Differentiation of human embryonic stem cells derived mesenchymal stem cells into corneal epithelial cells after being seeded on decellularized SMILE-derived lenticules
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摘要
AIM: To evaluate the feasibility of mesenchymal stem cells(MSCs) to differentiate into corneal epithelial cells after being seeded on the decellularized small incision lenticule extraction(SMILE)-derived lenticules. METHODS: The fresh lenticules procured from patients undergoing SMILE for the correction of myopia were decellularized. The MSCs were subsequently cultivated on those denuded lenticules. The MSCs without lenticules were used as a control. The proliferation activity of the MSCs after seeding 24 h was quantitatively determined with the Cell Counting Kit-8(CCK-8) assay. Immunofluorescence staining and quantitative reverse transcription polymerase chain reaction(qRT-PCR) were used to assess the marker expression in differentiated MSCs. RESULTS: The data showed that both fresh and decellularized lenticules could significantly promote the proliferation of MSCs, compared to that in control(P=0.02 for fresh lenticules, P=0.001 for decellularize ones, respectively). The MSCs seeded on both lenticules were positive for cytokeratin 3(CK3) staining. The expression of CK3 increased 5-fold in MSCs seeded on fresh lenticules and 18-fold on decellularized ones, compared to that in control. There was a significant difference in the expression of CK3 in MSCs seeded on fresh and decellularized lenticules(P<0.001). The expression of CK8 and CK18 was similar in pure MSCs and MSCs seeded on fresh lenticules(P>0.05), while the expression of these markers was decreased in MSCs seeded on decellularized ones. CONCLUSION: These results suggest that the decellularized lenticules might be more suitable for MSCs to differentiate into corneal epithelial cells, which offersthe prospect of a novel therapeutic modality of SMILEderived lenticules in regenerative corneal engineering.
AIM: To evaluate the feasibility of mesenchymal stem cells(MSCs) to differentiate into corneal epithelial cells after being seeded on the decellularized small incision lenticule extraction(SMILE)-derived lenticules. METHODS: The fresh lenticules procured from patients undergoing SMILE for the correction of myopia were decellularized. The MSCs were subsequently cultivated on those denuded lenticules. The MSCs without lenticules were used as a control. The proliferation activity of the MSCs after seeding 24 h was quantitatively determined with the Cell Counting Kit-8(CCK-8) assay. Immunofluorescence staining and quantitative reverse transcription polymerase chain reaction(qRT-PCR) were used to assess the marker expression in differentiated MSCs. RESULTS: The data showed that both fresh and decellularized lenticules could significantly promote the proliferation of MSCs, compared to that in control(P=0.02 for fresh lenticules, P=0.001 for decellularize ones, respectively). The MSCs seeded on both lenticules were positive for cytokeratin 3(CK3) staining. The expression of CK3 increased 5-fold in MSCs seeded on fresh lenticules and 18-fold on decellularized ones, compared to that in control. There was a significant difference in the expression of CK3 in MSCs seeded on fresh and decellularized lenticules(P<0.001). The expression of CK8 and CK18 was similar in pure MSCs and MSCs seeded on fresh lenticules(P>0.05), while the expression of these markers was decreased in MSCs seeded on decellularized ones. CONCLUSION: These results suggest that the decellularized lenticules might be more suitable for MSCs to differentiate into corneal epithelial cells, which offersthe prospect of a novel therapeutic modality of SMILEderived lenticules in regenerative corneal engineering.
AIM: To evaluate the feasibility of mesenchymal stem cells(MSCs) to differentiate into corneal epithelial cells after being seeded on the decellularized small incision lenticule extraction(SMILE)-derived lenticules. METHODS: The fresh lenticules procured from patients undergoing SMILE for the correction of myopia were decellularized. The MSCs were subsequently cultivated on those denuded lenticules. The MSCs without lenticules were used as a control. The proliferation activity of the MSCs after seeding 24 h was quantitatively determined with the Cell Counting Kit-8(CCK-8) assay. Immunofluorescence staining and quantitative reverse transcription polymerase chain reaction(qRT-PCR) were used to assess the marker expression in differentiated MSCs. RESULTS: The data showed that both fresh and decellularized lenticules could significantly promote the proliferation of MSCs, compared to that in control(P=0.02 for fresh lenticules, P=0.001 for decellularize ones, respectively). The MSCs seeded on both lenticules were positive for cytokeratin 3(CK3) staining. The expression of CK3 increased 5-fold in MSCs seeded on fresh lenticules and 18-fold on decellularized ones, compared to that in control. There was a significant difference in the expression of CK3 in MSCs seeded on fresh and decellularized lenticules(P<0.001). The expression of CK8 and CK18 was similar in pure MSCs and MSCs seeded on fresh lenticules(P>0.05), while the expression of these markers was decreased in MSCs seeded on decellularized ones. CONCLUSION: These results suggest that the decellularized lenticules might be more suitable for MSCs to differentiate into corneal epithelial cells, which offersthe prospect of a novel therapeutic modality of SMILEderived lenticules in regenerative corneal engineering.
引文
1 Xu SC,Chow J,Liu J,Li L,Maslin JS,Chadha N,Chen BH,Teng CC.Risk factors for visual impairment associated with corneal diseases in southern China.Clin Ophthalmol 2016;10:777-782.
2 Lynch AP,Ahearne M.Strategies for developing decellularized corneal scaffolds.Exp Eye Res 2013;108:42-47.
3 Kong B,Mi SL.Electrospun scaffolds for corneal tissue engineering:a review.Materials(Basel)2016;9(8):E614.
4 Wang B,Guo YW,Chen XF,Zeng C,Hu QK,Yin W,Li W,Xie H,Zhang BY,Huang XC,Yu FL.Nanoparticle-modified chitosan-agarosegelatin scaffold for sustained release of SDF-1 and BMP-2.Int JNanomedicine 2018;13:7395-7408.
5 Yang X,Li YY,He W,Huang QL,Zhang RR,Feng QL.Hydroxyapatite/collagen coating on PLGA electrospun fibers for osteogenic differentiation of bone marrow mesenchymal stem cells.J Biomed Mater Res A2018;106(11):2863-2870.
6 Sharma S,Mohanty S,Gupta D,Jassal M,Agrawal AK,Tandon R.Cellular response of limbal epithelial cells on electrospun poly-ε-caprolactone nanofibrous scaffolds for ocular surface bioengineering:a preliminary in vitro study.Mol Vis 2011;17:2898-2910.
7 Levis HJ,Brown RA,Daniels JT.Plastic compressed collagen as a biomimetic substrate for human limbal epithelial cell culture.Biomaterials2010;31(30):7726-7737.
8 Nguyen KN,Bobba S,Richardson A,Park M,Watson SL,Wakefield D,Di Girolamo N.Native and synthetic scaffolds for limbal epithelial stem cell transplantation.Acta Biomater 2018;65:21-35.
9 Shafiq MA,Gemeinhart RA,Yue BY,Djalilian AR.Decellularized human cornea for reconstructing the corneal epithelium and anterior stroma.Tissue Eng Part C Methods 2012;18(5):340-348.
10 Chen Z,You JJ,Liu X,Cooper S,Hodge C,Sutton G,Crook JM,Wallace GG.Biomaterials for corneal bioengineering.Biomed Mater2018;13(3):032002.
11 Yan H,Gong LY,Huang W,Peng YL.Clinical outcomes of small incision lenticule extraction versus femtosecond laser-assisted LASIK for myopia:a Meta-analysis.Int J Ophthalmol 2017;10(9):1436-1445.
12 Liu YC,Rosman M,Mehta JS.Enhancement after small-incision lenticule extraction:incidence,risk factors,and outcomes.Ophthalmology2017;124(6):813-821.
13 Pradhan KR,Reinstein DZ,Carp GI,Archer TJ,Gobbe M,Gurung R.Femtosecond laser-assisted keyhole endokeratophakia:correction of hyperopia by implantation of an allogeneic lenticule obtained by SMILEfrom a myopic donor.J Refract Surg 2013;29(11):777-782.
14 Ganesh S,Brar S,Rao PA.Cryopreservation of extracted corneal lenticules after small incision lenticule extraction for potential use in human subjects.Cornea 2014;33(12):1355-1362.
15 Ganesh S,Brar S.Femtosecond intrastromal lenticular implantation combined with accelerated collagen cross-linking for the treatment of keratoconus:initial clinical result in 6 eyes.Cornea 2015;34(10):1331-1339.
16 Sun L,Yao PJ,Li MY,Shen Y,Zhao J,Zhou XT.The safety and predictability of implanting autologous lenticule obtained by SMILE for hyperopia.J Refract Surg 2015;31(6):374-379.
17 Yam GH,Yusoff NZ,Goh TW,Setiawan M,Lee XW,Liu YC,Mehta JS.Decellularization of human stromal refractive lenticules for corneal tissue engineering.Sci Rep 2016;6:26339.
18 Bhandari V,Ganesh S,Brar S,Pandey R.Application of the SMILE-derived glued lenticule patch graft in microperforations and partialthickness corneal defects.Cornea 2016;35(3):408-412.
19 Wu F,Jin XM,Xu YS,Yang YB.Treatment of corneal perforation with lenticules from small incision lenticule extraction surgery:a preliminary study of 6 patients.Cornea 2015;34(6):658-663.
20 Yin HF,Qiu PJ,Wu F,Zhang W,Teng WQ,Qin ZW,Li C,Zhou JJ,Fang Z,Tang QM,Fu QL,Ma J,Yang YB.Construction of a corneal stromal equivalent with SMILE-derived lenticules and fibrin glue.Sci Rep2016;6:33848.
21 Chamberlain G,Fox J,Ashton B,Middleton J.Concise review:mesenchymal stem cells:their phenotype,differentiation capacity,immunological features,and potential for homing.Stem Cells2007;25(11):2739-2749.
22 Li XH,Yue SJ,Luo ZQ.Mesenchymal stem cells in idiopathic pulmonary fibrosis.Oncotarget 2017;8(60):102600-102616.
23 Gu SF,Xing CZ,Han JY,Tso MO,Hong J.Differentiation of rabbit bone marrow mesenchymal stem cells into corneal epithelial cells in vivo and ex vivo.Mol Vis 2009;15:99-107.
24 Katikireddy KR,Dana RZ,Jurkunas UV.Differentiation potential of limbal fibroblasts and bone marrow mesenchymal stem cells to corneal epithelial cells.Stem Cells 2014;32(3):717-729.
25 Brown PT,Squire MW,Li WJ.Characterization and evaluation of mesenchymal stem cells derived from human embryonic stem cells and bone marrow.Cell Tissue Res 2014;358(1):149-164.
26 Alio del Barrio JL,Chiesa M,Garagorri N,Garcia-Urquia N,Fernandez-Delgado J,Bataille L,Rodriguez A,Arnalich-Montiel F,Zarnowski T,álvarez de Toledo JP,Alio JL,De Miguel MP.Acellular human corneal matrix sheets seeded with human adipose-derived mesenchymal stem cells integrate functionally in an experimental animal model.Exp Eye Res 2015;132:91-100.
27 Lin XC,Hui YN,Wang YS,Meng H,Zhang YJ,Jin Y.Lamellar keratoplasty with a graft of lyophilized acellular porcine corneal stroma in the rabbit.Vet Ophthalmol 2008;11(2):61-66.
28 Pang KP,Du LQ,Wu XY.A rabbit anterior cornea replacement derived from acellular porcine cornea matrix,epithelial cells and keratocytes.Biomaterials 2010;31(28):7257-7265.
29 Yoeruek E,Bayyoud T,Maurus C,Hofmann J,Spitzer MS,BartzSchmidt KU,Szurman P.Decellularization of porcine corneas and repopulation with human corneal cells for tissue-engineered xenografts.Acta Ophthalmol 2012;90(2):e125-e131.
30 Yoeruek E,Bayyoud T,Maurus C,Hofmann J,Spitzer MS,BartzSchmidt KU,Szurman P.Reconstruction of corneal stroma with decellularized porcine xenografts in a rabbit model.Acta Ophthalmol2012;90(3):e206-e210.
31 Bayyoud T,Thaler S,Hofmann J,Maurus C,Spitzer MS,BartzSchmidt KU,Szurman P,Yoeruek E.Decellularized bovine corneal posterior lamellae as carrier matrix for cultivated human corneal endothelial cells.Curr Eye Res 2012;37(3):179-186.
32 Beyer Nardi N,da Silva Meirelles L.Mesenchymal stem cells:isolation,in vitro expansion and characterization.Handb Exp Pharmacol2006(174):249-282.
33 Gonzalez-Andrades M,de la Cruz Cardona J,Ionescu AM,Campos A,Del Mar Perez M,Alaminos M.Generation of bioengineered corneas with decellularized xenografts and human keratocytes.Invest Ophthalmol Vis Sci 2011;52(1):215-222.
34 Xiao JH,Duan HC,Liu Z,Wu Z,Lan YQ,Zhang W,Li CY,Chen F,Zhou Q,Wang XR,Huang JQ,Wang ZC.Construction of the recellularized corneal stroma using porous acellular corneal scaffold.Biomaterials 2011;32(29):6962-6971.
35 Murphy SV,Atala A.Organ engineering:combining stem cells,biomaterials,and bioreactors to produce bioengineered organs for transplantation.Bioessays 2013;35(3):163-172.
36 Du LQ,Wu XY,Pang KP,Yang YM.Histological evaluation and biomechanical characterisation of an acellular porcine cornea scaffold.Br J Ophthalmol 2011;95(3):410-414.
37 Zhang J,Huang C,Feng Y,Li Y,Wang W.Comparison of beneficial factors for corneal wound-healing of rat mesenchymal stem cells and corneal limbal stem cells on the xenogeneic acellular corneal matrix in vitro.Mol Vis 2012;18:161-173.
38 Hwang NS,Varghese S,Lee HJ,Zhang ZJ,Ye ZH,Bae J,Cheng LZ,Elisseeff J.In vivo commitment and functional tissue regeneration using human embryonic stem cell-derived mesenchymal cells.Proc Natl Acad Sci USA 2008;105(52):20641-20646.
39 Ma YL,Xu YS,Xiao ZF,Yang W,Zhang C,Song E,Du YQ,Li LS.Reconstruction of chemically burned rat corneal surface by bone marrowderived human mesenchymal stem cells.Stem Cells 2006;24(2):315-321.
40 Nieto-Miguel T,Galindo S,Reinoso R,Corell A,Martino M,PérezSimón JA,Calonge M.In vitro simulation of corneal epithelium microenvironment induces a corneal epithelial-like cell phenotype from human adipose tissue mesenchymal stem cells.Curr Eye Res2013;38(9):933-944.
41 Moll R,Divo M,Langbein L.The human keratins:biology and pathology.Histochem Cell Biol 2008;129(6):705-733.
42 Merjava S,Neuwirth A,Tanzerova M,Jirsova K.The spectrum of cytokeratins expressed in the adult human cornea,limbus and perilimbal conjunctiva.Histol Histopathol 2011;26(3):323-331.
43 Merjava S,Neuwirth A,Mandys V,Jirsova K.Cytokeratins 8 and 18in adult human corneal endothelium.Exp Eye Res 2009;89(3):426-431.
44 Pajoohesh-Ganji A,Pal-Ghosh S,Tadvalkar G,Stepp MA.Corneal goblet cells and their niche:implications for corneal stem cell deficiency.Stem Cells 2012;30(9):2032-2043.
45 Hashmani K,Branch MJ,Sidney LE,Dhillon PS,Verma M,McIntosh OD,Hopkinson A,Dua HS.Characterization of corneal stromal stem cells with the potential for epithelial transdifferentiation.Stem Cell Res Ther2013;4(3):75.
46 Merjava S,Brejchova K,Vernon A,Daniels JT,Jirsova K.Cytokeratin 8 is expressed in human corneoconjunctival epithelium,particularly in limbal epithelial cells.Invest Ophthalmol Vis Sci 2011;52(2):787-794.
2 Lynch AP,Ahearne M.Strategies for developing decellularized corneal scaffolds.Exp Eye Res 2013;108:42-47.
3 Kong B,Mi SL.Electrospun scaffolds for corneal tissue engineering:a review.Materials(Basel)2016;9(8):E614.
4 Wang B,Guo YW,Chen XF,Zeng C,Hu QK,Yin W,Li W,Xie H,Zhang BY,Huang XC,Yu FL.Nanoparticle-modified chitosan-agarosegelatin scaffold for sustained release of SDF-1 and BMP-2.Int JNanomedicine 2018;13:7395-7408.
5 Yang X,Li YY,He W,Huang QL,Zhang RR,Feng QL.Hydroxyapatite/collagen coating on PLGA electrospun fibers for osteogenic differentiation of bone marrow mesenchymal stem cells.J Biomed Mater Res A2018;106(11):2863-2870.
6 Sharma S,Mohanty S,Gupta D,Jassal M,Agrawal AK,Tandon R.Cellular response of limbal epithelial cells on electrospun poly-ε-caprolactone nanofibrous scaffolds for ocular surface bioengineering:a preliminary in vitro study.Mol Vis 2011;17:2898-2910.
7 Levis HJ,Brown RA,Daniels JT.Plastic compressed collagen as a biomimetic substrate for human limbal epithelial cell culture.Biomaterials2010;31(30):7726-7737.
8 Nguyen KN,Bobba S,Richardson A,Park M,Watson SL,Wakefield D,Di Girolamo N.Native and synthetic scaffolds for limbal epithelial stem cell transplantation.Acta Biomater 2018;65:21-35.
9 Shafiq MA,Gemeinhart RA,Yue BY,Djalilian AR.Decellularized human cornea for reconstructing the corneal epithelium and anterior stroma.Tissue Eng Part C Methods 2012;18(5):340-348.
10 Chen Z,You JJ,Liu X,Cooper S,Hodge C,Sutton G,Crook JM,Wallace GG.Biomaterials for corneal bioengineering.Biomed Mater2018;13(3):032002.
11 Yan H,Gong LY,Huang W,Peng YL.Clinical outcomes of small incision lenticule extraction versus femtosecond laser-assisted LASIK for myopia:a Meta-analysis.Int J Ophthalmol 2017;10(9):1436-1445.
12 Liu YC,Rosman M,Mehta JS.Enhancement after small-incision lenticule extraction:incidence,risk factors,and outcomes.Ophthalmology2017;124(6):813-821.
13 Pradhan KR,Reinstein DZ,Carp GI,Archer TJ,Gobbe M,Gurung R.Femtosecond laser-assisted keyhole endokeratophakia:correction of hyperopia by implantation of an allogeneic lenticule obtained by SMILEfrom a myopic donor.J Refract Surg 2013;29(11):777-782.
14 Ganesh S,Brar S,Rao PA.Cryopreservation of extracted corneal lenticules after small incision lenticule extraction for potential use in human subjects.Cornea 2014;33(12):1355-1362.
15 Ganesh S,Brar S.Femtosecond intrastromal lenticular implantation combined with accelerated collagen cross-linking for the treatment of keratoconus:initial clinical result in 6 eyes.Cornea 2015;34(10):1331-1339.
16 Sun L,Yao PJ,Li MY,Shen Y,Zhao J,Zhou XT.The safety and predictability of implanting autologous lenticule obtained by SMILE for hyperopia.J Refract Surg 2015;31(6):374-379.
17 Yam GH,Yusoff NZ,Goh TW,Setiawan M,Lee XW,Liu YC,Mehta JS.Decellularization of human stromal refractive lenticules for corneal tissue engineering.Sci Rep 2016;6:26339.
18 Bhandari V,Ganesh S,Brar S,Pandey R.Application of the SMILE-derived glued lenticule patch graft in microperforations and partialthickness corneal defects.Cornea 2016;35(3):408-412.
19 Wu F,Jin XM,Xu YS,Yang YB.Treatment of corneal perforation with lenticules from small incision lenticule extraction surgery:a preliminary study of 6 patients.Cornea 2015;34(6):658-663.
20 Yin HF,Qiu PJ,Wu F,Zhang W,Teng WQ,Qin ZW,Li C,Zhou JJ,Fang Z,Tang QM,Fu QL,Ma J,Yang YB.Construction of a corneal stromal equivalent with SMILE-derived lenticules and fibrin glue.Sci Rep2016;6:33848.
21 Chamberlain G,Fox J,Ashton B,Middleton J.Concise review:mesenchymal stem cells:their phenotype,differentiation capacity,immunological features,and potential for homing.Stem Cells2007;25(11):2739-2749.
22 Li XH,Yue SJ,Luo ZQ.Mesenchymal stem cells in idiopathic pulmonary fibrosis.Oncotarget 2017;8(60):102600-102616.
23 Gu SF,Xing CZ,Han JY,Tso MO,Hong J.Differentiation of rabbit bone marrow mesenchymal stem cells into corneal epithelial cells in vivo and ex vivo.Mol Vis 2009;15:99-107.
24 Katikireddy KR,Dana RZ,Jurkunas UV.Differentiation potential of limbal fibroblasts and bone marrow mesenchymal stem cells to corneal epithelial cells.Stem Cells 2014;32(3):717-729.
25 Brown PT,Squire MW,Li WJ.Characterization and evaluation of mesenchymal stem cells derived from human embryonic stem cells and bone marrow.Cell Tissue Res 2014;358(1):149-164.
26 Alio del Barrio JL,Chiesa M,Garagorri N,Garcia-Urquia N,Fernandez-Delgado J,Bataille L,Rodriguez A,Arnalich-Montiel F,Zarnowski T,álvarez de Toledo JP,Alio JL,De Miguel MP.Acellular human corneal matrix sheets seeded with human adipose-derived mesenchymal stem cells integrate functionally in an experimental animal model.Exp Eye Res 2015;132:91-100.
27 Lin XC,Hui YN,Wang YS,Meng H,Zhang YJ,Jin Y.Lamellar keratoplasty with a graft of lyophilized acellular porcine corneal stroma in the rabbit.Vet Ophthalmol 2008;11(2):61-66.
28 Pang KP,Du LQ,Wu XY.A rabbit anterior cornea replacement derived from acellular porcine cornea matrix,epithelial cells and keratocytes.Biomaterials 2010;31(28):7257-7265.
29 Yoeruek E,Bayyoud T,Maurus C,Hofmann J,Spitzer MS,BartzSchmidt KU,Szurman P.Decellularization of porcine corneas and repopulation with human corneal cells for tissue-engineered xenografts.Acta Ophthalmol 2012;90(2):e125-e131.
30 Yoeruek E,Bayyoud T,Maurus C,Hofmann J,Spitzer MS,BartzSchmidt KU,Szurman P.Reconstruction of corneal stroma with decellularized porcine xenografts in a rabbit model.Acta Ophthalmol2012;90(3):e206-e210.
31 Bayyoud T,Thaler S,Hofmann J,Maurus C,Spitzer MS,BartzSchmidt KU,Szurman P,Yoeruek E.Decellularized bovine corneal posterior lamellae as carrier matrix for cultivated human corneal endothelial cells.Curr Eye Res 2012;37(3):179-186.
32 Beyer Nardi N,da Silva Meirelles L.Mesenchymal stem cells:isolation,in vitro expansion and characterization.Handb Exp Pharmacol2006(174):249-282.
33 Gonzalez-Andrades M,de la Cruz Cardona J,Ionescu AM,Campos A,Del Mar Perez M,Alaminos M.Generation of bioengineered corneas with decellularized xenografts and human keratocytes.Invest Ophthalmol Vis Sci 2011;52(1):215-222.
34 Xiao JH,Duan HC,Liu Z,Wu Z,Lan YQ,Zhang W,Li CY,Chen F,Zhou Q,Wang XR,Huang JQ,Wang ZC.Construction of the recellularized corneal stroma using porous acellular corneal scaffold.Biomaterials 2011;32(29):6962-6971.
35 Murphy SV,Atala A.Organ engineering:combining stem cells,biomaterials,and bioreactors to produce bioengineered organs for transplantation.Bioessays 2013;35(3):163-172.
36 Du LQ,Wu XY,Pang KP,Yang YM.Histological evaluation and biomechanical characterisation of an acellular porcine cornea scaffold.Br J Ophthalmol 2011;95(3):410-414.
37 Zhang J,Huang C,Feng Y,Li Y,Wang W.Comparison of beneficial factors for corneal wound-healing of rat mesenchymal stem cells and corneal limbal stem cells on the xenogeneic acellular corneal matrix in vitro.Mol Vis 2012;18:161-173.
38 Hwang NS,Varghese S,Lee HJ,Zhang ZJ,Ye ZH,Bae J,Cheng LZ,Elisseeff J.In vivo commitment and functional tissue regeneration using human embryonic stem cell-derived mesenchymal cells.Proc Natl Acad Sci USA 2008;105(52):20641-20646.
39 Ma YL,Xu YS,Xiao ZF,Yang W,Zhang C,Song E,Du YQ,Li LS.Reconstruction of chemically burned rat corneal surface by bone marrowderived human mesenchymal stem cells.Stem Cells 2006;24(2):315-321.
40 Nieto-Miguel T,Galindo S,Reinoso R,Corell A,Martino M,PérezSimón JA,Calonge M.In vitro simulation of corneal epithelium microenvironment induces a corneal epithelial-like cell phenotype from human adipose tissue mesenchymal stem cells.Curr Eye Res2013;38(9):933-944.
41 Moll R,Divo M,Langbein L.The human keratins:biology and pathology.Histochem Cell Biol 2008;129(6):705-733.
42 Merjava S,Neuwirth A,Tanzerova M,Jirsova K.The spectrum of cytokeratins expressed in the adult human cornea,limbus and perilimbal conjunctiva.Histol Histopathol 2011;26(3):323-331.
43 Merjava S,Neuwirth A,Mandys V,Jirsova K.Cytokeratins 8 and 18in adult human corneal endothelium.Exp Eye Res 2009;89(3):426-431.
44 Pajoohesh-Ganji A,Pal-Ghosh S,Tadvalkar G,Stepp MA.Corneal goblet cells and their niche:implications for corneal stem cell deficiency.Stem Cells 2012;30(9):2032-2043.
45 Hashmani K,Branch MJ,Sidney LE,Dhillon PS,Verma M,McIntosh OD,Hopkinson A,Dua HS.Characterization of corneal stromal stem cells with the potential for epithelial transdifferentiation.Stem Cell Res Ther2013;4(3):75.
46 Merjava S,Brejchova K,Vernon A,Daniels JT,Jirsova K.Cytokeratin 8 is expressed in human corneoconjunctival epithelium,particularly in limbal epithelial cells.Invest Ophthalmol Vis Sci 2011;52(2):787-794.