DIFFERENTIATION OF ADIPOSE-DERIVED MESENCHYMAL STEM CELLS AFTER TRANSFECTION WITH Pax6 GENE_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

LIUWeihua ¹ ,  LIUYilun ^1,2 , LIUHairong ¹ , LUOYonghui ¹ , XUJinmei ¹

1. Experiment Research Center, the First Affiliated Hospital of Chengdu Medical College, Chengdu Sichuan, 610500, P. R. China;
2. Department of Burn and Plastic Surgery, the First Affiliated Hospital of Chengdu Medical College, Chengdu Sichuan, 610500, P. R. China;

Corresponding author：

LIUYilun, Email: 382818934@qq.com

Keywords：

Tissue engineered cornea; Adipose-derived mesenchymal stem cells; Pax6 gene; Induction and differentiation; Corneal epithelium-like cells; Mice

DOI：

10.7507/1002-1892.20140221

Video：

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Objective To investigate the feasibility of adipose-derived mesenchymal stem cells (ADMSCs) differentiating into corneal epithelium-like cells after transfection with Pax6 gene. Methods The adipose tissue from bilateral inguinal of healthy C57BL/6 mice (5-6 weeks old) was used to isolate and culture ADMSCs.The 3rd passage ADMSCs were subjected to treatments of non-transfection (group A),pcDNA3.1 empty vector transfection (group B),and recombinant plasmid of pcDNA3.1-Pax6 transfection (group C),respectively.At 48 hours after transfection,the cells in groups B and C were selected with G418.The cell morphology changes were observed under the inverted microscope.Pax6 protein and level of corneal epithelial cells specific molecular-cytokeratin 12 (CK-12) were measured by Western blot.Real-time fluorescence quantitative PCR was applied to measure the mRNA expression of CK-12. Results No morphology change was observed in groups A and B.Two different cell clones were found in group C.No.1 selected clone showed a flagstone-like appearance that was similar to that of corneal epithelial cells;No.2 selected clone showed a net-like appearance,with 3-7 cell processes.The Western blot results showed the Pax6 protein expression in 2 clones of group C,but no expression in groups A and B; and CK-12 protein expression was only observed in No.1 selected clone of group C,and no expression in the others.The real-time fluorescence quantitative PCR results showed that the CK-12 mRNA expression level of No.1 selected clone of group C was 8.64±0.73,which was significantly higher than that of No.2 selected clone of group C (0.55±0.42),group B (1.36±0.40),and group A (1.00±0.00) (P<0.05),and there was no significant difference among groups A,B and No.2 selected clone of group C (P>0.05). Conclusion Pax6 gene transfection could induce differentiation of ADMSCs into corneal epithelium-like cells which express CK-12 at both the mRNA and protein levels.This result provides a promising strategy of generating corneal epithelilcm-like cells for construction of tissue engineered cornea.

Citation： LIUWeihua, LIUYilun, LIUHairong, LUOYonghui, XUJinmei. DIFFERENTIATION OF ADIPOSE-DERIVED MESENCHYMAL STEM CELLS AFTER TRANSFECTION WITH Pax6 GENE. Chinese Journal of Reparative and Reconstructive Surgery, 2014, 28(8): 1004-1008. doi: 10.7507/1002-1892.20140221 Copy

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12.	Davis J,Duncan MK,Robison WG Jr,et al.Requirement for Pax6 in corneal morphogenesis:a role in adhesion.J Cell Sci,2003,116(Pt 11):2157-2167.
13.	Shubham K,Mishra R.Pax6 interacts with SPARC and TGF-β in murine eyes.Mol Vis,2012,18:951-956.
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15.	Davis-Silberman N,Kalich T,Oron-Karni V,et al.Genetic dissection of Pax6 dosage requirements in the developing mouse eye.Hum Mol Genet,2005,14(15):2265-2276.
16.	García-Villegas R,Escamilla J,Sánchez-Guzmán E,et al.Pax-6 is expressed early in the differentiation of a corneal epithelial model system.J Cell Physiol,2009,220(2):348-356.
17.	Rezanejad H,Soheili ZS,Haddad F,et al.In vitro differentiation of adipose-tissue-derived mesenchymal stem cells into neural retinal cells through expression of human PAX6 (5a) gene.Cell Tissue Res,2014,356(1):65-75.
18.	Huang P,He Z,Ji S,et al.Induction of functional hepatocyte-like cells from mouse fibroblasts by defined factors.Nature,2011,475(7356):386-389.
19.	Wang Q,Ye L,Liu H,et al.Reprogramming of bone marrow-derived mesenchymal stem cells into functional insulin-producing cells by chemical regimen.Am J Stem Cells,2012,1(2):128-137.
20.	Fuchs E,Tumbar T,Guasch G.Socializing with the neighbors:stem cells and their niche.Cell,2004,116(6):769-778.
21.	Xie T,Li L.Stem cells and their niche:an inseparable relationship.Development,2007,134(11):2001-2006.

1. Wang HY,Wei RH,Zhao SZ.Evaluation of corneal cell growth on tissue engineering materials as artificial cornea scaffolds.Int J Ophthalmol,2013,6(6):873-878.
2. Elisseeff J,Madrid MG,Lu Q,et al.Future perspectives for regenerative medicine in ophthalmology.Middle East Afr J Ophthalmol,2013,20(1):38-45.
3. 徐舒怡,侯光辉.骨髓间充质干细胞在组织工程角膜方面的研究进展.中华实验眼科杂志,2013,31(2):196-200.
4. Wilson A,Butler PE,Seifalian AM.Adipose-derived stem cells for clinical applications:a review.Cell Prolif,2011,44(1):86-98.
5. Zhu Y,Liu T,Song K,et al.Adipose-derived stem cell:a better stem cell than BMSC.Cell Biochem Funct,2008,26(6):664-675.
6. Ueno H,Kurokawa MS,Kayama M,et al.Experimental transplantation of corneal epithelium-like cells induced by Pax6 gene transfection of mouse embryonic stem cells.Cornea,2007,26(10):1220-1227.
7. Pittenger MF,Mackay AM,Beck SC,et al.Multilineage potential of adult human mesenchymal stem cells.Science,1999,284(5411):143-147.
8. 谢遂亮,杨达胜,毕凌云,等.小鼠脂肪源间充质干细胞的培养鉴定及增强绿色荧光蛋白-慢病毒载体转染.新乡医学院学报,2013,30(11):865-870.
9. Livak KJ,Schmittgen TD.Analysis of relative gene expression data using real-time quantitative PCR and the 2 (-Delta Delta C(T)) method.Methods,2001,25(4):402-408.
10. 徐舒怡,侯光辉,吴静,等.诱导人脐带间充质干细胞分化为角膜上皮样细胞的研究.中华实验眼科杂志,2012,30(10):882-887.
11. 姜廷帅,蔡莉,惠延年,等.大鼠骨髓间充质干细胞体外可诱导分化为角膜上皮细胞.国际眼科杂志,2007,7(2):339-341.
12. Davis J,Duncan MK,Robison WG Jr,et al.Requirement for Pax6 in corneal morphogenesis:a role in adhesion.J Cell Sci,2003,116(Pt 11):2157-2167.
13. Shubham K,Mishra R.Pax6 interacts with SPARC and TGF-β in murine eyes.Mol Vis,2012,18:951-956.
14. Gehring WJ.The evolution of vision.Wiley Interdiscip Rev Dev Biol,2014,3(1):1-40.
15. Davis-Silberman N,Kalich T,Oron-Karni V,et al.Genetic dissection of Pax6 dosage requirements in the developing mouse eye.Hum Mol Genet,2005,14(15):2265-2276.
16. García-Villegas R,Escamilla J,Sánchez-Guzmán E,et al.Pax-6 is expressed early in the differentiation of a corneal epithelial model system.J Cell Physiol,2009,220(2):348-356.
17. Rezanejad H,Soheili ZS,Haddad F,et al.In vitro differentiation of adipose-tissue-derived mesenchymal stem cells into neural retinal cells through expression of human PAX6 (5a) gene.Cell Tissue Res,2014,356(1):65-75.
18. Huang P,He Z,Ji S,et al.Induction of functional hepatocyte-like cells from mouse fibroblasts by defined factors.Nature,2011,475(7356):386-389.
19. Wang Q,Ye L,Liu H,et al.Reprogramming of bone marrow-derived mesenchymal stem cells into functional insulin-producing cells by chemical regimen.Am J Stem Cells,2012,1(2):128-137.
20. Fuchs E,Tumbar T,Guasch G.Socializing with the neighbors:stem cells and their niche.Cell,2004,116(6):769-778.
21. Xie T,Li L.Stem cells and their niche:an inseparable relationship.Development,2007,134(11):2001-2006.

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Chinese Journal of Reparative and Reconstructive Surgery

DIFFERENTIATION OF ADIPOSE-DERIVED MESENCHYMAL STEM CELLS AFTER TRANSFECTION WITH Pax6 GENE

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