A new method for constructing engineered corneal epithelium based on embryonic stem cells
Qu Yangluowa, Ou Shangkun, Liu Tingting, Zhang Liying, Zou Dulei, Li Juan, He Hui, Jia Changkai, Zuo Chengyou, Zhang Minjie, He Xin, Liu Zuguo, Li Wei
Abstract
ObjectiveTo construct engineered corneal epithelium from embryonic stem cells (ESCs) using Rock inhibitor combined with hypoxia-normoxia culture condition.
MethodsHuman ESC line H1 was induced to differentiate into epithelial-like cells by addition of retinoic acid (RA) and bone morphogenetic protein 4(BMP4) in the differentiation medium under the adherent culture condition.The ESCs derived epithelial-like cells were expanded in the mixed medium of SHEM and KSFM with the mixture ratio of 1∶2 with or without Rock inhibitor Y27632.The H1 derived epithelial-like cells were seeded on the denuded ammonic membrane to construct engineered corneal epithelium under hypoxia, normoxia and hypoxia-normoxia culture conditions, respectively.The inducted effect of ESCs into epithelial-like cells, the expansion ability of the epithelial-like cells and the characteristics of the constructed engineered corneal epithelium were evaluated by morphological observation, real-time fluorescent quantitative polymerase chain reaction (RTFQ-PCR) and immunofluorescence technology.
ResultsCompared with the control group, the relative expressions of ESCs marker Oct4 mRNA, Notch signaling pathway related factors Notch1 and Jagged1 mRNA, and Wnt signaling pathways related factors c-myc and Cyclin D1 mRNA were significantly reduced, and the relative expressions of cutaneous ectoderm markers p63 and K18 mRNA were significantly increased at day 8 after induction in the induced group, with significant differences between them (t =14.63, 20.15, 93.50, 11.60, 19.30, 18.44, 22.63; all at P<0.05). Compared with the without Y27632 group, the relative expressions of p63 and K14 mRNA, Notch signal pathway receptor Notch1 and Jagged1 mRNA were significantly increased, and Wnt signaling pathways downstream targeted gene c-myc and CylinD1 mRNA were significantly decreased at day 8 after induction in the Y27632 group, with significant differences between them (t =20.29, 59.22, 2.90, 39.59, 5.32, 10.14; all at P<0.05), and the relative expression of K18 mRNA in the two groups was not significantly changed(t=1.38, P>0.05). The ESCs derived epithelium and constructed under hypoxia-normoxia culture condition showed more obvious stratification and tighter cell arrangement in comparison with those cells cultured in consistent hypoxia culture condition or normoxia culture condition.Epithelial markers Pan-CK and K18 as well as epithelial progenitor cell markers p63 and K14 expressed in the whole cell layers of the ESCs derived epithelium constructed under hypoxia-normoxia culture condition.
ConclusionsThe addition of Y27632 enhances the proliferation ability of H1 derived epithelial cells and actives Notch signaling pathway and inhibits Wnt signaling pathway.The culture and construction in the expansion medium with Y27632 under the hypoxia-normoxia culture condition can promote the stratification of H1 derived engineered corneal epithelium.
Key words:
Embryonic stem cells; Engineered corneal epithlium; Rock inhibitor; Hypoxia-normoxia culture
Contributor Information
Qu Yangluowa
Eye Institute of Xiamen University, Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Medical College of Xiamen University, Xiamen 361102, China
Ou Shangkun
Eye Institute of Xiamen University, Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Medical College of Xiamen University, Xiamen 361102, China
Liu Tingting
Eye Institute of Xiamen University, Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Medical College of Xiamen University, Xiamen 361102, China
Zhang Liying
Eye Institute of Xiamen University, Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Medical College of Xiamen University, Xiamen 361102, China
Zou Dulei
Eye Institute of Xiamen University, Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Medical College of Xiamen University, Xiamen 361102, China
Li Juan
Eye Institute of Xiamen University, Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Medical College of Xiamen University, Xiamen 361102, China
He Hui
Eye Institute of Xiamen University, Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Medical College of Xiamen University, Xiamen 361102, China
Jia Changkai
Eye Institute of Xiamen University, Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Medical College of Xiamen University, Xiamen 361102, China
Zuo Chengyou
Eye Institute of Xiamen University, Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Medical College of Xiamen University, Xiamen 361102, China
Zhang Minjie
Eye Institute of Xiamen University, Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Medical College of Xiamen University, Xiamen 361102, China
He Xin
Eye Institute of Xiamen University, Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Medical College of Xiamen University, Xiamen 361102, China
Liu Zuguo
Eye Institute of Xiamen University, Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Medical College of Xiamen University, Xiang'an Hospital Affiliated to Xiamen University, Xiamen 361102, China
Li Wei
Eye Institute of Xiamen University, Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Medical College of Xiamen University, Xiang'an Hospital Affiliated to Xiamen University, Xiamen 361102, China
