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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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Effect of 3 culture media on ZO1 and N-cadherin localization in MSCs associated with damaged HCEC. Fluorescence confocal microscopic images show that, in wounded corneas incubated in MSCBM, ZO1 and N-cadherin were localized diffusely within the cytoplasm of the MSCs. In wounded corneas incubated in LECBM or LECCM, ZO1 and N-cadherin tended to localize at the lateral borders of MSCs. Arrows in A-C show edges of the damaged endothelium. Red: ZO1. Green: N-cadherin. Original magnification: 40×.
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f9: Effect of 3 culture media on ZO1 and N-cadherin localization in MSCs associated with damaged HCEC. Fluorescence confocal microscopic images show that, in wounded corneas incubated in MSCBM, ZO1 and N-cadherin were localized diffusely within the cytoplasm of the MSCs. In wounded corneas incubated in LECBM or LECCM, ZO1 and N-cadherin tended to localize at the lateral borders of MSCs. Arrows in A-C show edges of the damaged endothelium. Red: ZO1. Green: N-cadherin. Original magnification: 40×.

Mentions: Experiments were then conducted to determine the effect of the three culture media on the localization of ZO1 and N-cadherin when UCB1 MSCs were added to ex vivo corneal endothelial crush wounds. A similar protocol was used for these studies as for the ex vivo studies discussed above. The only difference was that the tissue was processed for both ZO1 and N-cadherin immunostaining. Images in Figure 9 show that both ZO1 and N-cadherin were expressed under all 3 medium conditions; however, as observed in the tissue culture studies, the relative localization of both proteins differed depending on the culture medium. In UCB1 MSCs incubated in MSCBM (Figure 9A,D), ZO1 and N-cadherin were both diffusely distributed within the cytoplasm. Both proteins showed a greater association with cell borders when MSCs were incubated in LECBM (Figure 9B,E), but the overall arrangement of the cells was relatively random. MSCs incubated in LECCM (Figure 9C,F) showed the greatest association of both ZO1 and N-cadherin at cell borders. Cells also formed a relatively tight sheet of parallel cells within the wound area.


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)

Effect of 3 culture media on ZO1 and N-cadherin localization in MSCs associated with damaged HCEC. Fluorescence confocal microscopic images show that, in wounded corneas incubated in MSCBM, ZO1 and N-cadherin were localized diffusely within the cytoplasm of the MSCs. In wounded corneas incubated in LECBM or LECCM, ZO1 and N-cadherin tended to localize at the lateral borders of MSCs. Arrows in A-C show edges of the damaged endothelium. Red: ZO1. Green: N-cadherin. Original magnification: 40×.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f9: Effect of 3 culture media on ZO1 and N-cadherin localization in MSCs associated with damaged HCEC. Fluorescence confocal microscopic images show that, in wounded corneas incubated in MSCBM, ZO1 and N-cadherin were localized diffusely within the cytoplasm of the MSCs. In wounded corneas incubated in LECBM or LECCM, ZO1 and N-cadherin tended to localize at the lateral borders of MSCs. Arrows in A-C show edges of the damaged endothelium. Red: ZO1. Green: N-cadherin. Original magnification: 40×.
Mentions: Experiments were then conducted to determine the effect of the three culture media on the localization of ZO1 and N-cadherin when UCB1 MSCs were added to ex vivo corneal endothelial crush wounds. A similar protocol was used for these studies as for the ex vivo studies discussed above. The only difference was that the tissue was processed for both ZO1 and N-cadherin immunostaining. Images in Figure 9 show that both ZO1 and N-cadherin were expressed under all 3 medium conditions; however, as observed in the tissue culture studies, the relative localization of both proteins differed depending on the culture medium. In UCB1 MSCs incubated in MSCBM (Figure 9A,D), ZO1 and N-cadherin were both diffusely distributed within the cytoplasm. Both proteins showed a greater association with cell borders when MSCs were incubated in LECBM (Figure 9B,E), but the overall arrangement of the cells was relatively random. MSCs incubated in LECCM (Figure 9C,F) showed the greatest association of both ZO1 and N-cadherin at cell borders. Cells also formed a relatively tight sheet of parallel cells within the wound area.

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