Lentiviral transfection of pigment epithelial derived factor gene into human umbilical cord mesenchymal stem cells_Chinese Journal of Ocular Fundus Diseases

Authors：

Duan Hongtao ,  Chen Song , Dong Meng , Wang Yuexin , Kong Jiahui , Song Jian , Li Zedong , Wang Jiming

Tianjin Eye Hospital，Clinical College of Ophthalmology of Tianjin Medical University, Tianjin Key Laboratory of Ophthalmology and Vision Science, Tianjin Institute of Ophthalmology, Tianjin 300020, China;

Corresponding author：

Chen Song, Email: chensong9999@126.com

Keywords：

Vascular endothelial growth factors; Mesenchymal stem cells; Lentivirus infections; Cells, cultured

DOI：

10.3760/cma.j.issn.1005-1015.2017.06.016

Video：

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Objective To build the lentiviral vectors of pigment epithelial derived factor (PEDF) gene, and investigate their expression in human umbilical cord mesenchymal stem cells (hUCMSCs). Methods The PEDF lentiviral vectors (LV-PEDF) were built by DNA recombination and confirmed by DNA sequencing. hUCMSCs were transfected by LV-PEDF with MOI 10, 30, 50, respectively. The transfection efficiency was observed under fluorescence microscope. Cell immunofluorescence, immunocytochemistry and real-time PCR methods were used for detecting the expression of PEDF and VEGF. Results The PEDF cDNA was sub-cloned into pCDH-CMV-MCS-EF1-copGFP vector successfully. DNA sequencing analysis confirmed that PEDF gene sequence was exactly the same with that reported in GenBank. pCDH-PEDF infected cells could show green fluorescence under fluorescence microscope. The transfection efficiency was 72.1% in PEDF-MSCs. Immunofluorescence and immunochemical staining confirmed that PEDF protein was overexpressed in hUCMSCs. The relative expression of PEDF mRNA in experimental group and control group was (0.170±0.028) and (0.015±0.007) respectively by RT-PCR, the difference was statistically significant (P＜0.001). The relative expression levels of VEGF mRNA in the two groups were (0.265±0.022) and (0.285±0.049), respectively, with no significant difference (P＞0.05). Conclusions We successfully built a lentivirus vector carrying PEDF gene and obtained hUCMSCs with overexpressed PEDF.

Citation： Duan Hongtao, Chen Song, Dong Meng, Wang Yuexin, Kong Jiahui, Song Jian, Li Zedong, Wang Jiming. Lentiviral transfection of pigment epithelial derived factor gene into human umbilical cord mesenchymal stem cells. Chinese Journal of Ocular Fundus Diseases, 2017, 33(6): 621-625. doi: 10.3760/cma.j.issn.1005-1015.2017.06.016 Copy

1.	Fong CY, Subramanian A, Biswas A, et al. Derivation efficiency, cell proliferation, freeze-thaw survival, stem-cell properties and differentiation of human Wharton's jelly stem cells[J]. Reprod Biomed Online, 2010, 21(3): 391-401. DOI: 10.1016/j.rbmo.2010.04.010.
2.	Akiyama T, Dass CR, Shinoda Y, et al. PEDF regulates osteoclasts via osteoprotegerin and RANKL[J]. Biochem Biophys Res Commun, 2010, 391(1): 789-794. DOI: 10.1016/j. bbrc. 2009.11.139.
3.	Sanchez-Sanchez F, Aroca-Aguilar JD, Segura I, et al. Expression and purification of functional recombinant human pigment epithelium-derived factor (PEDF) secreted by the yeast Pichia pastoris[J]. J Biotechnol, 2008, 134(1-2): 193-201. DOI: 10.1016/j.jbiotec.2008.01. 005.
4.	Broadhead ML, Dass CR, Choong PF. In vitro and in vivo biological activity of PEDF against a range of tumors[J]. Expert Opin Ther Targets, 2009, 13(12): 1429-1438. DOI: 10.1517/14728220903307475.
5.	Liao W, Xie J, Zhong J, et al. Therapeutic effect of human umbilical cord multipotent mesenchymal stromal cells in a rat model of stroke[J]. Transplantation, 2009, 87(3): 350-359. DOI: 10.1097/TP.0b013e318195742e.
6.	Huang C, Zhang J, Ao M, et al. Combination of retinal pigment epithelium cell-conditioned medium and photoreceptor outer segments stimulate mesenchymal stem cell differentiation toward a functional retinal pigment epithelium cell phenotype[J]. J Cell Biochem, 2012, 113(2): 590-598. DOI: 10.1002/jcb.23383.
7.	Yang Z, Li K, Yan X, et al. Amelioration of diabetic retinopathy by engrafted human adipose-derived mesenchymal stem cells in streptozotocin diabetic rats[J]. Graefe's Arch Clin Exp Ophthalmol, 2010, 248(10): 1415-1422. DOI: 10.1007/s00417-010-1384-z.
8.	Scalinci SZ, Scorolli L, Corradetti G, et al. Potential role of intravitreal human placental stem cell implants in inhibiting progression of diabetic retinopathy in type 2 diabetes: neuroprotective growth factors in the vitreous[J]. Clin Ophthalmol, 2011, 5: 691-696. DOI: 10.2147/OPTH.S21161.
9.	Jin W, Xing YQ, Yang AH. Epidermal growth factor promotes the differentiation of stem cells derived from human umbilical cord blood into neuron-like cells via taurine induction in vitro[J]. In Vitro Cell Dev Biol Anim, 2009, 45(7): 321-327. DOI: 10.1007/s11626-009-9184-7.
10.	Park HY, Kim JH, Sun KH, et al. Stem cell-based delivery of brain-derived neurotrophic factor gene in the rat retina[J]. Brain Res, 2012, 1469: 10-23. DOI: 10.1016/j.brainres.2012.06.006.
11.	Huo DM, Dong FT, Yu WH, et al. Differentiation of mesenchymal stem cell in the microenviroment of retinitis pigmentosa[J]. Int J Ophthalmol, 2010, 3(3): 216-219. DOI: 10.3980/j.issn.2222-3959.2010.03.08.
12.	Tan ML, Choong PF, Dass CR. Anti-chondrosarcoma effects of PEDF mediated via molecules important to apoptosis, cell cycling, adhesion and invasion[J]. Biochem Biophys Res Commun, 2010, 398(4): 613-618. DOI: 10.1016/j.bbrc.2010.05.098.
13.	Boehm BO, Lang G, Volpert O, et al. Low content of the natural ocular anti-angiogenic agent pigment epithelium-derived factor (PEDF) in aqueous humor predicts progression of diabetic retinopathy[J]. Diabetologia, 2003, 46(3): 394-400. DOI: 10.1007/s00125-003-1040-9.
14.	Yokoi M, Yamagishi S, Saito A, et al. Positive association of pigment epithelium-derived factor with total antioxidant capacity in the vitreous fluid of patients with proliferative diabetic retinopathy[J]. Br J Ophthalmol, 2007, 91(7): 885-887. DOI: 10.1136/bjo.2006.110890.
15.	Liu F, Akiyama Y, Tai S, et al. Changes in the expression of CD106, osteogenic genes, and transcription factors involved in the osteogenic differentiation of human bone marrow mesenchymal stem cells[J]. J Bone Miner Metab, 2008, 26(4): 312-320. DOI: 10.1007/s00774-007-0842-0.
16.	Kakinuma S, Tanaka Y, Chinzei R, et al. Human umbilical cord blood as a source of transplantable hepatic progenitor cells[J]. Stem Cells, 2003, 21(2): 217-227. DOI: 10.1634/stemcells.21-2-217.
17.	Naldini L, Blomer U, Gallay P, et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector[J]. Science, 1996, 272(5259): 263-267.
18.	胡明, 马远征, 李大伟, 等. 慢病毒介导的RNA干扰技术构建人胚椎间盘髓核上皮膜蛋白-1基因低表达细胞模型[J]. 中国骨伤, 2012, 25(10): 842-845. DOI: 10.3969/j.issn.1003-0034.2012.10.014.Hu M, Ma YZ, Li DW, et al. Construction of Epithelia Membrane Protein 1 gene-deficient in human fetal nucleus pulposus cell model by lentivirus-mediated RNA interference[J]. China J Onhop Trauma, 2012, 25(10): 842-845. DOI: 10.3969/j.issn.1003-0034.2012.10.014.
19.	Ji F, Duan HG, Zheng CQ, et al. Comparison of chloromethyl-dialkylcarbocyanine and green fluorescent protein for labeling human umbilical mesenchymal stem cells[J]. Biotechnol Lett, 2015, 37(2): 437-447. DOI: 10.1007/s10529-014-1692-1.
20.	Fan W, Crawford R, Xiao Y. The ratio of VEGF/PEDF expression in bone marrow mesenchymal stem cells regulates neovascularization[J]. Differentiation, 2011, 81(3): 181-191. DOI: 10.1016/j.diff.2010.12.003.
21.	Otsu K, Das S, Houser S D, et al. Concentration-dependent inhibition of angiogenesis by mesenchymal stem cells[J]. Blood, 2009, 113(18): 4197-4205. DOI: 10.1182/blood-2008-09-176198.

1. Fong CY, Subramanian A, Biswas A, et al. Derivation efficiency, cell proliferation, freeze-thaw survival, stem-cell properties and differentiation of human Wharton's jelly stem cells[J]. Reprod Biomed Online, 2010, 21(3): 391-401. DOI: 10.1016/j.rbmo.2010.04.010.
2. Akiyama T, Dass CR, Shinoda Y, et al. PEDF regulates osteoclasts via osteoprotegerin and RANKL[J]. Biochem Biophys Res Commun, 2010, 391(1): 789-794. DOI: 10.1016/j. bbrc. 2009.11.139.
3. Sanchez-Sanchez F, Aroca-Aguilar JD, Segura I, et al. Expression and purification of functional recombinant human pigment epithelium-derived factor (PEDF) secreted by the yeast Pichia pastoris[J]. J Biotechnol, 2008, 134(1-2): 193-201. DOI: 10.1016/j.jbiotec.2008.01. 005.
4. Broadhead ML, Dass CR, Choong PF. In vitro and in vivo biological activity of PEDF against a range of tumors[J]. Expert Opin Ther Targets, 2009, 13(12): 1429-1438. DOI: 10.1517/14728220903307475.
5. Liao W, Xie J, Zhong J, et al. Therapeutic effect of human umbilical cord multipotent mesenchymal stromal cells in a rat model of stroke[J]. Transplantation, 2009, 87(3): 350-359. DOI: 10.1097/TP.0b013e318195742e.
6. Huang C, Zhang J, Ao M, et al. Combination of retinal pigment epithelium cell-conditioned medium and photoreceptor outer segments stimulate mesenchymal stem cell differentiation toward a functional retinal pigment epithelium cell phenotype[J]. J Cell Biochem, 2012, 113(2): 590-598. DOI: 10.1002/jcb.23383.
7. Yang Z, Li K, Yan X, et al. Amelioration of diabetic retinopathy by engrafted human adipose-derived mesenchymal stem cells in streptozotocin diabetic rats[J]. Graefe's Arch Clin Exp Ophthalmol, 2010, 248(10): 1415-1422. DOI: 10.1007/s00417-010-1384-z.
8. Scalinci SZ, Scorolli L, Corradetti G, et al. Potential role of intravitreal human placental stem cell implants in inhibiting progression of diabetic retinopathy in type 2 diabetes: neuroprotective growth factors in the vitreous[J]. Clin Ophthalmol, 2011, 5: 691-696. DOI: 10.2147/OPTH.S21161.
9. Jin W, Xing YQ, Yang AH. Epidermal growth factor promotes the differentiation of stem cells derived from human umbilical cord blood into neuron-like cells via taurine induction in vitro[J]. In Vitro Cell Dev Biol Anim, 2009, 45(7): 321-327. DOI: 10.1007/s11626-009-9184-7.
10. Park HY, Kim JH, Sun KH, et al. Stem cell-based delivery of brain-derived neurotrophic factor gene in the rat retina[J]. Brain Res, 2012, 1469: 10-23. DOI: 10.1016/j.brainres.2012.06.006.
11. Huo DM, Dong FT, Yu WH, et al. Differentiation of mesenchymal stem cell in the microenviroment of retinitis pigmentosa[J]. Int J Ophthalmol, 2010, 3(3): 216-219. DOI: 10.3980/j.issn.2222-3959.2010.03.08.
12. Tan ML, Choong PF, Dass CR. Anti-chondrosarcoma effects of PEDF mediated via molecules important to apoptosis, cell cycling, adhesion and invasion[J]. Biochem Biophys Res Commun, 2010, 398(4): 613-618. DOI: 10.1016/j.bbrc.2010.05.098.
13. Boehm BO, Lang G, Volpert O, et al. Low content of the natural ocular anti-angiogenic agent pigment epithelium-derived factor (PEDF) in aqueous humor predicts progression of diabetic retinopathy[J]. Diabetologia, 2003, 46(3): 394-400. DOI: 10.1007/s00125-003-1040-9.
14. Yokoi M, Yamagishi S, Saito A, et al. Positive association of pigment epithelium-derived factor with total antioxidant capacity in the vitreous fluid of patients with proliferative diabetic retinopathy[J]. Br J Ophthalmol, 2007, 91(7): 885-887. DOI: 10.1136/bjo.2006.110890.
15. Liu F, Akiyama Y, Tai S, et al. Changes in the expression of CD106, osteogenic genes, and transcription factors involved in the osteogenic differentiation of human bone marrow mesenchymal stem cells[J]. J Bone Miner Metab, 2008, 26(4): 312-320. DOI: 10.1007/s00774-007-0842-0.
16. Kakinuma S, Tanaka Y, Chinzei R, et al. Human umbilical cord blood as a source of transplantable hepatic progenitor cells[J]. Stem Cells, 2003, 21(2): 217-227. DOI: 10.1634/stemcells.21-2-217.
17. Naldini L, Blomer U, Gallay P, et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector[J]. Science, 1996, 272(5259): 263-267.
18. 胡明, 马远征, 李大伟, 等. 慢病毒介导的RNA干扰技术构建人胚椎间盘髓核上皮膜蛋白-1基因低表达细胞模型[J]. 中国骨伤, 2012, 25(10): 842-845. DOI: 10.3969/j.issn.1003-0034.2012.10.014.Hu M, Ma YZ, Li DW, et al. Construction of Epithelia Membrane Protein 1 gene-deficient in human fetal nucleus pulposus cell model by lentivirus-mediated RNA interference[J]. China J Onhop Trauma, 2012, 25(10): 842-845. DOI: 10.3969/j.issn.1003-0034.2012.10.014.
19. Ji F, Duan HG, Zheng CQ, et al. Comparison of chloromethyl-dialkylcarbocyanine and green fluorescent protein for labeling human umbilical mesenchymal stem cells[J]. Biotechnol Lett, 2015, 37(2): 437-447. DOI: 10.1007/s10529-014-1692-1.
20. Fan W, Crawford R, Xiao Y. The ratio of VEGF/PEDF expression in bone marrow mesenchymal stem cells regulates neovascularization[J]. Differentiation, 2011, 81(3): 181-191. DOI: 10.1016/j.diff.2010.12.003.
21. Otsu K, Das S, Houser S D, et al. Concentration-dependent inhibition of angiogenesis by mesenchymal stem cells[J]. Blood, 2009, 113(18): 4197-4205. DOI: 10.1182/blood-2008-09-176198.

Chinese Journal of Ocular Fundus Diseases

Lentiviral transfection of pigment epithelial derived factor gene into human umbilical cord mesenchymal stem cells

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