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Potential of human umbilical cord blood mesenchymal stem cells to heal damaged corneal endothelium.

Joyce NC, Harris DL, Markov V, Zhang Z, Saitta B - Mol. Vis. (2012)

Bottom Line: Immunolocalization of the junction-associated proteins, zonula occludins-1 (ZO1) and N-cadherin, was visualized by fluorescence confocal microscopy.This localization was lost when extracellular calcium levels were reduced by treatment with EGTA.Further study is needed to identify the specific microenvironmental conditions that would permit tissue engineering of UCB MSCs to replace damaged or diseased corneal endothelium.

View Article: PubMed Central - PubMed

Affiliation: Schepens Eye Research Institute, Boston, MA 02114, USA. nancy.joyce@schepens.harvard.edu

ABSTRACT

Purpose: To test the feasibility of altering the phenotype of umbilical cord blood mesenchymal stem cells (UCB MSCs) toward that of human corneal endothelial cells (HCEC) and to determine whether UCB MSCs can "home" to sites of corneal endothelial cell injury using an ex vivo corneal wound model.

Methods: RNA was isolated and purified from UCB MSCs and HCECs. Baseline information regarding the relative gene expression of UCB MSCs and HCEC was obtained by microarray analysis. Quantitative real-time PCR (q-PCR) verified the microarray findings for a subset of genes. The ability of different culture media to direct UCB MSCs toward a more HCEC-like phenotype was tested in both tissue culture and ex vivo corneal endothelial wound models using three different media: MSC basal medium (MSCBM), a basal medium used to culture lens epithelial cells (LECBM), or lens epithelial cell-conditioned medium (LECCM). Morphology of the MSCs was observed by phase-contrast microscopy or by light microscopic observation of crystal violet-stained cells. Immunolocalization of the junction-associated proteins, zonula occludins-1 (ZO1) and N-cadherin, was visualized by fluorescence confocal microscopy. Formation of cell-cell junctions was tested by treatment with the calcium chelator, EGTA. A second microarray analysis compared gene expression between UCB MSCs grown in LECBM and LECCM to identify changes induced by the lens epithelial cell-conditioned culture medium. The ability of UCB MSCs to "home" to areas of endothelial injury was determined using ZO1 immunolocalization patterns in ex vivo corneal endothelial wounds.

Results: Baseline microarray analysis provided information regarding relative gene expression in UCB MSCs and HCECs. MSCs attached to damaged, but not intact, corneal endothelium in ex vivo corneal wounds. The morphology of MSCs was consistently altered when cells were grown in the presence of LECCM. In tissue culture and in ex vivo corneal wounds, UCB MSC treated with LECCM were elongated and formed parallel sheets of closely apposed cells. In both tissue culture and ex vivo corneal endothelial wounds, ZO1 and N-cadherin localized mainly to the cytoplasm of UCB MSCs in the presence of MSCBM. However, both proteins localized to cell borders when UCB MSCs were grown in either LECBM or LECCM. This localization was lost when extracellular calcium levels were reduced by treatment with EGTA. A second microarray analysis showed that, when UCB MSCs were grown in LECCM instead of LECBM, the relative expression of a subset of genes markedly differed, suggestive of a more HCEC-like phenotype.

Conclusions: Results indicate that UCB MSCs are able to "home" to areas of injured corneal endothelium and that the phenotype of UCB MSCs can be altered toward that of HCEC-like cells. Further study is needed to identify the specific microenvironmental conditions that would permit tissue engineering of UCB MSCs to replace damaged or diseased corneal endothelium.

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Cluster dendrogram and heat map results. Cluster dendrogram (A) shows relationships in gene expression among UCB1, UCB4, young HCEC, and older HCEC samples. The heatmap in (B) shows relative levels of gene expression at a p-value of 0.001 between UCB1 and UCB4 MSCs and HCECs from young and older donors. The relative levels of gene expression are depicted using a color scale where red represents the lowest and green represents the highest level of expression.
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f2: Cluster dendrogram and heat map results. Cluster dendrogram (A) shows relationships in gene expression among UCB1, UCB4, young HCEC, and older HCEC samples. The heatmap in (B) shows relative levels of gene expression at a p-value of 0.001 between UCB1 and UCB4 MSCs and HCECs from young and older donors. The relative levels of gene expression are depicted using a color scale where red represents the lowest and green represents the highest level of expression.

Mentions: Microarray analysis was used to compare gene expression between MSCs and HCEC. The cluster dendrogram in Figure 2A indicates relative similarity in gene expression among closely related groups, i.e., among young HCEC, older HCEC, UCB1, and UCB4 samples. As expected, relative gene expression in both sets of HCEC samples exhibited closer similarity to each other than to the two sets of UCB samples. The same is true of the two types of UCB samples versus HCEC. Significance Analysis of Microarrays (SAM) [41] was conducted to compare relative gene expression between UCB1 and UCB4 MSCs, each from two different passages (UCB1P6, UCB1P8, UCB4P4, and UCB4P9) and passage 2 HCECs isolated and cultured from three young (17, 26, and 29 years old) and three older donors (50, 51, and 56 years old). This comparison was made to establish a baseline of relative gene expression for UCB MSCs and HCECs before attempting to differentiate MSCs toward more HCEC-like cells. A list of the top 250 genes in which the mean expression level in UCB MSCs is significantly higher than in HCEC is presented in Appendix 1. Appendix 2 contains a list of the top 250 genes in which the mean expression level in UCB MSCs is significantly lower than in HCEC. Figure 2B presents a heat map comparing the relative expression of individual genes at an ANOVA p-value of 0.001. The heat map indicates significant differences in the relative level of individual gene expression among the UCB1, UCB4, young HCEC, and older HCEC samples. In general, UCB samples exhibited greater similarity in expression to each other than to either group of HCEC and vice versa.


Potential of human umbilical cord blood mesenchymal stem cells to heal damaged corneal endothelium.

Joyce NC, Harris DL, Markov V, Zhang Z, Saitta B - Mol. Vis. (2012)

Cluster dendrogram and heat map results. Cluster dendrogram (A) shows relationships in gene expression among UCB1, UCB4, young HCEC, and older HCEC samples. The heatmap in (B) shows relative levels of gene expression at a p-value of 0.001 between UCB1 and UCB4 MSCs and HCECs from young and older donors. The relative levels of gene expression are depicted using a color scale where red represents the lowest and green represents the highest level of expression.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3298421&req=5

f2: Cluster dendrogram and heat map results. Cluster dendrogram (A) shows relationships in gene expression among UCB1, UCB4, young HCEC, and older HCEC samples. The heatmap in (B) shows relative levels of gene expression at a p-value of 0.001 between UCB1 and UCB4 MSCs and HCECs from young and older donors. The relative levels of gene expression are depicted using a color scale where red represents the lowest and green represents the highest level of expression.
Mentions: Microarray analysis was used to compare gene expression between MSCs and HCEC. The cluster dendrogram in Figure 2A indicates relative similarity in gene expression among closely related groups, i.e., among young HCEC, older HCEC, UCB1, and UCB4 samples. As expected, relative gene expression in both sets of HCEC samples exhibited closer similarity to each other than to the two sets of UCB samples. The same is true of the two types of UCB samples versus HCEC. Significance Analysis of Microarrays (SAM) [41] was conducted to compare relative gene expression between UCB1 and UCB4 MSCs, each from two different passages (UCB1P6, UCB1P8, UCB4P4, and UCB4P9) and passage 2 HCECs isolated and cultured from three young (17, 26, and 29 years old) and three older donors (50, 51, and 56 years old). This comparison was made to establish a baseline of relative gene expression for UCB MSCs and HCECs before attempting to differentiate MSCs toward more HCEC-like cells. A list of the top 250 genes in which the mean expression level in UCB MSCs is significantly higher than in HCEC is presented in Appendix 1. Appendix 2 contains a list of the top 250 genes in which the mean expression level in UCB MSCs is significantly lower than in HCEC. Figure 2B presents a heat map comparing the relative expression of individual genes at an ANOVA p-value of 0.001. The heat map indicates significant differences in the relative level of individual gene expression among the UCB1, UCB4, young HCEC, and older HCEC samples. In general, UCB samples exhibited greater similarity in expression to each other than to either group of HCEC and vice versa.

Bottom Line: Immunolocalization of the junction-associated proteins, zonula occludins-1 (ZO1) and N-cadherin, was visualized by fluorescence confocal microscopy.This localization was lost when extracellular calcium levels were reduced by treatment with EGTA.Further study is needed to identify the specific microenvironmental conditions that would permit tissue engineering of UCB MSCs to replace damaged or diseased corneal endothelium.

View Article: PubMed Central - PubMed

Affiliation: Schepens Eye Research Institute, Boston, MA 02114, USA. nancy.joyce@schepens.harvard.edu

ABSTRACT

Purpose: To test the feasibility of altering the phenotype of umbilical cord blood mesenchymal stem cells (UCB MSCs) toward that of human corneal endothelial cells (HCEC) and to determine whether UCB MSCs can "home" to sites of corneal endothelial cell injury using an ex vivo corneal wound model.

Methods: RNA was isolated and purified from UCB MSCs and HCECs. Baseline information regarding the relative gene expression of UCB MSCs and HCEC was obtained by microarray analysis. Quantitative real-time PCR (q-PCR) verified the microarray findings for a subset of genes. The ability of different culture media to direct UCB MSCs toward a more HCEC-like phenotype was tested in both tissue culture and ex vivo corneal endothelial wound models using three different media: MSC basal medium (MSCBM), a basal medium used to culture lens epithelial cells (LECBM), or lens epithelial cell-conditioned medium (LECCM). Morphology of the MSCs was observed by phase-contrast microscopy or by light microscopic observation of crystal violet-stained cells. Immunolocalization of the junction-associated proteins, zonula occludins-1 (ZO1) and N-cadherin, was visualized by fluorescence confocal microscopy. Formation of cell-cell junctions was tested by treatment with the calcium chelator, EGTA. A second microarray analysis compared gene expression between UCB MSCs grown in LECBM and LECCM to identify changes induced by the lens epithelial cell-conditioned culture medium. The ability of UCB MSCs to "home" to areas of endothelial injury was determined using ZO1 immunolocalization patterns in ex vivo corneal endothelial wounds.

Results: Baseline microarray analysis provided information regarding relative gene expression in UCB MSCs and HCECs. MSCs attached to damaged, but not intact, corneal endothelium in ex vivo corneal wounds. The morphology of MSCs was consistently altered when cells were grown in the presence of LECCM. In tissue culture and in ex vivo corneal wounds, UCB MSC treated with LECCM were elongated and formed parallel sheets of closely apposed cells. In both tissue culture and ex vivo corneal endothelial wounds, ZO1 and N-cadherin localized mainly to the cytoplasm of UCB MSCs in the presence of MSCBM. However, both proteins localized to cell borders when UCB MSCs were grown in either LECBM or LECCM. This localization was lost when extracellular calcium levels were reduced by treatment with EGTA. A second microarray analysis showed that, when UCB MSCs were grown in LECCM instead of LECBM, the relative expression of a subset of genes markedly differed, suggestive of a more HCEC-like phenotype.

Conclusions: Results indicate that UCB MSCs are able to "home" to areas of injured corneal endothelium and that the phenotype of UCB MSCs can be altered toward that of HCEC-like cells. Further study is needed to identify the specific microenvironmental conditions that would permit tissue engineering of UCB MSCs to replace damaged or diseased corneal endothelium.

Show MeSH
Related in: MedlinePlus