Characterization of ex vivo cultured limbal, conjunctival, and oral mucosal cells: A comparative study with implications in transplantation medicine.
OBJECTIVE
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.
Stem Cell Research Laboratory, GROW Research Laboratory, Narayana Nethralaya Foundation, Bangalore, Karnataka, India ; School of Biosciences and Technology, VIT University, Vellore, Tamil Nadu, India.
Kamesh
K
Dhamodaran
Stem Cell Research Laboratory, GROW Research Laboratory, Narayana Nethralaya Foundation, Bangalore, Karnataka, India ; Post-graduate & Research Department of Biotechnology, Jamal Mohamed College, Bharathidasan University, Tiruchirappalli, India.
Murali
M
Subramani
GROW Research Laboratory, Narayana Nethralaya Foundation, Bangalore, Karnataka, India.
Nallathambi
N
Jeyabalan
Stem Cell Research Laboratory, GROW Research Laboratory, Narayana Nethralaya Foundation, Bangalore, Karnataka, India.
Murugeswari
M
Ponnalagu
GROW Research Laboratory, Narayana Nethralaya Foundation, Bangalore, Karnataka, India.
Priyanka
P
Chevour
Stem Cell Research Laboratory, GROW Research Laboratory, Narayana Nethralaya Foundation, Bangalore, Karnataka, India.
Reshma
R
Shetty
Department of Cornea and Refractive surgery, Narayana Nethralaya Post Graduate Institute of Ophthalmology, Narayana Nethralaya Foundation, Narayana Health City, Bommasandra, Bangalore, Karnataka, India.
Himanshu
H
Matalia
Department of Cornea and Refractive surgery, Narayana Nethralaya Post Graduate Institute of Ophthalmology, Narayana Nethralaya Foundation, Narayana Health City, Bommasandra, Bangalore, Karnataka, India.
Rohit
R
Shetty
School of Biosciences and Technology, VIT University, Vellore, Tamil Nadu, India.
Sabina Evan
SE
Prince
Stem Cell Research Laboratory, GROW Research Laboratory, Narayana Nethralaya Foundation, Bangalore, Karnataka, India.
Debashish
D
Das
D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000235:genetics; D049109:Cell Proliferation / Q000235:genetics; D017690:Cell Transplantation / Q000379:methods*; D064987:Cell- and Tissue-Based Therapy / Q000379:methods; D002478:Cells, Cultured; D003228:Conjunctiva / Q000166:cytology* / Q000378:metabolism; D003316:Corneal Diseases / Q000378:metabolism / Q000628:therapy; D015870:Gene Expression; D006801:Humans; D066298:In Vitro Techniques; D016850:Limbus Corneae / Q000166:cytology* / Q000378:metabolism; D009061:Mouth Mucosa / Q000166:cytology* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism
D012333:RNA, Messenger