Effects and mechanism of exogenous tumor necrosis factor α on differentiation of mesenchymal stem cells of mice into sweat gland cells in three-dimensional environment

Objective To explore the effects and molecular mechanism of tumor necrosis factor α (TNF-α) on differentiation of mesenchymal stem cells of mice into sweat gland cells in a three-dimensional environment. Methods (1) Five 6-8 week-old female C57BL/6 mice were used, with one 1 cm² deep partial-thickness to full-thickness scald wound being created on the back of each mouse with a scald apparatus. One day after injury, the full-thickness skin tissue of the wound was taken, and the concentration of TNF-α in the tissue was detected by enzyme-linked immunosorbent assay. (2) Gelatin in the mass of 0.9 g and 0.3 g sodium alginate were mixed and then dissolved in 30 mL phosphate buffer solution to make hydrogel. Full-thickness skin of the planta of 10 male and female one day newborn C57BL/6 mice was ground into dermal homogenate. The mesenchymal stem cells were isolated from femur and tibia of 10 male and female C57BL/6 mice born for 7 days and cultured. A final density of 1.5×10⁵ cells/mL of bioink was made of mixture of 8 mL pre-warmed hydrogel, 1 mL mouse foot dermal homogenate, and 1 mL the second or third passage of mesenchymal stem cell suspensions. The three-dimensional bioprinter was used to print 12 cylindrical blocks arranged in a crisscross pattern in petri dish. The printed blocks were cross-linked with 25 g/L calcium chloride solution for 10 min and then cultured for 12 hours by adding a medium for mesenchymal stem cells. Subsequently, the printed blocks were divided into blank control group and TNF-α treatment group according to the random number table, with 6 plates and 6 blocks in each group. Both groups of printed blocks were cultured with fresh sweat gland induction medium, and a final mass concentration of 20 ng/mL TNF-α was added into the medium of TNF-α treatment group. After 6 hours of culture, the mRNA expression of pluripotency marker Nanog in the mesenchymal stem cells of two plates of each group was detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction (RT-PCR), and the protein expression of Nanog in the mesenchymal stem cells of one plate of each group was detected by Western blotting, both with triplicate samples. After 14 days of culture, the mRNA expression of sweat gland cell markers cytokeratin 14 (CK14), CK18, sodium potassium adenosine triphosphatase protein a1 (ATP1a1), and aquaporin 5 (AQP5) was detected by real-time fluorescent quantitative RT-PCR in the mesenchymal stem cells of 2 plates of each group (n=3), and the protein expression distribution of CK14, CK18, ATP1a1, and AQP5 of the mesenchymal stem cells in one plate of each group was detected by immunofluorescence staining. Data were statistically analyzed with independent sample t test. Results (1) One day after injury, the mass concentration of TNF-α in the scald wound tissue of mouse was (19±3) ng/mL. (2) After 6 hours of culture, the mRNA and protein expression levels of Nanog in the mesenchymal stem cells of printed blocks in TNF-α treatment group were 0.39±0.04 and 0.36±0.03, respectively, which were significantly lower than 1.00±0.05 and 1.00±0.07 of blank control group (t=16.51, 14.56, P<0.01). (3) After 14 days of culture, the mRNA expression levels of CK18, CK14, ATP1a1, and AQP5 in the mesenchymal stem cells of printed blocks in TNF-α treatment group were 0.38±0.03, 0.42±0.11, 0.23±0.06, and 0.25±0.03, respectively, which were significantly less than 1.00±0.03, 1.00±0.05, 1.00±0.05, 1.00±0.07 of blank control group (t=25.31, 8.31, 17.07, 17.06, P<0.01). (4) After 14 days of culture, the CK18, CK14, ATP1a1, and AQP5 protein were widely distributed in the cytoplasm of mesenchymal stem cells in printed blocks of blank control group, while the distribution of CK18, CK14, ATP1a1, and AQP5 protein in the cytoplasm of mesenchymal stem cells in printed blocks of TNF-α treatment group were significantly reduced in comparison. Conclusions Exogenous TNF-α inhibits the directional differentiation of mesenchymal stem cells of mice into sweat gland cells in a three-dimensional environment, which may be related to the inhibition of the expression of Nanog mRNA and protein by TNF-α that subsequently results in the down-regulation of multi-directional differentiation potential of mesenchymal stem cells.