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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

mRNA expression of proliferative markers. mRNA expression profile of proliferative markers, PCNA, cyclin D1, Ki-67, and CDC20 genes of day 14 limbal, conjunctival, and oral cultured cells. Results were normalized with β-actin. All experiments were performed in triplicate using six samples per group (limbal, conjunctival, and oral). Significance denoted, p *<0.05, **<0.01, ***<0.005.
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f5: mRNA expression of proliferative markers. mRNA expression profile of proliferative markers, PCNA, cyclin D1, Ki-67, and CDC20 genes of day 14 limbal, conjunctival, and oral cultured cells. Results were normalized with β-actin. All experiments were performed in triplicate using six samples per group (limbal, conjunctival, and oral). Significance denoted, p *<0.05, **<0.01, ***<0.005.

Mentions: Cell proliferation and apoptotic levels were estimated for the day 14 limbal, conjunctival, and oral cultured cells. Proliferative markers (cyclin D1 (p=0.0039; p=0.0205), Ki-67 (p=0.0021) and CDC20 (p=0.0020; p=0.0485) showed significantly elevated expression levels in the cultured limbal epithelial cells compared to the conjunctival and oral cultured cells. Based on the expression level, the conjunctival cells showed the lowest proliferative status among all three types of ex vivo cultured epithelial cells (Figure 5).


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)

mRNA expression of proliferative markers. mRNA expression profile of proliferative markers, PCNA, cyclin D1, Ki-67, and CDC20 genes of day 14 limbal, conjunctival, and oral cultured cells. Results were normalized with β-actin. All experiments were performed in triplicate using six samples per group (limbal, conjunctival, and oral). 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

f5: mRNA expression of proliferative markers. mRNA expression profile of proliferative markers, PCNA, cyclin D1, Ki-67, and CDC20 genes of day 14 limbal, conjunctival, and oral cultured cells. Results were normalized with β-actin. All experiments were performed in triplicate using six samples per group (limbal, conjunctival, and oral). Significance denoted, p *<0.05, **<0.01, ***<0.005.
Mentions: Cell proliferation and apoptotic levels were estimated for the day 14 limbal, conjunctival, and oral cultured cells. Proliferative markers (cyclin D1 (p=0.0039; p=0.0205), Ki-67 (p=0.0021) and CDC20 (p=0.0020; p=0.0485) showed significantly elevated expression levels in the cultured limbal epithelial cells compared to the conjunctival and oral cultured cells. Based on the expression level, the conjunctival cells showed the lowest proliferative status among all three types of ex vivo cultured epithelial cells (Figure 5).

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