Limits...
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 Notch1 and Jagged1 expression. A: Quantitative PCR results for Notch1 and Jagged1 mRNA expression in the day 0 cultured limbal, conjunctival, and oral biopsies. B: mRNA expression of Notch1 and Jagged1in the day 14 limbal conjunctival and oral cultures. Results were calibrated with β-actin. All experiments were performed in triplicate using six samples per group (limbal, conjunctival, and oral). Statistical significance denoted, p *<0.05, **<0.01, ***<0.005.
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f2: Expression of Notch1 and Jagged1 expression. A: Quantitative PCR results for Notch1 and Jagged1 mRNA expression in the day 0 cultured limbal, conjunctival, and oral biopsies. B: mRNA expression of Notch1 and Jagged1in the day 14 limbal conjunctival and oral cultures. Results were calibrated with β-actin. All experiments were performed in triplicate using six samples per group (limbal, conjunctival, and oral). Statistical significance denoted, p *<0.05, **<0.01, ***<0.005.

Mentions: Notch signaling has been shown to have an important role during corneal epithelial proliferation and differentiation. Abnormal differentiation has been detected in the mouse corneal epithelium on deletion of Notch1 in the epidermal layer [19]. Recently, it has been proposed that blocking Notch signaling with dominant negative Mastermind did not have an effect on corneal epithelial differentiation [26]. Moreover, Notch activity is downregulated during the initial phase of corneal epithelial proliferation, whereas active Notch signaling is necessary for corneal epithelial differentiation [18,27]. There are contradictory reports on the role of Notch signaling in the epithelial differentiation of the cornea. Thus, we were interested in comparing Notch signaling by looking at the expression of Notch1, Jagged1, and Notch downstream targets Hes1, 3, and 5, and Hey1 in the day 0 (Figure 2A and Appendix 4A) and day 14 (Figure 2B and Appendix 4B) cultures. Cultured day 0 limbal cells showed elevated expression of Jagged1 compared to the cultured conjunctival (p=0.0002) and oral (p≤0.001) cells, but no difference in Notch1 expression was detected among all three cultures. The day 0 limbal epithelial cultures revealed a significantly lower expression of Hes1 compared to the conjunctival (p=0.002) and oral (p≤0.001) epithelial cultures. Moreover, Hes3 expression in the day 0 cultured limbal epithelial cells was significantly lower than in the day 0 cultured oral epithelial cells (p=0.0001). The day 14 cultured limbal epithelial cells revealed elevated mRNA expression of Notch1 (p=0.0004; p<0.005) and Jagged1 (p=0.0022; p<0.005) compared to the conjunctival and oral cultures, respectively. Expression of Notch1 was elevated significantly (p=0.0005) in the day 14 cultured oral mucosal epithelial cells compared to the cultured conjunctival cells. Concurrently, the elevated mRNA expression of Notch downstream targets Hes1, Hes3, Hes5, and Hey1 was noted in the day 14 cultures of all three biopsies. The day 14 cultured limbal epithelial cells revealed significantly higher Hes1 expression compared to the cultured conjunctival (p=0.008) and oral (p≤0.001) epithelial cells. Hey1 expression was also elevated in the day 14 limbal cultures compared to the conjunctival (p=0.0006) and oral (0.001) cultures. The day 14 cultured oral epithelial cells showed higher expression of Notch1 (p=0.0005), Jagged1 (p=0.0003), Hes1 (p=0.002), Hes5 (p=0.009), and Hey1 (p=0.001) expression compared to the conjunctival cultures. As a result, the day 14 limbal and oral cultures showed the highest expression of Notch signaling components compared to the day 14 cultured conjunctival cells.


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 Notch1 and Jagged1 expression. A: Quantitative PCR results for Notch1 and Jagged1 mRNA expression in the day 0 cultured limbal, conjunctival, and oral biopsies. B: mRNA expression of Notch1 and Jagged1in the day 14 limbal conjunctival and oral cultures. Results were calibrated with β-actin. All experiments were performed in triplicate using six samples per group (limbal, conjunctival, and oral). Statistical significance denoted, p *<0.05, **<0.01, ***<0.005.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Expression of Notch1 and Jagged1 expression. A: Quantitative PCR results for Notch1 and Jagged1 mRNA expression in the day 0 cultured limbal, conjunctival, and oral biopsies. B: mRNA expression of Notch1 and Jagged1in the day 14 limbal conjunctival and oral cultures. Results were calibrated with β-actin. All experiments were performed in triplicate using six samples per group (limbal, conjunctival, and oral). Statistical significance denoted, p *<0.05, **<0.01, ***<0.005.
Mentions: Notch signaling has been shown to have an important role during corneal epithelial proliferation and differentiation. Abnormal differentiation has been detected in the mouse corneal epithelium on deletion of Notch1 in the epidermal layer [19]. Recently, it has been proposed that blocking Notch signaling with dominant negative Mastermind did not have an effect on corneal epithelial differentiation [26]. Moreover, Notch activity is downregulated during the initial phase of corneal epithelial proliferation, whereas active Notch signaling is necessary for corneal epithelial differentiation [18,27]. There are contradictory reports on the role of Notch signaling in the epithelial differentiation of the cornea. Thus, we were interested in comparing Notch signaling by looking at the expression of Notch1, Jagged1, and Notch downstream targets Hes1, 3, and 5, and Hey1 in the day 0 (Figure 2A and Appendix 4A) and day 14 (Figure 2B and Appendix 4B) cultures. Cultured day 0 limbal cells showed elevated expression of Jagged1 compared to the cultured conjunctival (p=0.0002) and oral (p≤0.001) cells, but no difference in Notch1 expression was detected among all three cultures. The day 0 limbal epithelial cultures revealed a significantly lower expression of Hes1 compared to the conjunctival (p=0.002) and oral (p≤0.001) epithelial cultures. Moreover, Hes3 expression in the day 0 cultured limbal epithelial cells was significantly lower than in the day 0 cultured oral epithelial cells (p=0.0001). The day 14 cultured limbal epithelial cells revealed elevated mRNA expression of Notch1 (p=0.0004; p<0.005) and Jagged1 (p=0.0022; p<0.005) compared to the conjunctival and oral cultures, respectively. Expression of Notch1 was elevated significantly (p=0.0005) in the day 14 cultured oral mucosal epithelial cells compared to the cultured conjunctival cells. Concurrently, the elevated mRNA expression of Notch downstream targets Hes1, Hes3, Hes5, and Hey1 was noted in the day 14 cultures of all three biopsies. The day 14 cultured limbal epithelial cells revealed significantly higher Hes1 expression compared to the cultured conjunctival (p=0.008) and oral (p≤0.001) epithelial cells. Hey1 expression was also elevated in the day 14 limbal cultures compared to the conjunctival (p=0.0006) and oral (0.001) cultures. The day 14 cultured oral epithelial cells showed higher expression of Notch1 (p=0.0005), Jagged1 (p=0.0003), Hes1 (p=0.002), Hes5 (p=0.009), and Hey1 (p=0.001) expression compared to the conjunctival cultures. As a result, the day 14 limbal and oral cultures showed the highest expression of Notch signaling components compared to the day 14 cultured conjunctival cells.

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