To increase the epithelial proliferation of an auto-tissue-engineered lamellar cornea, 3.0×106 corneal epithelial cells (CECs) were combined with 3×105 mouse embryonic stem cells (ESCs) pretransfected with the HSV-tk gene (CECs+ESCs-TK group), and 3.3×106 corneal epithelial cells (CECs group) were seeded between the acellular porcine corneal stroma and the amniotic membrane using the centrifugal cell seeding method. After 4 days of perfusion culture (treatment with ganciclovir starting on day 2), a thicker corneal epithelium (four to five layers) formed in the CECs+ESCs-TK group compared with that observed in the CECs group (two to three layers). More stem/progenitor cell (K3 −, p63+, ABCG2+, and integrin-β1+) and proliferation phenotypes (Ki67+) were measured in the CECs+ESCs-TK group compared with the CECs group using immunofluorescence staining, real-time quantitative reverse transcription polymerase chain reaction, and flow cytometry. Consistent with these findings, the colony-forming efficiency and cellular doubling time were significantly different between the CECs+ESCs-TK group (16.18%±3.98%, 28.45±2.03 h) and CECs group (11.96%±2.60%, 36.3±1.15 h). In a rabbit lamellar transplantation model, the CECs+ESCs-TK group had better epithelial barrier functions and wound healing abilities compared with the CECs group. Furthermore, ESCs-TK could be completely and safely removed by ganciclovir. Thus, the ESCS-TK coculture system could serve as a potential strategy for corneal tissue engineering.
Get full access to this article
View all access options for this article.
References
1.
Casaroli-MaranoR.P., Nieto-NicolauN., and Martinez-ConesaE.M.Progenitor cells for ocular surface regenerative therapy. Ophthalmic Res, 49, 115, 2013.
2.
ShahA., BrugnanoJ., SunS., VaseA., and OrwinE.The development of a tissue-engineered cornea: biomaterials and culture methods. Pediatr Res, 63, 535, 2008.
3.
ZhangX., SunH., LiX., YuanX., ZhangL., and ZhaoS.Utilization of human limbal mesenchymal cells as feeder layers for human limbal stem cells cultured on amniotic membrane. J Tissue Eng Regen Med, 4, 38, 2010.
4.
RobertsonD.M., KalangaraJ.P., BaucomR.B., PetrollW.M., and CavanaghH.D.A reconstituted telomerase-immortalized human corneal epithelium in vivo: a pilot study. Curr Eye Res, 36, 706, 2011.
5.
LiuJ., SongG., WangZ., HuangB., GaoQ., LiuB., XuY., LiangX., MaP., GaoN., and GeJ.Establishment of a corneal epithelial cell line spontaneously derived from human limbal cells. Exp Eye Res, 84, 599, 2007.
6.
ZhouJ., ChenF., XiaoJ., LiC., LiuY., DingY., WanP., WangX., HuangJ., and WangZ.Enhanced functional properties of corneal epithelial cells by coculture with embryonic stem cells via the integrin beta1-FAK-PI3K/Akt pathway. Int J Biochem Cell Biol, 43, 1168, 2011.
7.
LiuZ., WanP., DuanH., ZhouJ., TanB., LiuY., ZhouQ., ZhouC., HuangZ., TianB., LiC., and WangZ.ES micro-environment enhances stemness and inhibits apoptosis in human limbal stem cells via the maintenance of telomerase activity. PLoS One, 8, e53576, 2013.
8.
LiuY., DingY., MaP., WuZ., DuanH., LiuZ., WanP., LuX., XiangP., GeJ., and WangZ.Enhancement of long-term proliferative capacity of rabbit corneal epithelial cells by embryonic stem cell conditioned medium. Tissue Eng Part C Methods, 16, 793, 2010.
9.
LuX., ChenD., LiuZ., LiC., LiuY., ZhouJ., WanP., MouY.G., and WangZ.Enhanced survival in vitro of human corneal endothelial cells using mouse embryonic stem cell conditioned medium. Mol Vis, 16, 611, 2010.
10.
MaD.H., LaiJ.Y., ChengH.Y., TsaiC.C., and YehL.K.Carbodiimide cross-linked amniotic membranes for cultivation of limbal epithelial cells. Biomaterials, 31, 6647, 2010.
11.
RamaP., MatuskaS., PaganoniG., SpinelliA., De LucaM., and PellegriniG.Limbal stem-cell therapy and long-term corneal regeneration. N Engl J Med, 363, 147, 2010.
12.
BrayL.J., GeorgeK.A., AinscoughS.L., HutmacherD.W., ChirilaT.V., and HarkinD.G.Human corneal epithelial equivalents constructed on Bombyx mori silk fibroin membranes. Biomaterials, 32, 5086, 2011.
13.
DuanX., McLaughlinC., GriffithM., and SheardownH.Biofunctionalization of collagen for improved biological response: scaffolds for corneal tissue engineering. Biomaterials, 28, 78, 2007.
14.
SongJ.J., and OttH.C.Organ engineering based on decellularized matrix scaffolds. Trends Mol Med, 17, 424, 2011.
15.
WuZ., ZhouY., LiN., HuangM., DuanH., GeJ., XiangP., and WangZ.The use of phospholipase A(2) to prepare acellular porcine corneal stroma as a tissue engineering scaffold. Biomaterials, 30, 3513, 2009.
GuoY., Graham-EvansB., and BroxmeyerH.E.Murine embryonic stem cells secrete cytokines/growth modulators that enhance cell survival/anti-apoptosis and stimulate colony formation of murine hematopoietic progenitor cells. Stem Cells, 24, 850, 2006.
24.
PeartonD.J., YangY., and DhouaillyD.Transdifferentiation of corneal epithelium into epidermis occurs by means of a multistep process triggered by dermal developmental signals. Proc Natl Acad Sci U S A, 102, 3714, 2005.
25.
JiangX.X., ZhangY., LiuB., ZhangS.X., WuY., YuX.D., and MaoN.Human mesenchymal stem cells inhibit differentiation and function of monocyte-derived dendritic cells. Blood, 105, 4120, 2005.
OrkinS.H., and HochedlingerK.Chromatin connections to pluripotency and cellular reprogramming. Cell, 145, 835, 2011.
28.
JonesR.R., HamleyI.W., and ConnonC.J.Ex vivo expansion of limbal stem cells is affected by substrate properties. Stem Cell Res, 8, 403, 2012.
29.
MatterK., and BaldaM.S.Signalling to and from tight junctions. Nat Rev Mol Cell Biol, 4, 225, 2003.
30.
CattanV., BernardG., RousseauA., BouhoutS., ChabaudS., AugerF.A., and BolducS.Mechanical stimuli-induced urothelial differentiation in a human tissue-engineered tubular genitourinary graft. Eur Urol, 60, 1291, 2011.
31.
NakamuraK., KurosakaD., Bissen-MiyajimaH., and TsubotaK.Intact corneal epithelium is essential for the prevention of stromal haze after laser assisted in situ keratomileusis. Br J Ophthalmol, 85, 209, 2001.
32.
ZhouC., HuangZ., LiP., LiW., LiuY., LiC., LiuZ., WangX., WanP., and WangZ.Safety and efficacy of embryonic stem cell microenvironment in a leukemia mouse model. Stem Cells Dev, 23, 1741, 2014.
33.
SchuldinerM., Itskovitz-EldorJ., and BenvenistyN.Selective ablation of human embryonic stem cells expressing a “suicide” gene. Stem Cells, 21, 257, 2003.
