بررسی وضعیت بقای سلولی و بیان نشانگرهای عصبی در سلول‌های حاصل از تمایز سلول‌های بنیادی مشتق از بافت چربی در سیستم کشت سه بعدی آلژینات

نوع مقاله : مقاله های پژوهشی

نویسندگان

1 دانشجوی دکتری، گروه علوم تشریحی و بیولوژی مولکولی، دانشکده‌ی پزشکی، دانشگاه علوم پزشکی تهران، تهران، ایران

2 استاد، گروه علوم تشریحی، دانشکده‌ی پزشکی، دانشگاه علوم پزشکی اصفهان، اصفهان، ایران

3 دانشیار، گروه علوم تشریحی، دانشکد‌ه‌ی پزشکی، دانشگاه علوم پزشکی اصفهان، اصفهان، ایران

4 دانشجوی دکتری، گروه ژنتیک و بیولوژی مولکولی، دانشکده‌ی پزشکی، دانشگاه علوم پزشکی اصفهان، اصفهان، ایران

چکیده

مقدمه: مطالعات متعددی برای فراهم کردن تعداد کافی سلول‌های عصبی جهت درمان آسیب‌های نورودژنراتیو انجام شده است. سلول‌های بنیادی جدا شده از بافت چربی، میزان تکثیر و مقاومت زیادی نسبت به آپوپتوز دارند. این سلول‌ها، قابلیت تمایز به دودمان‌های مختلف سلولی از قبیل سلول‌های عصبی را دارند. هیدروژل آلژینات، پلیمر پلی‌ساکاریدی است که دارای ویژگی‌های مناسبی از قبیل زیست‌سازگاری و عدم تحریک سیستم ایمنی می‌باشد. در این مطالعه، تأثیر هیدروژل آلژینات بر میزان بقا و بیان نشانگرهای عصبی سلول‌های حاصل از تمایز سلول‌های بنیادی جدا شده از بافت چربی انسانی بررسی شد.روش‌ها: سلول‌های بنیادی جدا شده از بافت چربی انسانی در محیط کشت عصبی القا و سپس در هیدروژل آلژینات انکپسوله شدند. میزان رشد و تمایز سلول‌ها به ترتیب با استفاده از ارزیابی Doubling time و Real time quantitative reverse transcription polymerase chain reaction (Real time RT-PCR) 7 روز پس از انکپسوله کردن سلول‌ها بررسی شد. آنالیز داده‌ها با استفاده از آزمون One way-ANOVA انجام گردید.یافته‌ها: میزان رشد سلول‌های انکپسوله در هیدروژل آلژینات، کاهش معنی‌داری را نسبت به گروه شاهد نشان داد (001/0 > P). در حالی که میانگین بیان ژن‌های GFAP (Glial fibrillary acidic protein)، Nestin و MAP2 (Microtubule-associated protein 2) در سلول‌های انکپسوله در مقایسه با گروه شاهد به طور معنی‌داری افزایش یافت (001/0 > P).نتیجه‌گیری: اگر چه سرعت تکثیر سلول‌های انکپسوله در هیدروژل آلژینات، در مقایسه با گروه شاهد به طور معنی‌داری کاهش یافت، اما هیدروژل آلژینات می‌تواند باعث افزایش تمایز عصبی سلول‌های انکپسوله شود.

کلیدواژه‌ها

عنوان مقاله [English]

Cell Viability and Neural Markers Expression of Human Adipose-Derived Stem Cells after Neural Induction in Alginate Tridimensional Cell Culture

نویسندگان [English]

  • Zahra Khosravizadeh 1
  • Shahnaz Razavi 2
  • Hamid Bahramian 3
  • Mohammad Kazemi 4
1 PhD Student, Department of Anatomical Sciencesand Molecular Biology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
2 Professor, Department of Anatomical Sciences, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
3 Associate Professor, Department of Anatomical Sciences, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
4 PhD Student, Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
چکیده [English]

Background: Different studies have been done to obtain sufficient number of neuronal cells for treatment of central nervous system injuries. Adipose-derived stem cells have higher proliferation rate and more resistant to apoptosis. These cells can be induced to differentiate along several multilineage cells such as neuron-like cells. Alginate hydrogel is a polysaccharide polymer that has proper properties including biocompatibility with no immunogenicity. In this study, the effect of alginate hydrogel on the cell viability, proliferation rate and neurogenic differentiation of human adipose derived stem cells was evaluated at 14 days after induction.Methods: Adipose-derived stem cells isolated from human, were cultured in neural induction media and seeded into alginate hydrogel. The cell viability and differentiation efficiency of encapsulated cells were evaluated via doubling time and real time quantitative reverse transcription polymerase chain reaction (real time RT-PCR) analysis, respectively. The collected data was analyzed using one-way ANOVA test.Findings: The rate of proliferation of encapsulated cells significantly decreased as compared with control group (P < 0.001). The expression of nestin, glial fibrillary acidic protein (GFAP) and microtubule-associated protein 2 (MAP2) markers significantly decreased as compared with control group (P < 0.001).Conclusion: Altogether cell viability and proliferation rate of encapsulated cells in alginate hydrogel significantly decreased as compared with control group, but alginate hydrogel can promote neural differentiation of adipose-derived stem cells.

کلیدواژه‌ها [English]

  • Alginate hydrogel
  • Adipose-derived stem cells
  • Neurogenic differentiation
  • Proliferation

مراجع

  1. Purcell EK, Singh A, Kipke DR. Alginate composition effects on a neural stem cell-seeded scaffold. Tissue Eng Part C Methods 2009; 15(4): 541-50.
  2. Hunt NC, Smith AM, Gbureck U, Shelton RM, Grover LM. Encapsulation of fibroblasts causes accelerated alginate hydrogel degradation. Acta Biomater 2010; 6(9): 3649-56.
  3. Barrilleaux B, Phinney DG, Prockop DJ, O'Connor KC. Review: ex vivo engineering of living tissues with adult stem cells. Tissue Eng 2006; 12(11): 3007-19.
  4. Rodriguez AM, Elabd C, Amri EZ, Ailhaud G, Dani C. The human adipose tissue is a source of multipotent stem cells. Biochimie 2005; 87(1): 125-8.
  5. Zuk PA, Zhu M, Ashjian P, De Ugarte DA, Huang JI, Mizuno H, et al. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell 2002; 13(12): 4279-95.
  6. Razavi S, Mardani M, Kazemi M, Esfandiari E, Narimani M, Esmaeili A, et al. Effect of leukemia inhibitory factor on the myelinogenic ability of Schwann-like cells induced from human adipose-derived stem cells. Cell Mol Neurobiol 2013; 33(2): 283-9.
  7. Xu Y, Liu Z, Liu L, Zhao C, Xiong F, Zhou C, et al. Neurospheres from rat adipose-derived stem cells could be induced into functional Schwann cell-like cells in vitro. BMC Neurosci 2008; 9: 21.
  8. Xie X, Tang Z, Chen J, Yang J, Zeng W, Liu N, et al. Neurogenesis of adipose-derived stem cells in hydrogel. J Huazhong Univ Sci Technolog Med Sci 2011; 31(2): 174-7.
  9. Kim MH, Hong HN, Hong JP, Park CJ, Kwon SW, Kim SH, et al. The effect of VEGF on the myogenic differentiation of adipose tissue derived stem cells within thermosensitive hydrogel matrices. Biomaterials 2010; 31(6): 1213-8.
  10. Jang S, Cho HH, Cho YB, Park JS, Jeong HS. Functional neural differentiation of human adipose tissue-derived stem cells using bFGF and forskolin. BMC Cell Biol 2010; 11: 25.
  11. Kalbermatten DF, Schaakxs D, Kingham PJ, Wiberg M. Neurotrophic activity of human adipose stem cells isolated from deep and superficial layers of abdominal fat. Cell Tissue Res 2011; 344(2): 251-60.
  12. Strem BM, Hedrick MH. The growing importance of fat in regenerative medicine. Trends Biotechnol 2005; 23(2): 64-6.
  13. Schipper BM, Marra KG, Zhang W, Donnenberg AD, Rubin JP. Regional anatomic and age effects on cell function of human adipose-derived stem cells. Ann Plast Surg 2008; 60(5): 538-44.
  14. Lopatina T, Kalinina N, Karagyaur M, Stambolsky D, Rubina K, Revischin A, et al. Adipose-derived stem cells stimulate regeneration of peripheral nerves: BDNF secreted by these cells promotes nerve healing and axon growth de novo. PLoS One 2011; 6(3): e17899.
  15. Razavi S, Razavi MR, Kheirollahi-Kouhestani M, Mardani M, Mostafavi FS. Co-culture with neurotrophic factor secreting cells induced from adipose-derived stem cells: promotes neurogenic differentiation. Biochem Biophys Res Commun 2013; 440(3): 381-7.
  16. Vermette M, Trottier V, Menard V, Saint-Pierre L, Roy A, Fradette J. Production of a new tissue-engineered adipose substitute from human adipose-derived stromal cells. Biomaterials 2007; 28(18): 2850-60.
  17. Boontheekul T, Kong HJ, Mooney DJ. Controlling alginate gel degradation utilizing partial oxidation and bimodal molecular weight distribution. Biomaterials 2005; 26(15): 2455-65.
  18. Gao C, Liu M, Chen J, Zhang X. Preparation and controlled degradation of oxidized sodium alginate hydrogel. Polym Degrad Stab 2009; 94(9): 1405-10.
  19. Matyash M, Despang F, Mandal R, Fiore D, Gelinsky M, Ikonomidou C. Novel soft alginate hydrogel strongly supports neurite growth and protects neurons against oxidative stress. Tissue Eng Part A 2012; 18(1-2): 55-66.
  20. West ER, Xu M, Woodruff TK, Shea LD. Physical properties of alginate hydrogels and their effects on in vitro follicle development. Biomaterials 2007; 28(30): 4439-48.
  21. Haug A, Larsen B, Smidsrod O, Eriksson G, Blinc R, Pausak S. Studies on the sequence of uronic acid residues in alginic acid. Acta Chemica Scandinavica 1967; 21(3): 691-704.
  22. Martinsen A, Skjak-Braek G, Smidsrod O. Alginate as immobilization material: I. Correlation between chemical and physical properties of alginate gel beads. Biotechnol Bioeng 1989; 33(1): 79-89.
  23. Draget KI, Skjak Braek G, Smidsrod O. Alginic acid gels: the effect of alginate chemical composition and molecular weight. Carbohydrate Polymers 1994; 25(1): 31-8.
  24. Smidsrod O, Haug A, Larsen B, Gronowitz S, Hoffman RA, Westerdahl A. The effect of divalent metals on the properties of alginate solutions. I. calcium ions. Acta Chemica Scandinavica 1965; 19(2): 329-40.
  25. Mulder L, Baaijens FPT, Bouten CVC, van Lieshout MI. Cell adhesion on alginate scaffolds for the tissue engineering of an aortic valve - a review. Eindhoven, Netherlands: Eindhoven University of Technology; 2002. p. 22-34.
  26. Rhiner C, Hengartner MO. Sugar antennae for guidance signals: syndecans and glypicans integrate directional cues for navigating neurons. ScientificWorldJournal 2006; 6: 1024-36.
  27. Maeda N, Noda M. Involvement of receptor-like protein tyrosine phosphatase zeta/RPTPbeta and its ligand pleiotrophin/heparin-binding growth-associated molecule (HB-GAM) in neuronal migration. J Cell Biol 1998; 142(1): 203-16.
  28. Elfenbein A, Simons M. Auxiliary and autonomous proteoglycan signaling networks. Methods Enzymol 2010; 480: 3-31.
  29. Barker CF, Billingham RE. Immunologically privileged sites. Adv Immunol 1977; 25: 1-54.
  30. Li X, Liu T, Song K, Yao L, Ge D, Bao C, et al. Culture of neural stem cells in calcium alginate beads. Biotechnol Prog 2006; 22(6): 1683-9.
  31. Read TA, Stensvaag V, Vindenes H, Ulvestad E, Bjerkvig R, Thorsen F. Cells encapsulated in alginate: a potential system for delivery of recombinant proteins to malignant brain tumours. Int J Dev Neurosci 1999; 17(5-6): 653-63.
  32. Rowley JA, Madlambayan G, Mooney DJ. Alginate hydrogels as synthetic extracellular matrix materials. Biomaterials 1999; 20(1): 45-53.
  33. Augst AD, Kong HJ, Mooney DJ. Alginate hydrogels as biomaterials. Macromol Biosci 2006; 6(8): 623-33.
  34. Drury JL, Mooney DJ. Hydrogels for tissue engineering: scaffold design variables and applications. Biomaterials 2003; 24(24): 4337-51.
  35. Coviello T, Matricardi P, Marianecci C, Alhaique F. Polysaccharide hydrogels for modified release formulations. J Control Release 2007; 119(1): 5-24.
  36. Simmons CA, Alsberg E, Hsiong S, Kim WJ, Mooney DJ. Dual growth factor delivery and controlled scaffold degradation enhance in vivo bone formation by transplanted bone marrow stromal cells. Bone 2004; 35(2): 562-9.
  37. Kong HJ, Mooney DJ. The effects of poly(ethyleneimine) (PEI) molecular weight on reinforcement of alginate hydrogels. Cell Transplant 2003; 12(7): 779-85.
  38. Alsberg E, Kong HJ, Hirano Y, Smith MK, Albeiruti A, Mooney DJ. Regulating bone formation via controlled scaffold degradation. J Dent Res 2003; 82(11): 903-8.
  39. Alsberg E, Anderson KW, Albeiruti A, Franceschi RT, Mooney DJ. Cell-interactive alginate hydrogels for bone tissue engineering. J Dent Res 2001; 80(11): 2025-9.
  40. Hsiong SX, Huebsch N, Fischbach C, Kong HJ, Mooney DJ. Integrin-adhesion ligand bond formation of preosteoblasts and stem cells in three-dimensional RGD presenting matrices. Biomacromolecules 2008; 9(7): 1843-51.
  41. Lee KY, Alsberg E, Mooney DJ. Degradable and injectable poly(aldehyde guluronate) hydrogels for bone tissue engineering. J Biomed Mater Res 2001; 56(2): 228-33.
  42. Petruzzo P, Cappai A, Ruiu G, Dessy E, Rescigno A, Brotzu G. Development of biocompatible barium alginate microcapsules. Transplant Proc 1997; 29(4): 2129-30.
  43. Schagemann JC, Mrosek EH, Landers R, Kurz H, Erggelet C. Morphology and function of ovine articular cartilage chondrocytes in 3-d hydrogel culture. Cells Tissues Organs 2006; 182(2): 89-97.
  44. Markusen JF, Mason C, Hull DA, Town MA, Tabor AB, Clements M, et al. Behavior of adult human mesenchymal stem cells entrapped in alginate-GRGDY beads. Tissue Eng 2006; 12(4): 821-30.
  45. Saha K, Keung AJ, Irwin EF, Li Y, Little L, Schaffer DV, et al. Substrate modulus directs neural stem cell behavior. Biophys J 2008; 95(9): 4426-38.
  46. Magyar JP, Nemir M, Ehler E, Suter N, Perriard JC, Eppenberger HM. Mass production of embryoid bodies in microbeads. Ann N Y Acad Sci 2001; 944: 135-43.
  47. de Groot M, Schuurs TA, van Schilfgaarde R. Causes of limited survival of microencapsulated pancreatic islet grafts. J Surg Res 2004; 121(1): 141-50.
  48. Orive G, Tam SK, Pedraz JL, Halle JP. Biocompatibility of alginate-poly-L-lysine microcapsules for cell therapy. Biomaterials 2006; 27(20): 3691-700.
  49. Dar A, Shachar M, Leor J, Cohen S. Optimization of cardiac cell seeding and distribution in 3D porous alginate scaffolds. Biotechnol Bioeng 2002; 80(3): 305-12.
  50. Ma HL, Hung SC, Lin SY, Chen YL, Lo WH. Chondrogenesis of human mesenchymal stem cells encapsulated in alginate beads. J Biomed Mater Res A 2003; 64(2): 273-81.
  51. Reddy GP, Tiarks CY, Pang L, Wuu J, Hsieh CC, Quesenberry PJ. Cell cycle analysis and synchronization of pluripotent hematopoietic progenitor stem cells. Blood 1997; 90(6): 2293-9.
  52. Bryant SJ, Chowdhury TT, Lee DA, Bader DL, Anseth KS. Crosslinking density influences chondrocyte metabolism in dynamically loaded photocrosslinked poly(ethylene glycol) hydrogels. Ann Biomed Eng 2004; 32(3): 407-17.
  53. Bryant SJ, Durand KL, Anseth KS. Manipulations in hydrogel chemistry control photoencapsulated chondrocyte behavior and their extracellular matrix production. J Biomed Mater Res A 2003; 67(4): 1430-6.
  54. Bryant SJ, Anseth KS. Hydrogel properties influence ECM production by chondrocytes photoencapsulated in poly(ethylene glycol) hydrogels. J Biomed Mater Res 2002; 59(1): 63-72.
  55. Bossio C, Mastrangelo R, Morini R, Tonna N, Coco S, Verderio C, et al. A simple method to generate adipose stem cell-derived neurons for screening purposes. J Mol Neurosci 2013; 51(2): 274-81.
  56. Razavi S, Razavi MR, Zarkesh EH, Kazemi M, Mostafavi FS. Comparing brain-derived neurotrophic factor and ciliary neurotrophic factor secretion of induced neurotrophic factor secreting cells from human adipose and bone marrow-derived stem cells. Dev Growth Differ 2013; 55(6): 648-55.
  57. Retta S, Ternullo M, Tarone G. Adhesion to matrix proteins. In: Dejana E, Corada M, editors. Adhesion protein protocols. 96th ed. Totowa, NJ: Humana Press; 1999. p. 125-30.
مجله دانشکده پزشکی اصفهان
دوره 33، شماره 350 - شماره پیاپی 350
مهر و آبان 1394
صفحه 1491-1505

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سابقه مقاله

  • تاریخ دریافت: 15 مرداد 1393
  • تاریخ بازنگری: 11 آبان 1403
  • تاریخ پذیرش: 17 فروردین 1401

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