Ex vivo Expansion and Differentiation of Mesenchymal Stem Cells from Goat Bone Marrow

Document Type: Original Article

Authors

1 Stem Cell Department, Cell Science Research Centre, Royan Institute, ACECR, Tehran, Iran

2 Royan Animal Facility, Karaj, Iran

Abstract

Objective(s)
Mesenchymal stem cells (MSCs) from large animals as goat which is genetically more closely related to human have rarely been gained attentions. The present study tried to isolate and characterize MSCs from goat bone marrow.
Materials and Methods
Fibroblastic cells appeared in goat marrow cell culture were expanded through several subcultures. Passaged-3 cells were then differentiated among the osteogenic, adipogenic and chondrogenic cell lineages to determine their MSC nature. Differentiations were determined by RT-PCR analysis of related gene expression. To identify the best culture conditions for propagation, passage-3 cells were plated either at varying cell densities or different fetal bovine serum (FBS) concentrations for a week, at the end of which the cultures were statistically compared with respect to the cell proliferation. In this study, we also determined goat MSC population doubling time (PDT) as the index of their in vitro expansion rate.
Results
Passage-3 fibroblastic cells tended to differentiate into skeletal cell lineages. This was evident in both specific staining as well as the specific gene expression profile. Moreover, there appeared to be more expansion when the cultures were initiated at 100 cells/cm[1] in a medium supplemented with 15% FBS. A relatively short PDT (24.94±2.67 hr) was a reflection of the goat MSC rapid rate of expansion.
Conclusion
Taken together, fibroblastic cells developed at goat marrow cell culture are able to differentiate into skeletal cell lineages. They undergo extensive proliferation when being plated at low cell density in 15% FBS concentration.

Keywords


1.Prochop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissue. Science 1997; 276:71-74.

2.Friedenstein AJ, Piatetzky-Shapiro II, Petrakova KV. Osteogenesis in transplants of bone marrow cells. J Embryol Exp Morphol 1966;16:381-390.

3.Digirolamo CM, Stokes D, Colter D, Phinney DG, Class R, Prockop DJ. Propagation and senescence of human marrow stromal cells in culture: a simple colony-forming assay identifies samples with the greatest potential to propagate and differentiate. Br J Haematol 1999; 107:275-281.

4.Javazon EH, Colter DC, Schwarz EJ, Prockop DJ. Rat marrow stromal cells are more sensitive to plating density and expand more rapidly from single-cell-derived colonies than human marrow stromal cells. Stem Cells 2001; 19:219-225.

5.Friedenstein AJ, Chailakhjan RK, Lalykina KS. The development of fibroblast colonies in monolayer cultures of guinea-pig bone marrow and spleen cells. Cell Tissue Kinet 1970; 3:393-403.

6.Rickard DJ, Sullivan TA, Shenker BJ, Leboy PS, Kazhdan I. Induction of rapid osteoblast differentiation in rat bone marrow stromal cell cultures by dexamethasone and BMP-2. Dev Biol 1994; 161:218-228.

7.Awad HA, Butler DL, Boivin GP, Smith FN, Malaviya P, Huibregtse B, et al. Autologous mesenchymal stem cell-mediated repair of tendon. Tissue Eng 1999; 5:267-277.

8.Baksh D, Song L, Tuan RS. Adult mesenchymal stem cells:characterization, differentiation and application in cell therapy. Mol Med 2004; 8:301-136.

9.Williams CG, Kim TK, Taboas A, Malik A, Manson P, Elisseeff J. In vitro chondrogenesis of bone marrow- derived mesenchymal stem cells in a photopolymerizing hydrogel. Tissue Eng 2003; 9:679-688.

10.Nair MB, Suresh Babu S, Varma HK, John A. A triphasic ceramic-coated porous hydroxyapatite for tissue engineering application. Acta Biomater 2008; 4:173-181.

11.Li Q, Wang J, Shahani S, Sun DD, Sharma B, Elisseeff JH, et al. Biodegradable and photocrosslinkable polyphosphoester hydrogel. Biomaterials 2006; 27:1027-10134.

12.Dai KR, Xu XL, Tang TT, Zhu ZA, Yu CF, Lou JR, et al. Repairing of goat tibial bone defects with BMP-2 gene-modified tissue-engineered bone. Calcif Tissue Int 2005; 77:55-61.

13.Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The international societry for cellular therapy position statement. Cytotherapy 2006; 4:315-317.

14.Eslaminejad MB, Nikmahzar A, Thagiyar L, Nadri S, Massumi M. Murine mesenchymal stem cells isolated by low density primary culture system. Dev Growth Differ 2006; 48: 361-370.

15.Eslaminejed MB, Mirzadeh H, Mohamadi Y, Nickmahzar A. Bone differentiation of the marrow-derived mesenchymal stem cells using tricalcium phosphate/alginate/gelatin scaffolds. J Tissue Eng Regen Med 2007; 6:417-424.

16.Eslaminejad MB, Nadri S, Hosseini RH. Expression of Thy 1.2 surface antigen increases significantly during the murine MSCs cultivation period. Dev Growth Differ 2007; 49:351-364.

17.Pioletti D, Montjovent MC, Zambelli PY, Applegate L. Bone tissue engineering using fetal cell therapy. Swiss Med Wkly 2007; 136:557-560.

18.Weissman IL. Translating stem and progenitor cell biology to the clinic: barrier and opportunities. Science 2000; 287:1442-1446.

19.Donahue RE, Dunbar CE. An update on the use of non-human primate models for preclinical testing of gene therapy approaches targeting hemotopoiesis cells. Hum Gene Ther 2001; 12:607-617.

20.Horn PA, Thomasson BM, Wood BL, Andrews RG, Morris JC, Kiem HP. Distinct hematopoietic stem/progenitor cell populations are responsible for repopulating NOD/SCID mice compared with nonhuman primates. Blood 2003; 102:4329-4335.

21.Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999; 284:143-147.

22.Spees JL, Gregory CA, Singh H, Tucker HA, Peister A, Lynch PJ, et al. Internalized antigens must be removed to prepare hypoimmunogenic mesenchymal stem cells for cell and gene therapy. Mol Ther 2004; 95:747-756. 

23.Bartmann C, Rohde E, Schallmoser K, Purstner P, Lanzer G, Linkesch W, et al. Relationship between donor age and the replicative lifespan of human cells in culture: a reevaluation. Proc Natl Acad Sci USA 1998; 95:10614-10619.

24.Kugi S, Wessels JT, Buhring HJ, Schilbach K, Schumm M, Seitz G, et al. Identification of a novel class of human adherent CD34- stem cells that give rise to SCID-repopulating cells. Blood 2003; 3:869-876.

25.Quirici N, Soligo D, Bossolasco P, Servida F, Lumini C, Deliliers GL. Isolation of bone marrow mesenchymal stem cells by anti-nerve growth factor receptor antibodies. Exp Hematol 2002; 7:783-791.

26.Gronthose S, Simmons PJ. The growth factor requirements of STRO-1-positive human bone marrow stromal precursors under serum-derived condisions in vitro. Blood 1995; 85:929-940.