ABSTRACT
BACKGROUND: Exosomes have the function of some mesenchymal stem cells. Understanding the substance composition that plays a representative role in mesenchymal stem cell exosomes will provide clues for further exploration of synthetic exosome analogues. OBJECTIVE: To investigate the difference of microRNA expression profiles in exosomes derived from passage 2 and 5 human umbilical cord mesenchymal stem cells (hUC-MSCs). METHODS: Exosomes in the supernatant of passage 2 and 5 hUC-MSCs were extracted by ultra-high speed centrifugation. The established library was sequenced by using high-throughput sequencing technology. Then we analyzed the sequence results so as to understand the microRNA expression between different groups, and finally did a cluster analysis. RESULTS AND CONCLUSION: 427 657 kinds of microRNAs were detected in the exosomes from passage 2 hUC-MSCs, accounting for 68.93% of the total microRNAs detected; and 119283 microRNAs were detected in the exosomes from passage 5 hUC-MSCs, accounting for 19.22% of the total microRNAs detected. There were 73 526 microRNAs shared between the exosomes from passage 2 and passage 5 hUC-MSCs, accounting for 11.85% of the total microRNAs detected. Bioinformatics analysis (cluster analysis) results showed that these miRNAs were likely to be involved in 161 biological processes, including cell repair, immune and anti-aging. The microRNAs in exosomes from passage 2 to passage 5 hUC-MSCs were largely different. Partial miRNAs exhibited significantly reduced copy numbers. The top five microRNAs with a higher amount, including has-miR-146a-5p, has-miR-191-5p, has-miR-493-3p, has-miR-423-5p, and has-miR-134-5p, have the potential to be the component of synthetic exosome analogues.
ABSTRACT
BACKGROUND: Studies have shown increasing risks and problems in the serum culture system, such as immune rejection, batch differences and virus risk. In addition, with the discovery and application of exosomes, the serum-free culture system is becoming an increasing concern. OBJECTIVE: To compare the similarities and differences between the serum-free culture system and the traditional serum culture system, which lays the foundation for the clinical transformation of human umbilical cord mesenchymal stem cells (hUCMSCs) and provides experimental data. METHODS: Umbilical cord was collected from term infants of cesarean section under aseptic condition, and hUCMSCs were isolated and cultured by explant tissue technique. hUCMSCs was cultured with 10% fetal bovine serum (FBS) and 15% serum substitutes (AGS) from the original generation. Then an inverted microscope was used to observe cell morphological changes. Flow cytometry was used to detect cell surface markers. Cell counting kit-8 was used to detect cell proliferation. Induced differentiation experiment was used to detect cell differentiation potential. Western Blot was used to detect the protein levels of oct4, nanog and sox2. RESULTS AND CONCLUSION: Under the inverted microscope, hUCMSCs cultured with AGS showed more uniform vortex-like growth, and those cultured with FBS gradually appeared with cell differentiation or aging with the increase of cell generations. hUCMSCs cultured by both methods expressed CD73,CD90 and CD105 but lowly expressed CD34 and CD45, and there was no significant difference between the two culture methods. FBS method was superior to AGS method in proliferation ability. Results from the induced differentiation experiments showed that hUCMSCs cultured by both methods had adipogenic, osteogenic and chondrogenic abilities, and there was no significant difference between the two culture methods. hUCMSC cultured by both methods expressed oct4 and nanog but showed no significant difference in level, while the expression of sox2 was significantly higher in the hUCMSCs cultured by AGS than by FBS (P < 0.05). To conclude, the hUCMSCs cultured with AGS are in accordance with the international standards of mesenchymal stem cells. The AGS method as an alternative to the FBS method can become a preferred method for hUCMSCs culture.