Limits...
Gene expression profile of epithelial cells and mesenchymal cells derived from limbal explant culture.

Polisetti N, Agarwal P, Khan I, Kondaiah P, Sangwan VS, Vemuganti GK - Mol. Vis. (2010)

Bottom Line: The microarray data was validated by using Realtime and semiquntitative reverse transcription polymerase chain reaction.We have also observed similar and differential gene expression between MC-L and MSC-BM.This study represents the first extensive gene expression analysis of limbal explant culture derived epithelial and mesenchymal cells and as such reveals new insight into the biology, ontogeny, and in vivo function of these cells.

View Article: PubMed Central - PubMed

Affiliation: C-TRACER, Hyderabad Eye Research Foundation, LV Prasad Eye Institute, Hyderabad, India.

ABSTRACT

Purpose: Limbal stem cell deficiency is a challenging clinical problem and the current treatment involves replenishing the depleted limbal stem cell (LSC) pool by either limbal tissue transplantation or use of cultivated limbal epithelial cells (LEC). Our experience of cultivating the LEC on denuded human amniotic membrane using a feeder cell free method, led to identification of mesenchymal cells of limbus (MC-L), which showed phenotypic resemblance to bone marrow derived mesenchymal stem cells (MSC-BM). To understand the transcriptional profile of these cells, microarray experiments were carried out.

Methods: RNA was isolated from cultured LEC, MC-L and MSC-BM and microarray experiments were carried out by using Agilent chip (4x44 k). The microarray data was validated by using Realtime and semiquntitative reverse transcription polymerase chain reaction.

Results: The microarray analysis revealed specific gene signature of LEC and MC-L, and also their complementary role related to cytokine and growth factor profile, thus supporting the nurturing roles of the MC-L. We have also observed similar and differential gene expression between MC-L and MSC-BM.

Conclusions: This study represents the first extensive gene expression analysis of limbal explant culture derived epithelial and mesenchymal cells and as such reveals new insight into the biology, ontogeny, and in vivo function of these cells.

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Validation of micro array data by semiquantitative RT–PCR. Reverse transcription polymerase chain reaction analysis of the selected differentially expressed genes. Ribosomal protein large 35 (RPL35) was served as an internal control. Abbreviations: LEC – limbal epithelial cells, MC-L – mesenchymal cells of limbus, MSC-BM – mesenchymal stem cells of bone marrow, -RT – no reverse trasncriptase, CDH1 – cadherin 1 (E-cadherin), COLVIA1 – collagen 6 alpha 1, COL4A2 – collagen 4 alpha 2, IL-1B – interleukin 1 beta, FN1 – fibronectin 1, MAL2 - T-cell differentiated antigen 2, CTGF – Connective tissue growth factor, FGFR1 – fibroblast growth factor receptor 1, BDNF – brain derived nerve growth factor, CCL2 – chemokine ligand 2, CHI3L1 – chitanase 3 like 1, MMP2 – matrix metallo peptidase 2, IL1A – interleukin 1 alpha, KRT7- cytokeratin 7, DCN – decorin, Ang 1 – angiopoientin, bFGF – basic fibroblast growth factor, NTRK2 – neurotrophin tyrosine kinase receptor 2.
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f4: Validation of micro array data by semiquantitative RT–PCR. Reverse transcription polymerase chain reaction analysis of the selected differentially expressed genes. Ribosomal protein large 35 (RPL35) was served as an internal control. Abbreviations: LEC – limbal epithelial cells, MC-L – mesenchymal cells of limbus, MSC-BM – mesenchymal stem cells of bone marrow, -RT – no reverse trasncriptase, CDH1 – cadherin 1 (E-cadherin), COLVIA1 – collagen 6 alpha 1, COL4A2 – collagen 4 alpha 2, IL-1B – interleukin 1 beta, FN1 – fibronectin 1, MAL2 - T-cell differentiated antigen 2, CTGF – Connective tissue growth factor, FGFR1 – fibroblast growth factor receptor 1, BDNF – brain derived nerve growth factor, CCL2 – chemokine ligand 2, CHI3L1 – chitanase 3 like 1, MMP2 – matrix metallo peptidase 2, IL1A – interleukin 1 alpha, KRT7- cytokeratin 7, DCN – decorin, Ang 1 – angiopoientin, bFGF – basic fibroblast growth factor, NTRK2 – neurotrophin tyrosine kinase receptor 2.

Mentions: To validate the gene expression profiles determined by the microarray analysis, the expression levels of selected genes were analyzed by real time PCR (10 genes; Figure 3) and semi quantitative RT–PCR (28 genes, Figure 4). The gene expression patterns obtained by the two techniques were in good agreement with that from the microarray analysis, indicating high fidelity in microarray data and analytical methods.


Gene expression profile of epithelial cells and mesenchymal cells derived from limbal explant culture.

Polisetti N, Agarwal P, Khan I, Kondaiah P, Sangwan VS, Vemuganti GK - Mol. Vis. (2010)

Validation of micro array data by semiquantitative RT–PCR. Reverse transcription polymerase chain reaction analysis of the selected differentially expressed genes. Ribosomal protein large 35 (RPL35) was served as an internal control. Abbreviations: LEC – limbal epithelial cells, MC-L – mesenchymal cells of limbus, MSC-BM – mesenchymal stem cells of bone marrow, -RT – no reverse trasncriptase, CDH1 – cadherin 1 (E-cadherin), COLVIA1 – collagen 6 alpha 1, COL4A2 – collagen 4 alpha 2, IL-1B – interleukin 1 beta, FN1 – fibronectin 1, MAL2 - T-cell differentiated antigen 2, CTGF – Connective tissue growth factor, FGFR1 – fibroblast growth factor receptor 1, BDNF – brain derived nerve growth factor, CCL2 – chemokine ligand 2, CHI3L1 – chitanase 3 like 1, MMP2 – matrix metallo peptidase 2, IL1A – interleukin 1 alpha, KRT7- cytokeratin 7, DCN – decorin, Ang 1 – angiopoientin, bFGF – basic fibroblast growth factor, NTRK2 – neurotrophin tyrosine kinase receptor 2.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2903463&req=5

f4: Validation of micro array data by semiquantitative RT–PCR. Reverse transcription polymerase chain reaction analysis of the selected differentially expressed genes. Ribosomal protein large 35 (RPL35) was served as an internal control. Abbreviations: LEC – limbal epithelial cells, MC-L – mesenchymal cells of limbus, MSC-BM – mesenchymal stem cells of bone marrow, -RT – no reverse trasncriptase, CDH1 – cadherin 1 (E-cadherin), COLVIA1 – collagen 6 alpha 1, COL4A2 – collagen 4 alpha 2, IL-1B – interleukin 1 beta, FN1 – fibronectin 1, MAL2 - T-cell differentiated antigen 2, CTGF – Connective tissue growth factor, FGFR1 – fibroblast growth factor receptor 1, BDNF – brain derived nerve growth factor, CCL2 – chemokine ligand 2, CHI3L1 – chitanase 3 like 1, MMP2 – matrix metallo peptidase 2, IL1A – interleukin 1 alpha, KRT7- cytokeratin 7, DCN – decorin, Ang 1 – angiopoientin, bFGF – basic fibroblast growth factor, NTRK2 – neurotrophin tyrosine kinase receptor 2.
Mentions: To validate the gene expression profiles determined by the microarray analysis, the expression levels of selected genes were analyzed by real time PCR (10 genes; Figure 3) and semi quantitative RT–PCR (28 genes, Figure 4). The gene expression patterns obtained by the two techniques were in good agreement with that from the microarray analysis, indicating high fidelity in microarray data and analytical methods.

Bottom Line: The microarray data was validated by using Realtime and semiquntitative reverse transcription polymerase chain reaction.We have also observed similar and differential gene expression between MC-L and MSC-BM.This study represents the first extensive gene expression analysis of limbal explant culture derived epithelial and mesenchymal cells and as such reveals new insight into the biology, ontogeny, and in vivo function of these cells.

View Article: PubMed Central - PubMed

Affiliation: C-TRACER, Hyderabad Eye Research Foundation, LV Prasad Eye Institute, Hyderabad, India.

ABSTRACT

Purpose: Limbal stem cell deficiency is a challenging clinical problem and the current treatment involves replenishing the depleted limbal stem cell (LSC) pool by either limbal tissue transplantation or use of cultivated limbal epithelial cells (LEC). Our experience of cultivating the LEC on denuded human amniotic membrane using a feeder cell free method, led to identification of mesenchymal cells of limbus (MC-L), which showed phenotypic resemblance to bone marrow derived mesenchymal stem cells (MSC-BM). To understand the transcriptional profile of these cells, microarray experiments were carried out.

Methods: RNA was isolated from cultured LEC, MC-L and MSC-BM and microarray experiments were carried out by using Agilent chip (4x44 k). The microarray data was validated by using Realtime and semiquntitative reverse transcription polymerase chain reaction.

Results: The microarray analysis revealed specific gene signature of LEC and MC-L, and also their complementary role related to cytokine and growth factor profile, thus supporting the nurturing roles of the MC-L. We have also observed similar and differential gene expression between MC-L and MSC-BM.

Conclusions: This study represents the first extensive gene expression analysis of limbal explant culture derived epithelial and mesenchymal cells and as such reveals new insight into the biology, ontogeny, and in vivo function of these cells.

Show MeSH
Related in: MedlinePlus