Optimal Selection of Cell Transfection Methods for Human Umbilical Cord Mesenchymal Stem Cells In Vivo_CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Authors：

 CHENZheng ,  KUANGQiao-ting , YANGJie , ZHANGQiang

Department of General Surgery, Affiliated Xiangyang Hospital, Hubei Medical College, Xiangyang 441000, Hubei Province, China;

Corresponding author：

KUANGQiao-ting, Email: qqqdkqt@163.com

Keywords：

Stem cells; Transplantation; Transfection; In vivo

DOI：

10.7507/1007-9424.20160242

Video：

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Objective To evaluate the most efficient method for transfection of human umbilical cord mesenchymal stem cells (HUMCSs) in vivo. Methods HUCMSCs were isolated from human umbilical cord and cultured, which were labelled by PKH26 and lentivirus-GFP, then were observed by using a fluorescence microscope. Sixty SD rats were randomly divided into PKH26 transfection group and lentivirus-GFP transfection group. The right hepatic lobe of rat was resected, then the transfected stem cells were injected into portal vein. The rats were sacrificed on day 3, 8, and 13 after transfection. The liver specimens were observed by using a fluorescence microscope. Flow cytometry was used to evaluate the percentage of transfected stem cells and the apoptotic stem cells. Results The third generation of HUCMSCs labelled by PKH26 and lentivirus-GFP were spindle shaped. PKH26 red dye was evenly distributed in the cell membrane of HUCMSCs and could be clearly labelled. The HUCMSCs labelled by lentivirus-GFP were green fluorescence under the fluorescence microscope, and it was clear and stable. The HUCMSCs were clear and could be clearly distinguished on day 3 after transfection by two methods in vivo. As the time went by, red was faded and blurred, then was gradually disappeared on day 13 after transfection in the HUCMSCs stansfected by PKH26; but the color in the HUCMSCs stansfected by lentivirus-GFP were clear at all the time points. The transfection rate of the lentivirus-GFP was significantly higher that that of the PKH26 (P < 0.05), the rate of apoptotic stem cells had no significant differences at all the time points between these two groups (P > 0.05). Conclusion Lentivirus-GFP transfection is a higher efficient method for stem cell labelling in vivo, it could be used to observe transplantation cells for a long time in future.

Citation： CHENZheng, KUANGQiao-ting, YANGJie, ZHANGQiang. Optimal Selection of Cell Transfection Methods for Human Umbilical Cord Mesenchymal Stem Cells In Vivo. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2016, 23(8): 922-925. doi: 10.7507/1007-9424.20160242 Copy

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2.	Yagi K, Kojima M, Oyagi S, et al. Application of mesenchymal stem cells to liver regenerative medicine. Yakugaku Zasshi, 2008, 128(1):3-9.
3.	Forraz N, McGuckin CP. The umbilical cord:a rich and ethical stem cell source to advance regenerative medicine. Cell Prolif, 2011, 44 Suppl 1:60-69.
4.	Cai J, Li W, Su H, et al. Generation of human induced pluripotent stem cells from umbilical cord matrix and amniotic membrane mesenchymal cells. J Biol Chem, 2010, 285(15):11227-11234.
5.	Tipnis S, Viswanathan C, Majumdar AS. Immunosuppressive properties of human umbilical cord-derived mesenchymal stem cells:role of B7-H1 and IDO. Immunol Cell Biol, 2010, 88(8):795-806.
6.	Weiss ML, Troyer DL. Stem cells in the umbilical cord. Stem Cell Rev, 2006, 2(2):155-162.
7.	Wang HS, Hung SC, Peng ST, et al. Mesenchymal stem cells in the Wharton, s jelly of the human umbilical cord. Stem Cells, 2004, 22(7):1330-1337.
8.	Lanfranchi A, Porta F, Chirico G. Stem cells and the frontiers of neonatology. Early Hum Dev, 2009, 85(10 Suppl):S15-S18.
9.	Pai M, Spalding D, Xi F, et al. Autologous bone marrow stem cells in the treatment of chronic liver disease. Int J Hepatol, 2012, 2012:307165.
10.	Sakai Y, Kaneko S. Mesenchymal stem cell therapy on murine model of nonalcoholic steatohepatitis. Methods Mol Biol, 2012, 826:217-223.
11.	Gilchrist ES, Plevris JN. Bone marrow-derived stem cells in liver repair:10 years down the line. Liver Transpl, 2010, 16(2):118-129.
12.	Shi M, Liu ZW, Wang FS. Immunomodulatory properties and therapeutic application of mesenchymal stem cells. Clin Exp Immunol, 2011, 164(1):1-8.
13.	滕军, 居胜红.开展干细胞标记和MR活体示踪研究推进分子影像学发展.中华放射学杂志, 2006, 40(2):117-119.
14.	Dickson EL, Vogel RI, Bliss RL, et al. Multiple-type human papillomavirus (HPV) infections:a cross-sectional analysis of the prevalence of specific types in 309 000 women referred for HPV testing at the time of cervical cytology. Int J Gynecol Cancer, 2013, 23(7):1295-1302.
15.	Dickson EL, Vogel RI, Geller MA, et al. Cervical cytology and multiple type HPV infection:a study of 8 182 women ages 31-65. Gynecol Oncol, 2014, 133(3):405-408.
16.	Baksh D, Yao R, Tuan RS. Comparison of proliferative and multilineage differentiation potential of human mesenchymal stem cells derived from umbilical cord and bone marrow. Stem Cells, 2007, 25 (6):1384-1392.
17.	Dominici M, Le Blanc K, Mueller I, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy, 2006, 8(4):315-317.
18.	Fan CG, Zhang QJ, Zhou JR. Therapeutic potentials of mesenchymal stem cells derived from human umbilical cord. Stem Cell Rev, 2011, 7(1):195-207.
19.	Liang XJ, Chen XJ, Yang DH, et al. Differentiation of human umbilical cord mesenchymal stem cells into hepatocyte-like cells by hTERT gene transfection in vitro. Cell Biol Int, 2012, 36(2):215-221.
20.	Ouyang HW, Cao T, Zou XH, et al. Mesenchymal stem cell sheets revitalize nonviable dense grafts:implications for repair of large-bone and tendon defects. Transplantation, 2006, 82(2):170-174.
21.	Bulte JW, Douglas T, Witwer B, et al. Magnetodendrimers allow endosomal magnetic labeling and in vivo tracking of stem cells. Nat Biotechnol, 2001, 19(12):1141-1147.
22.	Ji KH, Xiong J, Fan LX, et al. Rat marrow-derived multipotent adult progenitor cells differentiate into skin epidermal cells in vivo. J Dermatol, 2009, 36(7):403-409.
23.	Wallace PK, Muirhead KA. Cell tracking 2007:a proliferation of probes and applications. Immunol Invest, 2007, 36(5-6):527-561.
24.	Haas SJ, Bauer P, Rolfs A, et al. Immunocytochemical characterization of in vitro PKH26-labelled and intracerebrally transplanted neonatal cells. Acta Histochem, 2000, 102(3):273-280.
25.	Fox D, Kouris GJ, Blumofe KA, et al. Optimizing fluorescent labeling of endothelial cells for tracking during long-term studies of autologous transplantation. J Surg Res, 1999, 86(1):9-16.
26.	Bauer G, Dao MA, Case SS, et al. in vivo biosafety model to assess the risk of adverse events from retroviral and lentiviral vectors. Mol Ther, 2008, 16(7):1308-1315.
27.	Parolini O, Alviano F, Bagnara GP, et al. Concise review:isolation and characterization of cells from human term placenta:outcome of the first international Workshop on Placenta Derived Stem Cells. Stem Cells, 2008, 26(2):300-311.
28.	Nakamizo A, Marini F, Amano T, et al. Human bone marrow-derived mesenchymal stem cells in the treatment of gliomas. Cancer Res, 2005, 65(8):3307-3318.

1. Uranio MF, Valentini L, Lange-Consiglio A, et al. Isolation, proliferation, cytogenetic, and molecular characterization and in vitro differentiation potency of canine stem cells from foetal adnexa:A comparative study of amniotic fluid, amnion, and umbilical cord matrix. Mol Reprod Dev, 2011, 78(5):361-373.
2. Yagi K, Kojima M, Oyagi S, et al. Application of mesenchymal stem cells to liver regenerative medicine. Yakugaku Zasshi, 2008, 128(1):3-9.
3. Forraz N, McGuckin CP. The umbilical cord:a rich and ethical stem cell source to advance regenerative medicine. Cell Prolif, 2011, 44 Suppl 1:60-69.
4. Cai J, Li W, Su H, et al. Generation of human induced pluripotent stem cells from umbilical cord matrix and amniotic membrane mesenchymal cells. J Biol Chem, 2010, 285(15):11227-11234.
5. Tipnis S, Viswanathan C, Majumdar AS. Immunosuppressive properties of human umbilical cord-derived mesenchymal stem cells:role of B7-H1 and IDO. Immunol Cell Biol, 2010, 88(8):795-806.
6. Weiss ML, Troyer DL. Stem cells in the umbilical cord. Stem Cell Rev, 2006, 2(2):155-162.
7. Wang HS, Hung SC, Peng ST, et al. Mesenchymal stem cells in the Wharton, s jelly of the human umbilical cord. Stem Cells, 2004, 22(7):1330-1337.
8. Lanfranchi A, Porta F, Chirico G. Stem cells and the frontiers of neonatology. Early Hum Dev, 2009, 85(10 Suppl):S15-S18.
9. Pai M, Spalding D, Xi F, et al. Autologous bone marrow stem cells in the treatment of chronic liver disease. Int J Hepatol, 2012, 2012:307165.
10. Sakai Y, Kaneko S. Mesenchymal stem cell therapy on murine model of nonalcoholic steatohepatitis. Methods Mol Biol, 2012, 826:217-223.
11. Gilchrist ES, Plevris JN. Bone marrow-derived stem cells in liver repair:10 years down the line. Liver Transpl, 2010, 16(2):118-129.
12. Shi M, Liu ZW, Wang FS. Immunomodulatory properties and therapeutic application of mesenchymal stem cells. Clin Exp Immunol, 2011, 164(1):1-8.
13. 滕军, 居胜红.开展干细胞标记和MR活体示踪研究推进分子影像学发展.中华放射学杂志, 2006, 40(2):117-119.
14. Dickson EL, Vogel RI, Bliss RL, et al. Multiple-type human papillomavirus (HPV) infections:a cross-sectional analysis of the prevalence of specific types in 309 000 women referred for HPV testing at the time of cervical cytology. Int J Gynecol Cancer, 2013, 23(7):1295-1302.
15. Dickson EL, Vogel RI, Geller MA, et al. Cervical cytology and multiple type HPV infection:a study of 8 182 women ages 31-65. Gynecol Oncol, 2014, 133(3):405-408.
16. Baksh D, Yao R, Tuan RS. Comparison of proliferative and multilineage differentiation potential of human mesenchymal stem cells derived from umbilical cord and bone marrow. Stem Cells, 2007, 25 (6):1384-1392.
17. Dominici M, Le Blanc K, Mueller I, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy, 2006, 8(4):315-317.
18. Fan CG, Zhang QJ, Zhou JR. Therapeutic potentials of mesenchymal stem cells derived from human umbilical cord. Stem Cell Rev, 2011, 7(1):195-207.
19. Liang XJ, Chen XJ, Yang DH, et al. Differentiation of human umbilical cord mesenchymal stem cells into hepatocyte-like cells by hTERT gene transfection in vitro. Cell Biol Int, 2012, 36(2):215-221.
20. Ouyang HW, Cao T, Zou XH, et al. Mesenchymal stem cell sheets revitalize nonviable dense grafts:implications for repair of large-bone and tendon defects. Transplantation, 2006, 82(2):170-174.
21. Bulte JW, Douglas T, Witwer B, et al. Magnetodendrimers allow endosomal magnetic labeling and in vivo tracking of stem cells. Nat Biotechnol, 2001, 19(12):1141-1147.
22. Ji KH, Xiong J, Fan LX, et al. Rat marrow-derived multipotent adult progenitor cells differentiate into skin epidermal cells in vivo. J Dermatol, 2009, 36(7):403-409.
23. Wallace PK, Muirhead KA. Cell tracking 2007:a proliferation of probes and applications. Immunol Invest, 2007, 36(5-6):527-561.
24. Haas SJ, Bauer P, Rolfs A, et al. Immunocytochemical characterization of in vitro PKH26-labelled and intracerebrally transplanted neonatal cells. Acta Histochem, 2000, 102(3):273-280.
25. Fox D, Kouris GJ, Blumofe KA, et al. Optimizing fluorescent labeling of endothelial cells for tracking during long-term studies of autologous transplantation. J Surg Res, 1999, 86(1):9-16.
26. Bauer G, Dao MA, Case SS, et al. in vivo biosafety model to assess the risk of adverse events from retroviral and lentiviral vectors. Mol Ther, 2008, 16(7):1308-1315.
27. Parolini O, Alviano F, Bagnara GP, et al. Concise review:isolation and characterization of cells from human term placenta:outcome of the first international Workshop on Placenta Derived Stem Cells. Stem Cells, 2008, 26(2):300-311.
28. Nakamizo A, Marini F, Amano T, et al. Human bone marrow-derived mesenchymal stem cells in the treatment of gliomas. Cancer Res, 2005, 65(8):3307-3318.

CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Optimal Selection of Cell Transfection Methods for Human Umbilical Cord Mesenchymal Stem Cells In Vivo

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