Immortalized Human Bone Marrow Mesenchymal Stromal Cells-hTERT (iMSC3)

Transcriptomic Profiling of the Telomerase Transformed Mesenchymal Stromal Cells Derived Adipocytes in Response to Rosiglitazone

The escalating obesity epidemic calls for novel therapeutic interventions as current methods fall short of resolving excessive adipose accumulation and related health risks. Mesenchymal Stem Cells (MSCs) play a crucial role in obesity by differentiating into adipocytes, elucidating adipogenesis is fundamental for effective intervention. Al-Ali et al. employs mRNA sequencing to analyze transcriptomic profiles of iMSC3-derived adipocytes exposed to rosiglitazone, enriching our understanding of molecular regulations involved in adipogenesis.

The transcript abundance, expressed as Fragment Per Kilobase per Million mapped reads (FPKM) values, reflects gene expression levels calculated from mapped reads. These counts are proportional to gene expression, gene length, and sequencing depth. Using FPKM values, Differentially Expressed Genes (DEGs) were analyzed through DESeq2 for each comparison group. In comparing undifferentiated iMSC3 to fully differentiated adipocytes (A vs B), 1508 genes were differentially expressed-757 downregulated and 751 upregulated, indicating significant transcriptomic changes. Rosiglitazone presence during induction versus absence (C vs B) resulted in 86 DEGs (65 downregulated, 21 upregulated). Between only induction and both induction/maintenance with rosiglitazone (C vs D), 214 genes differed (64 downregulated, 150 upregulated). Notably, FABP4 showed significant differential expression between fully treated and untreated adipocytes (D vs B) (Fig. 1A). Volcano plots display DEG distribution (Fig. 1B).

Fig. 1. An overall presentation of differential gene expression analysis data (Al-Ali MM, Khan AA, et al., 2022).

miR-22-3p Decreased Adipocyte Formation and Lipid Content in Differentiated iMSC3

The expansion of adipocytes which drives obesity involves sophisticated signaling pathways such as MAPK and PI3-K while miRNAs like miR-22-3p control these pathways and impact cellular processes including proliferation and differentiation. Alomran et al. used transfection methods to study the impact of miR-22-3p on iMSC3 differentiation into adipocytes by examining changes in lipid content and signaling pathways.

iMSC3 cells develop morphological changes after differentiation into adipocytes through the creation of lipid droplets. Researchers used fluorescent microscopy and inverted light microscopy to examine lipid droplet formation in control samples, non-target miRNA transfected cells and miR-22-3p transfected cells. Oil red and Nile red stains specifically identified fat or lipid droplets while DAPI stained cell nuclei. The control groups (1,2,3,4) displayed greater numbers of larger lipid droplets than the miR-22-3p transfected groups (5,6,7,8) in Figure 2A. Images resulting from the combination of DAPI and Nile red stains demonstrated that lipid droplets surrounded cell nuclei. Transfected cells with miR-22-3p contained fewer lipids when compared to the negative control group. Oil red elution spectrophotometry revealed significantly reduced lipid levels in miR-22-3p cells (p<0.05) when compared to other cells (Fig. 2B). Additionally, average fat droplet numbers (Fig. 2C) were significantly lower (p<0.01, p<0.001) in miR-22-3p transfected cells.

Fig. 2. Morphological effects of miR-22-3p transfection (lipid content and lipid droplet count) on the differentiation of iMSC3 cells into adipocyte (Alomran SS, Khattak MNK, et al., 2021).