A NOVEL METHOD FOR ISOLATION AND TRILINEAGE DIFFERENTIATION OF RAT BONE MARROW DERIVED MESENCHYMAL STEM CELLS
Objective: Goal of this study is to find a simple method for isolation, colony formation and Trilineage differentiation of Mesenchymal stem cells from
bone marrow of Wistar Rat. Adherent capacity, morphology, trilineage differentiation and colony formation of bone marrow mesenchymal stem cells
were studied in low glucose, high glucose Dulbecco modified eagle medium with various concentration of fetal bovine serum.
Methods: Mesenchymal stem cells were isolated from bone marrow of Wistar rat by Ficoll (sigma) density gradient centrifugation with plastic
adherence method. Bone marrow was collected from femur and Tibia of 6-weeks-old Wistar rat; Bone marrow was cultured in Dulbeccoâ€™s modified
Eaglesâ€™s medium (DMEM) with low glucose, high glucose supplement (Invitrogen) and 10%, 15% of fetal bovine serum (FBS) at the density of
1 Ã— 10
, incubated at 37Â°C in 5% of CO
; adherent capacity, colony formation were studied. Differentiation of bone marrow mesenchymal stem cells
(BMMSCs) was monitored under suitable differentiation medium. The cells were refeed every 3-4 days and passaged when the cells reached 80-90%
Result: BMMSCs were adhered in tissue culture flask at 24 hrs, 48 hrs in high glucose and low glucose DMEM with 10% of FBS, respectively. Colony
formation was faster in high glucose and low glucose DMEM with 15% of FBS compared to high glucose and low glucose DMEM with10% FBS.
Morphological changes were observed in BMMSCs high glucose, low glucose DMEM with 10% FBS, but no changes were found in the differentiation
of BMMSCs in high glucose, low glucose DMEM. Differentiation of BMMSCs was nourishing in third passage cells.
Conclusion: High glucose DMEM with 10% FBS is a good supplement for adherence of cells whereas high glucose, low glucose DMEM with 15% FBS
can be utilized for rapid Colony formation. Third passage BMMSCs is fruitful for differentiation of BMMSCs.
Keywords: Wistar rat, Bone marrow, Mesenchymal stem cells, Adherent capacity, Colony formation, Differentiation, Osteoblast, Chondrocytes,
normal and irradiated mouse hematopoietic organs. Exp Hematol
2. Beyer Nardi N, da Silva Meirelles L. Mesenchymal stem cells: Isolation,
in vitro expansion and characterization. Handb Exp Pharmacol
3. Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic
tissues. Science 1997;276(5309):71-4.
4. Pittenger MF, Marshak DR. In: Marshak DR, Gardner RL, Gottlieb D,
editors. Stem Cell Biology. New York: Cold Spring Harbor Laboratory
Press, Cold Spring Harbor; 2001.
5. 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 society for cellular therapy position
statement. Cytotherapy 2006;8(4):315-7.
6. Kang XQ, Zang WJ, Song TS, Xu XL, Yu XJ, Li DL, et al. Rat bone
marrow mesenchymal stem cells differentiate into hepatocytes in vitro.
World J Gastroenterol 2005;11(22):3479-84.
7. Oswald J, Boxberger S, JÃ¸rgensen B, Feldmann S, Ehninger G,
BornhÃ¤user M, et al. Mesenchymal stem cells can be differentiated into
endothelial cells in vitro. Stem Cells 2004;22(3):377-84.
8. Polisetti N, Chaitanya VG, Babu PP, Vemuganti GK. Isolation,
characterization and differentiation potential of rat bone marrow
stromal cells. Neurol India 2010;58(2):201-8.
9. Ayatollahi M, Salmani MK, Geramizadeh B, Tabei SZ, Soleimani M,
Sanati MH. Conditions to improve expansion of human mesenchymal
stem cells based on rat samples. World J Stem Cells 2012;4(1):1-8.
10. Meirelles Lda S, Nardi NB. Murine marrow-derived mesenchymal stem
cell: Isolation, in vitro expansion, and characterization. Br J Haematol
11. Yameen Z, Leavesley Z, Upton Z, Xiao Y. Multileneage differentiation
potential of bone and cartilage cells derived from explant culture. Open
Stem Cell J 2009;1:10-9.
12. Wilson A, Trumpp A. Bone-marrow haematopoietic-stem-cell niches.
Nat Rev Immunol 2006;6(2):93-106.
13. Fu WL, Zhang JY, Fu X, Duan XN, Leung KK, Jia ZQ, et al.
Comparative study of the biological charecteristics of mesenchymal
stem cells from bone marrow and peripheral blood of rats. Tissue Eng
Part A 2012;18(17-18):1793-803.
14. Taran R, Mamidi MK, Singh G, Dutta S, Parhar IS, John JP, et al.
In vitro and in vivo neurogenic potential of mesenchymal stem cells
isolated from different sources. J Biosci 2014;39(1):157-69.
The publication is licensed under CC By and is open access. Copyright is with author and allowed to retain publishing rights without restrictions.