Immunological characteristics of human umbilical cord mesenchymal stem cells after hepatogenic differentiation

2.1. Preparation of hUCMSCs

We isolated and cultured hUCMSCs using the adherent method. The umbilical cord was disinfected and sterilized in 75% alcohol, and then rinsed three times in saline solution. Ligation at both ends of the umbilical cord was then removed, and the cord was cut into segments of 3–5-cm long. The segments were soaked in 4 °C phosphate-buffered saline (PBS), while the arteries (n = 2) and vein (n = 1) were removed. The tissues were then washed twice in PBS and cut into pieces approximately 1 mm in length. The tissue blocks were placed in six-well plates and incubated at 37 °C in a 5% CO₂ incubator for 30 minutes. Dulbecco’s modified Eagle medium (DMEM; 37 °C, 2 mL) was added to the six-well plates and refreshed every 3 days. When the confluence of cells reached 80%, it was subcultured.

2.2. Identification of hUCMSCs

The cell phenotype was determined by flow cytometry. Briefly, P3 generation cells were collected, and the fluorescence intensity of each marker was analyzed by monochromatic labeling for CD45, CD90, CD105, CD34, and octamer-binding transcription factor 4 (OCT-4). Osteogenic differentiation was then induced. Briefly, hUCMSCs of the P3 generation were collected and inoculated in 24-well plates (1 × 10⁴ cells/well). After 48 hours of adherent growth, an osteogenic induction reagent (dexamethasone 1 μmol/L, β-glycerol phosphate 10 nmol/L, and vitamin C 0.05 nmol/L) was added to induce osteogenic differentiation for 28 days. Alizarin red staining was performed, and the plates were observed under a microscope and photographed. Adipogenic differentiation was then induced. Briefly, hUCMSCs of the P3 generation were collected and inoculated in 24-well plates (1 × 10⁴ cells/well). After 48 hours of adherent growth, an osteogenic induction reagent was added (dexamethasone 1 μmol/L, rifampicin-1-1 methylxanthine 0.5 μmol/L, and indomethacin 100 μmol/L). The cells were cultured for 28 days. Oil red O staining was then performed, and the plates were observed under a microscope and photographed.

2.3. Liver orientation induced differentiation of hUCMSCs

To differentiate hUCMSCs from hepatocyte-like cells (HLCs), we performed in vitro pre-culture using staged cytokine induction. Briefly, after laying the plate, serum-free pretreatment medium (20 ng/mL epidermal growth factor and 10 ng/mL basic fibroblast growth factor [bFGF]-DMEM) were added to the cells when the cell fusion rate reached 80%–90%. After 2 days, the supernatant was discarded, and hepatocyte differentiation induction solution (20 ng/mL hepatocyte growth factor, 10 ng/mL bFGF, and 0.61 g/L nicotinamide) was added. The differentiation induction medium was cultured for 5 days and changed three times a week. The maturation induction medium contained 20 ng/mL hepatocyte growth factor, 10 ng/mL bFGF, and 0.61 g/L nicotinamide in DMEM. The cells were cultured in this medium for 7 days. The hepatocyte maturation induction medium contained 20 ng/mL OSM, 10 μmoL/L dexamethasone, and 10 μL/mL 1 × ITS + Premix, and was cultured for 14 days and refreshed three times a week. The control group was cultured in complete medium without the inducing factor for 21 days and changed three times a week.

2.4. Identification of HLCs

After liver-like induction for 21 days, the cell morphology of the induced HLCs was photographed and recorded. Differences between the induced hUCMSCs group and the control hUCMSCs group were observed by periodic acid–Schiff (PAS) staining.

2.5. HLC cell marker analysis

The cell surface markers CD34, CD45, CD90, CD105, and OCT-4 were analyzed by the paired t-test to compare their mean fluorescence intensity before and after induction.

2.6. In vivo HLC immunogenicity analysis

HLCs and hUCMSCs were collected, and the cell concentrations were adjusted to 2 × 10⁶ cells/mL in PBS. Female BALB/c mice (n = 25) were randomly divided into the induced group (I, 10 mice), control group (C, 10 mice), and placebo group (P, 5 mice). The mouse groups received an intraperitoneal injection of 0.5 mL HLC suspension (induced group), 0.5 mL hUCMSC cell suspension (control group), or 0.5 mL PBS (placebo group). After 4 hours, peritoneal lavage fluid was collected, and aseptic PBS flushing was performed (5 mL/mouse, centrifugation at 1400 rpm for 5 minutes). The supernatant was discarded, the cells were re-suspended in PBS, and the leukocyte count was analyzed. One-way analysis of variance was performed to compare leukocyte counts in each group. Post-hoc testing was performed using the Bonferroni test. The t-test was used to compare the percentages of CD45, CD3, CD4, and CD8 between the groups.

3.1. Isolation and culture of hUCMSCs

The isolated hUCMSCs were consistent in shape to that of typical hUCMSCs. P0 cells were irregular and fusiform (Fig. 1A, B), and P3 cells were regular and fusiform (Fig. 1C, D). The homogeneity of the cells was good.

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Fig. 1.

Morphological observation of hUCMSCs in different times of passage. A, B: Morphological features of P0 hUCMSCs (scale bar = 100 μm). C, D: Morphological features of P3 hUCMSCs (scale bar = 100 μm).

The cell surface markers of P3 cells were detected by flow cytometry (Fig. 2). P3 cells showed high expression of CD90 and CD105 (Fig. 2A, B), low expression of CD34 and CD45 (Fig. 2C, D), and high expression of OCT-4 (Fig. 2E). The percentages of CD90, CD105, CD45, CD34, and OCT-4 expression are shown in Fig. 2F (n = 50).

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Fig. 2.

Surface markers of hUCMSCs were detected by flow cytometry. A: High expression of CD90. B: High expression of CD105. C: Low expression of CD34. D: Low expression of CD45. E: High expression of OCT-4. F: Quantitative results of marker expression. Date are presented as mean ± standard deviation, n = 50.

Obvious calcium nodules were observed after alizarin red staining in the induction group (Fig. 3A). No obvious calcium nodules were observed after alizarin red staining in the control group (Fig. 3B). With regard to adipogenic differentiation, obvious oil droplets were observed in the cells of the induced group after oil red O staining (Fig. 3C). No oil droplet aggregation was observed in uninduced hUCMSCs stained with oil red O (Fig. 3D).

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Fig. 3.

Images of osteogenesis and induction of adipogenesis. A: Calcium nodules can be seen after the induction of osteogenesis (scale bar = 100 μm). B: No calcium nodules can be seen in the control group (scale bar = 100 μm). C: In the adipogenic induction group, fat droplets can be seen inside the cells (scale bar = 100 μm). D: No fat droplet has formed in the control group (scale bar = 100 μm).

After 21 days of hepatogenic differentiation, the morphology of the cells changed from a long fusiform shape to an irregular polygonal shape (Fig. 4). PAS staining showed a larger number of sugars in the induced group (purplish-red stain; Fig. 5C) compared with the control group (Fig. 5B, D).

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Fig. 4.

Change in morphology of hUCMSCs after induction treatment. A: The hUCMSCs in the control group still show a long spindle shape (scale bar = 100 μm). B: The induced cells were deformed from a long spindle shape to a polygon at 21 days (scale bar = 100 μm).

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Fig. 5.

PAS staining (scale bar = 100 μm). A, C: PAS staining is not present in the control group. B, D: The induced group is strongly positive for PAS staining (scale bar = 100 μm).

The mean fluorescence intensity of hUCMSCs cell surface markers was detected by flow cytometry before and after induction (Fig. 6). The mean fluorescence intensity of CD105, CD90, and OCT-4 in the control group was 20.37 ± 16.55, 90.2 ± 40.99, and 11.61 ± 9.98, respectively. The mean fluorescence intensity of these markers in the induction group was 8.12 ± 6.58, 47.17 ± 10.42, and 9.75 ± 6.61, respectively. Paired t-test analysis showed no significant differences in these markers between the control group and the induction group. There was no change in CD34 or CD45 between before and after induction (2.12 ± 1.07 and 1.91 ± 0.77 in the control group and 1.92 ± 1.25 and 1.85 ± 1.14 in the induction group, respectively). The expression patterns of CD34 and CD45 were low (Fig. 6).

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Fig. 6.

Comparison of the mean fluorescence intensity of cell surface markers before and after induction. The data are presented as mean ± standard deviation (n = 3).

The leukocyte count was significantly lower in the placebo group than in the control group and the induced group, but there was no significant difference between the control group and the induced group (Fig. 7). The positive percentages of CD3, CD4, CD8, and CD45 in the control group were 2.74 ± 0.81, 21.3 ± 2.71, 2.84 ± 2.21, and 95.52 ± 3.90, respectively. The positive percentages of CD3, CD4, CD8, and CD45 in the induction group were 2.77 ± 1.71, 19.76 ± 6.19, 5.70 ± 1.88, and 80.32 ± 16.62, respectively. There was no significant difference in CD3 or CD4 expression between the induction group and the control group (Fig. 8). CD8 expression in the induction group was significantly higher than that in the control group (P < 0.01; Fig. 8).

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Fig. 7.

Graph of the white blood cell count in the three groups. The white blood cell count in the P group was significantly lower than that in the C and I groups. Data are presented as mean ± standard deviation. ***P ≤ 0.01. P: placebo; C: control; I: induction.

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Fig. 8.

Flow cytometric results of peritoneal lavage fluid. Data are presented as mean ± standard deviation. ***P ≤ 0.01.