In vitro differentiation of human bone marrow stromal cells into neural stem cells in serum-free medium

Liye YANG; Wenyu LI; Jiakun ZHENG; Qiang CHEN; Chaoyang WANG; Xiaocong LIN

ABSTRACT

BACKGROUND:

At present,the most frequently agents used for neural induction of bone marrow stromal cells(BMSCs)in vitro are anti-oxidants,such as beta-mercaptoethanol and all trans-retinoids.The majorities of induction from BMSCs are neuron-like cells in these protocols;however,whether it has neuronal function or not should be further studied.

OBJECTIVE:

TO investigate the differentiated characteristics of inducing human BMSCs into neural cells in serum-free medium.

DESIGN:

Observational study.

SETTING:

Chaozhou Central Hospital.MATERIALSThe experiment was carried out in the Chaozhou Central Hospital from April 2004 to December 2005.Adult bone marrows were derived from femoral and tibial bone marrow of three patients with fracture.All patients provided the confirmed consent and were approved by the Ethics Committee of Chaozhou Central Hospital.DMEMIF12 medium(11),fetal bovine serum (FBS), glutamine, N2 supplements and B27 Supplements were from GIBCO/BRL Company;recombinant basic fibroblast growth factor(bFGF)and recombinant epidermal growth factor(EGF)from Sigma Company;monoclonal antibody for vimentin(1100),monoclonal antibody for myelin basic protein(MBP) (1100),monoclonal antibody for S1 00(11 00),monoclonal antibody for neuron specific enolase(NSE)(11 00),and monoclonal antibody for neurofilament 200(NF200)(11 00)from Beijing Zhongshan Company;monoclonal antibody for glial fibrillary acidic protein (GFAP)(1200)and polyclonal antibody for nestin(1100)from Boster Company(Wuhan);mouse monoclonal antibody for beta-tubulin 3(11 000)from Sigma Company;SP-9000 kits and quick AEC from Beijing Zhongshan Company; culture dishes and flasks from Coming Company.

METHODS:

BMSCs from human bone marrow were cultured in serum-containing medium.When the primary culture was established, BMSCs were transferred into serum-free medium containing N2 or B27 supplement with 20 μg/L basic fibroblast growth factor(bFGF)and epidermal growth factor(EGF),and cells were cultured in an incubator containing C02of 0.05 volume fraction at 37℃. Morphological changes of BMSCs in serum-free medium were observed under phase contrast microscope. And two days after culture. Expression of relative markers of BMSCs was detected withimmunocytochemistry.MAIN OUTCOME

MEASURES:

Morphological changes of BMSCs and expression of relative markers of nerve cells.

RESULTS:

A population of BMSCs could be isolated from adult human bone marrow,and they were processed to obtain a fibroblast-like population and were expanded as undifferentiated cells in culture for more than 10 passages.indicating their proliferative capacity.They could form spheroid state when they were sub-cultured in serum-free media supplemented with bFGF and EGF.these cells could express the markers for neural stem cells such as vimentin and nestin;they could expressed neuron specific enolase(NSE),beta-tubulin 3,TrkC and neurofilament 200(NF200)when they were plated on dishes with serum-containing medium; some cells exhibited the phenotypes for astrocytes.expressing gilal fibrillary acidic protein(GFAP)and S100 protein.

CONCLUSION:

The morphology,protein expression and differentiation ability of BMSCs in serum-free medium was similar to those of neural stem cells.The data support the hypothesis that adult bone marrow contains stem cells capable of differentiating into neural cells,the serum-free media make BMSCs overcome their mesenchymal commitment,showing the phenotypes for neural stem cells.