Genetic modification of human mesenchymal stem cells helps to reduce adiposity and improve glucose tolerance in an obese diabetic mouse model

Abstract

Introduction: Human mesenchymal stem cells (MSCs) are multipotent cells that can differentiate into fat, muscle, bone and cartilage cells. Exposure of subcutaneous abdominal adipose tissue derived AD-MSCs to high glucose (HG) leads to superoxide accumulation and up-regulation of inflammatory molecules. Our aim was to inquire how HG exposure affects MSCs differentiation and whether the mechanism is reversible.

Methods: We exposed human adipose tissue derived MSCs to HG (25 mM) and compared it to normal glucose (NG, 5.5 mM) exposed cells at 7, 10 and 14 days. We examined mitochondrial superoxide accumulation (Mitosox-Red), cellular oxygen consumption rate (OCR, Seahorse) and gene expression.

Results: HG increased reactive superoxide (ROS) accumulation noted by day 7 both in cytosol and mitochondria. The OCR between the NG and HG exposed groups however did not change until 10 days at which point OCR of HG exposed cells were reduced significantly. We noted that HG exposure upregulated mRNA expression of adipogenic (PPARG, FABP-4, CREBP alpha and beta), inflammatory (IL-6 and TNF alpha) and antioxidant (SOD2 and Catalase) genes. Next, we used AdSOD2 to upregulate SOD2 prior to HG exposure and thereby noted reduction in superoxide generation. SOD2 upregulation helped reduce mRNA over-expression of PPARG, FABP-4, IL-6 and TNFα. In a series of separate experiments, we delivered the eGFP and SOD2 upregulated MSCs (5 days post ex-vivo transduction) and saline intra-peritoneally (IP) to obese diabetic (db/db) mice. We confirmed homing-in of eGFP labeled MSCs, delivered IP, to different inflamed fat pockets, particularly omental fat. Mice receiving SOD2-MSCs showed progressive reduction in body weight and improved glucose tolerance (GTT) at 4 weeks, post MSCs transplantation compared to the GFP-MSC group (control).

Conclusions: High glucose evokes superoxide generation, OCR reduction and adipogenic differentiation. Mitochondrial superoxide dismutase upregulation quenches excess superoxide and reduces adipocyte inflammation. Delivery of superoxide dismutase (SOD2) using MSCs as a gene delivery vehicle reduces inflammation and improves glucose tolerance in vivo. Suppression of superoxide production and adipocyte inflammation using mitochondrial superoxide dismutase may be a novel and safe therapeutic tool to combat hyperglycemia mediated effects.

Fig. 1

MSCs in normal and high glucose: DMEM-normal glucose on the left and in DMEM- high glucose on the right. The MSCs in high glucose show multiple fat droplet accumulation, intra-cellularly, stained with oil red O stain. Magnification: 40×. MSCs mesenchymal stem cells, DMEM Dulbecco’s modified Eagle’s medium

Fig. 2

Effect of high glucose on MSC gene expression at Day 7 and 14. MSCs were exposed to normal and high glucose for 7 (black bars) and 14 (gray bars) days. Inflammatory, antioxidant, and adipogenic markers mRNA expression was analyzed by real time PCR. mRNA expression results of HG vs NG exposed cells were compared at the two different time points. Data are expressed as mean of three independents experiments ± SEM (paired t-test, *P < 0.05). MSC mesenchymal stem cell, PCR polymerase chain reaction, HG high glucose, NG normal glucose

Fig. 3

mRNA gene expression of Complex I- NDUFA1 (subunit of Complex I) of NG and HG cultured ADMSCs at day 14. NDUFA1 mRNA expression is statistically significantly lower in HG exposed cells NG normal glucose, HG high glucose, ADMSCs adipose tissue derived mesenchymal stem cells

Fig. 4

a Adipose tissue derived MSC (ADMSCs) cells in high glucose (HG) show mitochondrial Complex 1 (NDUFS3) dysfunction compared to normal glucose (NG). Complex 2 used as a loading control. A: high glucose exposed, B: normal glucose exposed, C: cultured in HG media following SOD2 upregulation, D: cultured in NG media following SOD2 upregulation. On comparing Panel A (HG, no SOD2) and panel C (HG with SOD2) we demonstrate that SOD2 up-regulation prior to HG exposure rescues NDUFS3 band. b Bone marrow derived MSC cells in HG show mitochondrial Complex 1 (NDUFS3) dysfunction compared to NG. Complex 2 used as a loading control. A: High glucose exposed, B: Normal glucose exposed, C: cultured in HG media following SOD2 upregulation, D: cultured in NG media following SOD2 upregulation. On comparing Panel A (HG, no SOD2) and panel C (HG with SOD2) we demonstrate that SOD2 up-regulation prior to HG exposure rescues NDUFS3 band. SOD2 superoxide dismutase

Fig. 5

Comparison of MSC basal respiration in the presence of normal glucose (NG-circles) and high glucose (HG-squares). No cell permeabilizer was added. The results are representative and cells were cultured during 7 (top), 10 (middle), and 14 (bottom) days. MSC mesenchymal stem cells

Fig. 6

a and b Bone marrow derived MSC showing oxygen consumption rate (OCR) in percentage in normal glucose (NG, squares) and high glucose (HG, triangles) using SeaHorse. a shows better OCR in MSCs cultured in NG compared to MSCs cultured in HG, at day 7 of culture. Additives marked with arrows, A: 1nM rPFO, B: 10 mM glutamate and malate +2uM FCCP (Complex-I substrate), c 10 mM succinate (Complex-II substrate). b the experiment has been repeated with prior SOD2 upregulation in HG exposed cells, showing SOD2 upregulation did improve mitochondrial respiration, compared to the panel on left

Fig. 7

Mitosox Red Dye to detect ROS in MSCs in HG at day-7 of culture: Using FACS Analyzer increased mitochondrial fluorescence was noted. This increased fluorescence may be due to increased mitochondrial swelling, possibly secondary to ROS in high glucose state (red vs green). No difference was noted at Day1 or Day4 exposure to HG. Blue- unstained cells in NG (negative control), Green- MSCs in normal glucose and Red- MSCs in high glucose. ROS reactive oxygen species, MSCs mesenchymal stem cells, HG high glucose, FACS fluorescence activated cell sorting

Fig. 8

a (left panel) and b (right panel): Mitosox Red dye assay for detection and quantification of mitochondria generated reactive oxygen species presence intra-cellularly: Post culture at day 7 in NG and HG. Left panel: ADMSCs plated in normal glucose (NG, 5.5 mM), Right panel: ADMSCs plated in high glucose (HG, 25 mM). The fluorescence peaked at day 7 of culture and decayed beyond 14 days. Magnification: 10×. c Showing relative MitoSox fluorescence of normal glucose vs high glucose exposed cells over two weeks. NG normal glucose, HG high glucose, ADMSCs adipose tissue derived mesenchymal stem cells

Fig. 9

Mitotraker Green Dye Assay in NG and HG: NG (Left) and HG (right): Helps to visualize mitochondrial mass in HG, which unlike NG shows fragmented fibrillary structures that are uniformly more fluorescent, at day 7 of culture; magnification: 20×. NG normal glucose, HG high glucose

Fig. 10

Mitosox Red Dye to detect ROS in MSCs in HG at Day-14 of culture, using FACS Analyzer: shows increased mitochondrial swelling, possibly secondary to ROS in high glucose state (red vs green). Blue- SCM (negative control), green- MSCs in normal glucose and red- MSCs in High Glucose, Orange: MSCs transduced with AdSOD2 and then exposed to high glucose. The figure on the right indicates that MSCs post SOD2 up-regulation (orange) have less fluorescence than without (red line). In both cases the MSCs were HG exposed. ROS reactive oxygen species, MSCs mesenchymal stem cells, FACS fluorescence activated cell sorting, SOD2 superoxide dismutase

Fig. 11

Gene expression of ADMSCs in high glucose, post transduction with Null or SOD2. ADMSC on culture for 14 days also showed reduction in expression of both fat/adipogenic genes and also inflammatory genes (Fig. 11) when compared to Fig. 2. MSC mesenchymal stem cells, SOD2 superoxide dismutase. ADMSCs adipose tissue derived mesenchymal stem cells

Fig. 12

Intracellular SOD assay: detection of SOD activity in SOD1 and SOD2 upregulated cells compared to AdNull transduced cells. SOD superoxide dismutase. To ensure that superoxide dismutase (SOD) has been truly upregulated intracellularly we assessed SOD activity (Fig. 12) and demonstrated that SOD2 upregulated MSCs have more dismutase activity and used less xanthine oxidase compared to SOD1 upregulated cells indicating that SOD2 upregulated cells have more SOD activity than SOD1. Xanthine oxidase (XO) is a key enzyme necessary to produce superoxide. AdNull transduced cells were associated with maximum XO, as expected, and thereby was associated with least SOD activity

Fig. 13

Comparison of ADMSC respiration in the presence of normal glucose (NG-circles) and high glucose (HG-squares), following transduction with GFP, SOD1 and SOD2 at day 10 of NG or HG culture. ADMSC adipose tissue derived mesenchymal stem cells, GFP green fluorescent protein, SOD superoxide dismutase

Fig. 14

a GFP detection in fat depots at week 1 (upper panel) and week 2 (DAB secondary) with brown staining (lower panel). b GFP detection in omental fat depots at week 1 and week 2 by direct laser confocal microscopy is also shown. GFP green fluorescent protein,DAB 3,3'-diaminobenzidine

Fig. 15

a Glucose tolerance test in db / db mice 28 days after MSC delivery, intraperitoneally. b Weight of mice over four weeks that received either GFP or SOD2 up-regulated MSCs. MSC mesenchymal stem cell, GFP green fluorescent protein, SOD2 superoxide dismutase 2