title: A hyaluronan hydrogel scaffold-based xeno-free culture system for ex vivo expansion of human corneal epithelial stem cells.
abstract: PurposeTo develop a hyaluronan hydrogel scaffold-based xeno-free culture system for ex vivo cultivation of human corneal epithelial stem cells (CESCs).Patients and MethodsCESCs were cultivated from donor limbal explants on the HyStem-C Hydrogel bio-scaffold in 12-well plates for 3 weeks. Group A used the traditional supplemented hormonal epidermal medium (SHEM) and group B used the defined SHEM (without fetal bovine serum and toxin A, adding 20% serum replacement). The growth and morphology of the cultured cells were assessed by phase contrast microscope. The expressions of specific cell markers were assessed by immunofluorescence staining and quantitative real-time PCR (qRT-PCR).ResultsSuccessful cultures of CESCs were obtained in both groups, resulting in multilayered stratified epithelia. Comparing to group A, the cells in group B was grown slightly slower and formed less cellular layers at the end of culture. The corneal specific cytokeratin (K) 12 and differentiation markers, involucrin, and connexin 43, were mainly expressed in the superficial cellular layers in both groups. Interestingly, certain basal cells were immune-positive to proposed stem cell markers such as K19, ABCG2, and integrin β1 in both groups. There was no significant difference between the two groups with regard to the gene expression levels of all these selected corneal markers (all P>0.05).ConclusionsThe hyaluronan hydrogel scaffold-based xeno-free culture system may support the expansion of regenerative CESCs without the risk of xeno component contamination. The regenerated epithelium maintains similar characteristics of native corneal epithelium.
authors.affiliation: Eye Hospital, School of Optometry and Ophthalmology, Wenzhou Medical University, Wenzhou, China.|Ocular Surface Center, Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, Houston, TX, USA.
authors.forename: D
authors.initials: D
authors.lastname: Chen
authors.affiliation: Ocular Surface Center, Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, Houston, TX, USA.|The Eye Institute, Xiamen University, Xiamen, China.
authors.forename: Y
authors.initials: Y
authors.lastname: Qu
authors.affiliation: Ocular Surface Center, Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, Houston, TX, USA.
authors.forename: X
authors.initials: X
authors.lastname: Hua
authors.affiliation: Ocular Surface Center, Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, Houston, TX, USA.
authors.forename: L
authors.initials: L
authors.lastname: Zhang
authors.affiliation: The Eye Institute, Xiamen University, Xiamen, China.
authors.forename: Z
authors.initials: Z
authors.lastname: Liu
authors.affiliation: Ocular Surface Center, Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, Houston, TX, USA.
authors.forename: S C
authors.initials: SC
authors.lastname: Pflugfelder
authors.affiliation: Ocular Surface Center, Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, Houston, TX, USA.
authors.forename: D-Q
authors.initials: DQ
authors.lastname: Li
mesh_terms: D000368:Aged; D018929:Cell Culture Techniques / Q000379:methods; D002478:Cells, Cultured; D018031:Connexin 43 / Q000096:biosynthesis / Q000235:genetics; D017077:Culture Media, Conditioned; D019573:Epithelium, Corneal / Q000166:cytology* / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation; D006801:Humans; D006820:Hyaluronic Acid / Q000494:pharmacology*; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate / Q000494:pharmacology*; D053551:Keratin-12 / Q000096:biosynthesis / Q000235:genetics; D016850:Limbus Corneae / Q000166:cytology* / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D011498:Protein Precursors / Q000096:biosynthesis / Q000235:genetics; D012313:RNA / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D054457:Tissue Scaffolds
chemical_list: D018031:Connexin 43; D017077:Culture Media, Conditioned; D053551:Keratin-12; D011498:Protein Precursors; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate; C032253:involucrin; D012313:RNA; D006820:Hyaluronic Acid