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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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Crystal violet-stained light microscopic images of UCB1 MSCs. Cells grown in MSC basal medium (MSCBM; A), lens epithelial cell basal medium (LECBM; B), or lens epithelial cell-conditioned medium (LECCM; C) show relative differences in cell shape and culture characteristics. Original magnification: 4×.
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f4: Crystal violet-stained light microscopic images of UCB1 MSCs. Cells grown in MSC basal medium (MSCBM; A), lens epithelial cell basal medium (LECBM; B), or lens epithelial cell-conditioned medium (LECCM; C) show relative differences in cell shape and culture characteristics. Original magnification: 4×.

Mentions: During corneal development, appropriate differentiation of neural crest mesenchymal cells to form corneal endothelium appears to be dependent on inductive signals from the lens [20-25]. For this reason, we tested whether soluble factors from cultured lens epithelial cells would affect the differentiation of UCB1 MSCs toward more HCEC-like cells using lens epithelial cell-conditioned medium (LECCM) prepared as indicated above. LECCM, the basal medium used for growth of lens epithelial cells (LECBM), and the basal medium used for culture of MSCs (MSCBM) were first compared for their effect on the morphology of UCB1 MSCs in culture. Crystal violet-stained images of UCB1 MSCs grown in the 3 culture media are shown in Figure 4. As described previously [37] and shown in Figure 1A,C, UCB1 MSCs grown in MSCBM (Figure 4A) exhibited elongated, fibroblastic-like shapes and tended to form a swirl pattern with evidence of cellular overlap. MSCs grown in LECBM (Figure 4B) also exhibited elongated, fibroblastic-like shapes, but the cultures tended to have more open areas between cell clusters where cells appeared to be somewhat more broad and flattened. Cells within clusters formed a random criss-cross pattern, indicating multiple cell layers. UCB1 MSCs grown in LECCM (Figure 4C) were consistently more highly elongated than cells grown in the other two media. Cells in the more open areas of the culture tended to form parallel arrays, while cells located within clusters showed evidence of multi-layering.


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)

Crystal violet-stained light microscopic images of UCB1 MSCs. Cells grown in MSC basal medium (MSCBM; A), lens epithelial cell basal medium (LECBM; B), or lens epithelial cell-conditioned medium (LECCM; C) show relative differences in cell shape and culture characteristics. Original magnification: 4×.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Crystal violet-stained light microscopic images of UCB1 MSCs. Cells grown in MSC basal medium (MSCBM; A), lens epithelial cell basal medium (LECBM; B), or lens epithelial cell-conditioned medium (LECCM; C) show relative differences in cell shape and culture characteristics. Original magnification: 4×.
Mentions: During corneal development, appropriate differentiation of neural crest mesenchymal cells to form corneal endothelium appears to be dependent on inductive signals from the lens [20-25]. For this reason, we tested whether soluble factors from cultured lens epithelial cells would affect the differentiation of UCB1 MSCs toward more HCEC-like cells using lens epithelial cell-conditioned medium (LECCM) prepared as indicated above. LECCM, the basal medium used for growth of lens epithelial cells (LECBM), and the basal medium used for culture of MSCs (MSCBM) were first compared for their effect on the morphology of UCB1 MSCs in culture. Crystal violet-stained images of UCB1 MSCs grown in the 3 culture media are shown in Figure 4. As described previously [37] and shown in Figure 1A,C, UCB1 MSCs grown in MSCBM (Figure 4A) exhibited elongated, fibroblastic-like shapes and tended to form a swirl pattern with evidence of cellular overlap. MSCs grown in LECBM (Figure 4B) also exhibited elongated, fibroblastic-like shapes, but the cultures tended to have more open areas between cell clusters where cells appeared to be somewhat more broad and flattened. Cells within clusters formed a random criss-cross pattern, indicating multiple cell layers. UCB1 MSCs grown in LECCM (Figure 4C) were consistently more highly elongated than cells grown in the other two media. Cells in the more open areas of the culture tended to form parallel arrays, while cells located within clusters showed evidence of multi-layering.

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