PROTECTIVE EFFECT OF BONE MARROW MESENCHYMAL STEM CELLS ON ISLETS FROM HYPOXIA/REOXYGENATION-INDUCED INJURY_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

ZHAO Jiuming , HE Sirong , WANG Chengshi , LIU Jingping , LI Shengfu , CHEN Younan , LU Yanrong , CHENG Jingqiu.

Ministry of Health, Regenerative Medicine Research Center, West China Hospital, Sichuan University, Chengdu Sichuan, 610041, P.R.China. Corresponding author: CHENG Jingqiu, E-mail: chengjingqiu2013@gmail.com;

Corresponding author：

Keywords：

Bone marrow mesenchymal stem cells Islet; Hypoxia/reoxygenation; Rhesus monkey; Porcine

DOI：

10.7507/1002-1892.20130201

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Objective To study the protective effects of bone marrow mesenchymal stem cells (BMSCs) of rhesus monkeys on porcine islets from hypoxia/reoxygenation (H/R)-induced injury. Methods BMSCs were isolated and cultured from the marrow of 5 adult rhesus monkeys (weighing, 6-10 kg) by adherent monocytes. Islets were isolated and purified from the pancreas of 5 neonatal porcine (3-5 days old) by collagenase V digestion method, and were cultured with or without BMSCs, and exposed to hypoxia (1%O2) for 12 hours and reoxygenation for 24 or 48 hours, respectively. The experiment was divided into 4 groups: normal islet group (group A), normal islet + BMSCs group (Group B), H/R islet group (group C), and H/R islet + BMSCs group (group D). The survival rate of islets was calculated by fluorescein diacetate/propidium iodide (PI) staining. The viability of the islet cells was detected by cell counting kit 8. Apoptotic rate of islet cells was tested using Annexin V-FITC/PI labeling and flow cytometry. The stimulation index (SI) of islet function was analyzed by glucose-stimulated insulin secretion assay. Results The islet cell cluster of group C was more dispersed than that of groups A and B, and group C had more death cells; and the islet cell cluster of group D was more complete and the survival rate was higher than those of group C. The survival rate of islet was 90.2% ± 9.1%, 88.3% ± 5.9%, 52.3% ± 12.1%, and 71.4% ± 11.5% in groups A, B, C, and D respectively, it was significantly lower in groups C and D than in groups A and B (P lt; 0.05), but it was significantly higher in group D than in group C (P lt; 0.05). After coculture of BMSCs and islet at the ratio of 1 ∶ 10 and 1 ∶ 20 in group D, the viability of islet cells was significantly higher than that in group C (P lt; 0.05). The apoptotic rate was 27.1% ± 3.2%, 24.0% ± 1.0%, 64.3% ± 1.8%, and 46.2% ± 1.4% in groups A, B, C, and D respectively, it was significantly higher in groups C and D than that in groups A and B (P lt; 0.05), but it was significantly lower in group D than in group C (P lt; 0.05). There was no significant difference in SI between groups A and B at each time point (P gt; 0.05), but it was significantly lower in group C than in groups A and B (P lt; 0.05); and it was significantly higher in group D than in group C at 24 and 72 hours (P lt; 0.05). Conclusion BMSCs of rhesus monkeys can protect islet vitality and function from H/R-induced injury.

Citation： ZHAO Jiuming,HE Sirong,WANG Chengshi,LIU Jingping,LI Shengfu,CHEN Younan,LU Yanrong,CHENG Jingqiu.. PROTECTIVE EFFECT OF BONE MARROW MESENCHYMAL STEM CELLS ON ISLETS FROM HYPOXIA/REOXYGENATION-INDUCED INJURY. Chinese Journal of Reparative and Reconstructive Surgery, 2013, 27(8): 910-915. doi: 10.7507/1002-1892.20130201 Copy

1.	Koh A, Senior P, Salam A, et al. Insulin-heparin infusions peritransplant substantially improve single-donor clinical islet transplant success. Transplantation, 2010, 89(4): 465-471.
2.	Langer RM. Islet transplantation: lessons learned since the Edmonton breakthrough. Transplant Proc, 2010, 42(5): 1421-1424.
3.	Ichii H, Ricordi C. Current status of islet cell transplantation. J Hepatobiliary Pancreat Surg, 2009, 16(2): 101-112.
4.	Kobayashi N. The current status of islet transplantation and its perspectives. Rev Diabet Stud, 2008, 5(3): 136-143.
5.	Lakey JR, Mirbolooki M, Shapiro AM. Current status of clinical islet cell transplantation. Methods Mol Biol, 2006, 333: 47-104.
6.	Korsgren O, Nilsson B, Berne C, et al. Current status of clinical islet transplantation. Transplantation, 2005, 79(10): 1289-1293.
7.	Markmann JF, Deng S, Desai NM, et al. The use of non-heart-beating donors for isolated pancreatic islet transplantation. Transplantation, 2003, 75(9): 1423-1429.
8.	Vardanyan M, Parkin E, Gruessner C, et al. Pancreas vs. islet transplantation: a call on the future. Curr Opin Organ Transplant, 2010, 15(1): 124-130.
9.	Carlsson PO, Palm F, Mattsson G. Low revascularization of experimentally transplanted human pancreatic islets. J Clin Endocrinol Metab, 2002, 87(12): 5418-5423.
10.	Steinert AF, Rackwitz L, Gilbert F, et al. Concise review: the clinical application of mesenchymal stem cells for musculoskeletal regeneration: current status and perspectives. Stem Cells Transl Med, 2012, 1(3): 237-247.
11.	Pozzobon M, Piccoli M, De Coppi P. Sources of mesenchymal stem cells: current and future clinical use. Adv Biochem Eng Biotechnol, 2012. [Epub ahead of print].
12.	Gregory CA, Prockop DJ, Spees JL. Non-hematopoietic bone marrow stem cells: molecular control of expansion and differentiation. Exp Cell Res, 2005, 306(2): 330-335.
13.	Korbutt GS, Elliott JF, Ao Z, et al. Large scale isolation, growth, and function of porcine neonatal islet cells. J Clin Invest, 1996, 97(9): 2119-2129.
14.	Ma X, Ye B, Gao F, et al. Tissue factor knockdown in porcine islets: an effective approach to suppressing the instant blood-mediated inflammatory reaction. Cell Transplant, 2012, 21(1): 61-71.
15.	Karaoz E, Genc ZS, Demircan PC, et al. Protection of rat pancreatic islet function and viability by coculture with rat bone marrow-derived mesenchymal stem cells. Cell Death Dis, 2010, 1: e36.
16.	朱海涛, 于良, 王博. 猪胰岛细胞异种移植治疗糖尿病的研究进展. 世界华人消化杂志, 2012, 20(9): 737-742.
17.	Skiles ML, Wilder NB, Sahai S, et al. Identifying HIF activity in three-dimensional cultures of islet-like clusters. Int J Artif Organs, 2013, 36(3): 175-183.
18.	Padmasekar M, Lingwal N, Samikannu B, et al. Exendin-4 protects hypoxic islets from oxidative stress and improves islet transplantation outcome. Endocrinology, 2013, 154(4): 1424-1433.
19.	Yook S, Jeong JH, Jung YS, et al. Molecularly engineered islet cell clusters for diabetes mellitus treatment. Cell Transplant, 2012, 21(8): 1775-1789.
20.	鲁亚成, 任甫. 胚胎干细胞体外定向诱导分化为胰岛细胞的相关研究. 中国组织工程研究与临床康复, 2007, 11(11): 2156-2159.
21.	李富荣, 龙爱梅, 齐晖, 等. 胰岛干细胞转分化胰岛样细胞与天然胰岛功能的比较. 中国组织工程研究与临床康复, 2008, 12(25): 4833-4836.
22.	孟林燕, 刘畅, 王欣燕, 等. 肌源性干细胞移植修复糖尿病胰岛功能的旁分泌机制研究. 中国医学工程, 2011, 19(5): 35-36, 39.
23.	周静, 李进, 林戈, 等. 诱导人孤雌胚胎干细胞分化为胰岛样细胞团. 中国组织工程研究与临床康复, 2010, 14(27): 5001-5005.
24.	张妍, 郭希民, 王常勇, 等. 硒促进小鼠胚胎干细胞分化为胰岛细胞的实验研究. 军事医学科学院院刊, 2004, 28(2): 126-129, 139.
25.	Wang JC, Xia L, Song XB, et al. Transplantation of hypoxia preconditioned bone marrow mesenchymal stem cells improves survival of ultra-long random skin flap. Chin Med J (Engl), 2011, 124(16): 2507-2511.
26.	Lennon DP, Caplan AI. Isolation of rat marrow-derived mesenchymal stem cells. Exp Hematol, 2006, 34(11): 1606-1607.
27.	Fehrer C, Brunauer R, Laschober G, et al. Reduced oxygen tension attenuates differentiation capacity of human mesenchymal stem cells and prolongs their lifespan. Aging Cell, 2007, 6(6): 745-757.
28.	Lu Y, Jin X, Chen Y, et al. Mesenchymal stem cells protect islets from hypoxia/reoxygenation-induced injury. Cell Biochem Funct, 2010, 28(8): 637-643.
29.	Grayson WL, Zhao F, Izadpanah R, et al. Effects of hypoxia on human mesenchymal stem cell expansion and plasticity in 3D constructs. J Cell Physiol, 2006, 207(2): 331-339.
30.	Xu YX, Chen L, Wang R, et al. Mesenchymal stem cell therapy for diabetes through paracrine mechanisms. Med Hypotheses, 2008, 71(3): 390-393.
31.	Figliuzzi M, Cornolti R, Perico N, et al. Bone marrow-derived mesenchymal stem cells improve islet graft function in diabetic rats. Transplant Proc, 2009, 41(5): 1797-1800.
32.	Li YY, Liu HH, Chen HL, et al. Adipose-derived mesenchymal stem cells ameliorate STZ-induced pancreas damage in type 1 diabetes. Biomed Mater Eng, 2012, 22(1-3): 97-103.
33.	Luo L, Badiavas E, Luo JZ, et al. Allogeneic bone marrow supports human islet beta cell survival and function over six months. Biochem Biophys Res Commun, 2007, 361(4): 859-864.
34.	Chao KC, Chao KF, Chen CF, et al. A novel human stem cell coculture system that maintains the survival and function of culture islet-like cell clusters. Cell Transplant, 2008, 17(6): 657-664.
35.	Cavallari G, Olivi E, Bianchi F, et al. Mesenchymal stem cells and islet cotransplantation in diabetic rats: improved islet graft revascularization and function by human adipose tissue-derived stem cells preconditioned with natural molecules. Cell Transplant, 2012, 21(12): 2771-2781.
36.	Clarkin CE, King AJ, Dhadda P, et al. Activin Receptor-like kinase 5 Inhibition Reverses Impairment of Endothelial Cell Viability by Endogenous Islet Mesenchymal Stromal Cells. Stem Cells, 2013, 31(3): 547-559.

1. Koh A, Senior P, Salam A, et al. Insulin-heparin infusions peritransplant substantially improve single-donor clinical islet transplant success. Transplantation, 2010, 89(4): 465-471.
2. Langer RM. Islet transplantation: lessons learned since the Edmonton breakthrough. Transplant Proc, 2010, 42(5): 1421-1424.
3. Ichii H, Ricordi C. Current status of islet cell transplantation. J Hepatobiliary Pancreat Surg, 2009, 16(2): 101-112.
4. Kobayashi N. The current status of islet transplantation and its perspectives. Rev Diabet Stud, 2008, 5(3): 136-143.
5. Lakey JR, Mirbolooki M, Shapiro AM. Current status of clinical islet cell transplantation. Methods Mol Biol, 2006, 333: 47-104.
6. Korsgren O, Nilsson B, Berne C, et al. Current status of clinical islet transplantation. Transplantation, 2005, 79(10): 1289-1293.
7. Markmann JF, Deng S, Desai NM, et al. The use of non-heart-beating donors for isolated pancreatic islet transplantation. Transplantation, 2003, 75(9): 1423-1429.
8. Vardanyan M, Parkin E, Gruessner C, et al. Pancreas vs. islet transplantation: a call on the future. Curr Opin Organ Transplant, 2010, 15(1): 124-130.
9. Carlsson PO, Palm F, Mattsson G. Low revascularization of experimentally transplanted human pancreatic islets. J Clin Endocrinol Metab, 2002, 87(12): 5418-5423.
10. Steinert AF, Rackwitz L, Gilbert F, et al. Concise review: the clinical application of mesenchymal stem cells for musculoskeletal regeneration: current status and perspectives. Stem Cells Transl Med, 2012, 1(3): 237-247.
11. Pozzobon M, Piccoli M, De Coppi P. Sources of mesenchymal stem cells: current and future clinical use. Adv Biochem Eng Biotechnol, 2012. [Epub ahead of print].
12. Gregory CA, Prockop DJ, Spees JL. Non-hematopoietic bone marrow stem cells: molecular control of expansion and differentiation. Exp Cell Res, 2005, 306(2): 330-335.
13. Korbutt GS, Elliott JF, Ao Z, et al. Large scale isolation, growth, and function of porcine neonatal islet cells. J Clin Invest, 1996, 97(9): 2119-2129.
14. Ma X, Ye B, Gao F, et al. Tissue factor knockdown in porcine islets: an effective approach to suppressing the instant blood-mediated inflammatory reaction. Cell Transplant, 2012, 21(1): 61-71.
15. Karaoz E, Genc ZS, Demircan PC, et al. Protection of rat pancreatic islet function and viability by coculture with rat bone marrow-derived mesenchymal stem cells. Cell Death Dis, 2010, 1: e36.
16. 朱海涛, 于良, 王博. 猪胰岛细胞异种移植治疗糖尿病的研究进展. 世界华人消化杂志, 2012, 20(9): 737-742.
17. Skiles ML, Wilder NB, Sahai S, et al. Identifying HIF activity in three-dimensional cultures of islet-like clusters. Int J Artif Organs, 2013, 36(3): 175-183.
18. Padmasekar M, Lingwal N, Samikannu B, et al. Exendin-4 protects hypoxic islets from oxidative stress and improves islet transplantation outcome. Endocrinology, 2013, 154(4): 1424-1433.
19. Yook S, Jeong JH, Jung YS, et al. Molecularly engineered islet cell clusters for diabetes mellitus treatment. Cell Transplant, 2012, 21(8): 1775-1789.
20. 鲁亚成, 任甫. 胚胎干细胞体外定向诱导分化为胰岛细胞的相关研究. 中国组织工程研究与临床康复, 2007, 11(11): 2156-2159.
21. 李富荣, 龙爱梅, 齐晖, 等. 胰岛干细胞转分化胰岛样细胞与天然胰岛功能的比较. 中国组织工程研究与临床康复, 2008, 12(25): 4833-4836.
22. 孟林燕, 刘畅, 王欣燕, 等. 肌源性干细胞移植修复糖尿病胰岛功能的旁分泌机制研究. 中国医学工程, 2011, 19(5): 35-36, 39.
23. 周静, 李进, 林戈, 等. 诱导人孤雌胚胎干细胞分化为胰岛样细胞团. 中国组织工程研究与临床康复, 2010, 14(27): 5001-5005.
24. 张妍, 郭希民, 王常勇, 等. 硒促进小鼠胚胎干细胞分化为胰岛细胞的实验研究. 军事医学科学院院刊, 2004, 28(2): 126-129, 139.
25. Wang JC, Xia L, Song XB, et al. Transplantation of hypoxia preconditioned bone marrow mesenchymal stem cells improves survival of ultra-long random skin flap. Chin Med J (Engl), 2011, 124(16): 2507-2511.
26. Lennon DP, Caplan AI. Isolation of rat marrow-derived mesenchymal stem cells. Exp Hematol, 2006, 34(11): 1606-1607.
27. Fehrer C, Brunauer R, Laschober G, et al. Reduced oxygen tension attenuates differentiation capacity of human mesenchymal stem cells and prolongs their lifespan. Aging Cell, 2007, 6(6): 745-757.
28. Lu Y, Jin X, Chen Y, et al. Mesenchymal stem cells protect islets from hypoxia/reoxygenation-induced injury. Cell Biochem Funct, 2010, 28(8): 637-643.
29. Grayson WL, Zhao F, Izadpanah R, et al. Effects of hypoxia on human mesenchymal stem cell expansion and plasticity in 3D constructs. J Cell Physiol, 2006, 207(2): 331-339.
30. Xu YX, Chen L, Wang R, et al. Mesenchymal stem cell therapy for diabetes through paracrine mechanisms. Med Hypotheses, 2008, 71(3): 390-393.
31. Figliuzzi M, Cornolti R, Perico N, et al. Bone marrow-derived mesenchymal stem cells improve islet graft function in diabetic rats. Transplant Proc, 2009, 41(5): 1797-1800.
32. Li YY, Liu HH, Chen HL, et al. Adipose-derived mesenchymal stem cells ameliorate STZ-induced pancreas damage in type 1 diabetes. Biomed Mater Eng, 2012, 22(1-3): 97-103.
33. Luo L, Badiavas E, Luo JZ, et al. Allogeneic bone marrow supports human islet beta cell survival and function over six months. Biochem Biophys Res Commun, 2007, 361(4): 859-864.
34. Chao KC, Chao KF, Chen CF, et al. A novel human stem cell coculture system that maintains the survival and function of culture islet-like cell clusters. Cell Transplant, 2008, 17(6): 657-664.
35. Cavallari G, Olivi E, Bianchi F, et al. Mesenchymal stem cells and islet cotransplantation in diabetic rats: improved islet graft revascularization and function by human adipose tissue-derived stem cells preconditioned with natural molecules. Cell Transplant, 2012, 21(12): 2771-2781.
36. Clarkin CE, King AJ, Dhadda P, et al. Activin Receptor-like kinase 5 Inhibition Reverses Impairment of Endothelial Cell Viability by Endogenous Islet Mesenchymal Stromal Cells. Stem Cells, 2013, 31(3): 547-559.

Chinese Journal of Reparative and Reconstructive Surgery

PROTECTIVE EFFECT OF BONE MARROW MESENCHYMAL STEM CELLS ON ISLETS FROM HYPOXIA/REOXYGENATION-INDUCED INJURY

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