Int J Stem Cells.  2016 May;9(1):115-123. 10.15283/ijsc.2016.9.1.115.

Isolation, Characterization and Growth Kinetic Comparison of Bone Marrow and Adipose Tissue Mesenchymal Stem Cells of Guinea Pig

• Aliborzi G
• Vahdati A
• Mehrabani
• Hosseini S
• Tamadon A

• KMID: 2164168
• DOI: http://doi.org/10.15283/ijsc.2016.9.1.115

Abstract

BACKGROUND
Mesenchymal stem cells (MSCs) from different sources have different characteristics. Moreover, MSCs are not isolated and characterized in Guinea pig for animal model of cell therapy. AIM OF THE WORK: was the isolating of bone marrow MSCs (BM-MSCs) and adipose tissue MSCs (AT-MSCs) from Guinea pig and assessing their characteristics. MATERIAL AND METHODS: In this study, bone marrow and adipose tissue were collected from three Guinea pigs and cultured and expanded through eight passages. BM-MSCs and AT-MSCs at passages 2, 5 and 8 were seeded in 24-well plates in triplicate. Cells were counted from each well 1~7 days after seeding to determine population doubling time (PDT) and cell growth curves. Cells of passage 3 were cultured in osteogenic and adipogenic differentiation media.
RESULTS
BM-MSCs and AT-MSCs attached to the culture flask and displayed spindle-shaped morphology. Proliferation rate of AT-MSCs in the analyzed passages was more than BM-MSCs. The increase in the PDT of MSCs occurs with the increase in the number of passages. Moreover, after culture of BM-MSCs and AT-MSCs in differentiation media, the cells differentiated toward osteoblasts and adipocytes as verified by Alizarin Red staining and Oil Red O staining, respectively.
CONCLUSION
BM-MSCs and AT-MSCs of Guinea pig could be valuable source of multipotent stem cells for use in experimental and preclinical studies in animal models.

Keyword

Mesenchymal stem cells; Guinea pig; Adipose tissue; Bone marrow; Characterization

MeSH Terms

Adipocytes
Adipose Tissue*
Animals
Bone Marrow*
Cell- and Tissue-Based Therapy
Guinea Pigs*
Guinea*
Mesenchymal Stromal Cells*
Models, Animal
Multipotent Stem Cells
Osteoblasts

Figure

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  Fig. 1 Morphologic characteristics of adipose tissue-derived and bone marrow-derived mesenchymal stem cells (AT-MSCs and BM-MSCs, respectively) of Guinea pig. Most MSCs showed fibroblastic morphology regardless of the cell source. (A) Primary culture of AT-MSCs (×40), (B) Passage 2 of AT-MSCs (×100), (C) passage 5 of AT-MSCs (×100), (D) passage 8 of AT-MSCs (×100), (E) Primary culture of BM-MSCs (×100), (F) passage 2 of BM-MSCs (×100), (G) passage 5 of BM-MSCs (×100), and (H) passage 8 of BM-MSCs (×200).

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  Fig. 2 Agarose gel electrophoresis of (A) bone marrow and (B) adipose tissue-derived mesenchymal stem cells RT-PCR products show positive expression for CD44 and CD90 (mesenchymal surface marker) and negative expression for CD34 (hematopoietic surface marker).

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  Fig. 3 Comparison of mean and standard error of cell counts between growth curves of guinea pig bone marrow-derived mesenchymal stem cells (BM-MSCs) and adipose tissue-derived mesenchymal stem cells (AT-MSCs) at passages 2, 5 and 8. Proliferation rate of AT-MSCs was more than BM-MSCs, (A) in passages 2, (B) in passages 5, and (C) in passages 8. a,bSuperscript letters show differences between the number of cells in each curve in the same day (p<0.05).

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  Fig. 4 Comparison of mean and standard error of cell counts in growth curves of guinea pig bone marrow-derived mesenchymal stem cells (BM-MSCs) and adipose tissue-derived mesenchymal stem cells (AT-MSCs) between passages 2, 5 and 8. The proliferation rate of AT-MSCs (A) and BM-MSCs (B) decreased gradually from passage 2 to passage 8. a,b,cSuperscript letters show differences between the number of cells in each curve in the same day (p<0.05).

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  Fig. 5 Osteogenic and adipogenic differentiations of bone marrow-derived (A and C, respectively) and adipose tissue-derived (B and D, respectively) mesenchymal stem cells (BM-MSCs and AT-MSCs) from passage 3 of subcultures by Alizarin Red staining and Oil Red staining, respectively.

Reference

References

1. Wang S, Qu X, Zhao RC. Clinical applications of mesenchymal stem cells. J Hematol Oncol. 2012; 5:19. DOI: 10.1186/1756-8722-5-19. PMID: 22546280. PMCID: 3416655.

2. Mehrabani D, Hassanshahi MA, Tamadon A, Zare S, Keshavarz S, Rahmanifar F, Dianatpour M, Khodabandeh Z, Jahromi I, Tanideh N, Ramzi M, Aqababa H, Kuhi-Hoseinabadi O. Adipose tissue-derived mesenchymal stem cells repair germinal cells of seminiferous tubules of busulfan-induced azoospermic rats. J Hum Reprod Sci. 2015; 8:103–110. DOI: 10.4103/0974-1208.158618. PMID: 26157302. PMCID: 4477447.

3. Friedenstein AJ, Gorskaja JF, Kulagina NN. Fibroblast precursors in normal and irradiated mouse hematopoietic organs. Exp Hematol. 1976; 4:267–274. PMID: 976387.

4. in ’t Anker PS, Noort WA, Scherjon SA, Kleijburgvan der Keur C, Kruisselbrink AB, van Bezooijen RL, Beekhuizen W, Willemze R, Kanhai HH, Fibbe WE. Mesenchymal stem cells in human second-trimester bone marrow, liver, lung, and spleen exhibit a similar immunophenotype but a heterogeneous multilineage differentiation potential. Haematologica. 2003; 88:845–852.

5. Mahdiyar P, Zare S, Robati R, Dianatpour M, Torabi K, Tamadon A, Razeghian Jahromi I, Tamadon A, Mehrabani D. Isolation, culture, and characterization of human dental pulp mesenchymal stem cells. Int J Pediatr. 2014; 2:44.

6. Ding DC, Chang YH, Shyu WC, Lin SZ. Human umbilical cord mesenchymal stem cells: a new era for stem cell therapy. Cell Transplant. 2015; 24:339–347. DOI: 10.3727/096368915X686841. PMID: 25622293.

7. Anasetti C, Logan BR, Lee SJ, Waller EK, Weisdorf DJ, Wingard JR, Cutler CS, Westervelt P, Woolfrey A, Couban S, Ehninger G, Johnston L, Maziarz RT, Pulsipher MA, Porter DL, Mineishi S, McCarty JM, Khan SP, Anderlini P, Bensinger WI, Leitman SF, Rowley SD, Bredeson C, Carter SL, Horowitz MM, Confer DL. Blood and Marrow Transplant Clinical Trials Network. Peripheral-blood stem cells versus bone marrow from unrelated donors. N Engl J Med. 2012; 367:1487–1496. DOI: 10.1056/NEJMoa1203517. PMID: 23075175. PMCID: 3816375.

8. Kisiel AH, McDuffee LA, Masaoud E, Bailey TR, Esparza Gonzalez BP, Nino-Fong R. Isolation, characterization, and in vitro proliferation of canine mesenchymal stem cells derived from bone marrow, adipose tissue, muscle, and periosteum. Am J Vet Res. 2012; 73:1305–1317. DOI: 10.2460/ajvr.73.8.1305. PMID: 22849692.

9. Baer PC, Geiger H. Adipose-derived mesenchymal stromal/stem cells: tissue localization, characterization, and heterogeneity. Stem Cells Int. 2012; 2012:812693. DOI: 10.1155/2012/812693. PMID: 22577397. PMCID: 3345279.

10. Rodbell M. The metabolism of isolated fat cells. IV. Regulation of release of protein by lipolytic hormones and insulin. J Biol Chem. 1966; 241:3909–3917. PMID: 4288359.

11. Asadi-Yousefabad SL, Khodakaram-Tafti A, Dianatpour M, Mehrabani D, Zare S, Tamadon A, Nikeghbalian S, Raayat-Jahromi A, Ahmadlou S. Genetic evaluation of bone marrow-derived mesenchymal stem cells by a modified karyotyping method. Comp Clin Pathol. 2015; 24:1361–1366. DOI: 10.1007/s00580-015-2081-4.

12. Al-Nbaheen M, Vishnubalaji R, Ali D, Bouslimi A, Al-Jassir F, Megges M, Prigione A, Adjaye J, Kassem M, Aldahmash A. Human stromal (mesenchymal) stem cells from bone marrow, adipose tissue and skin exhibit differences in molecular phenotype and differentiation potential. Stem Cell Rev. 2013; 9:32–43. DOI: 10.1007/s12015-012-9365-8. PMCID: 3563956.

13. Izadpanah R, Trygg C, Patel B, Kriedt C, Dufour J, Gimble JM, Bunnell BA. Biologic properties of mesenchymal stem cells derived from bone marrow and adipose tissue. J Cell Biochem. 2006; 99:1285–1297. DOI: 10.1002/jcb.20904. PMID: 16795045. PMCID: 4048742.

14. Peng L, Jia Z, Yin X, Zhang X, Liu Y, Chen P, Ma K, Zhou C. Comparative analysis of mesenchymal stem cells from bone marrow, cartilage, and adipose tissue. Stem Cells Dev. 2008; 17:761–773. DOI: 10.1089/scd.2007.0217. PMID: 18393634.

15. Baghaban Eslaminejad M, Mardpour S, Ebrahimi M. Growth kinetics and in vitro aging of mesenchymal stem cells isolated from rat adipose versus bone marrow tissues. Iran J Vet Surg. 2008; 3:9–20.

16. Barros BB, Andrade JS, Garcia LB, Pifaia GR, Cruz OL, Onishi ET, Penido Nde O. Micro-endoscopic ear anatomy of guinea pig applied to experimental surgery. Acta Cir Bras. 2014; 29( Suppl 1):7–11. DOI: 10.1590/S0102-86502014001300002. PMID: 25185049.

17. Eyanagi R, Toda A, Imoto M, Uchiyama H, Ishii Y, Kuroki H, Kuramoto Y, Soeda S, Shimeno H. Covalent binding of nitroso-sulfonamides to glutathione S-transferase in guinea pigs with delayed type hypersensitivity. Int Immunopharmacol. 2012; 12:694–700. DOI: 10.1016/j.intimp.2012.01.017. PMID: 22342371.

18. Tonge DP, Bardsley RG, Parr T, Maciewicz RA, Jones SW. Evidence of changes to skeletal muscle contractile properties during the initiation of disease in the ageing guinea pig model of osteoarthritis. Longev Healthspan. 2013; 2:15. DOI: 10.1186/2046-2395-2-15.

19. Mehrabani D, Mahboobi R, Dianatpour M, Zare S, Tamadon A, Hosseini SE. Establishment, culture, and characterization of Guinea pig fetal fibroblast cell. Vet Med Int. 2014; 2014:510328. DOI: 10.1155/2014/510328. PMID: 24790770. PMCID: 3984808.

20. Kakudo N, Morimoto N, Ogawa T, Kusumoto K. Potential of adipose-derived stem cells for regeneration medicine: clinical application and usefulness of fat grafting. J Stem Cell Res Ther. 2014; 4:204. DOI: 10.4172/2157-7633.1000204.

21. Yoshimura H, Muneta T, Nimura A, Yokoyama A, Koga H, Sekiya I. Comparison of rat mesenchymal stem cells derived from bone marrow, synovium, periosteum, adipose tissue, and muscle. Cell Tissue Res. 2007; 327:449–462. DOI: 10.1007/s00441-006-0308-z.

22. Bruder SP, Jaiswal N, Haynesworth SE. Growth kinetics, self-renewal, and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation. J Cell Biochem. 1997; 64:278–294. DOI: 10.1002/(SICI)1097-4644(199702)64:2<278::AID-JCB11>3.0.CO;2-F. PMID: 9027588.

23. Kang TJ, Yeom JE, Lee HJ, Rho SH, Han H, Chae GT. Growth kinetics of human mesenchymal stem cells from bone marrow and umbilical cord blood. Acta Haematol. 2004; 112:230–233. DOI: 10.1159/000081281. PMID: 15564740.

24. Kern S, Eichler H, Stoeve J, Klüter H, Bieback K. Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue. Stem Cells. 2006; 24:1294–1301. DOI: 10.1634/stemcells.2005-0342. PMID: 16410387.

25. Suchanek J, Soukup T, Visek B, Ivancakova R, Kucerova L, Mokry J. Dental pulp stem cells and their characterization. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2009; 153:31–35. DOI: 10.5507/bp.2009.005. PMID: 19365523.

26. 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; 60:538–544. DOI: 10.1097/SAP.0b013e3181723bbe. PMID: 18434829. PMCID: 4160894.

27. Wagner W, Wein F, Seckinger A, Frankhauser M, Wirkner U, Krause U, Blake J, Schwager C, Eckstein V, Ansorge W, Ho AD. Comparative characteristics of mesenchymal stem cells from human bone marrow, adipose tissue, and umbilical cord blood. Exp Hematol. 2005; 33:1402–1416. DOI: 10.1016/j.exphem.2005.07.003. PMID: 16263424.

28. Alipour F, Parham A, Kazemi Mehrjerdi H, Dehghani H. Equine adipose-derived mesenchymal stem cells: phenotype and growth characteristics, gene expression profile and differentiation potentials. Cell J. 2015; 16:456–465. PMID: 25685736. PMCID: 4297484.

29. Colleoni S, Bottani E, Tessaro I, Mari G, Merlo B, Romagnoli N, Spadari A, Galli C, Lazzari G. Isolation, growth and differentiation of equine mesenchymal stem cells: effect of donor, source, amount of tissue and supplementation with basic fibroblast growth factor. Vet Res Commun. 2009; 33:811–821. DOI: 10.1007/s11259-009-9229-0. PMID: 19472068.

30. Pascucci L, Mercati F, Marini C, Ceccarelli P, Dall’Aglio C, Pedini V, Gargiulo AM. Ultrastructural morphology of equine adipose-derived mesenchymal stem cells. Histol Histopathol. 2010; 25:1277–1285. PMID: 20712012.