All UCB samples (age, 24–38 years) were collected from the Third Affiliated Hospital of Xinxiang Medical University, Xinxiang, China between February 2018 and December 2018. Typically, 30–60 ml of UCB samples were collected through the umbilical cord by way of gravity drainage. UCB was collected from the umbilical cord vein with written informed consent of the mother and in strict accordance with the ethical standards of the local ethics committee. All blood samples were processed 4–6 h after collection. To isolate and expand MSCs from the UCB, MNCs were isolated using a lymphocyte separation medium (ρ=1.077 g/l; TBDscience). D-Hanks balanced salt solution (Beijing Solarbio Science & Technology Co., Ltd.) was used to dilute 15–30 ml of fresh UCB at a 1:1 ratio, then the diluted blood was slowly added to the lymphocyte separation medium (taking care not to disturb the liquid surface). The diluted UCB was mixed with the lymphocyte separation media with a volume ratio of 2:1. After centrifugation at 1,317 × g for 20 min at room temperature, the white cloud-like MNCs in the interface layer were carefully aspirated, centrifuged as aforementioned, washed twice with PBS at 750 × g for 10 min at room temperature, and washed once with the DMEM (Gibco; Thermo Fisher Scientific, Inc.) at the same speed. After centrifugation, DMEM supplemented with 10% FBS (Hangzhou Sijiqing Biological Engineering Materials Co., Ltd.) and 1% penicillin/streptomycin was added to the collected cells to prepare a single cell suspension. The cells were counted and inoculated into an FBS-coated T25 cell culture flask (area of 25 cm² required 1–1.5 ml FBS) at a density of 1×10⁷ cells/ml and incubated at 37°C in a humidified atmosphere with 5% CO₂ (19). Upon reaching 70–80% confluency, the cells were digested with 0.1% trypsin and further subcultured. The growth and morphological characteristics of the primary cells were observed daily under an inverted light microscope (Leica Microsystems GmbH; magnification, ×200).

In the present study, the culture efficiency of hUCB-MSCs was compared in two types of media. DMEM supplemented with 10% FBS (n=16; DMEM group) and DMEM supplemented with mesenchymal stem cell growth supplement (ScienCell Research Laboratories, Inc.) and 10% FBS [n=22; the mesenchymal stem cell medium (MSCM) group]. To reduce costs, a sequential culture method consisting of two media was used in the MSCM group. hUCB-MSCs in the MSCM group were cultured in DMEM supplemented with 10% mesenchymal stem cell growth supplement (ScienCell Research Laboratories, Inc.) and 10% FBS for three passages, and on the fourth passage the culture media was replaced with DMEM supplemented with 10% FBS. The isolated hUCB-MSCs were inoculated into an FBS-coated T25 cell culture flask at a density of 1×10⁷/ml (5 ml/flask) and cultured at 37°C in a humidified atmosphere with 5% CO₂. The growth and morphological characteristics of the primary cells were observed daily under an inverted microscope and images were captured. The numbers of hUCB-MSCs in the two groups were counted on the 5, 7 and 14th day under the same magnification using a light microscope. After uniform spindle fibroblast-like cells, which grew in a whirled manner, were cultured successfully, the culture success rates and effects of the different types of media on the culture of hUCB-MSCs were compared.

MNCs from nine blood samples were divided equally into three subgroups and were inoculated in T25 cell culture flasks at cell densities of 1×10⁶, 1×10⁷ and 1×10⁸/ml (with each density representing one subgroup). The cells were cultured in DMEM supplemented with mesenchymal stem cell growth supplement and 10% FBS. The growth of hUCB-MSCs was observed daily under an inverted microscope, and the extension time and primary culture time were recorded.

MNCs from nine blood samples were divided equally and inoculated in a T25 cell culture flask at a cell density of 1×10⁷/ml; the cells were cultured in DMEM supplemented with mesenchymal stem cell growth supplement and 10% FBS. The medium was changed on the 3, 4, 5 and 7th day after inoculation. The media was transferred to a new culture flask for >5 days and further observed to determine whether MSCs could grow, and to determine the time of first medium change.

Passage 3 and 10 of hUCB-MSCs were prepared into single cell suspensions, the cell concentrations were adjusted and the cells were inoculated into a 24-well plate. The number of cells added to each well was 1×10³. Cells were cultured in DMEM supplemented with 10% FBS in a 37°C humidified atmosphere with 5% CO₂. Cells were counted twice per day and the mean was used as the final cell number. Any increase in the number of cells was calculated for 12 consecutive days. Growth curves of hUCB-MSCs were plotted according to the number of cells, and the population doubling time was calculated from the growth curves. All experiments were performed in triplicate.

hUCB-MSCs from passage 3 and 10 were digested with 0.1% trypsin. After centrifugation at 750 × g for 10 min at room temperature, the supernatant was removed and washed with PBS. Aliquots of ~1×10⁶ cells for each antibody were obtained. The harvested cells were stained with phycoerythrin-mouse anti-human CD29 (cat. no. 561795; 20 µl/10⁶ cells; BD Biosciences), FITC mouse anti-human CD44 (cat. no. 560977; 20 µl/10⁶ cells; BD Biosciences) and FITC mouse anti-human CD45 (cat. no. 560976; 20 µl/10⁶ cells; BD Biosciences). After 30 min of incubation at room temperature in the dark, the cells were centrifuged at 750 × g for 5 min at room temperature and the unconjugated antibody was removed. The stained cells were resuspended in 500 µl PBS and analyzed using a flow cytometer (Beckman-Coulter, Inc.), and CytExpert software 2.0 (Beckman-Coulter, Inc.) was used for data analysis. All experiments were performed in triplicate.

hUCB-MSCs (1×10⁶) from passage 3 and 10 were harvested and the total RNA was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions, and the RNA concentrations were determined. RNA sample (500 ng) was reverse transcribed into cDNA using a PrimeScript™ RT Reagent kit (Takara Biotechnology Co., Ltd.). The temperature protocol was as follows: 37°C for 30 min and then 85°C for 5 sec. Primers were designed according to the sequences of octamer-binding transcription factor 4 (Oct4), Sex determining region Y-box 2 (Sox2) and the homeobox protein Nanog (Table I). The PCR protocol was as follows: 94°C for 5 min, followed by 35 cycles of 94°C for 30 sec, 60°C for 30 sec and 72°C for 40 sec. The reaction was completed with a final extension at 72°C for 5 min. GAPDH was used as a positive control. PCR products were visualized with ethidium bromide on a 1% agarose gel.

The cDNA from passage 3 and 10 of the hUCB-MSCs was used for RT-qPCR using the SYBR Premix Ex Taq (Vazyme) using the ABI 7500 real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The reaction mixture (20 µl) consisted of 2 µl template (50 ng/µl), 10 µl 2X SYBR mix (Vazyme), 0.5 µl each primer (10 µmol/l) and 7 µl deionized water. The primer sequences were the same and are listed in Table I. The thermocycling conditions were as follows: 95°C for 5 min, followed by 40 cycles of 9°C for 10 sec and 60°C for 30 sec. The relative gene expression level was determined using the 2^−ΔΔCq method (22). GAPDH was used for normalization.

hUCB-MSCs from passage 3 and 10 were used for adipogenic differentiation. Briefly, for the differentiation assay, passage 3 and 10 of hUCB-MSCs were seeded in a 6-well plate at a density of 2×10⁴ cells/well and grown to 80% confluence. Subsequently, the growth medium was substituted for adipogenic differentiation medium (Cyagen Biosciences, Inc.) containing 1 µmol/l dexamethasone (Sigma-Aldrich; Merch KGaA), 10 µg/ml insulin (Sigma-Aldrich; Merck KGaA), 200 µmol/l indomethacin (Sigma-Aldrich; Merck KGaA) and 0.5 mmol/l 3-isobutyl-1-methylxanthine (Sigma-Aldrich; Merck KGaA), the medium was replaced every 3 days. After 14 days, the cells were washed with PBS and fixed using 4% formaldehyde for 30 min at room temperature. Adipogenic differentiation was determined using 0.3% Oil Red O staining for 30 min at room temperature. All experiments were performed in triplicate.

All experiments were performed in triplicate. All data were analysed using SPSS 22.0 software (IBM Corp.). Data were expressed as the mean ± SD. Student's t-tests were performed to compare differences between groups, whereas the differences among multiple groups were determined using one-way ANOVA, followed by post hoc Duncan's tests. P<0.05 was considered to indicate a statistically significant difference.

Using the same isolation procedure, 5 of 16 samples in the DMEM group obtained a uniform culture of hUCB-MSCs, indicating a culture success rate of 31.25%, whereas 18 of 22 samples in the MSCM group obtained a uniform culture of hUCB-MSCs, indicating a culture success rate of 81.81%, which was significantly higher than the DMEM group (P<0.05). The numbers of hUCB-MSCs in the MSCM group on the 5, 7 and 14th days were significantly higher than the DMEM group on the same days (P<0.05), and the hUCB-MSCs grew rapidly in a whirled manner in the MSCM group (Table II). In the DMEM group, the MNCs were round on the first day (Fig. 1A), after 7 days, the hUCB-MSCs began to expand (Fig. 1B); the primary cultured cells reached a confluency of 10–20% on day 14 (Fig. 1C) and 80–90% on day 40. In the MSCM group, the MNCs were round on the first day (Fig. 1D), then after five days, the hUCB-MSCs began to expand; the primary cultured cells reached a confluency of 10–15% on day 7 (Fig. 1E), 30–40% on day 14 (Fig. 1F) and 80–90% on day 22. The cultured cells were passaged twice to obtain a more uniform culture of hUCB-MSCs at passage 3. The UCB samples that were not cultured successfully contained a small numbers of MSCs. In addition, the volume of MSCs in the unsuccessful cultures was not as large as the volume of osteoclast-like cells, and a small number of MSCs were mixed with the osteoclast-like cells and could not expand normally. Osteoclast-like cells occupied the bottom of the culture flask and reached 80% confluence after 3–4 weeks; the cells could not be trypsinized from the base of the flask. After passaging, MSCs in the cultures initially determined to be unsuccessful could no longer be cultured, ultimately leading to cell death.

DMEM supplemented with mesenchymal stem cell growth supplement was used to compare the culture efficiency using three inoculation densities. As shown in Table III, an inoculation density of 1×10⁷ cells/ml (2×10⁶ cells/cm²) resulted in a shorter extension time and primary culture time than the other densities tested; the difference was statistically significant (P<0.05), indicating that this density was good for the culture of hUCB-MSCs.

The medium was changed for the first time on the 3, 4, 5 and 7th day after inoculation. Changing the media on the 7th day did not result in the growth of MSCs in the media removed; only a small number of MSCs grew in the new culture flask on the 5th day, and achieving a high cell density was difficult (Fig. 2A). When media was changed on the 3rd or 4th day MSCs could be obtained following long-term culture, therefore, the first medium change was set at four days after inoculation (Fig. 2B). Compared with changing the media on day 5 after inoculation, the subculturing efficiency was significantly higher at day 4 after inoculation, with a more uniform MSC population obtained when subcultured at passage 3 (Fig. 2C).

The cell expansion rate of hUCB-MSCs before passage 3 was slower than that of passage 4 or of later passages. As hUCB-MSCs were gradually purified, the osteoclast-like cells were removed, and the doubling rate of each passage became more stable. Furthermore, to reduce costs, starting with passage 4, conventional DMEM was also used to subculture hUCB-MSCs in the MSCM group. After several rounds of passaging, the growth rate of cells was almost equivalent as in the stem cell media and there were no significant differences in the morphological characteristics of the cells (Fig. 3). The growth curves of hUCB-MSCs showed that the growth latency was 1–3 days. Starting on the 4th day, cells entered the logarithmic phase of growth and began to proliferate, the cell density increased rapidly. The proliferation rate reached a peak on the 10th day and began to slow, and plateaued. The population doubling time for the entire hUCB-MSCs population was 68 h. There were no significant differences in the proliferation rate and growth cycle of hUCB-MSCs between passages 3 and 10 (Fig. 4).

Flow cytometry was used to detect the cell surface markers of hUCB-MSCs. hUCB-MSCs from passage 3 showed little to no expression of the hematopoietic marker CD45 (0.06%), however, the cells did stably express the stem cell markers CD29 (98.81%) and CD44 (98.41%). hUCB-MSCs stably expressed CD29 (99.29%) and CD44 (98.59%) until passage 10, and did not express CD45 (0.10%; Fig. 5). The expression levels of the cell surface markers of the passage 10 cells was almost equivalent to the passage 3 cells, which is consistent with a previous study investigating bone marrow derived mesenchymal stem cells (BM-MSCs) by Nagamura-Inoue and He (15).

RT-PCR was used to detect the gene expression levels of the embryonic stem cell-specific genes Oct4, Sox2 and Nanog in hUCB-MSCs. It was found that Oct4, Sox2 and Nanog mRNAs were present in the cells from passage 3 (Fig. 6A) and 10 (Fig. 6B). Moreover, RT-qPCR was used to analyze the gene expression levels of Oct4, Sox2 and Nanog in hUCB-MSCs. As shown in Fig. 6C, the gene expression levels of Oct4, Sox2 and Nanog showed no significant difference between passage 3 and 10 (P>0.05), indicating that the hUCB-MSCs had stem cell characteristics.

An adipogenic differentiation assay confirmed that hUCB-MSCs from passage 3 and 10 could undergo adipogenic differentiation after being exposed to specific induction media. The formation of lipid droplets was observed under an inverted microscope following Oil Red O staining (Fig. 7), further indicating that the hUCB-MSCs had multi-directional differentiation potential.