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Characterization of ex vivo cultured limbal, conjunctival, and oral mucosal cells: A comparative study with implications in transplantation medicine.

Dhamodaran K, Subramani M, Jeyabalan N, Ponnalagu M, Chevour P, Shetty R, Matalia H, Shetty R, Prince SE, Das D - Mol. Vis. (2015)

Bottom Line: The gene expression levels of the autophagy markers (LC3A, LC3B, and LAMP1) were significantly increased in the limbal cultures compared to the oral and conjunctival cultures.In conclusion, the limbal epithelial cultures showed higher expression of proliferative, limbal stem cell marker, Notch signaling, and autophagy markers suggesting a role in stem cell maintenance and differentiation.This implicates the probable factors that might drive a successful transplantation.

View Article: PubMed Central - PubMed

Affiliation: Stem Cell Research Laboratory, GROW Research Laboratory, Narayana Nethralaya Foundation, Bangalore, Karnataka, India ; School of Biosciences and Technology, VIT University, Vellore, Tamil Nadu, India.

ABSTRACT

Purpose: Limbal epithelial stem cell deficiency is caused by exposure of the cornea to thermal, chemical, or radiation burns or by diseases (aniridia and Stevens-Johnson syndrome). Autologous cell transplantation is a widely used therapeutic modality for restoring the corneal surface in such pathological conditions. Ex vivo cultured limbal, conjunctival, and oral biopsies have been widely used to reconstruct the corneal surface with variable outcomes. Culture characterization of the ex vivo cultured cells would provide insight and clues into the underlying signaling mechanisms that would aid in determining the probable transplantation outcome. Comparison of the vital proteins and genes among the three ex vivo cultured tissues has implications in clinical practice. To address this issue, we characterized and compared the proliferative and differentiated properties of ex vivo cultured limbal, conjunctival, and oral biopsies used for cell-based therapy for corneal surface restoration.

Methods: Limbal, conjunctival, and oral biopsies were collected with informed patient consent. Explant cultures were established on the denuded human amniotic membrane with corneal lineage differentiation medium. The day 14 cultures were characterized for epithelial and corneal lineage-specific markers using reverse transcription (RT)-PCR for cytokeratin 3, 4, 12, 13, 15, connexin 43, vimentin, p63α, and ABCG2 markers. mRNA expression was estimated in day 14 cultures with real-time quantitative real time (qRT)-PCR for pluripotency markers (OCT4, SOX2, NANOG), putative corneal stem cell markers (ABCG2 and p63α), proliferation markers (cyclin d1, Ki-67, PCNA, and CDC20), apoptotic markers (BCL2, BAX, caspase 3, and caspase 9), Notch signaling pathway markers (Notch1, Jagged1, Hes1, Hes3, Hes5, and Hey1), and autophagic markers (LC3A, LC3B, ATG7, RAB7, LAMP1, and LAMP2). Fluorescence-activated cell sorter profiling was performed for pluripotent markers and putative corneal stem cell markers ABCG2 and p63α.

Results: The protein and mRNA expression levels of the pluripotent markers were lower, whereas those of the putative stem/progenitor markers ABCG2, ΔNp63α, and Notch signaling molecules (Notch1 and Jagged1) were elevated in limbal cultures. The gene expression levels of the autophagy markers (LC3A, LC3B, and LAMP1) were significantly increased in the limbal cultures compared to the oral and conjunctival cultures.

Conclusions: In conclusion, the limbal epithelial cultures showed higher expression of proliferative, limbal stem cell marker, Notch signaling, and autophagy markers suggesting a role in stem cell maintenance and differentiation. This implicates the probable factors that might drive a successful transplantation. Our findings provide the initial steps toward understanding transplantation medicine in an ex vivo model.

No MeSH data available.


Related in: MedlinePlus

Expression of pluripotent markers. A: Quantitative PCR results for day 14 cultured limbal, conjunctival, and oral cells for OCT4, SOX2, and NANOG mRNA expression. Results were normalized with β-actin. All experiments were performed in triplicate using six samples per group (limbal, conjunctival, and oral). B: Graphical representation of the fluorescent activated cell sorting (FACS) data obtained for three different tissue origins, showing positive staining with antibodies against OCT4, SOX2, and NANOG. Statistical significance denoted, p *<0.05, **<0.01, ***<0.005.
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f1: Expression of pluripotent markers. A: Quantitative PCR results for day 14 cultured limbal, conjunctival, and oral cells for OCT4, SOX2, and NANOG mRNA expression. Results were normalized with β-actin. All experiments were performed in triplicate using six samples per group (limbal, conjunctival, and oral). B: Graphical representation of the fluorescent activated cell sorting (FACS) data obtained for three different tissue origins, showing positive staining with antibodies against OCT4, SOX2, and NANOG. Statistical significance denoted, p *<0.05, **<0.01, ***<0.005.

Mentions: We then checked the pluripotent marker gene expression profile (OCT4, SOX2, and NANOG) in day 0 and day 14 ex vivo cultured limbal, conjunctival, and oral epithelial cells. The mRNA expression profile of the day 0 cells was comparable across the different cell types (Appendix 3A) except a significant decrease in SOX2 expression in the oral biopsy (p=0.0286). Interestingly, the pluripotent markers were decreased in the day 14 limbal cultures compared to the conjunctival and oral cultures. There was a significant decrease in the expression of OCT4 (p=0.028) and NANOG (p=0.005), but no difference was observed in level of SOX2 expression in the limbal cultured cells compared to the oral cultures. The day 14 cultured conjunctival cells did not show any differences in the expression levels of the OCT4 and SOX2 genes, but NANOG (p=0.0085) was significantly elevated in comparison to the cultured limbal epithelial cells (Figure 1A).


Characterization of ex vivo cultured limbal, conjunctival, and oral mucosal cells: A comparative study with implications in transplantation medicine.

Dhamodaran K, Subramani M, Jeyabalan N, Ponnalagu M, Chevour P, Shetty R, Matalia H, Shetty R, Prince SE, Das D - Mol. Vis. (2015)

Expression of pluripotent markers. A: Quantitative PCR results for day 14 cultured limbal, conjunctival, and oral cells for OCT4, SOX2, and NANOG mRNA expression. Results were normalized with β-actin. All experiments were performed in triplicate using six samples per group (limbal, conjunctival, and oral). B: Graphical representation of the fluorescent activated cell sorting (FACS) data obtained for three different tissue origins, showing positive staining with antibodies against OCT4, SOX2, and NANOG. Statistical significance denoted, p *<0.05, **<0.01, ***<0.005.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4522244&req=5

f1: Expression of pluripotent markers. A: Quantitative PCR results for day 14 cultured limbal, conjunctival, and oral cells for OCT4, SOX2, and NANOG mRNA expression. Results were normalized with β-actin. All experiments were performed in triplicate using six samples per group (limbal, conjunctival, and oral). B: Graphical representation of the fluorescent activated cell sorting (FACS) data obtained for three different tissue origins, showing positive staining with antibodies against OCT4, SOX2, and NANOG. Statistical significance denoted, p *<0.05, **<0.01, ***<0.005.
Mentions: We then checked the pluripotent marker gene expression profile (OCT4, SOX2, and NANOG) in day 0 and day 14 ex vivo cultured limbal, conjunctival, and oral epithelial cells. The mRNA expression profile of the day 0 cells was comparable across the different cell types (Appendix 3A) except a significant decrease in SOX2 expression in the oral biopsy (p=0.0286). Interestingly, the pluripotent markers were decreased in the day 14 limbal cultures compared to the conjunctival and oral cultures. There was a significant decrease in the expression of OCT4 (p=0.028) and NANOG (p=0.005), but no difference was observed in level of SOX2 expression in the limbal cultured cells compared to the oral cultures. The day 14 cultured conjunctival cells did not show any differences in the expression levels of the OCT4 and SOX2 genes, but NANOG (p=0.0085) was significantly elevated in comparison to the cultured limbal epithelial cells (Figure 1A).

Bottom Line: The gene expression levels of the autophagy markers (LC3A, LC3B, and LAMP1) were significantly increased in the limbal cultures compared to the oral and conjunctival cultures.In conclusion, the limbal epithelial cultures showed higher expression of proliferative, limbal stem cell marker, Notch signaling, and autophagy markers suggesting a role in stem cell maintenance and differentiation.This implicates the probable factors that might drive a successful transplantation.

View Article: PubMed Central - PubMed

Affiliation: Stem Cell Research Laboratory, GROW Research Laboratory, Narayana Nethralaya Foundation, Bangalore, Karnataka, India ; School of Biosciences and Technology, VIT University, Vellore, Tamil Nadu, India.

ABSTRACT

Purpose: Limbal epithelial stem cell deficiency is caused by exposure of the cornea to thermal, chemical, or radiation burns or by diseases (aniridia and Stevens-Johnson syndrome). Autologous cell transplantation is a widely used therapeutic modality for restoring the corneal surface in such pathological conditions. Ex vivo cultured limbal, conjunctival, and oral biopsies have been widely used to reconstruct the corneal surface with variable outcomes. Culture characterization of the ex vivo cultured cells would provide insight and clues into the underlying signaling mechanisms that would aid in determining the probable transplantation outcome. Comparison of the vital proteins and genes among the three ex vivo cultured tissues has implications in clinical practice. To address this issue, we characterized and compared the proliferative and differentiated properties of ex vivo cultured limbal, conjunctival, and oral biopsies used for cell-based therapy for corneal surface restoration.

Methods: Limbal, conjunctival, and oral biopsies were collected with informed patient consent. Explant cultures were established on the denuded human amniotic membrane with corneal lineage differentiation medium. The day 14 cultures were characterized for epithelial and corneal lineage-specific markers using reverse transcription (RT)-PCR for cytokeratin 3, 4, 12, 13, 15, connexin 43, vimentin, p63α, and ABCG2 markers. mRNA expression was estimated in day 14 cultures with real-time quantitative real time (qRT)-PCR for pluripotency markers (OCT4, SOX2, NANOG), putative corneal stem cell markers (ABCG2 and p63α), proliferation markers (cyclin d1, Ki-67, PCNA, and CDC20), apoptotic markers (BCL2, BAX, caspase 3, and caspase 9), Notch signaling pathway markers (Notch1, Jagged1, Hes1, Hes3, Hes5, and Hey1), and autophagic markers (LC3A, LC3B, ATG7, RAB7, LAMP1, and LAMP2). Fluorescence-activated cell sorter profiling was performed for pluripotent markers and putative corneal stem cell markers ABCG2 and p63α.

Results: The protein and mRNA expression levels of the pluripotent markers were lower, whereas those of the putative stem/progenitor markers ABCG2, ΔNp63α, and Notch signaling molecules (Notch1 and Jagged1) were elevated in limbal cultures. The gene expression levels of the autophagy markers (LC3A, LC3B, and LAMP1) were significantly increased in the limbal cultures compared to the oral and conjunctival cultures.

Conclusions: In conclusion, the limbal epithelial cultures showed higher expression of proliferative, limbal stem cell marker, Notch signaling, and autophagy markers suggesting a role in stem cell maintenance and differentiation. This implicates the probable factors that might drive a successful transplantation. Our findings provide the initial steps toward understanding transplantation medicine in an ex vivo model.

No MeSH data available.


Related in: MedlinePlus