Anti-aging protective effect of L-carnitine as clinical agent in regenerative medicine through increasing telomerase activity and change in the hTERT promoter CpG island methylation status of adipose tissue-derived mesenchymal stem cells

doi:10.1016/j.tice.2018.08.012

Tissue and Cell

Volume 54, October 2018, Pages 105-113

https://doi.org/10.1016/j.tice.2018.08.012 Get rights and content

Highlights

•
LC could decrease the aging of ADSCs via an increase of and the epigenetic modification of the hTERT gene promoter.
•
LC at a concentration of 0.2 mM could be used as a good candidate for extending the replicative life-spans of ADSCs.
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LC could potentially beneficial for enhancing the application of aged-MSCs in cell therapy and regenerative medicine.

Abstract

The identification of factors that reduce the senescent tendency of the mesenchymal stem cells (MSCs) upon expansion has great potential for cellular therapies in regenerative medicine. Previous studies have shown the aging protective effect of L-carnitine (LC). On the other hand, reduction in proliferation potential and age-dependent decline in number and functions of MSCs were accompanied by telomere shortening, reduction in telomerase activity and epigenetic changes. The aim of this study was to evaluate the effects of LC on aging of MSCs through telomerase activity assessment and the investigation of methylation status of the hTERT gene promoter. Telomerase activity and hTERT promoter methylation investigation was performed with PCR-ELISA TRAP assay and methylation specific PCR (MSP), respectively. Also, beta-galactosidase (SA-ß-gal) staining was used to calculate the percentage of senescent cells. The results showed that the LC could efficiently promote the telomerase activity. In addition, the percentage of senescent cells had significantly decreased and changes in the methylation status of the CpG islands in the hTERT promoter region under treatment with LC were seen. In conclusion, it seems that LC could improve the aging-related features due to increasing the telomerase activity, decreasing aging, and changing the methylation status of hTERT promoter; it could potentially beneficial for enhancing the application of aged-MSCs in regenerative medicine.

Graphical abstract

Introduction

Adipose tissue-derived mesenchymal stem cells (ADSCs) are very important adult stem cell population with a broad potential application in tissue engineering and regenerative medicine (Aqmasheh et al., 2017; Katsara et al., 2011). The proliferation rate and potential differentiation of these cells into various cell lineages including cartilage, fat, bone etc., are strongly affected by aging (Ejtehadifar et al., 2015; Fathi and Farahzadi, 2016; Yu and Kang, 2013). As a result, the use of mesenchymal stem cells (MSCs) from older donors is lower than younger donors, which restricts clinical applications such as regenerative medicine or cell therapy (Baker et al., 2015). The effects of age on the MSCs are contradictory, probably due to differences in experimental parameters such as the type of donor, age, sex, cell division and cell culture protocols (Huibregtse et al., 2000). Asumda and Chase (2011) indicated the significant differences in the proliferation, population-doubling time, population size, morphology, and differentiation potential of the bone marrow-derived mesenchymal stem cells (BMSCs) of young rats in comparison to the BMSCs of old rats (Asumda and Chase, 2011). On the other hand, telomere, as terminal regions of chromosomes, shortening is one of the molecular mechanisms underlying ageing of MSCs (Brazvan et al., 2016; Flores et al., 2008). Telomerase as a ribonucleoprotein enzyme, is responsible for adding telomeric repeats to the ends of chromosomes and prevents progressive telomere loss (Fernandez-Marcelo et al., 2016; Lu et al., 2013). Therefore, the regulation of telomerase activity in aged cells is very important (Sampedro Camarena et al., 2007). In addition, the presence of a large CpG island with dense CG-rich content in the human TERT (hTERT) gene promoter suggests that DNA methylation may play a role in the regulation of the telomerase activity and it’s typically associated with gene silencing. Based on the theories of aging, it is reported that aging is the result of the accumulation of oxidative damage caused by free radicals generated as by-products during normal metabolism (Syslova et al., 2014). The free radical theory of aging assumes that oxidative stress is one of the major causes of age related cellular and molecular damage, and this phenomenon is implicated in the pathogenesis of a variety of human and animal diseases. For this purpose, the use of antioxidants to prevent cellular aging is important. L-carnitine (3-hydroxy-4-N-trimethylammonium-butyrate) (LC), an amino acid derivative is one of powerful antioxidants that have important role in the metabolism of fatty acids, consumption of ketone bodies, and reconstruction of erythrocyte membrane phospholipids (Fathi and Farahzadi, 2014). Also, LC cause to significant increase in the activities of antioxidant enzymes, such as superoxide dismutase, catalase, and glutathione peroxidase, constitute a natural defense system against the activity of oxidants (Wynd et al., 2015). LC has been investigated in maintenance of mental and physical function and reversal of decline with aging. Bonavita et al. determined intensive Acetyl-L-carnitine (ALC), an acetyl derivative of LC, treatment could influence a significant improvement of the main mental parameters of the senile brain, without any of side effects (Bonavita, 1986). In the following, Hollister et al. reported that LC leads to a modest decrease in the rate of progression of Alzheimer's disease (Hollister and Gruber, 1996). In another study, Kobayashi et al. demonstrated that ALC increases synaptic neurotransmission in the brain and consequently improves learning capacity in aging rats. Also, in a previous study, Mobarak et al. (2017) reported that LC at the concentration of 0.2 mM would be a good antioxidant to improve lifespan of rat adipose tissue-derived MSCs (rADSCs) due to the decrease in population doubling time and aging (Mobarak et al., 2017). In another publication by Farahzadi et al. (2016), it was shown that LC could be used as a good candidate for extending the replicative life-spans of aged MSCs by increasing hTERT gene expression and telomere length (Farahzadi et al., 2016). In addition to the role of LC in aging, its role in differentiation of MSCs has been shown. In this regard, Fathi et al. (2017) indicated that 200 μM LC induced the neurogenic differentiation of rat adipose tissue-derived MSCs via Wnt/β-catenin and PKA signaling pathways (Fathi et al., 2017).

However, it remains unclear whether LC supplementation can increase the telomerase activity and change the methylation status in the hTERT gene promoter in hMSCs, and whether there exists a relationship between the telomerase activity and the hypomethylation or hypermethylation of CpG islands in the hTERT promoter region. Therefore, this study was carried out to investigate the effect of LC on the telomerase activity, the percentage of senescent cells, and the methylation status of CpG islands in the hTERT promoter region in hMSCs.

Section snippets

Reagents

All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA), unless otherwise stated. All tissue culture plastic ware was from SPL Life Sciences (Pocheon, Korea).

Isolation and culture of MSC from human adipose tissue

Adipose tissue samples were collected from the six normal women (45–58 years old) after undergoing liposuction surgery as previously described in another paper by Farahzadi et al. (2016) (Farahzadi et al., 2016). The detailed methodology of the study was described by another paper in this series (Farahzadi et al., 2017), so

Multi-lineage differentiation and immunophenotype characterization of hADSCs

The initial adherent spindle-shaped cells appear as individual cells on the second-third day in phase-contrast microscopy. As shown in Fig. 1A, the isolated human adipose tissue derived-MSCs population displayed a spindle-shaped morphology like fibroblast cells. Also, the hADSCs isolated are not contaminated with hematopoietic cell lineages, readily differentiated into mesenchymal lineages. For this purpose, the osteogenic and adipogenic differentiation was evident in alizarin red and oil red O

Discussion

Age related changes in MSCs through the cell culture period and during the consecutive cell passages is one of the most important factors in reducing the clinical application of MSCs in cell transplantation (Ejtehadifar et al., 2016; Katsara et al., 2011). Aging is an inherently complex process that is regulated at multiple levels such as molecular, genetic, cellular and tissue (de Gonzalo-Calvo et al., 2010). It seems that hTERT gene expression and the regulation of telomerase activity as well

Conclusion

In conclusion, this research reports the preliminary evidence to support that LC could decrease the aging of ADSCs via an increase of the telomerase activity, and a decrease in the percentage of senescent cells and the epigenetic modification of the hTERT gene promoter. This mentioned effect of LC at a concentration of 0.2 mM could be used as a good candidate for extending the replicative life-spans of ADSCs in cell therapy and regenerative medicine.

Ethical approval

All participants provide their written informed consent to participate in this study. This stage of the project was approved by the ethics committee of Tabriz University of Medical Sciences, Tabriz, Iran.

Declaration of interest

The authors have no conflicting financial interest.

Acknowledgements

Phenotypical characterization and multi-lineage differentiation of hADSCs, calculation of PDT, SA-ß-gal staining, and PCR-ELISA TRAP assay, financially supported by University of Tabriz (No.16-855) and funding regarding the cultivation of hADSCs, and methylation specific PCR was supported by Tarbiat Modares University (No.d/52/1872).

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Anti-aging protective effect of L-carnitine as clinical agent in regenerative medicine through increasing telomerase activity and change in the hTERT promoter CpG island methylation status of adipose tissue-derived mesenchymal stem cells

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Reagents

Isolation and culture of MSC from human adipose tissue

Multi-lineage differentiation and immunophenotype characterization of hADSCs

Discussion

Conclusion

Ethical approval

Declaration of interest

Acknowledgements

Bone

Exp. Hematol.

Free Radic. Biol. Med.

Urology

Exp. Cell Res.

Curr. Opin. Genet. Dev.

Gene

Effects of mesenchymal stem cell derivatives on hematopoiesis and hematopoietic stem cells

Adv. Pharm. Bull.

Age-related changes in rat bone-marrow mesenchymal stem cell plasticity

BMC Cell Biol.

Introducing a single-cell-derived human mesenchymal stem cell line expressing hTERT after lentiviral gene transfer

J. Cell. Mol. Med.

Study of the efficacy and tolerability of L-acetylcarnitine therapy in the senile brain

Int. J. Clin. Pharmacol. Ther. Toxicol.

Key immune cell cytokines affects the telomere activity of cord blood cells in vitro

Adv. Pharm. Bull.

DNA methylation analysis of the promoter region of the human telomerase reverse transcriptase (hTERT) gene

Cancer Res.

The effect of hypoxia on mesenchymal stem cell biology

Adv. Pharm. Bull.

The effects of hypoxia on U937 cell line in mesenchymal stem cells co-culture system

Adv. Pharm. Bull.

L-carnitine effectively induces hTERT gene expression of human adipose tissue-derived mesenchymal stem cells obtained from the aged subjects

Int. J. Stem Cells

Zinc sulfate contributes to promote telomere length extension via increasing telomerase gene expression, telomerase activity and change in the TERT gene promoter CpG island methylation status of human adipose-derived mesenchymal stem cells

PLoS One

Application of L-carnitine as nutritional supplement in veterinary medicine

Rom. J. Biochem.

Isolation, culturing, characterization and aging of adipose tissue-derived mesenchymal stem cells: a brief overview

Braz. Arch. Biol. Technol.