal cells. Life Sci 2009:85:291-295.
102.Parr AM, Tator CH, Keating A. Bone marrow-derived mesenchymal stromal cells for the repair of central nervous system injury. Bone Marrow Transpl 2007:40:609-619.
103. Karussis D, Kassis I, Kurkalli BG, et al. Immunomodulation and neuroprotection with mesenchymal bone marrow stem cells (MSCs):A proposed treatment for multiple sclerosis and other neuroimmunological/neurodegenerative diseases. J Neurol Sci 2008;265:131-135.
104. Torrente Y, Polli E. Mesenchymal stem cell transplantation for neurodegenerative diseases. Cell Transplant 2008:17:1103-1113.
105. Caplan AI, Dennis JE. Mesenchymal stem cells as trophic mediators. J Cell Biochem 2006:98(5):1076-1084.
106. Alexanian AR. Epigenetic modifiers promote efficient generation of neural-like cells from bone marrow-derived mesenchymal cells grown in neural environment. J Cell Biochem 2007;100(2):362-371.
107. Ueda H, Fujita R, Yoshida A, Matsunaga H, Ueda M. Identification of prothymosin-alphal, the necrosis-apoptosis switch molecule in cortical neuronal cultures,J Cell Biol 2007;176(6):853-862.
108.Li L, Jiang J. Regulatory factors of mesenchymal stem cell migration into injured tissues and their signal transduction mechanisms. Front Med 2011;5(1):33-39.
109. Li GG, Chen SY, Xie HT, Zhu YT, Tseng SC. Angiogenesis potential of human limbal stromal niche cells. Invest Ophthalmol Vis Sci 2012;53(7):3357-3367.
1. Ren G, Chen X, Dong F, et al. Concise review:mesenchymal stem cells and translational medicine:emergingissues. Stem Cells Transl Med 2012;1(1):51-58.
2. Yu J, Vodyanik MA, Smuga-Otto K, et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 2007;318:1917-1920.
3. Miura K, Okada Y, Aoi T, et al. Variation in the safety of induced pluripotent stem cell lines. Nat Biotechnol 2009:27:743-745.
4. Gutierrez-Arandal, Ramos-MejiaV, BuenoC, et al. Human induced pluripotent stem cells develop teratoma more efficiently and faster than human embryonic stem cells regardless the site of injection. Stem Cells 2010;28:1568-1570.
5. Wong DJ, Liu H, Ridky TW, et al. Module map of stem cell genes guides creation of epithelial cancer stem cells. Cell Stem Cell 2008;2:333-344.
6. Fink DW Jr. FDA regulation of stem cell-based products. Science 2009; 324:1662-1663.
7. Zhang Y, Shen W, Hua J, et al. Pancreatic islet-like clusters from bone marrow mesenchymal stem cells of human first-trimester abortus can cure streptozocin-induced mouse diabetes. Rejuvenation Res 2010;13(6):695-706.
8. Crisan M, Yap S, Casteilla L, et al. A perivascular origin for mesenchymal stem cells in multiple human organs. Cell Stem Cell 2008;3:301-313.
9. Dellavalle A, Sampaolesi M, Tonlorenzi R, et al. Pericytes of human skeletal muscle are myogenic precursors distinct from satellite cells. Nat Cell Biol 2007:9:255-267.
10. Farrington-Rock C, Crofts NJ, Doherty MJ, et al. Chondrogenic and adipogenic potential of microvascular pericytes. Circulation 2004;110:2226-2232.
11. Collett GD, Canfield AE. Angiogenesis and pericytes in the initiation of ectopic calcification. Circ Res 2005;96:930-938.
12. Horwitz EM, Gordon PL, Koo WK, et al. Isolated allogeneic bone marrow-derived mesenchymal cells engraft and stimulate growth in children with osteogenesis imperfecta:Implications for cell therapy of bone. Proc Natl Acad SciUSA 2002;99:8932-8937.
13. Liechty KW, MacKenzie TC, Shaaban AF, et al. Human mesenchymal stem cells engraft and demonstrate site-specific differentiation after in utero transplantation in sheep. Nat Med 2000;6:1282-1286.
14. Kawada H, Fujita J, Kinjo K, et al. Nonhematopoietic mesenchymal stem cells can be mobilized and differentiate into cardiomyocytes after myocardial infarction. Blood 2004:104:3581-3587.
15. Dennis JE, Konstantakos EK, Arm D, et al. In vivo osteogenesis assay:A rapid method for quantitative analysis. Biomaterials 1998;19:1323-1328.
16. Ohgushi H, Kotobuki N, Funaoka H, et al. Tissue engineered ceramic artificial joint-ex vivo osteogenic differentiation of patient mesenchymal cells on total ankle joints for treat-ment of osteoarthritis. Biomaterials 2005:26:4654-4661.
17. Kon E, Muraglia A, Corsi A, et al. Autologous bone marrow stromal cells loaded onto porous hydroxyapatite ceramic accelerate bone repair in critical-size defects of sheep long bones. J Biomed Mater Res 2000:49:328-337.
18. Solchaga LA, Temenoff JS, Gao J, et al. Repair of osteochondral defects with hyaluronan-and polyester-based scaffolds. Osteoarthritis Cartilage 2005:13:297-309.
19. Horwitz EM, Gordon PL, Koo WK, et al. Isolated allogeneic bone marrow-derived mesenchymal cells engraft and stimulate growth in children with osteogenesis imperfecta:Implications for cell therapy of bone. Proc Natl Acad SciUSA 2002:99:8932-8937.
20. Mahmood A, Lu D, Lu M, et al. Treatment of traumatic brain injury in adult rats with intravenous administration of human bone marrow stromal cells. Neurosurgery 2003:53:697-702, discussion 702-693.
21. Ueda H, Fujita R, Yoshida A, Matsunaga H, Ueda M. Identification of prothymosin-alphal, the necrosis-apoptosis switch molecule in cortical neuronal cultures. J Cell Biol 2007;176(6):853-862.
22. Xie HT, Chen SY, Li GG, Tseng SC. Limbal epithelial stem/progenitor cells attract stromal niche cells by SDF-1/CXCR4signaling to prevent differentiation. Stem Cells 2011:29(11):1874-1885.
23. Wynn RF, Hart CA, Corradi-Perini C, et al. A small proportion of mesenchymal stem cells strongly expresses functionally active CXCR4 receptor capable of promoting migration to bone marrow. Blood 2004;104:2643-2645.
24. Belema-Bedada F, Uchida S, Martire A, et al. Efficient homing of multipotent adult mesenchymal stem cells depends on FROUNT-mediated clustering of CCR2. Cell Stem Cell 2008:2:566-575.
25. Cheng Z, Ou L, Zhou X, et al. Targeted migration of mesenchymal stem cells modified with CXCR4 gene to infarcted myocardium improves cardiac performance. Mol Ther 2008:16:571-579.
26. Hatch HM, Zheng D, Jorgensen ML, et al. SDF-lalpha/CXCR4:A mechanism for hepatic oval cell activation and bone marrow stem cell recruitment to the injured liver of rats. Cloning Stem Cells 2002:4:339-351.
27. Sordi V, Malosio ML, Marchesi F, et al. Bone marrow mesenchymal stem cells express a restricted set of functionally active chernokine receptors capable of promoting migration to pancreatic islets. Blood 2005;106:419-427.
28. Ma J, Shi M, Li J, Liao L, et al. Regulation of CXCR4 expression in human mesenchymal stem cells by cytokine treat-ment:Role in homing efficiency in NOD/SCID mice. Haematologica 2007;92:897-904.
29. Ruster B, Gottig S, Ludwig RJ, et al. Mesenchymal stem cells display coordinated rolling and adhesion behavior on endothelial cells. Blood 2006:108:3938-3944.
30. Ko IK, Kim BG, Awadallah A, et al. Targeting improves MSC treatment of inflammatory bowel disease. Mol Ther 2010;18:1365-1372.
31. Shi M, Li J, Liao L, et al. Regulation of CXCR4 expression in human mesenchymal stem cells by cytokine treatment:Role in homing efficiency in NOD/SCID mice. Haematologica 2007;92:897-904.
32. Ren G, Zhao X, Zhang L, et al. Inflammatory cytokine-induced intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in mesenchymal stem cells are critical for immunosuppression. J Immunol 2010;184:2321-2328.
33. Crisostomo PR, Wang Y, Markel TA, et al. Human mesenchymal stem cells stimulated by TNF-a, LPS, or hypoxia produce growth factors by an NF κB-but not JNK-dependent mechanism. Am J Physiol Cell Physiol 2008;294:C675-C682.
34. Caplan AI, Dennis JE. Mesenchymal stem cells as trophic mediators. J Cell Biochem 2006:98:1076-1084.
35. Xu G, Zhang Y, Zhang L, et al. The role of IL-6 in inhibition of lymphocyte apoptosis by mesenchymal stem cells. Biochem Biophys Res Commun 2007:361:745-750.
36. Cao W, Yang Y, Wang Z, et al. Leukemia inhibitory factor inhibits T helper 17 cell differentiation and confers treatment effects of neural progenitor cell therapy in autoimmune disease. Immunity 2011:35:273-284.
37. Hahn JY, Cho HJ, Kang HJ, et al. Pre-treatment of mesenchymal stem cells with a combination of growth factors enhances gap junction formation, cytoprotective effect on cardiomyocytes, and therapeutic efficacy for myocardial infarction. J Am Coll Cardiol 2008:51:933-943.
38. Schnabel LV, Lynch ME, van der Meulen MC, et al. Mesenchymal stem cells and insulin-like growth factor-I gene-enhanced mesenchymal stem cells improve structural aspects of healing in equine flexor digitorum superficialis tendons. J Orthop Res 2009:27:1392-1398.
39. Uccelli A, Moretta L, Pistoia V. Mesenchymal stem cells in health and disease. Nat Rev Immunol 2008:8:726-736.
40. Barry FP, Murphy JM, English K, et al. Immunogenicity of adult mesenchymal stem cells:Lessons from the fetal allograft. Stem Cells Dev 2005:14:252-265.
41. Toubai T, Paczesny S, Shono Y, et al. Mesenchymal stem cells for treatment and prevention of graft-versus-host disease after allogeneic hematopoietic cell transplantation. Curr Stem Cell Res Ther 2009;4:252-259.
42. Osawah H, Maruyama K, Streilein JW. CD95 ligand expression on corneal epithelium and endothelium influences the fates of orthotopic and heterotopic corneal allografts in mice. Invest Ophthalmol Vis Sci 2004:45:1908-1915.
43. Kwidzinskie E, Bunse J, Kovac AD, et al. Indolamine 2,3-dioxygenase is expressed in the CNS and down-regulates autoimmune inflammation. FASEB J 2005:19:1347-1349.
44. Waterman RS, Tomchuck SL, Henkle SL, et al. A new mesenchymal stem cell (MSC) paradigm:Polarization into a pro-inflammatory MSC1 or an immunosuppressive MSC2 phenotype. PLoS One.2010;5:e10088.
45. Ren G, Su J, Zhang L, et al. Species variation in the mechanisms of mesenchymal stem cell-mediated immunosuppression. Stem Cells.2009:27:1954-1962.
46. Allison M. Genzyme backs Osiris, despite Prochymal flop. Nat Biotechnol. 2009:27:966-967.
47. Tomita M, Adachi Y, Yamada H, et al. Bone marrow-derived stem cells can differentiate into retinal cells in injured rat retina. Stem Cells 2002;20:279-283.
48. Kicic A, Shen WY, Wilson AS, et al. Differentiation of marrow stromal cells into photoreceptors in the rat eye. J Neurosci 2003;23:7742-7749.
49. Tomita M, Mori T, Maruyama K, et al. A comparison of neural differentiation and retinal transplantation with bone marrow-derived cells and retinal progenitor cells. Stem Cells 2006:24:2270-2278.
50. Gong L, Wu Q, Song B, et al. Differentiation of rat mesenchymal stem cells transplanted into the subretinal space of sodium iodate-injected rats. Clin Experiment Ophthalmol 2008;36:666-671.
51. Castanheira P, Torquetti L, Nehemy MB, et al. Retinal incorporation and differentiation of mesenchymal stem cells intravitreally injected in the injured retina of rats. Arq Bras Oftalmol 2008:71:644-650.
52. Zhang ZQ, Dong FT. In vitro differentiation of rat mesenchymal stem cells into photoreceptors. Zhonghua Yan Ke Za Zhi 2008;44:540-544.
53. Vossmerbaeumer U, Ohnesorge S, Kuehl S, et al. Retinal pigment epithelial phenotype induced in human adipose tissue-derived mesenchymal stromal cells. Cytotherapy 2009:11:177-188.
54. Arnhold S, Heiduschka P, Klein H, et al. Adenovirally transduced bone marrow stromal cells differentiate into pigment epithelial cells and induce rescue effects in RCS rats. Invest Ophthalmol Vis Sci 2006;47:4121-4129.
55. Yu S, Tanabe T, Dezawa M, et al. Effects of bone marrow stromal cell injection in an experimental glaucoma model. Biochem Biophys Res Commun 2006:344:1071-1079.
56. Azizi SA, Stokes D, Augelli BJ, et al. Engraftment and migration of human bone marrow stromal cells implanted in the brains of albino rats—Similarities to astrocyte grafts. Proc Natl Acad Sci U S A 1998;95:3908-3913.
57. Li N, Li XR, Yuan JQ. Effects of bone-marrow mesenchymal stem cells transplanted into vitreous cavity of rat injured by ischemia/reperfusion. Graefes Arch Clin Exp Ophthalmol 2009;247:503-514.
58. Inoue Y, Iriyama A, Ueno S, et al. Subretinal transplantation of bone marrow mesenchymal stem cells delays retinal degeneration in the RCS rat model of retinal degeneration. Exp Eye Res 2007;85:234-241.
59. Wang S, Lu B, Girman S, et al. Non-invasive stem cell therapy in a rat model for retinal degeneration and vascular pathology. PLoS One 2010;5:e9200.
60. McIntosh Ambrose W, Schein O, Elisseeff J. A tale of two tissues:Stem cells in cartilage and corneal tissue engineering. Curr Stem Cell Res Ther 2010;5:37-48.
61. De Miguel MP, Alio JL, Arnalich-Montiel F, et al. cornea and ocular surface treatment. Curr Stem Cell Res Ther 2010;5:195-204.
62. Ma Y, Xu Y, Xiao Z, et al. Reconstruction of chemically burned rat corneal surface by bone marrow-derived human mesenchymal stem cells. Stem Cells 2006;24:315-321.
63. Oh JY, Kim MK, Shin MS, et al. The anti-inflammatory and anti-angiogenic role of mesenchymal stem cells in corneal wound healing following chemical injury. Stem Cells 2008;26:1047-1055.
64. Oh JY, Kim MK, Shin MS, et al. Cytokine secretion by human mesenchymal stem cells cocultured with damaged corneal epithelial cells. Cytokine 2009:46:100-103.
65. Oh JY, Kim MK, Ko JH, et al. Rat allogeneic mesenchymal stem cells did not prolong the survival of corneal xenograft in a pig-to-rat model. Vet Ophthalmol 2009:12:35-40.
66. Zoukhri D. Mechanisms involved in injury and repair of the murine lacrimal gland: Role of programmed cell death and mesenchymal stem cells. Ocul Surf 2010:8:60-69.
67. Zoukhri D, Fix A, Alroy J, et al. Mechanisms of murine lacrimal gland repair after experimentally induced inflammation. Invest Ophthalmol Vis Sci 2008:49:4399-4406.
68. Pease ME, McKinnon SJ, Quigley HA, et al. Obstructe axonal transport of BDNF and its receptor TrkB in experimental glaucoma. Invest Ophthalmol Vis Sci 2000;41:764-774.
69. Murphy JA, Clarke DB. Target-derived neurotrophin may influence the survival of adult retinal ganglio cells when local neurotrophic support is disrupted: Implications for glaucoma. Med Hypotheses 2006:67:1208-1212.
70. Yu S, Tanabe T, Dezawa M, et al. Effects of bone marrow stromal cell injection in an experimental glaucoma model. Biochem Biophys Res Commun 2006:344:1071-1079.
71. Osborne NN. Recent clinical findings with memantine should not mean that the idea of neuroprotection in glaucoma is abandoned. Acta Ophthalmol 2009:87:450-454.
72. Caspi RR. Th1 and Th2 responses in pathogenesis and regulation of experimental Autoimmune uveoretinitis. Int Rev Immunol 2002,21(2-3):197-208.
73. Gonzalez MA, Gonzalez-Rey E, Rico L, et al. Treatment of experimental arthritis by inducing immune tolerance with human adipose-derived mesenchymal stem cells. Arthritis Rheum 2009:60:1006-1019.
74. Parekkadan B, Tilles AW, Yarmush ML. Bone marrow-derived mesenchymal stem cells ameliorate autoimmune enteropathy independently of regulatory T cells. Stem Cells 2008; 26:1913.
75. Fiorina P, Jurewicz M, Augello A, et al. Immunomodulatory function of bone marrow-derived mesenchymal stem cells in experimental autoimmune type 1 diabetes. J Immunol 2009;183:993.
76. Marmont AM, Gualandi F, Piaggio G, et al. Allogeneic bone marrow transplantation (BMT) for refractory Behcet's disease with severe CNS involvement. Bone Marrow Transplant 2006;37:1061-1063.
77. Erokhin VV, Vasil'eva IA, Konopliannikov AG, et al. Systemic transplantation of autologous mesenchymal stem cells of the bone marrow in the treatment of patients with multidrug-resistant pulmonary tuberculosis. Probl Tuberk Bolezn Legk 2008;10:3-6.
78. Sacchetti B, Funari A, Michienzi S, et al. Self-renewing osteoprogenitors in bone marrow sinusoids can organize a hematopoietic microenvironment. Cell 2007:131:324-336.
79. Kuo CK, Tuan RS. Tissue engineering with mesenchymal stem cells. IEEE Eng Med Biol Mag 2003:22:51-56.
80. Chen F, Tuan R. Adult stem cells for cartilage tissue engineering and regeneration. Curr Rhematol Rev 2008;4:149-154.
81. Patel M, Betz MW, Geibel E, et al. Cyclic acetal hydroxyapatite nanocomposites for orbital bone regeneration. Tissue Eng Part A 2010;16:55-65.
82.82. Wright V, Peng H, Usas A, et al. BMP4-expressing muscle-derived stem cells differentiate into osteogenic lineage and improve bone healing in immunocompetent mice. Mol Ther 2002;6:169-178.
83. Peng H, Usas A, Olshanski A, et al. VEGF improves, whereas sFltl inhibits, BMP2-induced bone formation and bone healing through modulation of angiogenesis. J Bone Miner Res 2005;20:2017-2027.
84. Montarras D, Morgan J, Collins C, et al. Direct isolation of satellite cells for skeletal muscle regeneration. Science 2005;309:2064-2067.
85. Kuang S, Kuroda K, Le Grand F, et al. Asymmetric self-renewal and commitment of satellite stem cells in muscle. Cell 2007;129:999-1010.
86. Scheef EA, Sorenson CM, Sheibani N. Attenuation of proliferation and migration of retinal pericytes in the absence of thrombospondin-1. Am J Physiol Cell Physiol 2009;296:C724-C734.
87. Li Y, Huard J. Differentiation of muscle-derived cells into myofibroblasts in injured skeletal muscle. Am J Pathol 2002;161:895-907.
88. Quintero AJ, Wright VJ, Fu FH, et al. Stem cells for the treatment of skeletal muscle injury. Clin Sports Med 2009;28:1-11.
89. Li Y, Foster W, Deasy BM, et al. Transforming growth factor-betal induces the differentiation of myogenic cells into fibrotic cells in injured skeletal muscle: A key event in muscle fibrogenesis. Am J Pathol 2004;164:1007-1019.
90. Lu L, Saulis AS, Liu WR, et al. The temporal effects of anti-TGF-betal,2, and 3 monoclonal antibody on wound healing and hypertrophic scar formation. J Am Coll Surg 2005:201:391-397.
91. Rodeheffer MS, Birsoy K, Friedman JM. Identification of white adipocyte progenitor cells in vivo. Cell 2008;135:240-249.
92. Tang W, Zeve D, Suh JM, et al. White fat progenitor cells reside in the adipose vasculature. Science 2008;322:583-586.
93. Perros P, Krassas GE. Graves orbitopathy:A perspective. Nat Rev Endocrinol 2009:5:312-318.
94. Stenn K, Parimoo S, Zheng Y, et al. Bioengineering the hair follicle. Organogenesis 2007;3:6-13.
95. Nakagawa H, Akita S, Fukui M, et al. Human mesenchymal stem cells successfully improve skin-substitute wound healing. Br J Dermatol 2005:153:29-36.
96. Borue X, Lee S, Grove J, et al. Bone marrow-derived cells contribute to epithelial engraftment during wound healing. Am J Pathol 2004:165:1767-1772.