The role of serum amyloid A1 in the adipogenic differentiation of human adipose-derived stem cells basing on single-cell RNA sequencing analysis.

BACKGROUND

Adipose-derived stem cells (ASCs) are obtained from a variety of sources in vivo where they present in large quantities. These cells are suitable for use in autologous transplantation and the construction of tissue-engineered adipose tissue. Studies have shown that ASCs differentiation is in a high degree of heterogeneity, yet the molecular basis including key regulators of differentiation remains to clarify.

METHODS

We performed single-cell RNA sequencing and bioinformatics analysis on both undifferentiated (ASC-GM group) and adipogenically differentiated human ASCs (ASC-AD group, ASCs were cultured in adipogenic inducing medium for 1 week). And then, we verified the results of serum amyloid A1 (SAA1) with western blotting, immunofluorescence staining, oil red O staining. After these experiments, we down-regulated the expression of serum amyloid A1 (SAA1) gene to verify the adipogenic differentiation ability of ASCs.

RESULTS

In single-cell RNA sequence analyzing, we obtained 4415 cells in the ASC-GM group and 4634 cells in the ASC-AD group. The integrated sample cells could be divided into 11 subgroups (0-10 cluster). The cells in cluster 0, 2, 5 were came from ASC-GM group and the cells in cluster 1, 3, 7 came from ASC-AD group. The cells of cluster 4 and 6 came from both ASC-GM and ASC-AD groups. Fatty acid binding protein 4, fatty acid binding protein 5, complement factor D, fatty acid desaturase 1, and insulin like growth factor binding protein 5 were high expressed in category 1 and 7. Regulation of inflammatory response is the rank 1 biological processes. And cellular responses to external stimuli, negative regulation of defense response and acute inflammatory response are included in top 20 biological processes. Based on the MCODE results, we found that SAA1, C-C Motif Chemokine Ligand 5 (CCL5), and Annexin A1 (ANXA1) significantly highly expressed during adipogenic differentiation. Western blot and immunofluorescent staining results showed that SAA1 increased during adipogenesis. And the area of ORO positive staining in siSAA1 cells was significantly lower than in the siControl (negative control) cells.

CONCLUSIONS

Our results also indicated that our adipogenic induction was successful, and there was great heterogeneity in the adipogenic differentiation of ASCs. SAA1 with the regulation of inflammatory response were involved in adipogenesis of ASCs based on single-cell RNA sequencing analysis. The data obtained will help to elucidate the intrinsic mechanism of heterogeneity in the differentiation process of stem cells, thus, guiding the regulation of self-renewal and differentiation of adult stem cells.