The metabolic syndrome alters the miRNA signature of porcine adipose tissue-derived mesenchymal stem cells

Abstract

Autologous transplantation of mesenchymal stem cells (MSCs) is a viable option for the treatment of several diseases. Evidence indicates that MSCs release extracellular vesicles (EVs) and that EVs shuttle miRNAs to damaged parenchymal cells to activate an endogenous repair program. We hypothesize that comorbidities may interfere with the packaging of cargo in MSC-derived EVs. Therefore, we examined whether metabolic syndrome (MetS) modulates the miRNA content packed within MSC-derived EVs. MSCs were collected from swine abdominal adipose tissue after 16 weeks of lean or obese diet (n = 7 each). Next-generation RNA sequencing of miRNAs (miRNA-seq) was performed to identify miRNAs enriched in MSC-derived EVs and their predicted target genes. Functional pathway analysis of the top 50 target genes of the top 4 miRNAs enriched in each group was performed using gene ontology analysis. Lean- and MetS-EVs were enriched in, respectively, 14 and 8 distinct miRNAs. Target genes of miRNAs enriched in MetS-EVs were implicated in the development of MetS and its complications, including diabetes-related pathways, validated transcriptional targets of AP1 family members Fra1 and Fra2, Class A/1 (Rhodopsin-like receptors), and Peptide ligand-binding receptors. In contrast, miRNAs enriched in Lean EVs target primarily EphrinA-EPHA and the Rho family of GTPases. MetS alters the miRNA content of EVs derived from porcine adipose tissue MSCs. These alterations could impair the efficacy and limit the therapeutic use of autologous MSCs in subjects with MetS. Our findings may assist in developing adequate regenerative strategies to preserve the reparative potency of MSCs in individuals with MetS. © 2017 International Society for Advancement of Cytometry.

Keywords: extracellular vesicles; mesenchymal stem cells; metabolic syndrome; microRNA.

Figure 1. Overview of micro-RNA (miRNA) sequencing and functional analysis

High throughput miRNA sequencing analysis was performed in both swine adipose tissue-derived mesenchymal stem cells (MSCs) and their daughter extracellular vesicles (EVs) (n=7 each). Differentially expression analysis was performed to identify miRNAs enriched in EVs. miRNA predicted targets of top 4 miRNAs enriched in Lean- and MetS- EVs (highest fold change) were identified using TargetScan. Functional pathway analysis of top 50 target genes of top 4 miRNAs enriched in Lean- and MetS- EVs (total 200 miRNAs each) was performed using FunRich.

Figure 2. Characterization of MSC-derived EVs

A: Transmission electron microscopy of culture MSCs releasing EVs. B: Negative staining of MSC culture supernatants showing EV clusters (arrows) with the classic “cup-like” morphology. C: Characterization of Lean- and MetS- EVs by protein expression of common EV (CD9 and CD29) and MSC (CD105 and CD73) markers.

Figure 3. miRNAs enriched in Lean- and MetS- EVs

A: Of all annotated miRNAs (n=326), only 14 (miR-205, miR-486, miR-194a, miR-504, miR-342, miR-183, miR-192, miR-92a, miR-28-3p, miR-30d, miR-186, miR-320, miR-140-3p, miR-378) and 8 (miR-146a-3p, miR-30c-1-3p, miR-7, miR-148a-5p, miR-374a-3p, miR-23a, miR-132, miR-129b) distinct miRNAs were selectively enriched in Lean- and MetS- EVs, respectively. B: Total reads and fold change (EVs/MSCs) of top 4 (highest fold change) miRNAs enriched in Lean-EVs (top) and MetS-EVs (bottom).

Figure 4. Target genes of miRNAs enriched in Lean- and MetS- EVs

Quantitative polymerase chain reaction (qPCR) of candidate miRNAs followed the same patterns as the miRNAseq findings. miR-205, miR-486, and miR-194a were higher in Lean-EVs compared to their parent MSCs, whereas expression of miR-146a and miR-30c-1-3p were higher in MetS-EVs versus MetS-MSCs. B: Venn diagram showing distribution of miRNA target genes of miRNAs enriched in Lean- and MetS- EVs. A total of 194 and 198 target genes were identified for miRNAs enriched in Lean- and MetS- EVs, but only 7 EV-enriched miRNA targets overlapped between these 2 groups. A total of 194 and 198 target genes were identified for miRNAs enriched in Lean- and MetS- EVs, but only 7 EV-enriched miRNA targets overlapped between these 2 groups.

Figure 5. Functional pathway analysis of target genes of miRNAs enriched in Lean- and MetS- EVs

Classification of gene targets of miRNAs enriched in Lean- and MetS- EVs by cellular component, molecular function, biological process, and biological pathway.