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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Related in: MedlinePlus

Characterization of limbal explant culture derived epithelial (LEC) and mesenchymal like cells (MC-L). Following immunoctytochemical analysis (A-L) epithelial cells showing positive for ABCG2 (A), CK3/CK12 (B; green fluorescence), CK19 (C; red fluorescence), CK14 (D), E-Cadherin (E), vimentin (F; green fluroscence), double immunostaining for PAX-6 (green fluorescence) and vimentin (red fluorescence; G). Mesenchymal like cells of limbus (H-L) showing positive for vimentin (H) and negative for cytokeratin 3/12 (I), cytokeratin 14(J), CD34 (K), and nestin (L). Nuclear staining was performed with propidium iodide (red; A, B, D, E, H-L). Scale bar=20 µm (A-C, E-H, and J) and 10µm (D, I, K, L). Flow cytometry analysis (M-V) was performed by incubation of the mesenchymal like cells of limbus with the indicated antibodies. M: Isotype controls for FITC and PE, N: CD90 FITC and CD44 PE, O: CD40-FITC and HLA-ABC PE, P: CD11b FITC and HLA-ABC PE, Q: CD34 FITC and CD10 PE R: Isotype control for FITC and APC, S: CXCR4 APC and CD90 FITC, T: CD40L APC and CD40 FITC, U: Isotype control for FITC and PerCP, V: CD138 PerCP and CD13 FITC.
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f2: Characterization of limbal explant culture derived epithelial (LEC) and mesenchymal like cells (MC-L). Following immunoctytochemical analysis (A-L) epithelial cells showing positive for ABCG2 (A), CK3/CK12 (B; green fluorescence), CK19 (C; red fluorescence), CK14 (D), E-Cadherin (E), vimentin (F; green fluroscence), double immunostaining for PAX-6 (green fluorescence) and vimentin (red fluorescence; G). Mesenchymal like cells of limbus (H-L) showing positive for vimentin (H) and negative for cytokeratin 3/12 (I), cytokeratin 14(J), CD34 (K), and nestin (L). Nuclear staining was performed with propidium iodide (red; A, B, D, E, H-L). Scale bar=20 µm (A-C, E-H, and J) and 10µm (D, I, K, L). Flow cytometry analysis (M-V) was performed by incubation of the mesenchymal like cells of limbus with the indicated antibodies. M: Isotype controls for FITC and PE, N: CD90 FITC and CD44 PE, O: CD40-FITC and HLA-ABC PE, P: CD11b FITC and HLA-ABC PE, Q: CD34 FITC and CD10 PE R: Isotype control for FITC and APC, S: CXCR4 APC and CD90 FITC, T: CD40L APC and CD40 FITC, U: Isotype control for FITC and PerCP, V: CD138 PerCP and CD13 FITC.

Mentions: The LEC on immunocytochemical analysis, showed immunoreactivity toward ABCG2, CK3/CK12, CK14, PAX-6, CDH1, and vimentin. MC-L were found to be immunoreactive for vimentin and nestin and were negative cytokeratin 3/12 (KRT3/12), cytokeratin 14 (KRT14), and CD45 (Figure 2A-L).


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)

Characterization of limbal explant culture derived epithelial (LEC) and mesenchymal like cells (MC-L). Following immunoctytochemical analysis (A-L) epithelial cells showing positive for ABCG2 (A), CK3/CK12 (B; green fluorescence), CK19 (C; red fluorescence), CK14 (D), E-Cadherin (E), vimentin (F; green fluroscence), double immunostaining for PAX-6 (green fluorescence) and vimentin (red fluorescence; G). Mesenchymal like cells of limbus (H-L) showing positive for vimentin (H) and negative for cytokeratin 3/12 (I), cytokeratin 14(J), CD34 (K), and nestin (L). Nuclear staining was performed with propidium iodide (red; A, B, D, E, H-L). Scale bar=20 µm (A-C, E-H, and J) and 10µm (D, I, K, L). Flow cytometry analysis (M-V) was performed by incubation of the mesenchymal like cells of limbus with the indicated antibodies. M: Isotype controls for FITC and PE, N: CD90 FITC and CD44 PE, O: CD40-FITC and HLA-ABC PE, P: CD11b FITC and HLA-ABC PE, Q: CD34 FITC and CD10 PE R: Isotype control for FITC and APC, S: CXCR4 APC and CD90 FITC, T: CD40L APC and CD40 FITC, U: Isotype control for FITC and PerCP, V: CD138 PerCP and CD13 FITC.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Characterization of limbal explant culture derived epithelial (LEC) and mesenchymal like cells (MC-L). Following immunoctytochemical analysis (A-L) epithelial cells showing positive for ABCG2 (A), CK3/CK12 (B; green fluorescence), CK19 (C; red fluorescence), CK14 (D), E-Cadherin (E), vimentin (F; green fluroscence), double immunostaining for PAX-6 (green fluorescence) and vimentin (red fluorescence; G). Mesenchymal like cells of limbus (H-L) showing positive for vimentin (H) and negative for cytokeratin 3/12 (I), cytokeratin 14(J), CD34 (K), and nestin (L). Nuclear staining was performed with propidium iodide (red; A, B, D, E, H-L). Scale bar=20 µm (A-C, E-H, and J) and 10µm (D, I, K, L). Flow cytometry analysis (M-V) was performed by incubation of the mesenchymal like cells of limbus with the indicated antibodies. M: Isotype controls for FITC and PE, N: CD90 FITC and CD44 PE, O: CD40-FITC and HLA-ABC PE, P: CD11b FITC and HLA-ABC PE, Q: CD34 FITC and CD10 PE R: Isotype control for FITC and APC, S: CXCR4 APC and CD90 FITC, T: CD40L APC and CD40 FITC, U: Isotype control for FITC and PerCP, V: CD138 PerCP and CD13 FITC.
Mentions: The LEC on immunocytochemical analysis, showed immunoreactivity toward ABCG2, CK3/CK12, CK14, PAX-6, CDH1, and vimentin. MC-L were found to be immunoreactive for vimentin and nestin and were negative cytokeratin 3/12 (KRT3/12), cytokeratin 14 (KRT14), and CD45 (Figure 2A-L).

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