All chemical reagents and culture media were cell culture grade and obtained from Sigma Chemicals (America), while other sources were labeled. The plastic ware was obtained from Corning (America).
Umbilical cord collection
The umbilical cord tissue of healthy male infants without hepatitis, Acquired Immunodeficiency Syndrome (AIDS), and other infectious diseases was taken from the operation room of the Hohhot First Hospital. The ethics committee approved the study, and the family members provided informed consent. The tissue was collected and stored in the phosphate buffer containing 1% penicillin and streptomycin sulfate. The samples were immediately transported to the laboratory for father processing.
Cell isolation and culture
The umbilical cord in the clean bench was taken out, put into the Dulbecco’s Phosphate Buffered Saline (D-PBS, Gibco) with 1% penicillin/streptomycin (100 U/mL and 100 μg/mL; HyClone), and cleaned three times to remove the blood as much as possible. Alcohol was used for umbilical cord disinfection, following which the cord was placed in D-PBS with 1% penicillin/streptomycin for cleaning three times to remove the residual alcohol. The umbilical vein cavity was cut to remove two arteries and one vein using ophthalmic scissors. The umbilical cord tissue was divided into several 2-cm-long parts, and the Wharton’s jelly (WJ) was separated. The WJ was cut into 0.5- to 1-cm2 segments and several crisscross scratches were made on its surface, but it was not cut through the tissue block. The tissue block was placed in the culture dish. The tissue block was cultured with Dulbecco’s modified Eagle’s medium (DMEM)-F12 (Gibco, Rockville) supplemented with 10% fetal bovine serum (FBS, Gibco) and 0.1% penicillin/streptomycin at 37℃ in a 5% CO2 humidified incubator. The cells crawled out after 7-9 days. When the adherent cells reached 80%-90%, they were digested with trypsin (0.05% trypsin-EDTA, Gibco-Invitrogen) and then frozen or subculture. The culture medium was changed every 2-3 days.
Cryopreservation and reconstitution
The cell cryopreservation was performed as described by Guan (Guan et al. 2010) and Hao (Hao et al, 2012). When the cells were frozen, the cell density of each tube was 1x106 cell/mL. The cryopreserved solution consisted of a mixture of the following reagents: 70% DMEM/F12, 20% fetal bovine serum (FBS), and 10% dimethyl sulfoxide (Shanghai Chemical, Shanghai, China). When the cells were resuscitated, the cells were recovered by shaking the cryovial in 37℃ water baths for 1 min. The contents in the tube were transferred into a 1.5 mL centrifuge tube (Nunc) and centrifuged at 1400 rpm for 5 min. The supernatant was removed, mixed with culture solution, and then added to a new culture dish in a 1:3 ratio, followed by culturing at 37℃ with saturated humidity and 5% CO2.
Cell doubling method
After resuspension, the hUMSCs were cultured into a 24-well plate to achieve a cell density of 1x104 cells/well. The cells in three wells were counted every day, and the average value was taken. The growth curve was drawn after 8 days of calculation. The population-doubling time was calculated as t [lg2/ (lgNt - lgN0)] (N0 and Nt represent the cell counts after inoculation and t the hours after the start of culturing, respectively).
Reverse transcription polymerase chain reaction (RT-PCR)
The stem cell-specific gene expression in hUMSCs isolated and cultured in vitro was analyzed by RT-PCR (Yadav et al. 2011). Briefly, total RNA was isolated from hUMSCs at passage 3 using RNAiso Plus (TaKaRa, Otsu, Japan). Then, a 10 µL reverse transcription system was prepared following the instructions on the reverse transcription kit: enzyme (PrimeScript RT Master Mix, TaKaRa, Japan), 2 µL; and RNA template, 500-1000 ng. The system was supplemented with enzyme-free water to 10 µL. The conditions for RT were as follows: 37℃ for 15 min, 85℃ for 5 s, and 4℃ till the reaction was completed. The synthesized cDNA was stored at -20℃.
A 20-µL system was prepared according to the instructions of PCR kit: enzyme (Premix Taq, TaKaRa, Japan), 10 µL; cDNA template, 50-100 ng; and primer, 2 µL. The conditions for amplification were as follows: 35 cycles each consisting of denaturation at 94°C for 30 s, annealing at 58°C for 30 s, elongation at 72°C for 30 s, final extension at 72°C for 5 min, and at 4℃ till the reaction was completed. The PCR primers and the reaction conditions are summarized in Table 1. After the cDNA was amplified, agarose gel electrophoresis was performed and visualized under a gel documentation system (Gene genius, England).
Quantitative Real-time PCR
The expression of targeted genes in melanocyte cells was measured using a qPCR assay on Roche LightCycler 480 II Time PCR System (Basel, Switzerland). The PCR primers and the reaction conditions are summarized in Table 1. Total RNA was extracted with Trizol reagent and quality controlled with Agilent 2100 Bioanalyzer (America). cDNA reverse transcription was performed by using Synthesis SuperMix (TaKaRa, Otsu, Japan). The PCR system was 20 µL, including 10 µL of SYBR Green qPCR Master Mix (TaKaRa, Ostu, Japan), PCR forward primer, PCR reverse primer, 1 µL of template cDNA, and ddH2O, with the following reaction conditions: initial denaturation at 95℃ for 5 min, 40 cycles of denaturation at 95℃ for 3 s and annealing and extension at 60 ℃ for 30 s. GAPDH was used as reference gene, and the 2-ΔΔCT method was used to analyses the relative mRNA expression. Each experiment was conducted in triplicate.
Induced differentiation in vitro
The cells were cultured in six-well plates for passage 3. When the cell reached 70%-80%, they were added to the osteogenic induction fluid for osteogenic induction. A 50-mL system of an osteogenic induction medium was prepared as follows: 10% FBS, 10 mmol/L β-glycerophosphate, 20 nmol/L dexamethasone, and 50 μg/mL sodium 2-phosphate ascorbate in DMEM-F12). The cell differentiation was assessed morphologically, and they were stained with alizarin red and von Kossa (Zuk et al. 2001b).
To induce neural cells, hUMSCs were exposed to neuronal pre-induction media (a 50-mL system was prepared as follows: 20% FBS and 10-3 mol/L β-mercaptoethanol (Shanghai Chemical, Cat. #386) in DMEM). After 24 h, the cells were washed twice with D-PBS and placed in 5×10-3 mol/L β-mercaptoethanol in DMEM without FBS for 6 h (Palermo et al. 2005). The cells were stained with toluidine blue and observed under the microscope.
To induce melanocyte cells differentiation, a 50-mL system was prepared as follow: 10mL MCDB201 medium; 15mL DMEM; 2.5 mm dexamethasone 1 µL; 0.3 mg/ml cholera toxin 0.28 µL; 1mm TPA 25 µL; insulin transferrin selenium 500 µL; linoleic acid aluminum from bovine serum aluminum 500 µL; L-ascorbic acid 0.0009 g; Glutamax 500 µL; 10 μg/ml SCF 250 µL; 100 µm ET-3 50 µL; 4 μg/ml bFGF 50 µL; 25ml L-wnt-3a cell suspension and store in a dark place at 4 ° after sub-packaging. The solution was changed every 2 days, and the induction of cells ended after 28 days. The cell differentiation was assessed morphologically.
Table 1 Details of primers used for gene expression through RT-PCR and qPCR.
Base pair (bp); source for all primers is Homo sapiens arise from NCBI.
Staining methods
(1) Alizarin red staining. Medium was aspirated from culture wells. The cells were fixed in 4% paraformaldehyde for 1h, washed twice with water, and incubated with Alizarin red solution at room temperature for 30 min. After incubation, stain was removed. The cells were washed four times thoroughly with water, finally, ~1 mL of water was left to prevent cells from drying out. The cells were visualized under the phase contrast microscope. (2) Toluidine blue staining. The cells were rinsed two times with PBS, then fixed in 4% formalin and stained in a toluidine blue working solution for 2-3 min. Next, they were washed in running tap water for 2 min, rinsed in distilled water, and observed under the microscope.
Immunofluorescence
When the cell reached of 70%-80%, the culture solution was fixed with 4% paraformaldehyde. After 10 min, it was sealed with 1% bovine serum albumin at room temperature for 30 min. Futher, it was incubated with MITF (Mouse anti-human monoclonal antibodies to MITF), TYR (Mouse anti-human monoclonal antibodies) antibody (1:1000, Abcam, Cambridge, UK) at 4℃ overnight and then with FITC (Goat Anti-Mouse IgG H&L)-labeled secondary antibody (1;500, Abcam, Cambridge, UK) at 25℃ for 50min. 4’,6-diamidino-2-phenylindole (DAPI) was used to label the nucleus.