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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

Putative limbal stem/progenitor cell marker expression p63α. A: Quantitative PCR expression levels of ΔNp63 in limbal, conjunctival, and oral cultured cells from day 0 and day 14 cultures. Results were calibrated with β-actin. All experiments were performed in triplicate using six samples per group (limbal, conjunctival, and oral). B: Representative fluorescent activated cell sorting (FACS) analysis of the p63α-positive population in the limbal, conjunctival, and oral cultured cells from day 14 cultures. C: Summarizing graph represents the results of the FACS analysis of p63α in the limbal, conjunctival, and oral cultured cells, performed in triplicate. Significance denoted, p *<0.05, **<0.01, ***<0.005.
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f4: Putative limbal stem/progenitor cell marker expression p63α. A: Quantitative PCR expression levels of ΔNp63 in limbal, conjunctival, and oral cultured cells from day 0 and day 14 cultures. Results were calibrated with β-actin. All experiments were performed in triplicate using six samples per group (limbal, conjunctival, and oral). B: Representative fluorescent activated cell sorting (FACS) analysis of the p63α-positive population in the limbal, conjunctival, and oral cultured cells from day 14 cultures. C: Summarizing graph represents the results of the FACS analysis of p63α in the limbal, conjunctival, and oral cultured cells, performed in triplicate. Significance denoted, p *<0.05, **<0.01, ***<0.005.

Mentions: An ABC transporter protein is encoded by ABCG2, which showed no difference in mRNA expression level in the day 0 limbal, conjunctival, and oral cultures (Figure 3A). The day 14 limbal cultures showed higher expression of ABCG2 (p=0.0246 and p=0.0021) compared to the conjunctival and oral cultures, respectively (Figure 3A). FACS analysis revealed an increased number of ABCG2-positive cells in the day 14 limbal cultures compared to the conjunctival and oral cultures (Figure 3B,C). The number of ABCG2-positive cells was higher in the day 14 cultured oral cells compared to the cultured conjunctival cells (p=0.006). No difference was observed in the mRNA expression level of the ΔNp63α levels for day 0 and day 14 in the three cultured biopsies (Figure 4A). However, when compared the day 14 cultured limbal, conjunctival, and oral biopsies for p63 positivity with FACS staining, a significant difference was observed. The day 14 cultured limbal epithelial cells showed significant upregulation of p63α positivity compared to the day 14 cultured conjunctival cells (p=0.01) and the day 14 cultured oral epithelial cells (p=0.004; Figure 4B,C).


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)

Putative limbal stem/progenitor cell marker expression p63α. A: Quantitative PCR expression levels of ΔNp63 in limbal, conjunctival, and oral cultured cells from day 0 and day 14 cultures. Results were calibrated with β-actin. All experiments were performed in triplicate using six samples per group (limbal, conjunctival, and oral). B: Representative fluorescent activated cell sorting (FACS) analysis of the p63α-positive population in the limbal, conjunctival, and oral cultured cells from day 14 cultures. C: Summarizing graph represents the results of the FACS analysis of p63α in the limbal, conjunctival, and oral cultured cells, performed in triplicate. 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

f4: Putative limbal stem/progenitor cell marker expression p63α. A: Quantitative PCR expression levels of ΔNp63 in limbal, conjunctival, and oral cultured cells from day 0 and day 14 cultures. Results were calibrated with β-actin. All experiments were performed in triplicate using six samples per group (limbal, conjunctival, and oral). B: Representative fluorescent activated cell sorting (FACS) analysis of the p63α-positive population in the limbal, conjunctival, and oral cultured cells from day 14 cultures. C: Summarizing graph represents the results of the FACS analysis of p63α in the limbal, conjunctival, and oral cultured cells, performed in triplicate. Significance denoted, p *<0.05, **<0.01, ***<0.005.
Mentions: An ABC transporter protein is encoded by ABCG2, which showed no difference in mRNA expression level in the day 0 limbal, conjunctival, and oral cultures (Figure 3A). The day 14 limbal cultures showed higher expression of ABCG2 (p=0.0246 and p=0.0021) compared to the conjunctival and oral cultures, respectively (Figure 3A). FACS analysis revealed an increased number of ABCG2-positive cells in the day 14 limbal cultures compared to the conjunctival and oral cultures (Figure 3B,C). The number of ABCG2-positive cells was higher in the day 14 cultured oral cells compared to the cultured conjunctival cells (p=0.006). No difference was observed in the mRNA expression level of the ΔNp63α levels for day 0 and day 14 in the three cultured biopsies (Figure 4A). However, when compared the day 14 cultured limbal, conjunctival, and oral biopsies for p63 positivity with FACS staining, a significant difference was observed. The day 14 cultured limbal epithelial cells showed significant upregulation of p63α positivity compared to the day 14 cultured conjunctival cells (p=0.01) and the day 14 cultured oral epithelial cells (p=0.004; Figure 4B,C).

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