Limits...
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.

Show MeSH

Related in: MedlinePlus

Quantitative real-time PCR confirms differences in gene expression identified by microarray analysis in UCB1, UCB4, and HCEC. Relative expression levels were normalized to the housekeeping gene GAPDH and are shown relative to the highest value (0 to 1). Error bars represent one standard deviation for the four UCB (two UCB1 and two UCB4) and six HCEC biologic replicates tested. Robust multi-array average (RMA) estimated expression levels from the Affymetrix array, averaged for the biologic replicates within each cell type, are listed on the x-axis.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3298421&req=5

f3: Quantitative real-time PCR confirms differences in gene expression identified by microarray analysis in UCB1, UCB4, and HCEC. Relative expression levels were normalized to the housekeeping gene GAPDH and are shown relative to the highest value (0 to 1). Error bars represent one standard deviation for the four UCB (two UCB1 and two UCB4) and six HCEC biologic replicates tested. Robust multi-array average (RMA) estimated expression levels from the Affymetrix array, averaged for the biologic replicates within each cell type, are listed on the x-axis.

Mentions: Quantitative real time-PCR studies were then conducted using RNA isolated from the same UCB MSC and HCEC samples to verify results of the microarray analysis for a subset of genes. For these studies, data was averaged and comparison was made between UCB1 MSCs, UCB4 MSCs, and HCECs from both young and older donors (Figure 3). As observed in the microarray analysis, HCECs expressed significantly higher mRNA levels of both NCAM1 (neural cell adhesion molecule-1) and TFAP2B (transcription factor AP-2, beta) compared with either type of MSC. Both UCB1 and UCB4 MSCs expressed significantly higher mRNA levels of CDK15 (cyclin-dependent kinase-15) and MEIS1 (Meis homeobox1), as indicated by the microarray analysis. The relative expression of four additional mRNAs was tested. COL8A1 (collagen VIII, alpha-1) is expressed in corneal endothelial cells [42], as well as in UCB1 MSCs [37]. Results from qPCR indicated that, although all samples were positive, UCB1 MSCs expressed relatively low, but detectable, levels of COL8A1 in comparison with either UCB4 or HCECs. The cell surface antigen, THY1 (CD90), is an important marker of non-hematopoeitic UCB MSCs [37,43]. The qPCR results indicated that THY1 mRNA is not only expressed in UCB1 and UCB4 MSCs, but also in HCEC, with expression in HCEC being somewhat lower than in the MSCs. CDH2 (cadherin-2 / N-cadherin]) is expressed in corneal endothelial cells [44]. Expression of this cadherin isoform has also been detected in MSCs [45]. Results of the qPCR showed a relatively similar level of N-cadherin mRNA expression in the two UCB MSCs and HCEC samples. TJP1 (tight junction protein-1 / ZO1) is expressed in corneal endothelial cells and forms a discontinuous immunolocalization pattern at cell-cell borders [46,47]. This protein has also been detected in MSCs [45]. Comparisons for ZO1 mRNA indicated a similar expression in the two types of MSC samples tested, as well as in HCECs.


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)

Quantitative real-time PCR confirms differences in gene expression identified by microarray analysis in UCB1, UCB4, and HCEC. Relative expression levels were normalized to the housekeeping gene GAPDH and are shown relative to the highest value (0 to 1). Error bars represent one standard deviation for the four UCB (two UCB1 and two UCB4) and six HCEC biologic replicates tested. Robust multi-array average (RMA) estimated expression levels from the Affymetrix array, averaged for the biologic replicates within each cell type, are listed on the x-axis.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Quantitative real-time PCR confirms differences in gene expression identified by microarray analysis in UCB1, UCB4, and HCEC. Relative expression levels were normalized to the housekeeping gene GAPDH and are shown relative to the highest value (0 to 1). Error bars represent one standard deviation for the four UCB (two UCB1 and two UCB4) and six HCEC biologic replicates tested. Robust multi-array average (RMA) estimated expression levels from the Affymetrix array, averaged for the biologic replicates within each cell type, are listed on the x-axis.
Mentions: Quantitative real time-PCR studies were then conducted using RNA isolated from the same UCB MSC and HCEC samples to verify results of the microarray analysis for a subset of genes. For these studies, data was averaged and comparison was made between UCB1 MSCs, UCB4 MSCs, and HCECs from both young and older donors (Figure 3). As observed in the microarray analysis, HCECs expressed significantly higher mRNA levels of both NCAM1 (neural cell adhesion molecule-1) and TFAP2B (transcription factor AP-2, beta) compared with either type of MSC. Both UCB1 and UCB4 MSCs expressed significantly higher mRNA levels of CDK15 (cyclin-dependent kinase-15) and MEIS1 (Meis homeobox1), as indicated by the microarray analysis. The relative expression of four additional mRNAs was tested. COL8A1 (collagen VIII, alpha-1) is expressed in corneal endothelial cells [42], as well as in UCB1 MSCs [37]. Results from qPCR indicated that, although all samples were positive, UCB1 MSCs expressed relatively low, but detectable, levels of COL8A1 in comparison with either UCB4 or HCECs. The cell surface antigen, THY1 (CD90), is an important marker of non-hematopoeitic UCB MSCs [37,43]. The qPCR results indicated that THY1 mRNA is not only expressed in UCB1 and UCB4 MSCs, but also in HCEC, with expression in HCEC being somewhat lower than in the MSCs. CDH2 (cadherin-2 / N-cadherin]) is expressed in corneal endothelial cells [44]. Expression of this cadherin isoform has also been detected in MSCs [45]. Results of the qPCR showed a relatively similar level of N-cadherin mRNA expression in the two UCB MSCs and HCEC samples. TJP1 (tight junction protein-1 / ZO1) is expressed in corneal endothelial cells and forms a discontinuous immunolocalization pattern at cell-cell borders [46,47]. This protein has also been detected in MSCs [45]. Comparisons for ZO1 mRNA indicated a similar expression in the two types of MSC samples tested, as well as in HCECs.

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