Full-term pregnancy cesarean neonatal human umbilical cords were provided by Department Obstetrics/Gynecology, The Second Affiliated Hospital of Hebei Medical University (Hebei, China). This study was approved by the Ethics Committee of the Second Hospital of Hebei Medical University. The following reagents were obtained commercially: TMP (20 mg; purity: HPLC ≥98%; batch no. 120919 Chengdu Preferred Biological Technology Co., Ltd, Chengdu, China); low glucose Dulbecco's Modified Eagle's Medium (L-DMEM)/F-12, high glucose-DMEM (H-DMEM)/F-12 and fetal bovine serum (FBS) (Gibco, Grand Island, NY, USA); trypsin (Amresco, LLC, Solon, OH, USA); EDTA and DMSO (Sigma-Aldrich, St. Louis, MO, USA); Triton X-100 (Sinopharm Chemical Reagent Co., Ltd., Beijing, China); penicillin-streptomycin (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China); paraformaldehyde (Tianjin Institute of Chemical Preparations, Tianjin, China); epidermal growth factor (EGF) (human EGF, PHG0314; Beijing Jiamei North Biological Technology Co., Ltd., Beijing, China); fluorescein isothiocyanate (FITC)-CD19, FITC-CD34, phycoerythrin (PE)-CD11b, PE-CD73, PE-CD90, PE-CD45, PE-CD105 and glial fibrillary acidic protein (GFAP) (BD Biosciences, Franklin Lakes, NJ, USA); neurofilament protein (NF-H), neuron-specific enolase (NSE) (Cell Signaling Technology, Inc., Danvers, MA, USA); anti-rabbit immunoglobulin G (IgG) (1:2,000; Affinity Biosciences, Cincinnati, OH, USA); PS immunohistochemistry kit (Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China); Taq polymerase chain reaction (PCR) star mix (Beijing GenStar Biosolutions Co., Ltd., Beijing, China); EasyScript First-Strand cDNA synthesis supermix (TransGen Biotech Co., Ltd., Beijing, China); cell RIPA lysis buffer and phenylmethylsulfonyl fluoride (Beijing Solarbio Science & Technology Co., Ltd.).

Umbilical cords were placed in H-DMEM/F-12 culture medium under aseptic conditions, stored at 4°C and then timely transported to a cell culture room to carry out the following steps. Each umbilical cord was rinsed thoroughly with D-Hank's medium. After removing the blood sample, umbilical artery and umbilical vein, the umbilical cord mesenchymal tissue was cut into 1 mm³ pieces, digested with 0.2% collagenase II and placed in a culture flask containing 2 ng/ml EGF, 20% FBS, 25 mM L-Glu and 100 U/ml penicillin streptomycin/mixture at 37°C with 5% CO₂ and saturation humidity to obtain primary cells. Half of the culture medium was replaced after 24 h, and all the medium was replenished every 3 days. When the cells achieved 80–90% confluency, the medium was removed, and cells were rinsed two times with phosphate-buffered saline (PBS) and then digested with trypsin (0.25%)-EDTA (0.2 g/l) into single cells for passaging at the ratio of 1:3. The culture medium was H-DMEM/F-12 containing 100 U/ml of a penicillin-streptomycin mixture and 10% FBS.

In the logarithmic phase of growth, hUMSCs were digested with trypsin and rinsed with PBS, and then the single cell suspension was aliquotted into 10 microcentrifuge tubes at 1×10⁶/tube. Separately, mouse anti-human monoclonal antibodies CD11-PE, CD45-PE, CD73-PE, CD90-PE, CD105-PE, human leukocyte antigen (HLA)-DR-PE, CD19-FITC and CD34-FITC (each 5 μl) were added to eight of the tubes, while anti-mouse IgG1-PE and anti-mouse IgG1-FITC (each 7 μl) were added to the other two tubes as isotype controls. After mixing the contents thoroughly, the tubes were incubated at 4°C for 30 min. Thereafter, the cells were rinsed with PBS and then centrifuged. The supernatant was discarded, and the cells were resuspended with 400 μl of PBS for analysis by flow cytometry.

Cells were obtained from the 3rd passage in logarithmic growth phase with good growth conditions, hUMSCs were rinsed twice with PBS and digested with trypsin (0.25%)-EDTA (0.2 g/l). The digestion was terminated with FBS, and then the cells were centrifuged, resuspended in culture medium and seeded at 1×10⁵/ml into five 6-well cell culture plates coated with poly-lysine. The plates were divided randomly into groups A–D and then placed in the incubator. When the cells achieved 70–80% confluency, TMP induction medium was added to cells of groups A–C at the concentrations of 4.67, 2.34 and 1.17 mg/ml, respectively. Group D was treated with L-DMEM only and served as control. All the plates were cultured in an incubator at 37°C with 5% CO₂ and saturation humidity for 6 h. Morphological changes were observed with an inverted phase contrast microscope each 0.5 h.

After induction for 6 h, expression levels of neuronal-specific proteins NSE, NF-H and GFAP were detected by immunocytochemistry. Briefly, after discarding the culture medium from the culture plates, the cells were rinsed once with PBS gently and fixed with 4% paraformaldehyde for 20 min at room temperature. After this step and subsequent steps, the cells were rinsed with PBS three times for 5 min each time, unless otherwise noted. The cell membranes were disrupted with 0.5% Triton X-100 in PBS for 15 min at room temperature away from light and then incubated with 3% H₂O₂ at room temperature for 5 min. Normal goat serum was added to the cells for blocking at room temperature for 15 min and then removed without washing. Primary antibodies to NSE, GFAP and NF-H were added at a 1:200 dilution. After incubation at 4°C overnight, the cells were incubated with the appropriate biotinylated secondary antibody at room temperature for 15 min, followed by horseradish peroxidase-conjugated streptavidin at room temperature for 15 min. Finally, the cells were stained with freshly prepared DAB for 1 min and counterstained with hematoxylin before finally rinsed repeatedly with water.

Total RNA was extracted by TriPure Reagent (Aidlab Biotechnologies Co., Ltd., Beijing, China) following the manufacturer's instructions and quantified with a Ultramicro ultraviolet visible light meter (Gene Company, Ltd., Hong Kong, China). cDNA was synthesized from total RNA with an EasyScript First-Strand cDNA synthesis supermix (TransGen Biotech Co., Ltd.) following the manufacturer's instructions. Semi-quantitative PCR was performed using Taq PCR star mix (Beijing GenStar Biosolutions Co., Ltd.) with the following reaction conditions: 1 cycle of 94°C for 3 min; 30 cycles of 94°C for 30 sec, and 72°C for 30 sec; and a final extension at 72°C for 10 min. A 10 μl sample of the PCR product was analyzed by electrophoresis, and the gene expression levels were calculated using GAPDH as the internal standard. The primers were designed using Primer 5.0 software and are shown in Table I.

Cell lysates were prepared with cell RIPA lysis buffer and phenylmethylsulfonyl fluoride (Beijing Solarbio Science & Technology Co., Ltd.), and protein concentrations were determined with an ultramicro-ultraviolet visible light meter (Gene Company, Ltd.). Total protein (16 mg) of each lysate was electrophoresed in a 10% SDS-PAGE gel and transferred to a polyvinylidene fluoride membrane (Beijing Solarbio Science & Technology Co., Ltd.). The membranes were blocked in 10% non-fat milk for 2 h and incubated with the GFAP, NF-H and NSE primary antibodies at 4°C overnight, After rinsing with PBS for 5 min, the membranes were incubated with anti-rabbit IgG (1:2,000; Affinity Biosciences) for 2 h at room temperature and visualized by SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA, USA) using the Image AlphaEaseFC system (Alpha Innotech Co., San Leandro, CA, USA).

A total of 10 non-overlapping representative fields under an inverted microscope were selected in each group, in which the total number of cells and neuron-like cells were counted. The proportion of neuron-like cells was calculated and averaged from the 10 fields for each group. Results were presented as mean ± standard deviation.

Statistical analysis was performed by using SPSS 19.0 software (SPSS, Inc., Chicago, IL, USA). Comparison on the experimental data with a completely randomized design was tested by analysis of variance among groups and the Student-Newman-Keuls method (q-test). Difference in the proportion of neuron-like cells among groups was analyzed by the Chi-square test. P<0.05 was considered to indicate a statistically significant difference.

Primary cultured cells were passaged at the ratio of 1:1, and most cells were adherent after 12 h. However, the adherent cells were not outstretched, but appeared triangular or diamond in shape. The cell culture medium was replaced to remove non-adherent cells after 24 h. The adherent cells proliferated rapidly and became significantly larger and more uniform, long fusiform cells after 48 h of culture. They achieved 80–90% confluency on the 7th day as a monolayer arranged in a radial or spiral shaped pattern. After digestion, the hUMSCs cells floated as spherical single cells in the culture medium and were passaged at the ratio of 1:3 after centrifugation. Most cells were adherent 24 h after passaging and achieved 80–90% confluency on the 7th day with a radial or spiral shaped arrangement (Fig. 1A). The hUMSCs of the 10th passaged maintained a strong proliferative capacity.

We detected the cellular phenotype of the 2nd, 5th and 10th passages of hUMSCs by flow cytometry and found that all generations of the cells tested co-expressed CD105, CD90 and CD73, but not CD11b, CD34, CD19, CD45 and histocompatibility antigen HLA-DR (major histocompatibility complex class II) (Fig. 2).

After treatment with TMP for 0.5 h, 40% of cells in group A contracted into an oval shape and extended protuberances from their cell bodies (Fig. 1B). After treatment for 1 h, the ratio of neuron-like cells reached 55%, with the cell bodies contracted and the protuberances lengthened further. At this time, the cells appeared connected in a net-like pattern. After treatment for 1.5 h, ~85% of the cells appeared as neuron-like cells. After treatment for 4 h, some of the neuron-like cells began to detach, and the ratio was reduced to ~80% in the 6th h. In group B, ~45% of the cells were neuron-like cells after treated with TMP for 1.5 h, and the ratio increased to 80% until the 2nd h. Thereafter, some of the neuron-like cells started to become non-adherent, and the ratio was reduced to 70% in the 6th h. In group C, ~30% of the cells were neuron-like cells after treatment with TMP for 4.5 h; however, some became detached, and the ratio was ~55% at the 6th h. The control group D had no significant changes before or after treated with medium alone. As shown in Fig. 3, the proportion of TMP-induced neuron-like cells was, from high to low, group A >group B >group C at each time point, and it declined gradually over the induction time indicating that the neuron-like cells were continually becoming detached. The neuron-like cells in group C required more time to reach the highest ratio, and the detaching cells could also be seen during the process of differentiation. Treatment of TMP at the concentration of 4.67 mg/ml in group A was employed in the subsequent experiment to detect the neuronal-specific markers by western blotting.

Results from immunocytochemical staining showed that most neuron-like cells treated by different concentrations of TMP for 6 h were positive for NF-H and NSE, but negative for GFAP (Fig. 4). By contrast, no cells were found positively expressing these markers in the control group. Negative staining was evidenced when PBS was used to replace the primary antibody (data not shown). As shown in Table II, differences in the expression levels of NF-H and NSE among these groups were significant (P<0.01).

On the basis of neuron-like cell positive rate, we selected the TMP treatment at a concentration of 4.67 mg/ml to detect the expression of NSE, NF-H, GFAP using western blotting. Protein expression of cells expressing neuronal markers as well as GAPDG was observed after TMP treatment. The calculated relative transcript level for protein expression of NSE was 0, 0.717±0.097, 0.919±0.056, 1.097±0.143 and 1.157±0.055 before treatment, for 1, 2, 4 and 6 h, respectively; whereas, the relative level for NH-F expression was 0, 0.780±0.10, 0.973±0.150, 1.053±0.107 and 0.753±0.094, respectively. No expression of GFAP was detected before or after induction, whereas all tested samples showed high expression of GAPDH after TMP treatment (Fig. 5).

RT-PCR was used to detect the expression of NSE, NF-H, and GFAP of the group C in each time point after TMP treatment. The mRNA expression of NSE was 0, 1.303±0.031, 1.558±0.025, 1.927±0.019 and 2.415±0.033 in proper order. The mRNA expression of NF-H was 0, 1.429±0.025, 1.551±0.024, 1.930±0.042 and 1.398±0.014 in proper order. There was no expression of GFAP before or after induction and all the groups showed high expression of GAPDH at each time point (Fig. 6).