2022, Vol. 26 ›› Issue (24): 3802-3807

Isolation, identification, comparison of the differentiation ability of multilineage-differentiating stress-enduring cells from human umbilical cord derived and adipose derived mesenchymal stem cells

Zhang Kun1, 2, Li Fang1, 2, Xiao Dongjie1, 2, Wang Yunshan1, 2, Huang Guobao3, Liu hua1, 2   

Abstract: BACKGROUND: Multilineage-differentiating stress-enduring (Muse) cells possess self-renewal and multiple differentiation potential. Muse cells are able to differentiate into all three germ layers, and exhibit non-tumorigenicity and low telomerase activity. It is a seed cell in the fields of tissue engineering, cell transplantation and gene therapy, providing a broad prospect for the treatment of various clinical diseases.
OBJECTIVE: To study the biological characteristics of Muse cells from umbilical cord derived and adipose derived mesenchymal stem cells in vitro, and provide experimental data for obtaining sufficient Muse cells for clinical research.
METHODS: Umbilical cord derived and adipose derived mesenchymal stem cells were incubated with trypsin for different time. The cell survival rates and SSEA-3 expression were detected by Trypan Blue staining and Flow cytomertry. Muse cells were obtained by suspension adherence method. Alkaline phosphatase staining was used to obtain the pluripotency of Muse cells. Muse cells underwent multi-lineage differentiation under different induction conditions. Oil red O staining, Alizarin Red S staining, and Von Kossa staining were used to compare the adipogenic and osteogenic differentiation. RT-PCR was used to detect the changes in pluripotent stem cell marker genes Oct-3/4, Nanog, SSEA-3 and Muse cells induced differentiation related genes doublecortin, neuron-specific enolase, lipoprotein lipase, osteopontin, and alpha-fetoprotein expression.
RESULTS AND CONCLUSION: (1) Under trypsin incubation extension, survival rates of umbilical cord derived mesenchymal stem cells and adipose derived mesenchymal stem cells were decreased, accompanied with SSEA-3 expression increased. (2) Alkaline phosphatase staining was positive in Muse cells and negative in mesenchymal stem cells. (3) SSEA-3, Nanog, and Oct-3/4 were highly expressed in Muse cells compared with mesenchymal stem cells. Nanog and Oct-3/4 were highly expressed in adipose-Muse cells compared with umbilical cord-Muse cells. Oil red O, Alizarin Red S, and Von Kossa staining results showed that Muse cells had stronger adipogenetic and osteogenetic abilities than mesenchymal stem cells. (4) Mesenchymal stem cells and Muse cells had different differentiation efficiencies under the action of adipogenesis, osteogenic induction culture medium, neuronal cell induction liquid, and hepatocyte induction liquid, but they all highly expressed lipoprotein lipase, osteopontin, neuron-specific enolase, doublecortin, and alpha-fetoprotein. The above genes were highly expressed in Muse cells compared with mesenchymal stem cells. (5) To conclude, umbilical cord derived and adipose derived mesenchymal stem cells showed the best SSEA-3 positive rate and survival rate after trypsin digestion for 4 hours. The SSEA-3 positive rate of adipose derived mesenchymal stem cells was higher than that of umbilical cord derived mesenchymal stem cells. Muse cells obtained from umbilical cord derived mesenchymal stem cells and adipose derived mesenchymal stem cells highly express pluripotent stem cell genes and are able to differentiate into three germ layers.
Key words: mesenchymal stem cells, umbilical cord, adipose, multilineage-differentiating stress-enduring cells, SSEA-3