EFFECTS OF LEUKEMIA INHIBITORY FACTOR COMBINED WITH BASIC FIBROBLAST GROWTH FACTOR ON PROLIFERATION AND DIFFERENTIATION OF HUMAN BONE MARROW MESENCHYMAL STEM CELLS_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

SHIJianming ^1,2,3 ,  CAOJun ¹ , YINMing ³ , YINChangchang ¹ , CHENGXigao ³ , XIERonghui ^2,3 , LINSiwen ^2,3 , XUJunhuai ^2,3

1. Basic Medical College, Jiujiang University, Jiujiang Jiangxi, 332000, P. R. China;
2. Medicine Graduate School of Nanchang University;
3. Department of Orthopedics, the Second Affiliated Hospital of Nanchang University;

Corresponding?author：

CAOJun, Email: caojun002@aliyun.com

Keywords：

Leukemia inhibitory factor; Basic fibroblast growth factor; Human bone marrow mesenchymal; stem cells; Cell proliferation; Cell differentiation

DOI：

10.7507/1002-1892.20140249

Video：

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Objective To study the effects of leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF) on the proliferation and differentiation of human bone marrow mesenchymal stem cells (hBMSCs). Methods hBMSCs at passage 4 were divided into 4 groups according to different culture conditions:cells were treated with complete medium (α-MEM containing 10%FBS, group A), with complete medium containing 10 ng/mL LIF (group B), with complete medium containing 10 ng/mL bFGF (group C), and with complete medium containing 10 ng/mL LIF and 10 ng/mL bFGF (group D). The growth curves of hBMSCs at passage 4 in different groups were assayed by cell counting kit 8; cellular morphologic changes were observed under inverted phase contrast microscope; the surface markers of hBMSCs at passage 8 including CD44, CD90, CD19, and CD34 were detected by flow cytometry. Results The cell growth curves of each group were similar to the S-shape; the cell proliferation rates in 4 groups were in sequence of group D > group C > group B > group A. Obvious senescence and differentiation were observed very early in group A, cells in group B maintained good cellular morphology at the early stage, with slow proliferation and late senescence; a few cells in group C differentiated into nerve-like cells, with quick proliferation; and the cells in group D grew quickly and maintained cellular morphology of hBMSCs. The expressions of CD44 and CD90 in groups A and C at passage 8 cells were lower than those of groups B and D; the expressions of CD19 and CD34 were negative in 4 groups, exhibiting no obvious difference between groups. Conclusion LIF combined with bFGF can not only maintain multiple differentiation potential of hBMSCs, but also promote proliferation of hBMSCs.

Citation： SHIJianming, CAOJun, YINMing, YINChangchang, CHENGXigao, XIERonghui, LINSiwen, XUJunhuai. EFFECTS OF LEUKEMIA INHIBITORY FACTOR COMBINED WITH BASIC FIBROBLAST GROWTH FACTOR ON PROLIFERATION AND DIFFERENTIATION OF HUMAN BONE MARROW MESENCHYMAL STEM CELLS. Chinese Journal of Reparative and Reconstructive Surgery, 2014, 28(9): 1150-1155. doi: 10.7507/1002-1892.20140249 Copy

1.	Takahashi K, Tanabe K, Ohnuki M, et al.Induction of pluripotent stem cells from adult human fibroblasts by defined factors.Cell, 2007, 131(5):861-872.
2.	Yamanaka S.Induced pluripotent stem cells:past, present, and future.Cell Stem Cell, 2012, 10(6):678-684.
3.	Dominici M, Le Blanc K, Mueller I, et al.Minimal criteria for defining multipotent mesenchymal stromal cells.The International Society for Cellular Therapy position statement.Cytotherapy, 2006, 8(4):315-317.
4.	Li XH, Yu XY, Lin QX, et al.Bone marrow mesenchymal stem cells differentiate into functional cardiac phenotypes by cardiac microenvironment.J Mol Cell Cardiol, 2007, 42(2):295-303.
5.	Wang N, Xu Y, Qin T, et al.Myocardin-related transcription factor-A is a key regulator in retinoic acid-induced neural-like differentiation of adult bone marrow-derived mesenchymal stem cells.Gene, 2013, 523(2):178-186.
6.	鐘曉琳, 萬居易, 王忠瓊, 等.淤膽血清及肝細胞生長因子體外誘導BMSCs向肝細胞分化.中國修復重建外科雜志, 2010, 24(3):344-349.
7.	Shibata KR, Aoyama T, Shima Y, et al.Expression of the p16INK4A gene is associated closely with senescence of human mesenchymal stem cells and is potentially silenced by DNA methylation during in vitro expansion.Stem Cells, 2007, 25(9):2371-2382.
8.	Noh HB, Ahn HJ, Lee WJ, et al.The molecular signature of in vitro senescence in human mesenchymal stem cells.Genes&Genomics, 2010, 32(1):87-93.
9.	Tsai CC, Chen CL, Liu HC, et al.Overexpression of hTERT increases stem-like properties and decreases spontaneous differentiation in human mesenchymal stem cell lines.J Biomed Sci, 2010, 17:64.
10.	Sotiropoulou PA, Perez SA, Salagianni M, et al.Characterization of the optimal culture conditions for clinical scale production of human mesenchymal stem cells.Stem Cells, 2006, 24(2):462-471.
11.	Xiong N, Yang H, Liu L, et al.bFGF promotes the differentiation and effectiveness of human bone marrow mesenchymal stem cells in a rotenone model for Parkinson's disease.Environmental Toxicology and Pharmacology, 2013, 36(2):411-422.
12.	何丁文, 殷嫦嫦, 顧玉榮, 等.bFGF和EGF誘導大鼠骨髓間充質干細胞分化成神經樣細胞.基礎醫學與臨床, 2013, 33(4):444-449.
13.	Whitney MJ, Lee A, Ylostalo J, et al.Leukemia inhibitory factor secretion is a predictor and indicator of early progenitor status in adult bone marrow stromal cells.Tissue Engineering Part A, 2008, 15(1):33-44.
14.	Heinrich P, Behrmann I, Haan S, et al.Principles of interleukin (IL)-6-type cytokine signalling and its regulation.Biochem J, 2003, 374(Pt 1):1-20.
15.	Youngblood BA, Alfano R, Pettit SC, et al.Application of recombinant human leukemia inhibitory factor (LIF) produced in rice (Oryza sativa L.) for maintenance of mouse embryonic stem cells.J Biotechnol, 2014, 172(2):67-72.
16.	Gimble JM, Wanker F, Wang CS, et al.Regulation of bone marrow stromal cell differentiation by cytokines whose receptors share the gp130 protein.Journal of Cellular Biochemistry, 1994, 54(1):122-133.
17.	Malaval L, Gupta AK, Aubin JE.Leukemia inhibitory factor inhibits osteogenic differentiation in rat calvaria cell cultures.Endocrinology, 1995, 136(4):1411-1418.
18.	鄢文海, 許烜慧, 韓雪飛, 等.EGF和bFGF誘導骨髓間充質干細胞向神經細胞分化后對Tau和神經元特異性烯醇化酶表達的影響.解剖學雜志, 2005, 28(6):615-617, 640.
19.	Tropel P, Platet N, Platel JC, et al.Functional neuronal differentiation of bone marrow-derived mesenchymal stem cells.Stem Cells, 2006, 24(12):2868-2876.
20.	馮英, 王會軍, 黃錦濤, 等.β-巰基乙醇和bFGF分別誘導大鼠骨髓基質干細胞成神經元樣細胞的生物學性狀.中山大學學報:醫學科學版, 2010, 31(1):28-32.

1. Takahashi K, Tanabe K, Ohnuki M, et al.Induction of pluripotent stem cells from adult human fibroblasts by defined factors.Cell, 2007, 131(5):861-872.
2. Yamanaka S.Induced pluripotent stem cells:past, present, and future.Cell Stem Cell, 2012, 10(6):678-684.
3. Dominici M, Le Blanc K, Mueller I, et al.Minimal criteria for defining multipotent mesenchymal stromal cells.The International Society for Cellular Therapy position statement.Cytotherapy, 2006, 8(4):315-317.
4. Li XH, Yu XY, Lin QX, et al.Bone marrow mesenchymal stem cells differentiate into functional cardiac phenotypes by cardiac microenvironment.J Mol Cell Cardiol, 2007, 42(2):295-303.
5. Wang N, Xu Y, Qin T, et al.Myocardin-related transcription factor-A is a key regulator in retinoic acid-induced neural-like differentiation of adult bone marrow-derived mesenchymal stem cells.Gene, 2013, 523(2):178-186.
6. 鐘曉琳, 萬居易, 王忠瓊, 等.淤膽血清及肝細胞生長因子體外誘導BMSCs向肝細胞分化.中國修復重建外科雜志, 2010, 24(3):344-349.
7. Shibata KR, Aoyama T, Shima Y, et al.Expression of the p16INK4A gene is associated closely with senescence of human mesenchymal stem cells and is potentially silenced by DNA methylation during in vitro expansion.Stem Cells, 2007, 25(9):2371-2382.
8. Noh HB, Ahn HJ, Lee WJ, et al.The molecular signature of in vitro senescence in human mesenchymal stem cells.Genes&Genomics, 2010, 32(1):87-93.
9. Tsai CC, Chen CL, Liu HC, et al.Overexpression of hTERT increases stem-like properties and decreases spontaneous differentiation in human mesenchymal stem cell lines.J Biomed Sci, 2010, 17:64.
10. Sotiropoulou PA, Perez SA, Salagianni M, et al.Characterization of the optimal culture conditions for clinical scale production of human mesenchymal stem cells.Stem Cells, 2006, 24(2):462-471.
11. Xiong N, Yang H, Liu L, et al.bFGF promotes the differentiation and effectiveness of human bone marrow mesenchymal stem cells in a rotenone model for Parkinson's disease.Environmental Toxicology and Pharmacology, 2013, 36(2):411-422.
12. 何丁文, 殷嫦嫦, 顧玉榮, 等.bFGF和EGF誘導大鼠骨髓間充質干細胞分化成神經樣細胞.基礎醫學與臨床, 2013, 33(4):444-449.
13. Whitney MJ, Lee A, Ylostalo J, et al.Leukemia inhibitory factor secretion is a predictor and indicator of early progenitor status in adult bone marrow stromal cells.Tissue Engineering Part A, 2008, 15(1):33-44.
14. Heinrich P, Behrmann I, Haan S, et al.Principles of interleukin (IL)-6-type cytokine signalling and its regulation.Biochem J, 2003, 374(Pt 1):1-20.
15. Youngblood BA, Alfano R, Pettit SC, et al.Application of recombinant human leukemia inhibitory factor (LIF) produced in rice (Oryza sativa L.) for maintenance of mouse embryonic stem cells.J Biotechnol, 2014, 172(2):67-72.
16. Gimble JM, Wanker F, Wang CS, et al.Regulation of bone marrow stromal cell differentiation by cytokines whose receptors share the gp130 protein.Journal of Cellular Biochemistry, 1994, 54(1):122-133.
17. Malaval L, Gupta AK, Aubin JE.Leukemia inhibitory factor inhibits osteogenic differentiation in rat calvaria cell cultures.Endocrinology, 1995, 136(4):1411-1418.
18. 鄢文海, 許烜慧, 韓雪飛, 等.EGF和bFGF誘導骨髓間充質干細胞向神經細胞分化后對Tau和神經元特異性烯醇化酶表達的影響.解剖學雜志, 2005, 28(6):615-617, 640.
19. Tropel P, Platet N, Platel JC, et al.Functional neuronal differentiation of bone marrow-derived mesenchymal stem cells.Stem Cells, 2006, 24(12):2868-2876.
20. 馮英, 王會軍, 黃錦濤, 等.β-巰基乙醇和bFGF分別誘導大鼠骨髓基質干細胞成神經元樣細胞的生物學性狀.中山大學學報:醫學科學版, 2010, 31(1):28-32.

Chinese Journal of Reparative and Reconstructive Surgery

EFFECTS OF LEUKEMIA INHIBITORY FACTOR COMBINED WITH BASIC FIBROBLAST GROWTH FACTOR ON PROLIFERATION AND DIFFERENTIATION OF HUMAN BONE MARROW MESENCHYMAL STEM CELLS

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