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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 UCB1 MSC association with damaged endothelium. Fluorescence confocal microscopic images show the formation of MSC cell sheets in areas of damaged HCEC (arrows in A-C). Note that, in wounded corneas incubated in MSCBM, ZO1 is localized diffusely within the cytoplasm of the MSCs. In wounded corneas incubated in LECBM or LECCM, ZO1 tended to be localized at the lateral borders of MSCs. Red: ZO1. Blue: TO-PRO-3-stained nuclei. Original magnification: 40×.
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f8: Effect of 3 culture media on UCB1 MSC association with damaged endothelium. Fluorescence confocal microscopic images show the formation of MSC cell sheets in areas of damaged HCEC (arrows in A-C). Note that, in wounded corneas incubated in MSCBM, ZO1 is localized diffusely within the cytoplasm of the MSCs. In wounded corneas incubated in LECBM or LECCM, ZO1 tended to be localized at the lateral borders of MSCs. Red: ZO1. Blue: TO-PRO-3-stained nuclei. Original magnification: 40×.

Mentions: Studies were conducted to determine the effect of the three culture media on association of non-GFP-labeled UCB1 MSCs with damaged endothelium using the ex vivo crush wound model. For these studies, crush wounds were made in the endothelium of donor corneas. Corneas were incubated for 48 h in MSCBM plus 30 µg/ml 5-fluorouracil (5-FU) to inhibit proliferation of the HCEC and prevent premature closure of the wounds. After washing to remove the 5-FU, UCB1 MSCs were placed on the endothelial side of the corneas and incubated for 2 weeks in the presence of MSCBM, LECBM, or LECCM. Corneas were then processed for ZO1 staining and nuclear labeling with TO-PRO-3. As can be seen in Figure 8, UCB1 MSCs associated with damaged HCEC regardless of the culture medium used. However, there was a consistent difference noted in the relative arrangement of the cells based on the culture medium. In the presence of MSCBM (Figure 8A,D), MSCs showed a relatively random orientation within the damaged areas, whereas, when MSCs were incubated in LECBM (Figure 8B,E) or LECCM (Figure 8C,F), the MSCs tended to form a more oriented and more tightly associated cell sheet in 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 UCB1 MSC association with damaged endothelium. Fluorescence confocal microscopic images show the formation of MSC cell sheets in areas of damaged HCEC (arrows in A-C). Note that, in wounded corneas incubated in MSCBM, ZO1 is localized diffusely within the cytoplasm of the MSCs. In wounded corneas incubated in LECBM or LECCM, ZO1 tended to be localized at the lateral borders of MSCs. Red: ZO1. Blue: TO-PRO-3-stained nuclei. Original magnification: 40×.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f8: Effect of 3 culture media on UCB1 MSC association with damaged endothelium. Fluorescence confocal microscopic images show the formation of MSC cell sheets in areas of damaged HCEC (arrows in A-C). Note that, in wounded corneas incubated in MSCBM, ZO1 is localized diffusely within the cytoplasm of the MSCs. In wounded corneas incubated in LECBM or LECCM, ZO1 tended to be localized at the lateral borders of MSCs. Red: ZO1. Blue: TO-PRO-3-stained nuclei. Original magnification: 40×.
Mentions: Studies were conducted to determine the effect of the three culture media on association of non-GFP-labeled UCB1 MSCs with damaged endothelium using the ex vivo crush wound model. For these studies, crush wounds were made in the endothelium of donor corneas. Corneas were incubated for 48 h in MSCBM plus 30 µg/ml 5-fluorouracil (5-FU) to inhibit proliferation of the HCEC and prevent premature closure of the wounds. After washing to remove the 5-FU, UCB1 MSCs were placed on the endothelial side of the corneas and incubated for 2 weeks in the presence of MSCBM, LECBM, or LECCM. Corneas were then processed for ZO1 staining and nuclear labeling with TO-PRO-3. As can be seen in Figure 8, UCB1 MSCs associated with damaged HCEC regardless of the culture medium used. However, there was a consistent difference noted in the relative arrangement of the cells based on the culture medium. In the presence of MSCBM (Figure 8A,D), MSCs showed a relatively random orientation within the damaged areas, whereas, when MSCs were incubated in LECBM (Figure 8B,E) or LECCM (Figure 8C,F), the MSCs tended to form a more oriented and more tightly associated cell sheet in 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