Isolation and expansion of rat primary mesenchymal stem cells
Rat primary MSCs were isolated and expanded as described before . In brief, after the sacrifice of 4-week-old Sprague Dawley (SD) rats, the tibias and femurs were removed and cleaned of connective and soft tissues. Bone marrow was flushed out of the cut bones after removal of the epiphysis. Cells were cultured in Dulbecco's modified Eagle medium/Ham's Nutrient Mixture F12 (DMEM/F12; Gibico) supplemented with 10% fetal bovine serum (FBS; Gibico), 100 units/ml penicillin, and 100 μg/ml streptomycin at 37°C in a humidified incubator containing 5% CO2. Cells were digested with trypsin (Invitrogen) and passaged at approximately 90% confluence every 3-4 days. Cells between passages three and six were used for subsequent experiments.
Cultured cells were trypsinized and resuspended in PBS containing 1% bovine serum albumin (BSA). Cell suspensions were incubated with different antibodies including CD34-FITC (Santa Cruz), CD29-FITC (BD Biosciences), CD44-FITC (Santa Cruz), CD45-PE (Biolegend), CD90-PE (Biolegend), and CD106-PE (BD Biosciences) at recommended dilution for 30 min at room temperature in the dark. After washed with PBS twice, labeled cells were analyzed by FACSC-anto II system (BD Biosciences) and quantified by using CellQuest Pro software (BD Biosciences).
Construction of reversible immortalized mesenchymal stem cells
The retroviral vector SSR#69 that expresses SV40T antigen and hygromycin resistant gene flanked with loxP sites  and package vector pAmpho were co-transfected into human embryonic kidney 293 (HEK293) cell lines by using LipfectMINE 2000 to package retrovirus expressing SV40T antigen. Freshly isolated MSCs were seeded in T25 flask and infected with 2 mL of filtered retrovirus containing 4 μg/ml polybrene. After two days infection, MSCs were selected in the presence of 0.4 mg/ml hygromycin B (Invitrogen) for 14 days, colonies of selected MSCs emerged. Then the pool stable cell lines designed as immortalized mesenchymal stem cells (IMSCs) were expanded and frozen in liquid nitrogen tanks. The reversal of IMSCs was achieved by adenovirus containing Cre (Ad-Cre) mediated site-specific Cre/loxP recombination. After infection with Ad-Cre for 48 h, western blotting was carried out to confirm SV40T was excised.
Cell growth curve of primary mesenchymal stem cells and immortalized mesenchymal stem cells
Primary MSCs at passage 3 and IMSCs at passage 40 (7.5 × 104 per well) were inoculated into 6-well culture plates and incubated with complete DMEM/F12. Cells were trypsinized and counted with Trypan Blue exclusion assay (Sigma-Aldrich) as described  at 1, 2, 3, 4, 5 d after inoculation, respectively. Briefly, cell suspension was mixed with 2 × Trypan Blue solution. And then 10 μl of suspension mixture was added into hemocytometer and viable cells exclude trypan blue were counted twice under an inverted microscope (TE2000-S; Nikon). Three different independent experiments were executed in duplicate.
Osteogenic differentiation, adipogenic differentiation and chondrogenic differentiation of primary mesenchymal stem cells and immortalized mesenchymal stem cells
Cells were seeded and grown to approximately 70-80% confluence in 6-well culture plates. To initiate trilineage differentiation, complete DMEM⁄ F12 medium was removed, and tissue-specific differentiation medium was added and maintained for three weeks as described [21–23]. Concretely, osteogenic induction medium (Hyclone) was added to induce osteogenic differentiation and subsequently changed every 3-4 days. Three weeks later, cells were fixed in methanol at -20°C for 20 min and stained with 1% Alizarin Red S (Sigma-Aldrich) for 20 min to measure calcified extracellular matrix. For adipogenic differentiation, cells were induced by adipogenic induction medium (Hyclone) which changed every 3-4 days. Three weeks later, cells were fixed and stained with 0.3% Oil Red O (Sigma-Aldrich) in isopropanol for 60 min. Chondrogenic induction medium was used to induce chondrogenic differentiation and changed every 3-4 days in the whole process. After 28 days, cells were fixed and stained with 1% Alcian Blue 8-GX (Sigma-Aldrich) in 0.1 M HCl (pH 1.0) at 25°C for 12 h to detect sulfated glycosaminoglycans deposited in matrix. Qualitative analysis was carried out under an inverted microscope.
Neuronal induction of primary mesenchymal stem cells and immortalized mesenchymal stem cells
Firstly, cells were cultured in complete DMEM/F12 containing 1 μM all-trans-retinoic acid (ATRA) for 24 hr. When cultured cells confluenced approximately 70-80%, complete DMEM⁄ F12 was replaced by modified neuronal induction medium (MNM) [DMEM/F12/1.6% DMSO/160 μM butylated hydroxyanisole/20 mM KCl/1.6 mM valproic acid/8 μM forskolin/0.8 μM hydrocortisone/4 μg/ml insulin (all from Sigma)] to induce neuronal differentiation for 24 hr.
Total RNA was extracted from cells with RNA isolation Kit (Genemega Inc.) according to the manufacturer's manual. The RNA sample was treated with DNase I (Invitrogen) to absolutely remove residual DNA. The first strand cDNA was generated from purified RNA sample using PrimeScript RT reagent Kit (TaKaRa) according to the manufacturer's instruction. Briefly, 2 μl 5 × PrimeScript Buffer, 0.5 μl PrimeScript RT Enzyme Mix I, 0.5 μl Oligo dT Primer, 0.5 μl Random 6 mers, and 500 ng total mRNA were mixed together to a total volume of 10 μl with RNA free water. The reverse transcriptase reaction was performed as follows: 37°C for 15 min then 85°C for 5 sec. The cDNA products were diluted 10-fold as PCR templates. Real-time PCR reaction was performed using RealMasterMix kit (SYBR Green; TIANGEN BIOTECH) and Bio-Rad Real-time PCR instrument. Reaction protocol was carried out as follows: 94°C × 20 sec, 55°C × 20 sec, 72°C × 20 sec, reading plate for 40 cycles. Data were represented as Ct value and calculated to the fold change using the ratio of the relative quantity of target gene to the β-actin. Primer sequences for Nestin, neuron specific enolase (NSE), microtubule-associated protein-2 (MAP-2), glial cell line-derived neurotrophic factor (GDNF) and β-actin were designed by using the Primer Primier 5 software and listed as follows:
Nestin, forward: 5'-GGGCAAGTGGAACGTAGA-3'
NSE, forward: 5'-CTGTTTGCTGCTCAAGGTC-3'
MAP-2, forward: 5'-GTATCAGGAGACAGGGAGGAG-3'
GDNF, forward: 5'-CACTGACTTGGGTTTGGG-3'
β-actin, forward: 5'-TTTGAGACCTTCAACACCCC-3'
Immunofluorescence staining was performed as described before . Briefly, cells were fixed with methanol at -20°C for 20 min and washed twice with PBS. After blocked 1 hr with 5% BSA, the fixed cells were incubated with Nestin, neuron specific enolase (NSE), microtubule-associated protein-2 (MAP-2) (all from Abcam) and glial cell line-derived neurotrophic factor (GDNF) (Santa Cruz Biotechnology) primary antibody at 4°C overnight. After washed twice with PBS, all samples were incubated with appropriate secondary antibody conjugated to dylight 488 or 594 (Jackson ImmunoResearch Laboratories) for 1 hr. And then, 4'6'-diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma) was used to stain cell nucleus. At last, all samples were examined and imaged under a fluorescence microscope (TE2000-S; Nikon). Control IgG of corresponding primary antibodies were used as negative controls.
Western blotting was performed as described previously . Briefly, total protein was extracted by specific extraction kit (Bioteke Co. Ltd.). Protein concentrations of different samples were determined by using the BCA protein assay kit (Bioteke Co. Ltd.) and enzyme-labelling measuring instrument (Thermo Fisher Scientific). All protein samples were denatured by boiling and loaded onto a 10% SDS-polyacrylamide gel (Beyotime) with approximately 30 μg total proteins per lane. After electrophoretic separation, proteins were transferred to a polyvinylidene fluoride membrane (Millipore). After rinsed twice by tris buffered saline with tween-20 (TBST) (5 min per time), the membrane was blocked with 5% skimmed milk in TBST at 37°C for 1 h and probed with anti-NSE (Abcam) and anti-β-actin (Santa Cruz Biotechnology) primary antibodies at 4°C overnight then incubated with respective secondary antibody conjugated with horseradish peroxidase (Santa Cruz Biotechnology) at room temperature for 1 hr. The membrane was developed with an enhanced chemiluminescent kit (TIANGEN) and photographed by ECL Imaging System (Syngene GBOX, UK).
Resting membrane potential of cells was recorded using the whole-cell patch clamp technique at room temperature according to the methods described previously . Cells were seeded on 8 × 8-mm glass coverslips and then cultured in complete DMEM⁄ F12 or induced by MNM for 24 h before electrophysiology recording. Coverslips were placed in an acrylic chamber (RC-26L) of inverted microscope (TE-2000U, Nikon, Japan), then perfused at 1.0-1.8 ml/min. Borosilicate glass capillaries (TW150F-4, WPI, FL, USA) were calcined and stretched into approximately 1 μm tip diameter patch electrodes. Pipette resistances approximately ranged from 3 to 4 m Ω. Electrodes were filled with internal solution containing (in mmol/L) 140 KF, 10 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and 10 ethylene glycol tetraacetic acid (EGTA) and 5 adenosine triphosphate (ATP)-Na2. Internal solutions were adjusted to pH 7.2 with CsOH. The external bath solution contained (in mM) 140 NaCl, 5 KCl, 1.8 CaCl2, 1 MgCl2·6H2O, 10 D-glucose, and 10 HEPES were regulated to pH 7.4 with NaOH. After the formation of a gigaohm seal by negative pressure suction on single cell using patch electrodes, the whole-cell recording was established. Data acquisition was achieved by using Multiclamp 700B amplifier (Axon Instruments) and Digidata 1322 interface (Axon Instruments).
Animal experiment protocols were approved by Chongqing Medical University Institutional Animal Care and Use committee. Sprague Dawley (SD) rats and nude mice were gained from the Experimental Animal Centre of Chongqing Medical University [certificate: SCXK (Yu) 2007~0001]. All rats and mice were housed under specified pathogens free (SPF) level laboratory conditions. Rats and mice were maintained under optimal conditions of hygiene, temperature, humidity, photoperiods (12L: 12D) with food and water available ad libitum.
Tumorigenicity detection of primary mesenchymal stem cells and immortalized mesenchymal stem cells
Nude mice were used to detect tumorigenicity of primary MSCs and IMSCs. Cells suspended in PBS were transplanted into nude mice by subcutaneous injection. Rat yolk sac tumor cells (L2RYC) were used as positive control. Twenty four nude mice aging 8 weeks were divided into three groups equally. One group nude mice were subcutaneously injected primary MSCs (injected in the left front and rear notum of nude mice) and IMSCs (injected in the right front and rear notum of nude mice). Second group nude mice were subcutaneously injected rat yolk sac tumor cells (injected in the left front and rear notum of nude mice) and primary MSCs (injected in the right front and rear notum of nude mice). Third group nude mice were subcutaneously injected rat yolk sac tumor cells (injected in the left front and rear notum of nude mice) and IMSCs (injected in the right front and rear notum of nude mice). 1 × 106 cells were transplanted at each injection site. Six weeks later, all nude mice were executed by CO2 asphyxiation. Pictures of nude mice and gross specimen were taken using digital camera. Tumor nodules were removed and fixed in 4% paraformaldehyde solution. After fixation and paraffin embedded, tissues were cut into 5 μm thick sections. These tissue sections were deparaffinized in xylene and rehydrated using graded ethanol concentrations. The slides were stained by hematoxylin and eosin successively. And then, they were dehydrated and differentiated by ethanol. After dropping Canada balsam on the sections, coverslips were covered to seal them. Histological images were photographed with a microscope (Nikon ECLIPSE 55i, Japan) and a digital camera (LEIKA DFC 420C, Germany).
Hypoxic-ischemic brain-damage (HIBD) rat model
Neonatal hypoxic-ischemic brain damage model was established as previous methods . Briefly, the left carotid artery of seven day postnatal SD rat was isolated and ligated doubly. After 2 hours recovery with their mothers, offsprings were placed into a transparent plastic container which was ventilated with a constant flow of humidified mixture of 8% oxygen and 92% nitrogen for 2.5 hr. The container was placed in a 37°C water bath for maintaining proper temperature. 24 hr after hypoxia-ischemia, the offsprings were randomly divided into 3 groups and transfused with 1-2 × 106 MSCs or IMSCs in 100 μL of PBS by intraperitoneal injection.
Morris Water Maze test
At age of five weeks, 15 rats of each group were received Morris water maze  test training to evaluate their spatial learning and memory abilities. Morris water maze test system (MWM SLY-WMS 2.0, China) was used in the test. The maze was a 1.5-m-diam circular pool filled with clear tap water at a temperature of 20 ± 0.5°C. A 10-cm-diam platform was placed in the III quadrant 1 cm up or submerged 2 cm below the water surface during visible test and invisible test. The procedure consisted of 1 d of visible platform tests and followed by 4 d of hidden platform tests, plus a probe trial 24 h after the last hidden platform test. In the visible platform test and the hidden platform tests, rats were tested for 4 contiguous trials in respective four quadrants per day; with an intertrial interval of 30 min. Track of animal movement was recorded in the test. To assess memory consolidation, a probe trial was performed after the last hidden platform test. In this trial, the platform was removed from the pool and the pass times in target area were recorded within 60 s.
Shuttle Box test
At 35 days of age, 15 rats of each group were subjected to a shuttle box test (KE ZH-DSX2, China), comprising two compartments (50 × 20 × 25 cm). Experiments containing training and formal testing were performed to train the rats that consistent response to the electric shock. For each trial, a 10 sec conditioning stimulus (CS), comprising a buzzer and a light, was followed by 10 sec of unconditioned stimulus, comprising the CS plus a 0.6 mA electric current to the grid floor. All rats were trained for 1 day, 20 times a day, at intervals of 2-3 min. Formal test were operated at 2-5 d, 20 conditioning trials, separated by 2-3 min inter-trial intervals. If the rat run into the safe chamber within 10 s after sound and light, it was recorded for the active avoidance response, or fled into the safe chamber after got electric shocks, it was recorded for passive avoidance response; still not, it was record for no avoidance response. We analyzed the proportion of active avoidance response and no avoidance response, respectively.
Results were expressed as Means ± S.E.M. if not indicated otherwise. The data were analyzed using SPSS 18.0 software. Statistical analyses were performed using one-way analysis of variance (ANOVA) or student t test. P ≤ 0.05 was considered statistically significant.