Long-Term Cultures of Human Cornea Limbal Explants Form 3D Structures Ex Vivo – Implications for Tissue Engineering and Clinical Applications

Alexander V. Ljubimov, Dóra Júlia Szabó, Agate Noer, Richárd Nagymihály, Natasha Josifovska, Sofija Andjelic, Zoltán Veréb, Andrea Facskó, Morten C. Moe, Goran Petrovski

    Research output: Contribution to journalArticle

    14 Citations (Scopus)

    Abstract

    Long-term cultures of cornea limbal epithelial stem cells (LESCs) were developed and characterized for future tissue engineering and clinical applications. The limbal tissue explants were cultivated and expanded for more than 3 months in medium containing serum as the only growth supplement and without use of scaffolds. Viable 3D cell outgrowth from the explants was observed within 4 weeks of cultivation. The outgrowing cells were examined by immunofluorescent staining for putative markers of stemness (ABCG2, CK15, CK19 and Vimentin), proliferation (p63α, Ki-67), limbal basal epithelial cells (CK8/18) and differentiated cornea epithelial cells (CK3 and CK12). Morphological and immunostaining analyses revealed that long-term culturing can form stratified 3D tissue layers with a clear extracellular matrix deposition and organization (collagen I, IV and V). The LESCs showed robust expression of p63α, ABCG2, and their surface marker fingerprint (CD117/c-kit, CXCR4, CD146/MCAM, CD166/ALCAM) changed over time compared to short-term LESC cultures. Overall, we provide a model for generating stem cell-rich, long-standing 3D cultures from LESCs which can be used for further research purposes and clinical transplantation
    LanguageEnglish
    Pagese0143053
    JournalPLoS ONE
    Volume10
    Issue number11
    DOIs
    Publication statusPublished - 18 Nov 2015

    Fingerprint

    tissue engineering
    cornea
    Tissue Engineering
    Stem cells
    Tissue engineering
    Cornea
    explants
    epithelial cells
    Epithelial Cells
    stem cells
    Stem Cells
    Cell culture
    Activated-Leukocyte Cell Adhesion Molecule
    Tissue
    Vimentin
    Scaffolds
    vimentin
    Dermatoglyphics
    extracellular matrix
    Collagen

    Keywords

    • cornea
    • limbal epithelial stem cells
    • long-term cultures
    • 3D tissue explants

    Cite this

    Ljubimov, A. V., Szabó, D. J., Noer, A., Nagymihály, R., Josifovska, N., Andjelic, S., ... Petrovski, G. (2015). Long-Term Cultures of Human Cornea Limbal Explants Form 3D Structures Ex Vivo – Implications for Tissue Engineering and Clinical Applications. PLoS ONE, 10(11), e0143053. https://doi.org/10.1371/journal.pone.0143053
    Ljubimov, Alexander V. ; Szabó, Dóra Júlia ; Noer, Agate ; Nagymihály, Richárd ; Josifovska, Natasha ; Andjelic, Sofija ; Veréb, Zoltán ; Facskó, Andrea ; Moe, Morten C. ; Petrovski, Goran. / Long-Term Cultures of Human Cornea Limbal Explants Form 3D Structures Ex Vivo – Implications for Tissue Engineering and Clinical Applications. In: PLoS ONE. 2015 ; Vol. 10, No. 11. pp. e0143053.
    @article{7da687fe276041abbd66b6051af1ed60,
    title = "Long-Term Cultures of Human Cornea Limbal Explants Form 3D Structures Ex Vivo – Implications for Tissue Engineering and Clinical Applications",
    abstract = "Long-term cultures of cornea limbal epithelial stem cells (LESCs) were developed and characterized for future tissue engineering and clinical applications. The limbal tissue explants were cultivated and expanded for more than 3 months in medium containing serum as the only growth supplement and without use of scaffolds. Viable 3D cell outgrowth from the explants was observed within 4 weeks of cultivation. The outgrowing cells were examined by immunofluorescent staining for putative markers of stemness (ABCG2, CK15, CK19 and Vimentin), proliferation (p63α, Ki-67), limbal basal epithelial cells (CK8/18) and differentiated cornea epithelial cells (CK3 and CK12). Morphological and immunostaining analyses revealed that long-term culturing can form stratified 3D tissue layers with a clear extracellular matrix deposition and organization (collagen I, IV and V). The LESCs showed robust expression of p63α, ABCG2, and their surface marker fingerprint (CD117/c-kit, CXCR4, CD146/MCAM, CD166/ALCAM) changed over time compared to short-term LESC cultures. Overall, we provide a model for generating stem cell-rich, long-standing 3D cultures from LESCs which can be used for further research purposes and clinical transplantation",
    keywords = "cornea, limbal epithelial stem cells, long-term cultures, 3D tissue explants",
    author = "Ljubimov, {Alexander V.} and Szab{\'o}, {D{\'o}ra J{\'u}lia} and Agate Noer and Rich{\'a}rd Nagymih{\'a}ly and Natasha Josifovska and Sofija Andjelic and Zolt{\'a}n Ver{\'e}b and Andrea Facsk{\'o} and Moe, {Morten C.} and Goran Petrovski",
    note = "Reference text: 1. Shanmuganathan VA, Foster T, Kulkarni BB, Hopkinson A, Gray T, Powe DG, et al. Morphological characteristics of the limbal epithelial crypt. The British journal of ophthalmology. 2007;91(4):514–9. doi: 10.1136/bjo.2006.102640 ; PubMed Central PMCID: PMC1994762. [PMC free article] [PubMed] 2. Dua HS, Shanmuganathan VA, Powell-Richards AO, Tighe PJ, Joseph A. Limbal epithelial crypts: a novel anatomical structure and a putative limbal stem cell niche. The British journal of ophthalmology. 2005;89(5):529–32. doi: 10.1136/bjo.2004.049742 ; PubMed Central PMCID: PMC1772620. [PMC free article] [PubMed] 3. Chang CY, Green CR, McGhee CN, Sherwin T. Acute wound healing in the human central corneal epithelium appears to be independent of limbal stem cell influence. Investigative ophthalmology & visual science. 2008;49(12):5279–86. doi: 10.1167/iovs.07-1260 . [PubMed] 4. Pellegrini G, Rama P, Di Rocco A, Panaras A, De Luca M. Concise review: hurdles in a successful example of limbal stem cell-based regenerative medicine. Stem cells. 2014;32(1):26–34. doi: 10.1002/stem.1517 . [PubMed] 5. Pathak M, Cholidis S, Haug K, Shahdadfar A, Moe MC, Nicolaissen B, et al. Clinical transplantation of ex vivo expanded autologous limbal epithelial cells using a culture medium with human serum as single supplement: a retrospective case series. Acta ophthalmologica. 2013;91(8):769–75. doi: 10.1111/j.1755-3768.2012.02521.x . [PubMed] 6. Shahdadfar A, Haug K, Pathak M, Drolsum L, Olstad OK, Johnsen EO, et al. Ex vivo expanded autologous limbal epithelial cells on amniotic membrane using a culture medium with human serum as single supplement. Experimental eye research. 2012;97(1):1–9. doi: 10.1016/j.exer.2012.01.013 . [PubMed] 7. Lopez-Paniagua M, Nieto-Miguel T, de la Mata A, Galindo S, Herreras JM, Corrales RM, et al. Consecutive expansion of limbal epithelial stem cells from a single limbal biopsy. Current eye research. 2013;38(5):537–49. doi: 10.3109/02713683.2013.767350 . [PubMed] 8. Albert R, Vereb Z, Csomos K, Moe MC, Johnsen EO, Olstad OK, et al. Cultivation and characterization of cornea limbal epithelial stem cells on lens capsule in animal material-free medium. PloS one. 2012;7(10):e47187 doi: 10.1371/journal.pone.0047187 ; PubMed Central PMCID: PMC3467238. [PMC free article] [PubMed] 9. Gore A, Horwitz V, Gutman H, Tveria L, Cohen L, Cohen-Jacob O, et al. Cultivation and characterization of limbal epithelial stem cells on contact lenses with a feeder layer: toward the treatment of limbal stem cell deficiency. Cornea. 2014;33(1):65–71. doi: 10.1097/ICO.0000000000000002 . [PubMed] 10. Andjelic S, Lumi X, Vereb Z, Josifovska N, Facsko A, Hawlina M, et al. A simple method for establishing adherent ex vivo explant cultures from human eye pathologies for use in subsequent calcium imaging and inflammatory studies. Journal of immunology research. 2014;2014:232659 doi: 10.1155/2014/232659 ; PubMed Central PMCID: PMC4168039. [PMC free article] [PubMed] 11. European Parliament CotEU. [2015.09.21]. Available from: http://eur-lex.europa.eu/legal-content/EN/ALL/?uri=CELEX:32004L0023. 12. Joseph A, Powell-Richards AO, Shanmuganathan VA, Dua HS. Epithelial cell characteristics of cultured human limbal explants. The British journal of ophthalmology. 2004;88(3):393–8. ; PubMed Central PMCID: PMC1772026. [PMC free article] [PubMed] 13. Merjava S, Brejchova K, Vernon A, Daniels JT, Jirsova K. Cytokeratin 8 is expressed in human corneoconjunctival epithelium, particularly in limbal epithelial cells. Investigative ophthalmology & visual science. 2011;52(2):787–94. doi: 10.1167/iovs.10-5489 . [PubMed] 14. Ghoubay-Benallaoua D, Basli E, Goldschmidt P, Pecha F, Chaumeil C, Laroche L, et al. Human epithelial cell cultures from superficial limbal explants. Molecular vision. 2011;17:341–54. ; PubMed Central PMCID: PMC3033435. [PMC free article] [PubMed] 15. Chen Z, de Paiva CS, Luo L, Kretzer FL, Pflugfelder SC, Li DQ. Characterization of putative stem cell phenotype in human limbal epithelia. Stem cells. 2004;22(3):355–66. doi: 10.1634/stemcells.22-3-355 ; PubMed Central PMCID: PMC2906385. [PMC free article] [PubMed] 16. Bhattacharya S, Das A, Mallya K, Ahmad I. Maintenance of retinal stem cells by Abcg2 is regulated by notch signaling. Journal of cell science. 2007;120(Pt 15):2652–62. doi: 10.1242/jcs.008417 . [PubMed] 17. Priya CG, Prasad T, Prajna NV, Muthukkaruppan V. Identification of human corneal epithelial stem cells on the basis of high ABCG2 expression combined with a large N/C ratio. Microscopy research and technique. 2013;76(3):242–8. doi: 10.1002/jemt.22159 . [PubMed] 18. Nubile M, Curcio C, Dua HS, Calienno R, Lanzini M, Iezzi M, et al. Pathological changes of the anatomical structure and markers of the limbal stem cell niche due to inflammation. Molecular vision. 2013;19:516–25. ; PubMed Central PMCID: PMC3580971. [PMC free article] [PubMed] 19. Figueira EC, Di Girolamo N, Coroneo MT, Wakefield D. The phenotype of limbal epithelial stem cells. Investigative ophthalmology & visual science. 2007;48(1):144–56. doi: 10.1167/iovs.06-0346 . [PubMed] 20. Merjava S, Neuwirth A, Tanzerova M, Jirsova K. The spectrum of cytokeratins expressed in the adult human cornea, limbus and perilimbal conjunctiva. Histology and histopathology. 2011;26(3):323–31. . [PubMed] 21. Michel M, Torok N, Godbout MJ, Lussier M, Gaudreau P, Royal A, et al. Keratin 19 as a biochemical marker of skin stem cells in vivo and in vitro: keratin 19 expressing cells are differentially localized in function of anatomic sites, and their number varies with donor age and culture stage. Journal of cell science. 1996;109 (Pt 5):1017–28. . [PubMed] 22. Huang M, Wang B, Wan P, Liang X, Wang X, Liu Y, et al. Roles of limbal microvascular net and limbal stroma in regulating maintenance of limbal epithelial stem cells. Cell and tissue research. 2014. doi: 10.1007/s00441-014-2032-4 . [PubMed] 23. Utheim TP, Raeder S, Olstad OK, Utheim OA, de La Paz M, Cheng R, et al. Comparison of the histology, gene expression profile, and phenotype of cultured human limbal epithelial cells from different limbal regions. Investigative ophthalmology & visual science. 2009;50(11):5165–72. Epub 2009/07/07. doi: 10.1167/iovs.08-2884 iovs.08-2884 [pii]. . [PubMed] 24. Oldenborg PA. CD47: A Cell Surface Glycoprotein Which Regulates Multiple Functions of Hematopoietic Cells in Health and Disease. ISRN hematology. 2013;2013:614619 doi: 10.1155/2013/614619 ; PubMed Central PMCID: PMC3564380. [PMC free article] [PubMed] 25. McCracken MN, Cha AC, Weissman IL. Molecular Pathways: Activating T Cells After Cancer Cell Phagocytosis from Blockade of CD47 {"}Don't Eat Me{"} Signals. Clinical cancer research: an official journal of the American Association for Cancer Research. 2015. doi: 10.1158/1078-0432.CCR-14-2520 . [PMC free article] [PubMed] 26. Lv Z, Bian Z, Shi L, Niu S, Ha B, Tremblay A, et al. Loss of Cell Surface CD47 Clustering Formation and Binding Avidity to SIRPalpha Facilitate Apoptotic Cell Clearance by Macrophages. Journal of immunology. 2015. doi: 10.4049/jimmunol.1401719 . [PMC free article] [PubMed] 27. Kisselbach L, Merges M, Bossie A, Boyd A. CD90 Expression on human primary cells and elimination of contaminating fibroblasts from cell cultures. Cytotechnology. 2009;59(1):31–44. doi: 10.1007/s10616-009-9190-3 ; PubMed Central PMCID: PMC2677147. [PMC free article] [PubMed] 28. Yu FX, Guo J, Zhang Q. Expression and distribution of adhesion molecule CD44 in healing corneal epithelia. Investigative ophthalmology & visual science. 1998;39(5):710–7. . [PubMed] 29. Zhu SN, Nolle B, Duncker G. Expression of adhesion molecule CD44 on human corneas. The British journal of ophthalmology. 1997;81(1):80–4. ; PubMed Central PMCID: PMC1721988. [PMC free article] [PubMed] 30. Levis HJ, Menzel-Severing J, Drake RA, Daniels JT. Plastic compressed collagen constructs for ocular cell culture and transplantation: a new and improved technique of confined fluid loss. Current eye research. 2013;38(1):41–52. doi: 10.3109/02713683.2012.725799 . [PubMed] 31. McIntosh Ambrose W, Salahuddin A, So S, Ng S, Ponce Marquez S, Takezawa T, et al. Collagen Vitrigel membranes for the in vitro reconstruction of separate corneal epithelial, stromal, and endothelial cell layers. Journal of biomedical materials research Part B, Applied biomaterials. 2009;90(2):818–31. doi: 10.1002/jbm.b.31351 . [PubMed] 32. Mi S, Chen B, Wright B, Connon CJ. Ex vivo construction of an artificial ocular surface by combination of corneal limbal epithelial cells and a compressed collagen scaffold containing keratocytes. Tissue engineering Part A. 2010;16(6):2091–100. doi: 10.1089/ten.TEA.2009.0748 . [PubMed]",
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    pages = "e0143053",
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    Ljubimov, AV, Szabó, DJ, Noer, A, Nagymihály, R, Josifovska, N, Andjelic, S, Veréb, Z, Facskó, A, Moe, MC & Petrovski, G 2015, 'Long-Term Cultures of Human Cornea Limbal Explants Form 3D Structures Ex Vivo – Implications for Tissue Engineering and Clinical Applications', PLoS ONE, vol. 10, no. 11, pp. e0143053. https://doi.org/10.1371/journal.pone.0143053

    Long-Term Cultures of Human Cornea Limbal Explants Form 3D Structures Ex Vivo – Implications for Tissue Engineering and Clinical Applications. / Ljubimov, Alexander V.; Szabó, Dóra Júlia; Noer, Agate; Nagymihály, Richárd; Josifovska, Natasha; Andjelic, Sofija; Veréb, Zoltán; Facskó, Andrea; Moe, Morten C.; Petrovski, Goran.

    In: PLoS ONE, Vol. 10, No. 11, 18.11.2015, p. e0143053.

    Research output: Contribution to journalArticle

    TY - JOUR

    T1 - Long-Term Cultures of Human Cornea Limbal Explants Form 3D Structures Ex Vivo – Implications for Tissue Engineering and Clinical Applications

    AU - Ljubimov, Alexander V.

    AU - Szabó, Dóra Júlia

    AU - Noer, Agate

    AU - Nagymihály, Richárd

    AU - Josifovska, Natasha

    AU - Andjelic, Sofija

    AU - Veréb, Zoltán

    AU - Facskó, Andrea

    AU - Moe, Morten C.

    AU - Petrovski, Goran

    N1 - Reference text: 1. Shanmuganathan VA, Foster T, Kulkarni BB, Hopkinson A, Gray T, Powe DG, et al. Morphological characteristics of the limbal epithelial crypt. The British journal of ophthalmology. 2007;91(4):514–9. doi: 10.1136/bjo.2006.102640 ; PubMed Central PMCID: PMC1994762. [PMC free article] [PubMed] 2. Dua HS, Shanmuganathan VA, Powell-Richards AO, Tighe PJ, Joseph A. Limbal epithelial crypts: a novel anatomical structure and a putative limbal stem cell niche. The British journal of ophthalmology. 2005;89(5):529–32. doi: 10.1136/bjo.2004.049742 ; PubMed Central PMCID: PMC1772620. [PMC free article] [PubMed] 3. Chang CY, Green CR, McGhee CN, Sherwin T. Acute wound healing in the human central corneal epithelium appears to be independent of limbal stem cell influence. Investigative ophthalmology & visual science. 2008;49(12):5279–86. doi: 10.1167/iovs.07-1260 . [PubMed] 4. Pellegrini G, Rama P, Di Rocco A, Panaras A, De Luca M. Concise review: hurdles in a successful example of limbal stem cell-based regenerative medicine. Stem cells. 2014;32(1):26–34. doi: 10.1002/stem.1517 . [PubMed] 5. Pathak M, Cholidis S, Haug K, Shahdadfar A, Moe MC, Nicolaissen B, et al. Clinical transplantation of ex vivo expanded autologous limbal epithelial cells using a culture medium with human serum as single supplement: a retrospective case series. Acta ophthalmologica. 2013;91(8):769–75. doi: 10.1111/j.1755-3768.2012.02521.x . [PubMed] 6. Shahdadfar A, Haug K, Pathak M, Drolsum L, Olstad OK, Johnsen EO, et al. Ex vivo expanded autologous limbal epithelial cells on amniotic membrane using a culture medium with human serum as single supplement. Experimental eye research. 2012;97(1):1–9. doi: 10.1016/j.exer.2012.01.013 . [PubMed] 7. Lopez-Paniagua M, Nieto-Miguel T, de la Mata A, Galindo S, Herreras JM, Corrales RM, et al. Consecutive expansion of limbal epithelial stem cells from a single limbal biopsy. Current eye research. 2013;38(5):537–49. doi: 10.3109/02713683.2013.767350 . [PubMed] 8. Albert R, Vereb Z, Csomos K, Moe MC, Johnsen EO, Olstad OK, et al. Cultivation and characterization of cornea limbal epithelial stem cells on lens capsule in animal material-free medium. PloS one. 2012;7(10):e47187 doi: 10.1371/journal.pone.0047187 ; PubMed Central PMCID: PMC3467238. [PMC free article] [PubMed] 9. Gore A, Horwitz V, Gutman H, Tveria L, Cohen L, Cohen-Jacob O, et al. Cultivation and characterization of limbal epithelial stem cells on contact lenses with a feeder layer: toward the treatment of limbal stem cell deficiency. Cornea. 2014;33(1):65–71. doi: 10.1097/ICO.0000000000000002 . [PubMed] 10. Andjelic S, Lumi X, Vereb Z, Josifovska N, Facsko A, Hawlina M, et al. A simple method for establishing adherent ex vivo explant cultures from human eye pathologies for use in subsequent calcium imaging and inflammatory studies. Journal of immunology research. 2014;2014:232659 doi: 10.1155/2014/232659 ; PubMed Central PMCID: PMC4168039. [PMC free article] [PubMed] 11. European Parliament CotEU. [2015.09.21]. Available from: http://eur-lex.europa.eu/legal-content/EN/ALL/?uri=CELEX:32004L0023. 12. Joseph A, Powell-Richards AO, Shanmuganathan VA, Dua HS. Epithelial cell characteristics of cultured human limbal explants. The British journal of ophthalmology. 2004;88(3):393–8. ; PubMed Central PMCID: PMC1772026. [PMC free article] [PubMed] 13. Merjava S, Brejchova K, Vernon A, Daniels JT, Jirsova K. Cytokeratin 8 is expressed in human corneoconjunctival epithelium, particularly in limbal epithelial cells. Investigative ophthalmology & visual science. 2011;52(2):787–94. doi: 10.1167/iovs.10-5489 . [PubMed] 14. Ghoubay-Benallaoua D, Basli E, Goldschmidt P, Pecha F, Chaumeil C, Laroche L, et al. Human epithelial cell cultures from superficial limbal explants. Molecular vision. 2011;17:341–54. ; PubMed Central PMCID: PMC3033435. [PMC free article] [PubMed] 15. Chen Z, de Paiva CS, Luo L, Kretzer FL, Pflugfelder SC, Li DQ. Characterization of putative stem cell phenotype in human limbal epithelia. Stem cells. 2004;22(3):355–66. doi: 10.1634/stemcells.22-3-355 ; PubMed Central PMCID: PMC2906385. [PMC free article] [PubMed] 16. Bhattacharya S, Das A, Mallya K, Ahmad I. Maintenance of retinal stem cells by Abcg2 is regulated by notch signaling. Journal of cell science. 2007;120(Pt 15):2652–62. doi: 10.1242/jcs.008417 . [PubMed] 17. Priya CG, Prasad T, Prajna NV, Muthukkaruppan V. Identification of human corneal epithelial stem cells on the basis of high ABCG2 expression combined with a large N/C ratio. Microscopy research and technique. 2013;76(3):242–8. doi: 10.1002/jemt.22159 . [PubMed] 18. Nubile M, Curcio C, Dua HS, Calienno R, Lanzini M, Iezzi M, et al. Pathological changes of the anatomical structure and markers of the limbal stem cell niche due to inflammation. Molecular vision. 2013;19:516–25. ; PubMed Central PMCID: PMC3580971. [PMC free article] [PubMed] 19. Figueira EC, Di Girolamo N, Coroneo MT, Wakefield D. The phenotype of limbal epithelial stem cells. Investigative ophthalmology & visual science. 2007;48(1):144–56. doi: 10.1167/iovs.06-0346 . [PubMed] 20. Merjava S, Neuwirth A, Tanzerova M, Jirsova K. The spectrum of cytokeratins expressed in the adult human cornea, limbus and perilimbal conjunctiva. Histology and histopathology. 2011;26(3):323–31. . [PubMed] 21. Michel M, Torok N, Godbout MJ, Lussier M, Gaudreau P, Royal A, et al. Keratin 19 as a biochemical marker of skin stem cells in vivo and in vitro: keratin 19 expressing cells are differentially localized in function of anatomic sites, and their number varies with donor age and culture stage. Journal of cell science. 1996;109 (Pt 5):1017–28. . [PubMed] 22. Huang M, Wang B, Wan P, Liang X, Wang X, Liu Y, et al. Roles of limbal microvascular net and limbal stroma in regulating maintenance of limbal epithelial stem cells. Cell and tissue research. 2014. doi: 10.1007/s00441-014-2032-4 . [PubMed] 23. Utheim TP, Raeder S, Olstad OK, Utheim OA, de La Paz M, Cheng R, et al. Comparison of the histology, gene expression profile, and phenotype of cultured human limbal epithelial cells from different limbal regions. Investigative ophthalmology & visual science. 2009;50(11):5165–72. Epub 2009/07/07. doi: 10.1167/iovs.08-2884 iovs.08-2884 [pii]. . [PubMed] 24. Oldenborg PA. CD47: A Cell Surface Glycoprotein Which Regulates Multiple Functions of Hematopoietic Cells in Health and Disease. ISRN hematology. 2013;2013:614619 doi: 10.1155/2013/614619 ; PubMed Central PMCID: PMC3564380. [PMC free article] [PubMed] 25. McCracken MN, Cha AC, Weissman IL. Molecular Pathways: Activating T Cells After Cancer Cell Phagocytosis from Blockade of CD47 "Don't Eat Me" Signals. Clinical cancer research: an official journal of the American Association for Cancer Research. 2015. doi: 10.1158/1078-0432.CCR-14-2520 . [PMC free article] [PubMed] 26. Lv Z, Bian Z, Shi L, Niu S, Ha B, Tremblay A, et al. Loss of Cell Surface CD47 Clustering Formation and Binding Avidity to SIRPalpha Facilitate Apoptotic Cell Clearance by Macrophages. Journal of immunology. 2015. doi: 10.4049/jimmunol.1401719 . [PMC free article] [PubMed] 27. Kisselbach L, Merges M, Bossie A, Boyd A. CD90 Expression on human primary cells and elimination of contaminating fibroblasts from cell cultures. Cytotechnology. 2009;59(1):31–44. doi: 10.1007/s10616-009-9190-3 ; PubMed Central PMCID: PMC2677147. [PMC free article] [PubMed] 28. Yu FX, Guo J, Zhang Q. Expression and distribution of adhesion molecule CD44 in healing corneal epithelia. Investigative ophthalmology & visual science. 1998;39(5):710–7. . [PubMed] 29. Zhu SN, Nolle B, Duncker G. Expression of adhesion molecule CD44 on human corneas. The British journal of ophthalmology. 1997;81(1):80–4. ; PubMed Central PMCID: PMC1721988. [PMC free article] [PubMed] 30. Levis HJ, Menzel-Severing J, Drake RA, Daniels JT. Plastic compressed collagen constructs for ocular cell culture and transplantation: a new and improved technique of confined fluid loss. Current eye research. 2013;38(1):41–52. doi: 10.3109/02713683.2012.725799 . [PubMed] 31. McIntosh Ambrose W, Salahuddin A, So S, Ng S, Ponce Marquez S, Takezawa T, et al. Collagen Vitrigel membranes for the in vitro reconstruction of separate corneal epithelial, stromal, and endothelial cell layers. Journal of biomedical materials research Part B, Applied biomaterials. 2009;90(2):818–31. doi: 10.1002/jbm.b.31351 . [PubMed] 32. Mi S, Chen B, Wright B, Connon CJ. Ex vivo construction of an artificial ocular surface by combination of corneal limbal epithelial cells and a compressed collagen scaffold containing keratocytes. Tissue engineering Part A. 2010;16(6):2091–100. doi: 10.1089/ten.TEA.2009.0748 . [PubMed]

    PY - 2015/11/18

    Y1 - 2015/11/18

    N2 - Long-term cultures of cornea limbal epithelial stem cells (LESCs) were developed and characterized for future tissue engineering and clinical applications. The limbal tissue explants were cultivated and expanded for more than 3 months in medium containing serum as the only growth supplement and without use of scaffolds. Viable 3D cell outgrowth from the explants was observed within 4 weeks of cultivation. The outgrowing cells were examined by immunofluorescent staining for putative markers of stemness (ABCG2, CK15, CK19 and Vimentin), proliferation (p63α, Ki-67), limbal basal epithelial cells (CK8/18) and differentiated cornea epithelial cells (CK3 and CK12). Morphological and immunostaining analyses revealed that long-term culturing can form stratified 3D tissue layers with a clear extracellular matrix deposition and organization (collagen I, IV and V). The LESCs showed robust expression of p63α, ABCG2, and their surface marker fingerprint (CD117/c-kit, CXCR4, CD146/MCAM, CD166/ALCAM) changed over time compared to short-term LESC cultures. Overall, we provide a model for generating stem cell-rich, long-standing 3D cultures from LESCs which can be used for further research purposes and clinical transplantation

    AB - Long-term cultures of cornea limbal epithelial stem cells (LESCs) were developed and characterized for future tissue engineering and clinical applications. The limbal tissue explants were cultivated and expanded for more than 3 months in medium containing serum as the only growth supplement and without use of scaffolds. Viable 3D cell outgrowth from the explants was observed within 4 weeks of cultivation. The outgrowing cells were examined by immunofluorescent staining for putative markers of stemness (ABCG2, CK15, CK19 and Vimentin), proliferation (p63α, Ki-67), limbal basal epithelial cells (CK8/18) and differentiated cornea epithelial cells (CK3 and CK12). Morphological and immunostaining analyses revealed that long-term culturing can form stratified 3D tissue layers with a clear extracellular matrix deposition and organization (collagen I, IV and V). The LESCs showed robust expression of p63α, ABCG2, and their surface marker fingerprint (CD117/c-kit, CXCR4, CD146/MCAM, CD166/ALCAM) changed over time compared to short-term LESC cultures. Overall, we provide a model for generating stem cell-rich, long-standing 3D cultures from LESCs which can be used for further research purposes and clinical transplantation

    KW - cornea

    KW - limbal epithelial stem cells

    KW - long-term cultures

    KW - 3D tissue explants

    U2 - 10.1371/journal.pone.0143053

    DO - 10.1371/journal.pone.0143053

    M3 - Article

    VL - 10

    SP - e0143053

    JO - PLoS ONE

    T2 - PLoS ONE

    JF - PLoS ONE

    SN - 1932-6203

    IS - 11

    ER -