Supplementary Material for: Characterization and Beige Adipogenic Potential of Human Embryo White Adipose Tissue-Derived Stem Cells

Background/Aims: Brown and beige adipocytes are widely recognized as potential therapeutic targets to treat obesity and related metabolic disorders, and the recruitment of brown and beige adipocytes is an essential aspect that requires attention. Although many methods of activating brown adipocytes or generating beige adipocytes have been reported, the limited number and sources are the biggest challenges. The number of white adipocytes is much greater than the number of brown adipocytes, both in human adults and fetuses. Unfortunately, human adult white adipose tissue-derived stem cell (aWAsc) has little beige adipogenic potential. However, the characteristics and beige adipogenic potential of human embryo-derived white adipose stem cells (eWAsc) still need to be investigated. Methods: To analyze the characteristics and functionality of eWAsc, we analyzed the markers of adipose precursor cells by flow cytometry. Then, differentiation and browning/beiging were induced, and the identifying markers were analyzed by real-time PCR and immunoblot. In addition, more in-depth exploration was performed using RNA-SEQ on eWAsc and aWAsc. Results: eWAsc was isolated from human embryonic white adipose tissue, and aWAsc was isolated from adult white adipose tissue by collagenase treatment. eWAsc has extreme advantages in adipogenesis capacity and browning/beiging ability in comparison to aWAsc, indicating that eWAsc may possess some special regulatory factors to promote the generation of functional brown/beige adipocytes. Greater exploration was enabled by RNA-SEQ, revealing a large number of differences at the transcriptional levels, including 1263 differentially expressed genes, 657 down- and 605 upregulated, in eWAsc compared to aWAsc. Pathway analysis revealed enrichment in cell cycle, TGF-β signaling pathway, DNA replication, and Hippo signaling pathways. Interestingly, the expression levels of C/EBPα, FGF1 and FST gene, which are related to the maturation of adipocytes, Hippo signaling pathway and TGF-β signaling pathway, were significantly higher in eWAsc than in aWAsc. These may be potential candidates and possible regulatory targets for recruiting beige adipocytes in human adipose tissue. Conclusion: Overall, we have demonstrated the molecular characteristics and excellent beige adipogenic potential of eWAsc, providing a new reference for studying human adipocytes.