BACKGROUND:Bone marrow mesenchymal stem cells(MSCs) have been shown to differentiate into neuronal-like cells through the use of several factors,such as 2-mercaptoethanol,dimethyl sulfoxide,or monothioglycero However,these factors are not suitable for human use due to toxicity.Theoretically speaking,traditional Chinese medicine could be used as potential and safe factors. OBJECTIVE:To investigate the effect of natural cerebrolysin on neuronal-like differentiation of MSCs,based on protein and mRNA analyses. DESIGN,TIME AND SETTING:A parallel controlled,in vitro experiment was performed at the Institute or Integrated Chinese and Western Medicine,Shenzhen Hospital,Southern Medical University between June 2006 and April 2008. MATERIALS:Natural cerebrolysin was provided by Shenzhen Institute of Integrated Chinese and Western Medicine,China.It primarily consisted of Renshen(Radix Ginseng),Tianma(Rhizoma Gastrodiae),and Yinxingye(Ginkgo Leaf) at a proportion of 1:2:2.Natural cerebrolysin extract (1:20) was prepared using conventional water extraction methodology.Each gram of extract equaled 20 grams of the crude drug.Twelve adult,male,New Zealand rabbits were included,six of which underwent intragastric administration of natural cerebrolysin extract(0.976 g/kg per day) for 1 mon h for natural cerebrolysin-containing serum.The remaining six rabbits received intragastrie administration of equal volumes of physiological saline for normal blank serum. METHODS:Sprague Dawley male rats,6-8 weeks old,were used to harvest tibial and femoral bone marrow.Isolation and purification of MSCs were established from the whole bone marrow by removing the non-adherent cells in primary and passage cultures.For cellular identification,MSCs from four o five passages were co-cultured with LG-DMEM media containing 10% natural cerebrolys in.Simultaneously,MSCs cultured in LG-DMEM media containing 10%blank rabbit serum served as the control group. MAIN OUTCOME MEASURES:Morphology of MSCs and neurite outgrowth during differentiation was observed under inverted phase contrast microscope.Neurite-positive cells were classified by neurite length that was longer than 1.5×the cell body diameter.Immunocytochemistry was used to identify purity of MSCs following passage,as well as expression of nidogen,neuron-specific enolase,glial fibrillary acidic protein,and microtubule-associated protein 2 following treatment with natural cerebrolysin.mRNA expression of neuron-specific enolase and glial fibrillary acidic protein was detected using semi-quantitative RT-PCR. RESULTS:After MSCs were treated with natural cerebrolysin for 3-5 hours,the cell bodies were larger,and small neurites - similar to neuronal neurites - were observed.The number of neurite-positive cells significantly increased compared with the control group(P<0.05).After MSCs were treated with natural cerebrolysin for 12 hours,most expressed nidogen, neuron-specific enolase,and microtubule-associated protein 2 at higher levels than the control group(P<0.01).No evident expression of glial fibrillary acidic protein was found(P>0.05). CONCLUSION:Natural cerebrolysin promoted neurite outgrowth and induced neuronal-like differentiation of MSCs. BACKGROUND: Bone marrow mesenchymal stem cells (MSCs) have been shown to differentiate into neuronal-like cells through the use of several factors, such as 2-mercaptoethanol, dimethyl sulfoxide, or monothioglycero However, these factors are not suitable for human use due to Toxic.Theoretically speaking, traditional Chinese medicine could be used as potential and safe factors. OBJECTIVE: To investigate the effect of natural cerebrolysin on neuronal-like differentiation of MSCs, based on protein and mRNA analyzes. DESIGN, TIME AND SETTING: A parallel controlled , in vitro experiment was performed at the Institute or Integrated Chinese and Western Medicine, Shenzhen Hospital, Southern Medical University between June 2006 and April 2008. MATERIALS: Natural cerebrolysin was provided by Shenzhen Institute of Integrated Chinese and Western Medicine, China. of Renshen (Radix Ginseng), Tianma (Rhizoma Gastrodiae), and Yinxingye (Ginkgo Leaf) at a proportion of 1: 2: 2. Natural cerebrolysin extr act (1:20) was prepared using conventional water extraction methodology. Ecg of extract equaled 20 grams of the crude drug. Earweld adults, male, New Zealand rabbits were included, six of which underwent intragastric administration of natural cerebrolysin extract (0.976 g / kg per day) for 1 mon h for natural cerebrolysin-containing serum.The remaining six rabbits received intragastrie administration of equal volumes of physiological saline for normal blank serum. METHODS: Sprague Dawley male rats, 6-8 weeks old, were used to harvest tibial and femoral bone marrow. Isolation and purification of MSCs were established from the whole bone marrow by removing the non-adherent cells in primary and passage cultures. For cellular identification, MSCs from four o five passages were co-cultured with LG-DMEM media containing 10% natural cerebrolys in. Simultaneously, MSCs cultured in LG-DMEM media containing 10% blank rabbit serum served as the control group. MAIN OUTCOME MEASURES: Morphology of MSCs and neurite outgrowth during differentiation was observed under inverted phase contrast microscope. Neurite-positive cells were classified by neurite length that was longer than 1.5 × the cell body diameter. Immunocytochemistry was used to identify purity of MSCs following passage, as well as expression of nidogen, neuron-specific enolase, glial fibrillary acidic protein, and microtubule-associated protein 2 following treatment with natural cerebrolysin. mRNA expression of neuron-specific enolase and glial fibrillary acidic protein was detected using semi-quantitative RT-PCR. RESULTS: After MSCs were treated with natural cerebrolysin for 3-5 hours, the cell bodies were larger, and small neurites - were similar to neuronal neurites - were observed. The number of neurite-positive cells significantly increased compared to the control group (P <0.05). After MSCs were treated with natural cerebrolysin for 12 hours, most expressed nidogen, neuron-specific enolase, and microtubule-associated protein 2 at higher levels th an control group (P <0.01) .No evident expression of glial fibrillary acidic protein was found (P> 0.05). CONCLUSION: Natural cerebrolysin promoted neurite outgrowth and induced neuronal-like differentiation of MSCs.