Systemic Factors During Metabolic Disease Progression Contribute to the Functional Decline of Adipose Tissue-Derived Mesenchymal Stem Cells in Reproductive Aged Females

Abstract

It is known that advanced metabolic disorders such as type 2 diabetes compromise the functional and regenerative capacity of endogenous adipose-tissue resident stem cells (ADSCs). It is, however, still unclear at which stage of disease progression ADSCs become compromised and whether systemic factors contribute to their functional decline. It was therefore hypothesized that inflammatory changes in the systemic microenvironment during distinct stages of disease progression negatively affect the functional capacity of ADSCs. A total of forty-seven (n = 47) black African reproductive aged females (32 ± 8 years; mean ± SD) were included in this study and subdivided into: (a) healthy lean (C; body mass index, BMI ≤ 25 kg/m²), (b) healthy overweight/obese (OB; BMI ≥ 25 kg/m²), (c) obese metabolic syndrome (MetS; BMI ≥ 30 kg/m²), and (d) type 2 diabetes mellitus (T2DM; previously diagnosed and on treatment) groups. Participants underwent anthropometric assessments and a DXA scan to determine their body composition and adipose indices. Each persons' systemic metabolic- (cholesterol, HDL, LDL, triglycerides, and blood glucose) and inflammatory profiles (CRP, SDF1α, TNFα, IL6, IL8, IL10, and IFNy) were also evaluated. Participant-derived serum was then used to treat an ADSC cell line in vitro and its effect on viability (MTT-based assay), proliferation (BrdU), migration (wound healing assay), and osteogenic differentiation assessed. When exposed to serum derived from overweight/obese individuals (with or without metabolic syndrome), both the proliferative and migratory responses of ADSCs were less pronounced than when exposed to healthy control serum. Serum IL6 concentrations were identified as a factor influencing the proliferation of ADSCs, suggesting that long-term disruption to the systemic cytokine balance can potentially disrupt the proliferative responses of ADSCs. Obese participant-derived serum (with and without metabolic syndrome) furthermore resulted in lipid accumulation during osteogenic differentiation. This study, therefore demonstrated that systemic factors in obese individuals, regardless of the presence of metabolic syndrome, can be detrimental to the multifunctional properties of ADSCs.

Keywords: adipogenesis; adipose tissue; body composition; cytokines; interleukin 6; mesenchymal stem cells; obesity; osteogenesis.

FIGURE 1

Body composition and metabolic profile of participants within the healthy lean (C), overweight/obese (OB), metabolic syndrome (MetS), and type 2 diabetic (T2DM) groups. (A) Representative images of whole body DXA scans illustrating a normal (C), gynoid-shaped (OB), and android-shaped (MetS/T2DM) body composition. Body regions: R1, arms, R2, legs, R3, android, R4, head, R5, trunk, and R6, gynoid. (B) Body mass index (BMI; kg/m²). (C) Visceral adiposity measures: waist-to-hip ratio (WHR) and trunk-to-limb fat mass ratio (TF:LF). (D) Fasting blood glucose (FBG) levels (mmol/L). (E) Systolic and diastolic blood pressure (BP; mmHg). (F) Serum lipid levels: total cholesterol (mmol/L), low density lipoprotein (LDL; mmol/L), high density lipoprotein (HDL; mmol/L), and triglycerides (TGS; mmol/L). (G) Atherogenic index was calculated using the following formula: (total cholesterol–HDL)/HDL. Statistical analysis: analysis of co-variance (confounding factor: age) with Tukey post hoc test. ^∗p < 0.05 indicates significant difference from healthy lean (C) group. ^#p < 0.05 indicates significant difference between groups.

FIGURE 2

Serum inflammatory profile of participants within the healthy lean (C), overweight/obese (OB), metabolic syndrome (MetS), and type 2 diabetic (T2DM) groups. (A) Pro-inflammatory C-Reactive protein (CRP; pg/mL). (B) Anti-inflammatory interleukin 10 (IL10; pg/mL). Statistical analysis: non-parametric Dunn’s multiple comparisons test with Kruskal–Wallis post hoc test. ^∗p < 0.05 indicates significant difference from healthy lean (C) group. ^#p < 0.05 indicates significant difference between groups.

FIGURE 3

Circulating stem/progenitor cells and serum SDF1α levels of participants within the healthy lean (C), overweight/obese (OB), metabolic syndrome (MetS), and type 2 diabetic (T2DM) groups. (A,B) Representative flow cytometry plots of CD34 (APC conjugated) vs. CD45 (FITC conjugated) expression within the peripheral blood mononuclear cell (PBMC) population. Hematopoietic progenitor cells (HPCs) express both the stem cell marker CD34 as well as the hematopoietic lineage marker CD45, whereas stem cells do not express CD45. (C) Quantification of the percentage of stem cells (CD34 + CD45–) and HPCs (CD34 + CD45+) within the PBMC population. (D) Serum levels (pg/mL) of the chemokine, SDF1α. Statistical analysis: non-parametric Dunn’s multiple comparisons test with Kruskal–Wallis post hoc test. ^∗p < 0.05 indicates significant difference from healthy lean (C) group.

FIGURE 4

The effect of participant-derived serum on ADSC proliferation. (A) Cellular proliferation (bars) and serum IL6 concentrations (pg/mL; dotted line). Statistical analysis: non-parametric Dunn’s multiple comparisons test with Kruskal–Wallis post hoc test. ^∗p < 0.05 indicates significant difference from healthy lean (C) group. (B) Correlation between the IL6 concentrations in participant-derived serum and ADSC proliferative responses over a period of 24 h. (C) Correlation between increasing physiologically relevant concentrations of recombinant IL6 and ADSC proliferation (n = 3). Statistical analysis: Spearman’s ranked correlation analysis with two-tailed p-value. Level of significance accepted at p < 0.05.

FIGURE 5

The effect of participant-derived serum on ADSC migration. (A) The percentage wound closure over a period of 7 h in the presence of serum derived from participants within the healthy lean (C), overweight/obese (OB), metabolic syndrome (MetS), and type 2 diabetic (T2DM) groups. (B) IL8 concentrations (pg/mL) in participants’ serum. (C) Non-significant correlation between the IL8 concentrations in participant-derived serum and ADSC proliferative responses over a period of 24 h. (D) Representative images of ADSC migration at 0, 7, and 24 h in the presence of participant-derived serum. Statistical analysis: non-parametric Dunn’s multiple comparisons test with Kruskal–Wallis post hoc test. ^∗p < 0.05 indicates significant difference from healthy lean (C) group.

FIGURE 6

Differentiation capacity of ADSCs in presence of participant-derived sera. (A) The extent of mineralization (Alizarin Red staining) during osteogenesis (OM) over a period of 21 days. (B) Representative images of Alizarin Red S staining after 21 days of osteogenesis in the presence of sera derived from either healthy lean (C), overweight/obese (OB), metabolic syndrome (MetS), or type 2 diabetic (T2DM) participants. Arrows indicate the presence of lipid droplets within the mineralized matrix following osteogenesis. (C) Representative images of Oil Red O staining after 14 days of adipogenesis in the presence of sera derived from either C, OB, MetS, or T2DM participants. (D) The extent of lipid accumulation (Oil Red O staining) during adipogenesis (AM) over a period of 14 days. Statistical analysis: one-way ANOVA with Tukey post hoc test.