Human mesenchymal stem cells exhibit altered mitochondrial dynamics and poor survival in high glucose microenvironment

doi:10.21203/rs.3.rs-3117576/v1

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Human mesenchymal stem cells exhibit altered mitochondrial dynamics and poor survival in high glucose microenvironment

https://doi.org/10.21203/rs.3.rs-3117576/v1

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Background: Mesenchymal stem cells (MSCs) are reputable type of stem cells that has enchanted regenerative abilities and can be used to treat many chronic diseases. Diabetes mellitus (DM) is a frequently diagnosed chronic disease characterized by hyperglycemia which initiates many multisystem complications in the long-run. DM patients can benefit from MSCs transplantation to curb down the pathological consequences associated with hyperglycemia persistence and restore the function of damaged tissues. MSCs therapeutic outcomes are found to last for short period of time and ultimately these regenerative cells are eradicated and died in DM disease model. So it is necessary to investigate the impact of high glucose or hyperglycemia on the cellular and molecular characteristics of MSCs.

Materials and Methods: Human adipose tissue –derived MSCs (hAD-MSCs) were seeded in low (5.6 mmol/L of glucose) and high glucose (25 mmol/l of glucose) for 7 days. Cytotoxicity, viability, mitochondrial dynamics, and apoptosis were deplored using specific kits. Western blotting was performed to measure the protein expression of PI3K, TSC1, and mTOR in these cells.

Results: hAD-MSCs cultured in high glucose for 7 days demonstrated marked decrease in their viability. Mitcohondrial membrane potential and NAD+/NADH ratio were dropped in high glucose exposed hAD-MSCs indicating disturbed mitochondrial function. PI3K protein expression deceased in high glucose culture MSCs and it was coupled with upregulation in TSC 1 and downregulation in mTOR protein expression. Apoptosis was induced as a result of mitochondrial impairment and explained the poor survival of MSCs in high glucose.

Conclusion: high glucose impaired the mitochondrial dynamics and regulatory proteins in hAD-MSCs ensuing their poor survival and high apoptosis rate in hyperglycemic microenvironment.

Mesenchymal stem cells

high glucose

mitochondrial dynamics

apoptosis

poor survival

PI3K/ mTOR pathway

Mesenchymal stem cells (MSCs) are the most commonly studied forms of stem cells as they possess powerful regenerative and immunomodulatory abilities (1). MSCs have unique molecular machinery that empowers them to modulate many cellular signaling pathways and restore the functionality of damaged tissues rendering them suitable to treat many acute and chronic diseases (1). Despite their unprecedented abilities to adapt to many stress conditions, they can still behave unexpectedly under certain stress microenvironments. A lot of preclinical and clinical studies didn’t demonstrate only the positive outcomes after transplanting MSCs to injured sites (2, 3), but they also reported the short -lived therapeutic results in hostile microenvironments which need to be figured out (4). MSCs can smartly use different metabolic pathways to espouse their cellular and therapeutic functions (5). When MSCs are undifferentiated, they rely mainly on glycolysis which is considered an essential metabolic pathway to support their proliferation, expansion, and immunomodulation (6). When MSCs are programmed to differentiate, they switch to oxidative phosphorylation (OxPhos) (7), thereby, intact mitochondria are also important to maintain their multi-lineage differentiation capacities (7).

At present, with great improvement in the living standards and the efforts implemented to raise people awareness of healthy lifestyle, Diabetes mellitus (DM) incidence increases rapidly, and it is deemed among top five chronic diseases (8). DM is associated with serious multisystem complications that impair the life quality of many patients (9). DM is characterized mainly by chronic hyperglycemic state that causes many devastating, long-term complications (9). DM patients can benefit remarkably from MSCs therapy to repair and regenerate damaged tissues, restore their functionality, and reduce the severity of DM-related pathological complications (8). Several studies reported a significant improvement in glycemic parameters and cardiovascular complications after transplanting MSCs in DM patients (8, 10, 11); however, other studies reported that DM can accelerate the ecline in MSCs quantity and therapeutic quality, and impair their regenerative capabilities (12). When MSCs turn into a dysfunctioning cells, they may increase the severity of DM-related complications (12). Little is known about the impact of diabetic microenvironment, particularly hyperglycemia, on the molecular and cellular characteristics of transplanted MSCs. Thus, it is important to investigate the effect of high glucose on MSCs biological dynamics which are essential to understand their fate and performance in diabetic microenvironment, and provide new avenues on possible molecular targets to modulate their survival and therapeutic outcomes in DM patients.

2.1 Human mesenchymal stem cells

Adipose tissue -derived hMSCs (hAD-MSCs) were commercially purchased from Lonza (Cat# PT5006, Lot# 21TL138912) and expanded using Dulbecco’s Modified Eagle’s Medium Low Glucose (DMEM-Low Glucose, Euroclone) which contained 5.6 mmol/L glucose and were supplemented with 10% fetal bovine serum (FBS, Gibco) (13), 0.1 mg/ml streptomycin and 100 units/ml penicillin G in standard cell culture incubators (5% CO2/95% air; 37°C). Medium was changed every 72 hrs. and cells were sub-cultured when confluence exceeded 60%. For high glucose conditions, cells were cultured in Dulbecco’s Modified Eagle’s Medium High Glucose (DMEM-High Glucose, Euroclone) which contained 25 mmol/L glucose and were supplemented with 10% fetal bovine serum (FBS), 0.1 mg/ml streptomycin and 100 units/ml penicillin G for 3, 7, and 14 days in standard cell culture incubators (5% CO2/95% air; 37°C). High glucose complete medium was changed every 72 hrs. MSCs that were cultured in low glucose were considered as our study control.

2.2 Cytotoxicity assays:

To measure the level of cytotoxicity in human MSCs after being cultured in low and high glucose, we measured the lactate dehydrogenase (LDH) which was released from the damaged MSCs (LDH Assay Kit, Abcam, Cat# ab102526) and the absorbance values were obtained using Cytation 5 (BioTek, USA). The viability of MSCs in low and high glucose were also assessed using 0.4% Trypan blue and the percentage of viable cells as well as representative fluorescent images were obtained using Corning® CytoSmart Cell Counter.

2.3 TMRE assay:

Mitochondrial membrane potential was assessed using TMRE-Mitochondrial Membrane Potential Assay Kit (Abcam, Cat # ab113852). Briefly, human AD-MSCs were placed in 24-well plate at 1 × 10⁴ cells per well and allowed to adhere overnight. After that, cells were cultured either in low glucose or high glucose for 7 days. Then, media were aspirated, and cells were stained using 400 nM TMRE in culture media for 30 min in the incubator, and then media were replaced with 200 µl PBS per well. Fluorescence intensity was detected using Cytation 5 (BioTek, USA). (λex = 549 nm, λem = 575 nm), and TMRE fluorescent images were captured at Texas red filter using Cytation 5 (BioTek, USA).

2.4 NAD+/NADH assay:

NAD+/NADH ratio in AD-MSCs that were cultured in low and high glucose media was assessed using NAD/NADH-Glo™ assay kit (Promega, Cat# G9071). Briefly, cells were cultured in low and high glucose media for 7 days. After that, cells were detached and placed in 96 well plate at 5× 10⁵ in 50 µl of either low or high glucose media and were allowed to adhere overnight. 50µl of NAD/NADH-Glo™ Detection Reagent was added to each well and the plate was gently shaken to mix and lyse the cells and was incubated with the added reagent for 60 minutes at room temperature. The luminescence values were recorded using Cytation 5 (BioTek, USA) which were corresponding to the total amount of NAD + produced from NADH.

2.5 Western blotting:

The protein levels for PI3K (Santa Cruz Biotechnology Cat # sc-1637), TSC1 (Santa Cruz Biotechnology Cat # sc-377386), and mTOR (Santa Cruz Biotechnology Cat # sc-293133), and β-actin (Santa Cruz Biotechnology Cat # sc-47778 HRP) were measured by Western blot. Briefly, total protein levels were measured using NanoDrop™ Lite Spectrophotometer, and 40 µg of protein was loaded onto SDS–PAGE. Following electrophoresis, proteins were transferred to PVDF membrane and were incubated with appropriate primary and secondary antibodies. The membranes were visualized using VILBER FUSION Gel Documentation System, and bands were quantified using ImageJ for densitometry.

2.6 Apoptosis assay:

To detect apoptosis in human AD-SCs after being cultured in low and high glucose, we used RealTime-Glo™ Annexin V Apoptosis live assay (Promega, Cat# JA1011, Lot# 0000400486 following the manufacturer’s guidelines. The fluorescent images of green color which represented cells undergoing apoptosis were detected at GFP filter using Cytation 5 (BioTek, USA).

2.7 Statistical analysis

Data were reported as mean ± SD. Comparison of data between multiple groups was performed using one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc multiple comparison test, and analysis between two groups was made using Student’s t-test (two-tailed). Statistical significance is determined as p < 0.05. Each figure represents one of at least three independent quantifiable experiments.

3.1 Culturing MSCs in high glucose triggers massive cytotoxicity and poor survival:

MSCs are known to survive well in low glucose medium which is a physiological requirement to preserve their stemness features. We want to determine the viability of these cells in high glucose microenvironment. Human adipose tissue -derived MSCs (hAD-MSCs) were cultured in high glucose medium containing 25 mmol/L of glucose for 3, 7, and 14 days. The cytotoxicity was measured by determining the amount of lactate dehydrogenase (LDH) released and was found to be significantly greater in hAD-MSCs cultured in high glucose for 7 and 14 days compared with hAD-MSCs cultured in normal low glucose medium containing 5.6 mmol/L of glucose (Fig. 1a). To confirm these findings, the viability of hAD-MSCs was measured using 0.4% Trypan blue and found to be remarkably low in hAD-MSCs that were cultured in high glucose for 7 days compared to hAD-MSCs cultured in a low glucose medium (Fig. 1b)

3.2 The mitochondrial dynamics of MSCs impaired by high glucose:

Mitochondria are integral intracellular organelles promoting cells survival and viability. The variance of mitochondrial dynamics depends on the type of microenvironment where cells exist. Previous reports demonstrated that high glucose can induce the fission and fragmentation of mitochondria in glomeruli podocytes (14), but the knowledge surrounding the impact of high glucose on the mitochondrial dynamics of MSCs is still limited. The mitochondrial membrane potential (ΔΨm) generated by proton pumps (Complexes I, III and IV) is imperative to ensure proper ATP production by mitochondrial OxPhos. Abnormalities in the mitochondrial membrane potential (ΔΨm) can significantly elicit a decline in cells viability and trigger unwanted signaling pathways. We measured the mitochondrial membrane potential (ΔΨm) in hAD-MSCs after being grown in a high glucose medium for 7 days using tetramethylrhodamine ethyl ester (TMRE) which is sequestered by active mitochondria. Our results illustrated that the fluorescent intensity of TMRE was significantly low in hAD-MSCs cultured in a high glucose medium compared to hAD-MSCs cultured in a normal low glucose medium indicating an impairment in the mitochondrial membrane potential in these cells (Fig. 2a &b). Nicotinamide adenine dinucleotide (NAD+) and its reduced form (NADH) regulate many metabolic pathways, including mitochondrial OxPhos. Maintaining NAD+/NADH pool is required to ensure that the influx of electrons is coupled with the translocation of protons to the intermembrane space to generate the required membrane potential (ΔΨm) across the inner mitochondrial membrane to be harnessed by complex V to produce ATP (15). To investigate if the decrease in the mitochondrial membrane potential (ΔΨm) is correlated with abnormalities in NAD+/NADH pool, we measure the NAD+/NADH ratio in hAD-MSCs cultured in low and high glucose media (Fig. 3). The findings revealed a considerable drop in NAD+/NADH ratio when hAD-MSCs were cultured in high glucose. This may explain the disturbance in the inner mitochondrial potential detected by TMRE assay (Fig. 2a &b).

3.3 Disturbance of MSCs mitochondria dynamics parameters in high glucose engenders their apoptosis:

Apoptosis is a programmed cell death that can be physiological or pathological depending on the triggering factors that enkindle it. Mitochondrial dysfunction is a notable inciting factor which is able to fire up debilitating cellular apoptosis and impact the survival of numerous cells. The noticeable deterioration in the mitochondrial parameters detected in hAD-MSCs after being cultured in high glucose prompted us to examine the level of apoptosis in these cells. To achieve that, we used RealTime-Glo™ Annexin V Apoptosis kit to unveil the presence of apoptosis or not. The luminescence signal which is proportionally linked to apoptosis intensity was considerably higher in hAD-MSCs that were cultured in high glucose (Fig. 4). Fluorescent microscopic images showed that apoptosis (marked by green stained cells) was more in high glucose cultured hAD-MSCs (Fig. 4).

3.4 High glucose affects the level of PI3K and mTOR in MSCs:

Phosphatidylinositol 3-kinase (PI3K) orchestrates many vital signaling pathways and targets downstream factors that are required to maintain the vivacity of cells. PI3K controls integral proteins that are necessary to preserve mitochondrial dynamics (16, 17). PI3K is a known activator of mammalian target of rapamycin (mTOR) which is a central regulator of cell survival and metabolism. It has been reported that mTOR is important to preserve mitochondrial dynamics and generate the required mitochondrial potential to produce ATP. PI3K is required to remove the inhibitory effect of tuberous sclerosis tumor suppressor (TSC1) which binds mTOR and inactivates it. We performed Western blotting analysis to measure the expression levels of PI3K, TSC1, and mTOR in High glucose treated hAD-MSCs. Western blotting results demonstrated the loss of PI3K and mTOR in high glucose treated hAD-MSCs, while TSC1 is significantly increased in these cells (Fig. 5). Those findings indicated impaired PI3K/ mTOR Axis which may explain the disruption in mitochondrial parameters in high glucose cultured MSCs. Taking together, our findings highlight a new mechanism that can be released by high glucose causing the poor survival of MSCs in diabetic microenvironment.

MSCs are the most promising type of stem cells for regenerating damaged tissues and restoring their normal functions which can improve the quality of life of many patients who suffer from debilitating disorders (1). The miraculous abilities of MSCs to mitigate the course of many pathologies prompted many scientists to thoroughly investigate the therapeutic outcomes of transplanting MSCs in many disease models (2, 18). Several studies not only reported the remarkable improvements in several pathological markers after transplanting MSCs (19, 20), but they also unveiled the short-lived outcomes and poor survival rate which need extensive investigation (21, 22). Many pathologies have sharing hallmarks characterized by the existence of stress microenvironments, including hypoxia, inflammation, and nutritional imbalances which can significantly impact the expected outcomes of transplanting MSCs (23–26). These stress microenvironments can either inhibit or activate various cellular signaling pathways producing many molecular and biological changes in the transplanted cells. DM is one of frequently diagnosed chronic diseases that leads to multi-system complications which affect the quality of life of many patients (9). The major hallmark existing in DM disease is glucotoxicity that is featured by the presence of high glucose levels and impairments in insulin secretion and function (9). This glucotoxicity produces massive molecular changes in the residing cells which wreak havoc on them and cause the appearance of DM-related complications, such as cardiovascular and neurological complications (9). Both animal and clinical studies have demonstrated the great therapeutic efficacy of MSCs transplantation in alleviating chronic hyperglycemia by reversing insulin resistance, improving insulin sensitivity, controlling inflammation, relieving metabolic syndrome symptoms, and ameliorating β-cell destructive abilities (11, 27). Nevertheless, studies also revealed that MSCs exhibited short-lived outcomes due to their poor survival after being transplanted in DM disease model (4). It seems that diabetic microenvironment can trigger molecular and cellular changes in these transplanted cells. Several studies have asserted the importance of investigating the mechanisms leading to the poor survival of MSCs in hyperglycemia which is the major insult to DM patients (21, 28). Up to date little is known about the molecular and biological changes in MSCs under hyperglycemia. MSCs are capable of switching their metabolism on to support their regenerative and immunomodulation abilities (29). MSCs rely primarily on glycolysis when they are undifferentiated as it is important to preserve their stemness and proliferation (30). Several reports illustrated that glycolysis is required to preserve the immunosuppressive characteristics of MSCs (5). Upregulation in glycolysis has been found to increase the immunomodulatory abilities of MSCs and promote their capabilities to reprogram immune cells (31) .MSCs use mitochondrial OxPhos when they are induced to differentiate, thus the multi-lineage differentiation capacities of MSCs require intact mitochondria (7). Maintaining intact mitochondrial dynamics is not only required to support the differentiation capacities of MSCs, but it is also crucial for redox regulation and prevention of excessive production of reactive oxygen species (ROS) (32). Recently, it has been proposed by many studies that MSCs are capable of transferring mitochondrial DNA (mtDNA) to recipient cells and restoring the normal mitochondrial functions in these cells (33, 34). MSCs-mediated mitochondrial transfer further supports the necessity to conserve normal mitochondrial parameters (34). Previous reports revealed that the MSCs proliferation decreased in high glucose medium (35, 36). Furthermore, other studies showed that MSCs differentiation capacities might vary when they are cultured in high glucose conditions (37, 38). Our study illustrated that high glucose can reduce the survival rate of MSCs. The reduction in MSCs viability reported in our study is correlated with an impairment in the inner mitochondrial membrane potential (ΔΨm) which is an important parameter and the energy generated from this potential gradient will be utilized by complex V to produce ATP molecules. The maintenance of mitochondrial membrane potential depends on the presence of a plentiful amount of NAD + molecules (39). NAD + serves as an oxidoreductase cofactor that controls many vital energy production pathways. Maintaining normal NAD+/NADH ratio is integral to providing the required electron flux that will be used to generate proper mitochondrial membrane potential (ΔΨm) (39). High glucose insult causing a substantial drop in NAD+/NADH ratio in hAD-MSCs indicates a needed disturbance in the oxidization power to produce NAD + and donating electrons that will keep the functionality of mitochondrial complexes. Another important complex that is required to preserve the mitochondrial dynamics is mTOR complex (40). mTOR is a known regulator of many cellular processes, particularly the important mitochondrial biogenesis and physiological mitophagy (40). Several studies demonstrated that mTOR inhibition ensued mitochondrial hyperfusion and branching, which dysregulate the redox balancing and trigger intense apoptosis (40, 41). Herein, we have reported a significant decline in the protein level of mTOR in hAD-MSCs cultured in high glucose, which further explain the existence of mitochondrial dysfunction and the high apoptosis level detected in these cells. The effect of high glucose on modulating mitochondrial functions by targeting mTOR is barely investigated with controversial results being reported. Sen and co-authors found that high glucose activates mTOR pathway leading to myocardial hypertrophy and contractile dysfunction (42). Another study conducted by Pandey and co-authors revealed similar results that high glucose induced a massive oxidative stress due to mitochondrial function impairment in cardiomyocytes mediated by increased endothelin-1 and perturbed mTOR activity (43). Interestingly, studies also showed that insulin can activate mTOR pathway in DM patients and protect many cells from developing mitochondrial dysfunction (44), which indicate that high glucose can deactivate mTOR causing many pathological consequences, such as neurological disorders in DM patients (44). These discrepancies regarding the effect of high glucose on mTOR level and activity seem to differ depending on the type of cells and the upstream molecular regulators. Based on that, the role of high glucose in modulating the level of mTOR needs further investigation in different cell types. In our study, the upstream regulatory protein PI3K decreased remarkably in hAD-MSCs exposed to high glucose. A plentiful number of reports corroborated that PI3K-AKT pathway is required to activate mTOR by removing the inhibitory effect produced by TSC1 or Hamartin protein. We reported in this study that the reduction in PI3K protein level was coupled with marked elevation in TSC1 and downregulation in mTOR in hAD-MSCs cultured in high glucose. The findings of our study deserve consideration in broader context since it provides a mechanistic explanation of poor survival of MSCs in hyperglycemic microenvironment and provides new targets that can be modified to enhance the survival rate of MSCs in DM patients. Schematic summary of the study findings was illustrated in (Fig. 6).

MSCs are reputable type of stem cells that can regenerate damaged tissues and restore their functions. DM is a chronic disease that is linked to multisystem complications that can be serious and lethal. DM patients can hugely benefit from MSCs transplantation to abrogate the pathological consequences and improve their quality of life. The poor survival and short-lived positive outcomes following MSCs transplantation in DM patients are considered a stumbling blocks. High glucose impaired the mitochondrial function in MSCs by perturbing many mitochondrial regulatory factors, particularly mitochondrial membrane potential, NAD+/NADH pool, and mTOR protein. This mitochondrial dysfunction is associated with triggering apoptosis in MSCs. Preserving these factors may help in improving the survival rate of MSCs in diabetic microenvironment and initiate long-lasting therapeutic outcomes.

hAD-MSCS, human adipose tissue derived mesenchymal stem cells; OxPhos, oxidative phosphorylation; ΔΨm, mitochondrial membrane potential, TMRE, tetramethylrhodamine ethyl ester; NAD+, Nicotinamide adenine dinucleotide, PI3K, Phosphatidylinositol 3-kinase; TSC1, tuberous sclerosis tumor suppressor; mTOR, mammalian target of rapamycin.

Authors contributions:

E.A.E.R conceptualized the study. E.A.E.R, R.R.K, R.M, A.S, H.M, H.S, and M.M carried out the experiments. E.A.E.R and F.A.M analyzed the data. E.A.E.R, F.A.M, A.A, and D.G drafted and wrote the manuscript. All authors read and approved the final version of manuscript.

Acknowledgement:

This work was conducted in the Applied and Basic Medical Research Lab in the Faculty of Medicine at Yarmouk University. We would like to thank Mr. Muhammad Abu-El-Rub for his efforts in proof-editing and correcting the manuscript.

Funding:

This study is funded by grants from the Deanship of Scientific Research, Yarmouk University, Jordan (Grant # 29/2021, Grant # 11/2021, and Grant # 13/2022).

Ethical approval:

The ethical approval was not needed in this study. The human cell lines used for this study were commercially purchased.

Conflict of interest

The authors declare that they have no conflict of interests.

Data Availability:

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

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Human mesenchymal stem cells exhibit altered mitochondrial dynamics and poor survival in high glucose microenvironment

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Abstract

Figures

1. Introduction:

2. Materials and Methods:

2.1 Human mesenchymal stem cells

2.2 Cytotoxicity assays:

2.3 TMRE assay:

2.4 NAD+/NADH assay:

2.5 Western blotting:

2.6 Apoptosis assay:

2.7 Statistical analysis

3. Results:

3.1 Culturing MSCs in high glucose triggers massive cytotoxicity and poor survival:

3.2 The mitochondrial dynamics of MSCs impaired by high glucose:

3.3 Disturbance of MSCs mitochondria dynamics parameters in high glucose engenders their apoptosis:

3.4 High glucose affects the level of PI3K and mTOR in MSCs:

4. Discussion:

5. Conclusion:

Abbreviations

Declarations

References

Status:

Version 1