EFFECTS OF HUMAN URINE-DERIVED STEM CELL EXOSOMES ON THE PROLIFERATION AND DIFFERENTIATION OF NUCLEUS PULPOSUS-LIKE URINE-DERIVED STEM CELLS

Abstract

Objective To explore the relationship between degree of differentiation and time of differentiation of nucleus pulposus-like urine-derived stem cells (NP-USC) and the effects of human urine-derived stem cell exosomes (USC-EXO) on the proliferation and differentiation of NP-USC. Methods Nucleus pulposus cells (NPC) and urine-derived stem cells (USC) were extracted, identified, and cultured. According to the co-cultivation time, the cells were divided into group A (after 7 d of co-culture, the NP-USC were cultured in exosome-free USC medium for 14 d), group B (after 14 d of co-culture, the NP-USC were cultured in exosome-free USC medium for 7 d), group C (USC were cultured alone for 21 d), and NPC group (NPC were cultured alone for 21 d). Quantitative real-time PCR (RT-qPCR) and Western blot were used to measure the mRNA and protein expression of hypoxia-inducible factor 1α (HIF-1α), glucose transporter 1 (GLUT1), type Ⅱ collagen (COL2), and proteoglycan (ACAN) in the five groups. The cells in the five groups were cultured in exosome-free USC medium for 28 d, and CCK-8 was used to detect the cell proliferation on day 7, 14, 21, and 28. Results RT-qPCR and Western blot results showed that the relative mRNA and protein expression levels of HIF-1α, GLUT1, COL2, and ACAN increased sequentially in groups USC, A, B, and C (t=53.36-371.86,P<0.01). However, there was no significant difference in the relative expression levels of the four genes and proteins between groups C and NPC (P>0.05). There was no significant difference in the relative expression levels of the four genes in the cells of the five groups after adding USC-EXO (P>0.05). The results of CCK-8 showed that the cell viability decreased sequentially in groups USC, A, B, and C at each time point (F=3.77-103.58,P<0.05), with no significant difference between groups C and NPC (P>0.05). In each of the five groups, the cell viability gradually increased with the prolongation of culture time (F=9.96-121.68,P<0.05). Conclusion The longer the NPC-induced USC time, the higher the degree of NP-USC differentiation and the lower the proliferation rate of NP-USC. USC-EXO has the ability to promote NP-USC proliferation, but has no ability to promote differentiation. The higher the differentiation degree of NP-USC, the lower the proliferation-promoting ability of USC-EXO.

Keywords

• stem cell research|nucleus pulposus|exosomes|cell differentiation|cell proliferation|tissue engineering