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. 2023 Sep 3;37(5):2006–2017. doi: 10.21873/invivo.13298

The Role of Box A of HMGB1 in Enhancing Stem Cell Properties of Human Mesenchymal Cells: A Novel Approach for the Pursuit of Anti-aging Therapy

ZIN ZIN EI 1,2, APIWAT MUTIRANGURA 3,4, WANATCHAPORN ARUNMANEE 2,5, PITHI CHANVORACHOTE 1,2
PMCID: PMC10500522  PMID: 37652483

Abstract

Background/Aim

Box A is a highly conserved DNA-binding domain of high-mobility group box 1 (HMGB1) and has been shown to reverse senescence and aging features in many cell models. We investigated whether the activation of box A can influence stem cell properties.

Materials and Methods

Human dermal papilla (DP) cells and primary human white pre-adipocytes (HWPc) were employed as mesenchymal cell models. Box A-overexpressing plasmids were used to induce cellular box A expression. mRNA and protein levels of stemness markers POU class 5 homeobox 1 pseudogene 5 (OCT4, HGNC: 9221), Nanog homeobox (NANOG, HGNC: 20857), and SRY-box transcription factor 2 (SOX2, HGNC:11195) in DP cells and HWPc were measured by real-time polymerase chain reaction and immunofluorescence analysis, respectively.

Results

Transfection efficiency of box A-overexpressing plasmid was 80% and 50% in DP cells and HWPc, respectively. The proliferative rate of both cell types significantly increased 72 h after transfection. Levels of OCT4, NANOG and SOX2 mRNA and protein expression were significantly increased in box A-transfected DP cells and HWPc compared to empty plasmid-transfected cells. Immunofluorescence analysis confirmed the induction of OCT4, NANOG and SOX2 protein expression in response to box A in DP cells and HWPc. OCT4 and SOX2 were expressed in both the nuclear and cytoplasmic compartments, while NANOG was intensely located in the nucleus of box A-transfected cells.

Conclusion

Our findings suggest that box A may potentially enhance stemness, which may have significant benefits in improving stem cell function due to aging processes and disease. This research may have implications for regenerative medicine applications.

Keywords: Box A, stemness, aging, DNA damage, regenerative medicine

Aging is a natural biological process in all living organisms but can have significant impacts on the health and function of cells (1). DNA damage is one of the hallmarks of aging and is a major contributor to the decline in cellular function that occurs as cells age (2,3). DNA damage and activation of the DNA-damage response (DDR) are major contributors to aging processes, including negative effects on cellular function such as cellular senescence or apoptosis and telomere shortening (2). Moreover, the accumulation of DNA damage can lead to changes in cellular function and gene expression. Therefore, the activation of the DDR pathway can lead to aging and age-related disease at a clinical level by promoting cellular senescence and cell-cycle arrest (4,5).

High-mobility group box 1 (HMGB1) is a protein involved in DNA organization and repair. Box A is one of the highly conserved DNA-binding domains of the HMGB1 gene which is responsible for binding to DNA and interacting with other proteins. DNA tension and DNA damage in aging cells occur due to the reduction of naturally occurring DNA gaps, so-called youth DNA gaps. Intranuclear box A increases DNA durability by producing DNA gaps, thereby limiting endogenous DNA damage and the DDR (6,7). Box A-bearing plasmid was used to rejuvenate senescent cells and reduce β-galactose-induced aging and natural aging in rats (6). Limiting DDR ameliorated the expression of all aging-related markers (8).

The aging of cells may cause DNA damage, telomere shorting, and other changes that can lead to decline in cellular function and an increased risk of aging disease. Cell rejuvenation strategies, specifically focused on stem cell therapy, aim to enhance the proliferation of new cells and promote their growth (9). According to previous research, SRY-box transcription factor 2 (SOX2), a cell fate-determining transcription factor crucial in regulation of the maintenance of neural progenitors, is directly regulated by cyclin-dependent kinase inhibitor 1A (p21). SOX2 expression in neural stem cells is negatively regulated by p21, which binds to a SOX2 enhancer directly for controlling the cell cycle and long-term self-renewal. Moreover, the tumour-suppressor proteins p19Arf and p53 may accumulate in neural stem cells through this mechanism (10).

Stemness of stem cells refers to their ability for self-renew and differentiation into various cell lineages (11-13). These abilities fundamentally affect tissue generation, maintenance, and function (14-16). In the skin and hair, mesenchymal stem cells (MSCs) are found at various sites such as the epidermis stratum basale, deep down in the hair follicles, and inside the subcutaneous fat layer (17). These stem cells not only proliferate and differentiate to maintain tissue homeostasis and repair damage, but also secrete cytokines and growth factors to ensure the full functionality of the surrounding cells (18-21).

In the aging process, skin stem cells were found to decline in terms of number and activity, significantly affecting the overall health and appearance of the skin and hair. The stem cell properties of dermal papilla (DP) cells are responsible for hair follicle regeneration and new hair formation after loss. DP cells and their secreted substances have potential for use in regenerative medicine (22,23). In the hypodermal layer of the skin, white adipose tissue was found to be important for hair growth and cycle regulation, and wound repair (24). Moreover, white adipose tissue contains pre-adipocyte stem cells that are valuable in wound-healing and regenerative approaches (25). Adipose-derived stem cells (ADSCs) are MSCs obtained from adipose tissue. ADSCs have differentiative roles for suppressing immune cell activity and reducing inflammation for treating rheumatoid arthritis and multiple sclerosis. Consequently, ADSCs also have potential for use in regenerative medicine (26,27).

Therefore, we investigated whether the induction of box A within the HMGB1 gene influences the expression of stem cell markers POU class 5 homeobox 1 pseudogene 5 (OCT4, HGNC: 9221), Nanog homeobox (NANOG, HGNC: 20857), and SOX2 in DP cells and primary human white pre-adipocytes (HWPc), which may benefit the development of regenerative approaches.

Materials and Methods

Construction of hHMGB1-boxA plasmids. Expression plasmids encoding HMGB1-boxA gene were constructed, including a fusion gene for green fluorescent protein (GFP), picornavirus-derived self-cleaving peptide P2A and hHMGB1-boxA gene. The gene encoding hHMGB1-boxA was synthesized by Twist Bioscience (South San Francisco, CA, USA), All genes were amplified by polymerase chain reaction (PCR). The resulting gene fragments were cloned into pVAX1 vector (Thermo Fisher Scientific, Invitrogen, MA, USA) at NheI/NotI sites. All genes were inserted between the cytomegalovirus promoter and the bovine growth hormone polyadenylation signals.

The cloning strategies for all constructs were as follows. The fusion gene pVAX1–eGFP–P2A–boxA was constructed by the amplification of the eGFP gene from pSpCas9(BB)-2A-GFP (PX458, plasmid # 48138, Addgene, Watertown, MA, USA). The eGFP fragments were then assembled with the gene fragment containing P2A and hHMGB1-boxA using Gibson Assembly Master Mix kit (Thermo Fisher Scientific, Waltham, MA, USA). The DNA sequences were verified by Sanger DNA sequencing (Bionics, Republic of Korea). The plasmids with correct sequences were extracted from transformed Escherichia coli DH5 using Geneaid™ Midi Plasmid Kit (Geneaid, New Taipei, Taiwan, ROC) and quantified using EzDrop 1000 Micro-Volume Spectrophotometer (Blue-ray Biotech, New Taipei, Taiwan, ROC).

Cells and reagents. Human DP cells were obtained from Applied Biological Materials Inc. (Richmond, BC, Canada). DP cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, NY, USA) with 10% inactivated foetal bovine serum (Gibco, Gaithersburg, MA, USA) supplemented with 2 mM of glutamine and 1% antibiotic-antimycotic (Gibco, Gaithersburg, MA, USA). DP cells were incubated at 37˚C with 5% CO2.

HWPc were obtained from PromoCell (PromoCell GmbH, Heidelberg, Germany). HWPc were grown in a culture medium for pre-adipocytes containing a supplement mix provided by PromoCell GmbH. The cells were cultured under conditions without antibiotics and antimycotics, and the incubation took place at a temperature of 37˚C with 5% CO2. The cells were utilized for experiments until they reached multiple passages, with a maximum of 10 population doublings. Lipofectamine™ 3000 reagent, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and Alexa Fluor™ 594 goat anti-rabbit IgG (H+L)-conjugated secondary antibody were purchased from Thermo Fisher Scientific (Carlsbad, CA, USA). Hoechst 33342 was obtained from Molecular Probes Inc. (Eugene, OR, USA).

All OCT4, NANOG, SOX2 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) primers were obtained from Eurofins Genomics (Louisville, KY, USA). Rabbit monoclonal antibodies to OCT4 (ab19857), NANOG (ab80892) and SOX2 (ab97959) were obtained from Abcam (Waltham, MA, USA).

Plasmid transfection. DP cells and HWPc (3×105 cells/ml) were seeded into 6 well plates containing specific growth medium overnight. The Lipofectamine™ 3000 (Thermo Fisher Scientific, CarIsbad, CA, USA) (3.75 μl) and Opti medium (125 μl) were prepared in an Eppendorf tube. In another Eppendorf tube, pVAX1–eGFP–P2A–boxA plasmids (2,500 ng) were put into P3000™ Reagent (5 μl) and Opti medium (125 μl). After that, the plasmids and transfection reagent were mixed and incubated for 15 min to promote micelle formation in the plasmid–lipid complex. The plasmid–lipid complex was added to the cells (at 70% confluence) for transfection. pVAX1–eGFP–P2A–boxA-transfected cells were visualized by taking a photograph under a fluorescence microscope at 48 h after transfection. Empty vector plasmids, pVAX1 were used as the control.

Proliferation assay. The DP cells and HWPc (3×104 cell/well) were seeded into 6-well plates in an appropriate medium. On the next day, the cells were transfected with Lipofectamine™ 3000 (Thermo Fisher Scientific) for 48 h. After that, the native DP cells and HWPc, and cells transfected with plasmids (pVAX1 and pVAX1–eGFP–P2A-boxA) were seeded (5×103 cells/well) in 96-well plates and incubated at 37˚C for 24 h. Cell proliferation was determined for 3 consecutive days using 4 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma–Aldrich, St. Louis, MO, USA) and measuring the absorbance at 570 nm. The proliferative rate was calculated as: Percentage viability=(Average A570 of transfected cells/Average A570 of control cells)×100.

Real-time polymerase chain reaction (RT-PCR). The DP cells and HWPc (3×104 cells/well) were seeded in appropriate medium into 6-well plates. On the next day, the cells were transfected with Lipofectamine™ 3000 for 48 h. The total RNA from transfected cells was extracted with RNeasy kit (Qiagen, Germantown, MD, USA). After that, cDNA was synthesized from total RNA using SuperScript III reverse transcriptase (Thermo Fisher Scientific, Invitrogen, Carlsbad, CA, USA). After synthesizing cDNA, 100 ng of cDNA was used for RT-PCR with Luna Universal qPCR Master Mix (New England Biolabs GmbH, Frankfurt am Main, Germany) with a final volume of 20 μl. The reaction was carried out using a CFX 96 Real-time PCR system (Bio-Rad, Hercules, CA, USA). The conditions for RT-PCR were an initial denaturation step at 95˚C for 1 min, followed by 45 cycles of denaturation at 95˚C for 15 s, and primer annealing at 60˚C for 30 s. The primers used for targeting genes were OCT4: forward: TCGAGAACCGAGTGAGAGG, melting temperature (Tm)=58.8˚C, reverse: GAACCACA CTCGGACCACA Tm=58.8˚C; NANOG: forward: ATGCCTCA CACGGAGACTGT, Tm=59.4˚C, reverse: AAGTGGGTTGTTT GCCTTTG, Tm=55.3˚C; SOX2: forward TGATGGAGACG GAGCTGAA, Tm=56.7˚C, reverse: GGGCTGTTTTTCTGGTTGC, Tm=56.7˚C; GAPDH: forward: CCACCCATGGCAAAT TCCATGGCA, Tm=67˚C, reverse: TCTAGACGGCAGGTCA GGTCCACC, Tm=70.4˚C. Melting curve analysis was used to determine primer specificity. The expression level of each gene was normalized with that of GAPDH as an internal control. The relative mRNA expression level of each gene was calculated from comparative Cq values.

Immunofluorescence. The transfected DP cells and HWPc were seeded at 1×104 cells/well in a 96-well culture plate. After that, the cells were fixed with 4% paraformaldehyde for 15 min. The cells were permeabilized with 0.5% triton-X for 5 min and followed by blocking the non-specific protein with 10% foetal bovine serum in 0.1% Triton–phosphate-buffered saline for 1 h at room temperature. The cells were incubated with 1:400 of primary antibodies against OCT4, NANOG and SOX2 at 4˚C overnight. On the following day, the cells were incubated with 1:500 secondary Alexa Fluor 597-conjugated goat anti-rabbit IgG (H+L) (Invitrogen) and stained for nuclei with Hoechst33342 for 1 h at room temperature. The cells were then washed with phosphate-buffered saline and covered with 50% glycerol. Images of cells were captured through a fluorescence microscope (Olympus IX 51 with DP70; Olympus America Inc., Center Valley, PA, USA). The fluorescence intensity was measured by Image J software (National Institutes of Health, Bethesda, MA, USA). Moreover, the localization of stemness marker in box A-transfected DP cells and HWPc was determined by capturing images with a confocal microscope at 40× (Zeiss Microscopy LSM 900; Carl Zeiss, Maple Grove, MN, USA).

Statistical analysis. Multiple groups of sample comparisons were made by one-way analysis of variance with a post hoc test. Statistical analyses were carried out with Graph Pad Prism software 9.0 (GraphPad Software, La Jolla, CA, USA) and the statistical significance differences between groups were considered at p≤0.05. The results are reported as the mean±standard deviation from at least three biologically independent experiments.

Results

Transfection of box-A of HMGB1 gene to human DP cells and HWPc. We aimed to investigate the stemness properties of human mesenchymal cells affected by the induction of box A of HMGB1 gene. The box A-overexpressing plasmid was generated as presented in the Materials and Methods using a fusion gene GFP–P2A–boxA. This plasmid induces the overexpression of GFP and box A simultaneously in the system. The mesenchymal DP cells and primary HWPc were transfected with either peGFP–p2A–BoxA or PVAX1 (the backbone blank plasmid) by Lipofectamine 3000. The transfection efficiency of peGFP–p2A–BoxA-transfected cells was evaluated, and the results showed 80% and 50% transfection efficiency in DP cells and HWPc, respectively (Figure 1).

Figure 1. Dermal papilla (DP) cells (A) and human white pre-adipocytes (HWPc) (B) were efficiently transfected by Lipofectamine™ 3000 reagent. Enhanced green fluorescence protein–peptide 2A–boxA (peGFP-p2A-BoxA) or control (pVAX1) plasmids (2500 ng) were transfected into DP cells and HWPc with 3.75 μl Lipofectamine™ 3000. Images of transfected cells under phase-contrast (upper panel) and fluorescence (lower panel) microscopy are shown. Scale bar=100 μm.

Figure 1

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Analysis of proliferation in box A-transfected mesenchymal cells. The senescence of stem cells was demonstrated to have a negative impact on the regeneration of tissue as well as increasing pro-inflammatory cytokines (28). As cellular senescence is linked with a reduced rate of cell proliferation, proliferation assays were performed on these mesenchymal cells to investigate the effect of box A transfection on senescence phenotype over a period of 72 h. Our results showed that the proliferation of cells transfected with peGFP–p2A–BoxA plasmids increased slowly until 24 h and was significantly higher at 48 and 72 h in both DP cells (Figure 2A) and HWPc (Figure 2B).

Figure 2. Box A increased proliferation of mesenchymal cells. Cell proliferation was assessed for three consecutive days after transfection of dermal papilla (DP) cells (A) and human white pre-adipocytes (HWPc) (B) with enhanced green fluorescence protein–peptide 2A–boxA (peGFP–p2A–BoxA) or control (pVAX1) plasmids. For all the data, all experiments were from three independent biological samples. Data are presented as mean±SD. *Significantly different at p≤0.05 from cells transfected with pVAX1 empty vector with t-test. Scale bar=100 μm.

Figure 2

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Box A increased mRNA expression of pluripotency transcription factors. The pluripotency transcription factors NANOG, OCT4 and SOX2 are known to regulate stem cell properties and stemness in stem cells (29,30). Herein, we further analysed the possible effect of box A overexpression on pluripotency transcription factors using real-time RT-PCR analysis. According to our results, the box A-transfected DP cells and HWPc exhibited high mRNA expression levels of stemness markers OCT4, NANOG and SOX2.

Following transfection of DP cells with target vectors as aforementioned, it was found that the expression of the pluripotency transcription factor OCT4 was notably augmented (roughly a 2-fold increase). Moreover, the box A-transfected cells exhibited a significant 5-fold increase of NANOG mRNA in comparison to control-transfected cells, as depicted in Figure 3A. Notably, the mRNA level of SOX2 in box A-transfected cells was dramatically enhanced (8-fold increase) compared to control-transfected DP cells (Figure 3A).

Figure 3. Real-time polymerase chain reaction analysis shows the effect of box A transfection on the mRNA level of pluripotency transcription factors POU class 5 homeobox 1 pseudogene 5 (OCT4), Nanog homeobox (NANOG), and SRY-box transcription factor 2 (SOX2) in dermal papilla (DP) cells (A) and human white pre-adipocytes (HWPc) (B). DP cells transfected with enhanced green fluorescence protein–peptide 2A–boxA (peGFP–p2A–BoxA) exhibited amplified expression of pluripotency stem cell markers OCT4, NANOG, and SOX2. HWPc transfected with box A similarly showed a significant increase in the expression of OCT4, NANOG and SOX2. All data were obtained from three independent biological samples, and the results are presented as the mean±SD. Statistically significantly different at *p≤0.05 and ***p≤0.001 when compared to empty vector-transfected cells (pVAX1), as determined by t-test.

Figure 3

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Consistent with the results in DP cells, HWPc exhibited induction of stem cell markers, as evidenced by a 2.5-fold increase in OCT4 mRNA expression in box A-transfected HWPc. Furthermore, box A resulted in a 2-fold up-regulation of NANOG in HWPc compared to control-transfected cells (Figure 3B). In addition, SOX2 mRNA expression was elevated by 10-fold in response to box A in HWPc cells (Figure 3B).

Based on the results of mRNA expression, it can be concluded that box A expression in these human mesenchymal cells induced gene expression of stem cell-related pluripotency transcription factors. Among these, SOX2 exhibited the highest expression levels after targeted transfection of both DP cells and HWPc.

Box A increased protein expression of pluripotency transcription factors. Box A is a regulatory element of HMGB1 that controls gene expression by binding to specific transcription factors at a conserved DNA sequence (31). Having shown that box A induced mRNA expression of pluripotency transcription factors, we wanted to confirm this effect by evaluating the protein expression levels by immunofluorescence.

Figure 4 shows the effect of box A transfection on the protein expression of pluripotency transcription factors. In DP cells, OCT4 was observed to increase by approximately 2-fold compared to empty vector-transfected cells. Additionally, the NANOG fluorescent signal was found to be augmented 3-fold and more intensely localized in the nucleus of box A-transfected cells than in empty vector-transfected cells. The protein expression of SOX2 was also enhanced 2-fold in box A-transfected cells compared to empty vector-transfected cells (Figure 4A). Similarly, in box A-transfected HWPc cells, the protein expression of OCT4 was found to increase by approximately 3-fold compared to empty vector-transfected cells (Figure 4B). The NANOG signal was also induced 3-fold and was more intensely located in the nucleus of box A-transfected HWPc compared to empty vector-transfected cells. Furthermore, the protein expression level of OCT4 was observed to increase by 2.5-fold in box A-transfected HWPc compared to empty vector-transfected cells (Figure 4B).

Figure 4. The expression of pluripotency transcription factors POU class 5 homeobox 1 pseudogene 5 (OCT4), Nanog homeobox (NANOG), and SRY-box transcription factor 2 (SOX2) in box A-transfected dermal papilla (DP) cells (A) and human white pre-adipocytes (HWPc) (B) as evaluated by immunofluorescence. The intensity of immunofluorescence of pluripotency stemness markers was quantified as described in the Materials and Methods section. Scale bar=10 μm. pVAX1: Empty vector; peGFP-p2A-BoxA: enhanced green fluorescence protein–peptide 2A–boxA.

Figure 4

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Localization of pluripotency stem cells markers in box A-transfected DP cells and HWPc by confocal microscopy. To provide more insight into the nature of the changes induced by box A transfection, we utilized confocal microscopy to assess the nuclear/cytoplasmic localization of the studied transcription factors. The cells were transfected with either box A-containing vector or control empty vector and then stained with specific antibody for each marker as described in the Materials and Methods. As shown in Figure 5, OCT4 was found in both the cytoplasm and nucleus of box A-transfected cells, as was SOX2, whilst NANOG was found to be intensely scattered throughout the nucleus.

Figure 5. Localization of pluripotency stemness markers POU class 5 homeobox 1 pseudogene 5 (OCT4), Nanog homeobox (NANOG), and SRYbox transcription factor 2 (SOX2) in box A-transfected dermal papilla (DP) cells and human white pre-adipocytes (HWPc) by confocal microscopy at 40×. Arrows indicate where stemness markers were located. In box A-transfected cells, OCT4 and SOX2 protein was seen in the nucleus and cytoplasm, and NANOG was intensely scattered in the nucleus. Scale bar=20 μm.

Figure 5

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Discussion

Ageing is an important factor in illnesses such as cancer, cardiovascular disease, neurological diseases, and organ failure (1,32,33); cellular ageing is the primary cause of disease (6). Several mechanisms contribute to the pathophysiology of cellular aging, including mitochondrial malfunction, oxidative stress, DNA damage, DNA instability, telomerase shortening, and epigenetic alterations (6).

DNA holds genetic information and controls the operation and properties of living things, including humans. However, hallmarks of aging cells include DNA damage, epigenetic modifications, and instability. In ageing cells and tissues, this DNA instability can result in alterations to both the structure and function of cells (2,34-36).

Ageing can reduce the effectiveness of the DDR system, increasing DNA damage and mutations. Mutations may become more prevalent over time, speeding up ageing and the onset of age-related diseases (2,37-39). The main targets for rejuvenation strategies are ageing processes within cells (2,6,40,41). Consequently, in this study, we investigated whether preserving the integrity of the DDR pathway is necessary for preventing ageing and lowering the prevalence of age-related disorders.

Younger cells have higher rates of DNA methylation and naturally occurring DNA gaps than older cells, which can aid in protecting DNA. Expanding gaps in DNA can promote DNA stability and durability, and lead to rejuvenation. These DNA gaps are produced by cellular enzymes and prevent DNA damage. The levels of these so-called youth DNA gaps are notably diminished in aged cells (6). Notably, youth DNA gaps were also significantly reduced in cells lacking either HMGB1 or the NAD-dependent histone deacetylase silent information regulator (SIR2), leading to the discovery that box A of HMGB1 can generate DNA gaps and prevent DNA double-strand breaks (6,42-44). Furthermore, several studies have demonstrated that DNA gaps induced by box A can reverse ageing features both in vitro and in vivo (45-48). Moreover, the box A domain in HMGB1 functions as molecular scissors to create DNA gaps that increase DNA stability and protect DNA from damage (6,7,44). While DNA gap formation via box A may be a promising strategy for changing the ageing process (7), there is currently no evidence to suggest it has any impact on the stemness of human stem cells.

Senescent MSCs not only hamper tissue repair through senescence-associated stem cell exhaustion, but also mediate tissue degeneration by initiating and spreading senescence-associated inflammation. Lui and Chen suggested new strategies of MSC-based cell therapy to remove, rejuvenate, or replace senescent MSCs (28). According to previous research, expression of pluripotency-related genes were altered in MSCs when they were exposed to a DNA methylation inhibitor, and OCT4 and NANOG work together to stimulate DNA methyltransferase 1 expression to control the proliferation of undifferentiated MSCs and the reversal of the senescence caused through genes such as cyclin-dependent kinase inhibitor 2B, cyclin D1 and histone deacetylase 1 (49,50).

For this reason, in this study, we transfected HMGB1 box A plasmids into DP cells and HWPc, as depicted in Figure 1. Box A transfection led to enhanced DNA stability, reduced DNA damage, increased the presence of DNA gaps, and diminished the DDR (6,7,44). Box A HMGB1 prevented the DNA double-strand break-induced damage response and increased resistance to radiation. The formation of DNA gaps after transfection of box A contributes to the preservation of older DNA and extends its longevity (6). Molecules like box A of HMGB1 are known as genomic stability molecules rejuvenating DNA (REDGEM).

Furthermore, as shown in Figure 3 and Figure 4, box A-transfected cells demonstrated elevated mRNA and protein expression of pluripotency stem cell markers OCT4, NANOG and SOX2. These markers were localized in both the cytoplasm and nucleus of DP cells and HWPc transfected with box A (Figure 5). OCT4 is crucial for the pluripotency and self-renewal of stem cells. OCT4 has distinct isomers, with OCT4A being primarily located in the nucleus and OCT4B in the cytoplasm and nucleus (51-53). SOX2 is crucial for stemness, self-renewal, and differentiation of stem cells. Jeong et al. reported SOX2 was also localized in both the cytoplasm and nucleus (52) in transfected DP cells and HWPc. SOX2 also plays a role in cell proliferation and protects against cellular oxidative stress (54). NANOG, a key transcription factor responsible for cell self-renewal and pluripotency, was primarily found in the nucleus of box A-transfected DP cells and HWPc. Overall, the transfection of MSCs with box A can potentially demonstrate anti-ageing characteristics attributed to the overexpression of stemness markers.

This study revealed a novel finding of box A in induction of stemness as described in the schematic of Figure 6. It is promising that box A of HMGB1 can promote DNA stability and reduce DNA damage and DDR, which might help to promote cell regeneration and potentially be used in epigenetic therapy for age-related diseases. With the addition of stemness-inducing activity, this new approach might help to rejuvenate ageing cells by promoting stem cell properties.

Figure 6. The scheme illustrates the proposed role of box A in enhancing the stemness of human mesenchymal stem cells. A: The aging of cells is influenced by the accumulation of DNA damage and deterioration over time. This can be prevented by transfection of box A of high-mobility group box 1 (HMGB1) gene into dermal papilla cells (DP) and human white pre-adipocytes (HWPc). These two cell lines were utilized as cellular models to investigate how box A induction promotes stemness in age-related diseases. B: Box A-transfected DP cells and HWPc can produce DNA gaps through molecular scissoring activity. Here we provide new insight by demonstrating that box A can additionally induce the expression of pluripotency stemness markers, such as POU class 5 homeobox 1 pseudogene 5 (OCT4), Nanog homeobox (NANOG), and SRY-box transcription factor 2 (SOX2). Induction of stem cell transcription factors enhances stemness of stem cells, increasing their ability to self-renew and differentiate into various cell lineages. In closing, box A of HMGB1 acts as a genomic stability molecule rejuvenating DNA (REDGEM) and enhances the expression of pluripotency factors, potentially providing a novel, alternative epigenetic approach for therapy of age-related diseases. DDR: DNA damage response.

Figure 6

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Thus, box A of HMGB1 may have a genome-stabilizing function, which might have significant implications for the development of new therapies for age-related diseases. This present work has shown the activity of box A in inducing stem cell properties in cell models. We believe that in vivo investigations of such activities should be done in future work. In addition, the potential toxicity, as well as the appropriate plasmid concentration to be used, are also required.

Funding

This research was supported by the National Research Council of Thailand (NRCT) (N41A640075).

Conflicts of Interest

The Authors declare that there are no conflicts of interest.

Authors’ Contributions

Conceptualization and validation, P.C.; formal analysis, Z.Z.E. and P.C.; investigation, Z.Z.E. and P.C.; resources and methodology, A.M. and W.A.; writing—original draft preparation, Z.Z.E.; writing—review and editing, P.C.; supervision, P.C. All Authors have read and agreed to the published version of the article.

Acknowledgements

The Authors would like to thank Dr. Pichit Suvanprakorn (Division of Dermatology, Chulabhorn International College of Medicine, Thammasat University, Pathum Thani, Thailand) and Pan Rajdhevee Group Public Co., Ltd. Pathum Thani, Thailand) for his guidance and support and Sedthawut Laotee (Department of Biochemistry and Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand). Z.Z.E. is grateful to the Second Century Fund (C2F) for a postdoctoral fellowship, Chulalongkorn University.

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