Fibronectin and vitronectin alleviate adipose-derived stem cells senescence during long-term culture through the akt/mdm2/p53 pathway

ABSTRACT Cellular senescence plays a role in the development of aging-associated degenerative diseases. Cell therapy is recognized as a candidate treatment for degenerative diseases. To

achieve the goal of cell therapy, the quality and good characteristics of cells are concerned. Cell expansion relies on two-dimensional culture, which leads to replicative senescence of

expanded cells. This study aimed to investigate the effect of cell culture surface modification using fibronectin (FN) and vitronectin (VN) in adipose-derived stem cells (ADSCs) during

long-term expansion. Our results showed that ADSCs cultured in FN and VN coatings significantly enhanced adhesion, proliferation, and slow progression of cellular senescence as indicated by

lower SA-β-gal activities and decreased expression levels of genes including p16, p21, and p53. The upregulation of integrin α5 and αv genes influences phosphatidylinositol 4,5-bisphosphate

3-kinase (PI3K), and AKT proteins. FN and VN coatings upregulated AKT and MDM2 leading to p53 degradation. Additionally, MDM2 inhibition by Nutlin-3a markedly elevated p53 and p21

expression, increased cellular senescence, and induced the expression of inflammatory molecules including HMGB1 and IL-6. The understanding of FN and VN coating surface influencing ADSCs,

especially senescence characteristics, offers a promising and practical point for the cultivation of ADSCs for future use in cell-based therapies. SIMILAR CONTENT BEING VIEWED BY OTHERS

CHONDROGENIC DIFFERENTIATION INDUCED BY EXTRACELLULAR VESICLES BOUND TO A NANOFIBROUS SUBSTRATE Article Open access 19 November 2021 ATELOCOLLAGEN PROMOTES CHONDROGENIC DIFFERENTIATION OF

HUMAN ADIPOSE-DERIVED MESENCHYMAL STEM CELLS Article Open access 30 June 2020 EXTREMELY LOW FREQUENCY–ELECTROMAGNETIC FIELDS PROMOTE CHONDROGENIC DIFFERENTIATION OF ADIPOSE-DERIVED

MESENCHYMAL STEM CELLS THROUGH A CONVENTIONAL GENETIC PROGRAM Article Open access 03 May 2024 INTRODUCTION Senescence, a key process that causes aging, was first discovered by Hayflick and

colleagues, who reported that fibroblasts during in vitro display phenotypic changes and stop growing1. Based on their study, the Hayflick limit was established, i.e., every cell type has

its own limitation of growth2. The senescent characteristics during the aging process have been determined and include telomere shortening, the expression of cell-cycle inhibitor protein,

the upregulation of senescent-associated genes, and changes in signaling pathways involved in cell growth and differentiation3. The accumulation of senescent cells leads to tissue and organ

dysfunctions, which are associated with several degenerative diseases. Currently, treatment for degenerative diseases is challenging, but cell therapy has shown promising results in this

regard. Cell therapy is recognized as a treatment approach for tissue regeneration in which living cells are used to treat, prevent, or cure diseases or injuries. The type of cell used in

cell therapy depends on the condition being treated, e.g., diseases of the immune, blood, and stem cells4. Mesenchymal stem/stromal cells (MSCs) have shown promising therapeutic outcomes in

the field of regenerative medicine5. Adipose-derived stem cells (ADSCs) are an alternative source of MSCs that can be isolated from fat tissue by lipoaspiration or fat biopsy. They showed

high potential in rapid proliferation and immunosuppressive capabilities, allowing modulation of immune responses and potentially minimizing immune rejection in cell therapy applications5,6.

For clinical use of ADSCs, cell processing is performed under restricted regulation according to good manufacturing process (GMP)7. Many factors within culture conditions can influence

cellular properties, including culture medium, temperature, physical support, pH, and culture vessels. These factors should be considered and validated for continuing of reproducibility and

traceability. In vitro cell expansion influences the properties of ADSCs demonstrating by replicative senescent of ADSCs during culture prolongation. The extensive growth of ADSCs to reach

the therapeutic dose slowed the progression of cell growth and prolonged the population doubling time. The morphological alteration in abnormalities of the nuclear morphology8 and cell size

is indicated by the large, flattened cytoplasm with granules9. In addition, aging ADSCs increased the expression of senescence-associated β-galactosidase (SA-β-gal) and secreted

proinflammatory factors known as senescence-associated secretory phenotype (SASP) that influence the surrounding microenvironment through cell or tissue aging9. The unsatisfactory outcome of

the clinical study was raised as a major concern, and the quality of ADSCs was shown to impact the treatment outcome, leading to unsuccessful results10. However, replicative senescence can

be prevented by optimizing culture conditions, genetic modification, antioxidant supplementation, and senescence inhibitors11,12,13,14. These methods can be used individually or in

combination, depending on the specific cell culture system and desired outcomes. Growing cells in culture vessels coated with adhesion molecules aimed to increase cell adhesion15,16.

Typically, adhesion molecules, such as collagen, fibronectin (FN), laminin, and vitronectin (VN), are used as extracellular matrix (ECM) coatings17,18,19, but these are expensive and mostly

obtained from animal sources. FN and VN are found in fresh human plasma and can be easily purified20,21. FN can attach to cells through specific binding sites and integrin receptors on the

cell surface via the cell-binding domain, particularly prominent on α5β1 integrin through the arginine-glycine-aspartic acid (RGD) sequence in tenth fibronectin type III22. Besides, VN binds

to cells through its interaction with integrin receptors on the cell surface. Specifically, it binds to αVβ3 and αVβ5 integrin through the RGD sequence and promotes cell adhesion,

spreading, and migration23. A recent study demonstrated that FN and VN play crucial roles in regulating cell cycle progression and apoptosis. FN induces cell cycle arrest by upregulating p21

and can modulate cell survival or apoptosis depending on cellular context. FN signaling through integrins activating prosurvival pathways like PI3K-AKT to inhibit apoptosis24,25. Similarly,

VN has dual roles in cell survival and apoptosis regulation by influencing cell cycle progression and apoptosis through the PI3K-AKT pathway activated by integrin receptors. It has also

been associated with apoptosis-inducing processes like Anoikis, which is triggered by loss of cell adhesion26,27. Additionally, FN and VN have the potential to prevent cellular senescence by

modulating the PI3K/AKT signaling pathway24,25,26,27. Interestingly, coating with these human-derived proteins can improve the cell expansion process, enhance cell survival, and boost cell

growth performance. However, there is a lack of clarity regarding its role in cellular senescence. Currently, the process of cell processing especially ADSCs needs cell expansion steps to

provide a vast number of therapeutic cells. The standard practice for this process is under development and shows variability. To meet the good quality of ADSCs, the optimized cell culture

surface modification using adhesion molecules, including FN and VN might be a suitable process to reduce the senescent cells. In this study, we investigated the senescent characteristics of

ADSCs during long-term expansion. To explore the impact of these adhesion molecules on cellular senescence, the aging-related cellular signaling pathway was investigated. The use of adhesion

molecules is expected to promote cell adhesion, improve proliferation, and reduce replicative senescence, thereby enhancing the overall MSCs culture process. The finding of this study will

support the role of adhesion molecules in therapeutic cell growth, with the encouragement of a good protocol for cell culture in a clinical setting. RESULTS ADSCS CULTURED WITH FN AND VN

COATING ENHANCED ADHESION AND PROLIFERATION To determine the cell properties of ADSCs culture in FN and VN coating, adhesion rate and PDT was performed. The various concentrations of FN and

VN were investigated to determine the appropriate concentration for this study (Supplementary Table 1). The coated surface with the lowest concentration of FN and VN which promoted adhesion

and PDT was employed in this study. The adhesion rates of ADSCs cultured in FN and VN were investigated after cell seeding for 12 h. The number of adherence cells was counted and presented

as the percentage of the seeding cell number. Our results showed that the adhesion rates of cells in wells coated with FN (91.46% ± 3.60%) and VN (93.72% ± 3.60%) were significantly higher

than the control group (CTRL) (83.22% ± 3.62) (_p_ < 0.05) (Fig. 1A). The time interval spent in response to cell-cycle activity of ADSCs was determined using PDT assay. The PDT values of

ADSCs growth in FN- and VN-coated at P5, P7, and P10 are presented in Fig. 1B which was significantly shorter than the control group in the indicated passages. ADSCS CULTURED WITH FN AND VN

COATING SLOWS THE PROGRESSION OF CELLULAR SENESCENCE ADSCs cultured with the conventional method (control) and adhesion molecules at P5, P7, and P10 were determined for the senescence cells

using SA-β-gal staining (Fig. 2A). The number of SA-β-gal–stained cells was counted and presented in percentage. ADSCs in the control showed increased SA-β-gal positive cells at P5, P7, and

P10 in a passage-dependent manner. ADSCs cultured with FN coating at P5, P7, and P10 exhibited 12.78 ± 2.66, 18.33 ± 1.41, and 20.24 ± 2.82 numbers of SA-β-gal–stained cells, respectively.

Similarly, ADSCs cultured with VN coating at P5, P7, and P10 showed 14.05 ± 1.48, 19.89 ± 3.18, and 24.92 ± 5.11 numbers of SA-β-gal positive cells, respectively (Fig. 2B). Furthermore, the

hallmarks of cellular senescence, including p16 and p53 that play essential functions in senescence-associated cell-cycle arrest, were evaluated. The expression of _p16_ in ADSCs (P5, P7,

and P10) cultured with FN and VN coatings was lower than that in the control group. Besides, the expressional levels of _p21_ and _p53_ in ADSCs cultured with adhesion molecules coating were

significantly decreased in the indicated passages compared to the control group (Fig. 2C). ALTERATION OF AKT/MDM2/P53 IN ADSCS CULTURED WITH FN AND VN MEDIATED BY INTEGRIN Α5 AND ΑV The

expressions of AKT, MDM2, and P53 were investigated in ADSCs at P5, P7, and P10 to determine AKT/MDM2/p53 pathways involved in cellular senescence during in vitro expansion of ADSCs (Fig. 

3A–C). ADSCs cultured with FN and VN coatings resulted in an increase in AKT protein, particularly in P10 (Fig. 3C). In addition, both FN and VN coatings were found to reduce p53 expression,

which is a tumor-suppressor protein involved in cell-cycle arrest, senescence, and apoptosis. The expression of p53 was significantly decreased in ADSCs cultured with FN and VN at P10 (Fig.

 3C). Moreover, MDM2 expression was also observed in ADSCs at P10 which higher in ADSCs cultured with FN and VN coatings than in the control (Fig. 3C). Besides, the expressions of integrin

α5 and αv mediated AKT activation which enhancing p53 degradation. Herein, we investigated alterations in the gene expression of integrin subunits α5 (_ITGA5_) and β1 (_ITGB1_) in ADSCs

cultured with FN. The gene expression of integrin subunits αv (_ITGAV_), β3 (_ITGB3_), and β5 (_ITGB5_) was also observed in ADSCs cultured with VN. As shown in Figs. 3D and E, the

expression of _ITGA5_ and _ITGB1_ in ADSCs cultured with FN coating showed a significant increase compared to the control group. Moreover, the expressions of _ITGAV_, _ITGB3_, and _ITGB5_

(Fig. 3F–H) were upregulated in ADSCs cultured with VN coating. Furthermore, gene expression of _ITGAV, ITGB3_, and _ITGB5_ in ADSCs cultured with FN coating and _ITGA5_ and _ITGB1_ in ADSCs

cultured with VN coating were examined and were not different from the control (Supplementary Fig. S1). THE ALTERATION OF SENESCENT-ASSOCIATED PROTEIN IN ADSCS THROUGH AKT/MDM2/P53 PATHWAY

To investigate the regulation of cell senescence through AKT/MDM2/p53, ADSCs cultured without and with FN and VN coatings were treated with Nutlin-3a which is an MDM2 inhibitor, that

significantly suppresses p53 function by disrupting the p53–MDM2 interaction (Fig. 4A), Nutlin-3a was added to ADSCs culture at P10 to determine whether AKT regulated p53 expression through

MDM2. The expression of AKT in ADSCs from control, FN, and VN coating was similar with each ADSCs treated with Nutlin-3a. (Fig. 4B,C). The MDM2 expression was higher in ADSCs (CTRL, FN, VN)

treated with Nutlin-3a, which showed significant differences with non-Nutlin-3a-treated across all groups (Fig. 4B,D). The expression of MDM2 between the CTRL, FN, and VN (without Nutlin-3a)

in P10 revealed significantly increased MDM2 expression in FN and VN coating (Supplementary Fig. S2). According to the regulatory role of MDM2 to p53, all groups treated with Nutlin-3a

showed higher p53 expression (Fig. 4B,E). The expression of CDK inhibitor, p21, which is downstream of p53 and involved in cellular senescence, was significantly increased in ADSCs treated

with MDM2 inhibitor (Fig. 4B,F). Taken together, ADSCs senescent occurring was controlled by MDM2-p53 interaction which subsequently affect p21 expression. THE SENESCENT PHENOTYPE IN ADSCS

INFLUENCED BY AKT/MDM2/P53 PATHWAY To demonstrate whether MDM2-p53 influences the senescent characteristic, ADSCs in the control group and cultured with FN and VN coatings were treated with

Nutlin-3a. SA-β-gal staining and immunofluorescence analysis were applied. The SA-β-gal staining of ADSCs in control and FN, VN coatings revealed lower number of SA-β-gal positive cells than

Nutlin-3a treated cells (Fig. 5A). The percentage of SA-β-gal was significantly higher in ADSCs treated with Nutlin-3a across all groups (Fig. 5B). Remarkably, High mobility group box 1

(HMGB1) and interleukin-6 (IL-6) are implicated in senescence by promoting inflammation and SASP. Consequently, the study of HMGB1 and IL-6 expression using immunofluorescence was performed.

Immunostaining revealed that cells treated with Nutlin-3a showed HMGB1 localized in the cytoplasm compared with the non-Nutlin-3a-treated cells in which HMGB1 localized in the nucleus (Fig.

 5C). Furthermore, Nutlin-3a-treated cells also showed increased expression of IL-6 (Fig. 5C). Simultaneously, ADSCs cultured with FN, and VN coatings showed a reduction in the expression of

HMGB1 and secretion of IL-6 into a cytoplasm compared with the control. Moreover, the mRNA expression levels of SASP, _IL-1β, IL-6_, and _IL-10,_ were determined (Supplementary Fig. S3).

The results indicated a slight decrease of _IL-1β, IL-6,_ and _IL-10_ in ADSCs cultured with FN and VN coatings. Taken together, the senescent phenotype in ADSCs was regulated through

AKT/MDM2/p53 pathway, in which ADSCs cultured with FN, VN exhibited the slow progression in this event. DISCUSSION Cellular aging is the progressive deterioration and functional decline of

cells. This complex process is observed in vivo and _in vitro_28,29. Cell culture is the main process for cell processing performed using cells from autologous or allogeneic sources in cell

therapy. Here, the challenge remains in the standardization of cell processing and ensuring the stability and yield of cell products in achieving optimal therapeutic doses30. In cell-based

therapy, we found a certain number of clinical studies using ADSCs revealed a different clinical outcome, this variability might be from cellular components. Several factors can affect the

cell's properties including essential nutrients, physio-chemical environment, substrate and surface properties, and cell density and confluence31. The quality of cell products is also a

concern as the purity, potency, and proneness to senescence of MSCs used in cell therapy are critical32. Cellular senescence impairs replicative capacity and proliferation, affecting cell

production. Senescent MSCs display phenotypic changes and release SASPs, including proinflammatory cytokines33. SASP can lead to chronic inflammation and affect neighboring cell behavior,

proliferation, migration, and tissue regeneration capacity34. Therefore, the cell expansion process for cell therapy requires a good protocol to meet the high quality and quantity of MSCs.

FN and VN are extracellular matrix proteins widely used in cell culture aimed at promoting cell–matrix interaction. Cells with their specific integrin receptors can interact with FN and VN

via the RGD sequence, which enhances cell attachment and promotes cell growth22,23. Additionally, other ECM proteins, like laminin and collagen also contribute significantly to cell adhesion

and proliferation, showing similar effects on cell behavior when used as surface coatings. Notably, a previous study demonstrated growing bladder cancer cells in collagen cultured induced

premature senescence via integrin β1/AKT35. Recent investigations showed the benefits of FN and VN in cell adhesion, resulting in a good option for use in clinical applications36,37,38.

According to a recent study, by regulating the PI3K/AKT signaling pathway, FN and VN have the ability to modulate cell-cycle progression and prevent cellular senescence and

apoptosis24,25,26,27. Moreover, binding of cells with FN and VN also influences the biological properties of cells through signaling pathways, such as activation of downstream signaling

FAK–Src–PI3K39,40,41, AKT/mTOR pathway activation40,41, and MAPK/ERK pathway activation42,43. However, the roles of FN and VN in cell senescence have not been fully explored. Therefore, this

study aimed to investigate the roles of FN and VN coatings in the senescence of ADSCs culture. The adhesion rate of ADSCs cultured with FN and VN coatings showed a significant increase.

These coatings also contribute to an increased cell proliferation rate, as evidenced by the shorter PDT, which is frequently employed as a measure of proliferation capacity44. Shorter PDT

represents the cell cycle for a population of cells to double in number through cell division. Culturing ADSCs with FN and VN coatings reduces cellular senescence, as evidenced by lower

percentages of SA-β-gal positive cells at each passage compared to the control. Besides, the lower expression of senescence-associated markers, including _p16_ and _p53-p21_, was reduced in

ADSCs cultured with FN and VN coatings. The lower expression of these genes is associated with the attenuation of cellular senescence. Studies have also reported that the inactivation of

_p16_45,46 and _p53-p21_46,47 signaling pathways can effectively inhibit cellular senescence. The AKT/MDM2/p53 pathway is involved in cellular senescence and regulates the levels and

activity of the p53 protein. AKT phosphorylates MDM2, leading to p53 degradation under normal conditions48,49. However, cellular stress inhibits AKT activity, resulting in reduced

MDM2-mediated degradation of p53. Therefore, p53 can accumulate and induce senescence-associated cell-cycle arrest through p21. FN and VN coatings in ADSCs culture upregulated AKT expression

and potentially enhanced cell survival and therapeutic efficacy. These coatings also influenced MDM2 regulation and reduced p53 expression, suggesting an impact on senescence in ADSCs.

Overall, these results suggest that FN and VN coatings modulate AKT, MDM2, and p53 expressions and potentially regulate cellular senescence. In addition, FN-coated ADSCs upregulated α5 and

β1 integrin genes, while VN-coated ADSCs upregulated αv, β3, and β5 integrin genes. This finding suggests that these integrins may play a role in cellular processes by activating AKT

pathway. The increased expression of these integrin subunits may have implications for cell adhesion, migration, and survival. Nutlin-3a, an MDM2 inhibitor50, was used to investigate the

relationship between AKT, MDM2, and p53 in regulating cellular processes in ADSCs. Nutlin-3a functions by competitively inhibiting the binding of MDM2 to p53, preventing the ubiquitination

and degradation of p53 which is leading to significantly increased p53 expression50. Nutlin-3a has been shown to induce cellular senescence through the MDM2-p53 pathway in various cell

types, making it a valuable experimental model for studying the mechanisms underlying cellular senescence51,52. The results showed that the expression of AKT was reduced in ADSCs culture at

passage 10 and increased in ADSC cultured with either FN or VN. Furthermore, ADSCs cultured with FN or VN also express integrin receptors such as α5 and αV, which can influence the

activation of AKT22,23. Increased AKT activation may significantly enhance MDM2 activity, finally promoting the degradation of P53. Interestingly, ADSCs cultured with FN and VN coatings

promoted AKT and MDM2 expressions while reducing p53 and p21 expressions. This observation suggests that the coatings may modulate these factors and impact cellular processes related to

senescence and survival. In addition, Nutlin-3a treatment increased the percentage of SA-β-gal positive cells, lead to cytoplasmic localization of HMGB1, and increased the expression of IL-6

in ADSCs culture. In contrast, ADSCs cultured with FN and VN coatings showed a lower percentage of SA-β-gal positive cells, reduced expression of HMGB1, and secretion of IL-6. These

findings indicate that FN and VN coatings impact the cellular senescence markers and the expression of inflammatory molecules, such as HMGB1 and IL-6. Furthermore, various promising

strategies rely on the prevention of proinflammatory-induced senescent cells. Recent studies are focused on the discovery of pharmacological agents that induce apoptosis in senescent cells.

These substances are commonly referred to senolytic drugs or senolytics53. However, specific methodologies or treatment approaches for the cell culture process in this context have not been

established. This study demonstrated a promising procedure to cultivate ADSCs using FN and VN coatings. ADSCs cultured in these coatings exhibited improved cell adhesion, proliferation, and

modulated senescence-associated markers. These results also suggest the potential role of FN and VN coatings in regulating cell senescence via AKT/MDM2/p53 pathway mediated by integrin α5

and αv. Our results showed culturing ADSCs with FN and VN coating is an easy and practical strategy that should be recommended as a good protocol for the preparation of ADSCs for therapeutic

use. The impact of cellular senescence on the clinical outcomes of cell-based therapy can be significant. Therefore, further studies to investigate the impact of senescent cells on

therapeutic outcomes should be performed. In addition, meta-analysis serves as a valuable tool in addressing this challenge and bridging the gap between laboratory studies and clinical

applications. METHODS ISOLATION AND CULTURE OF ADSCS The lipoaspiration technique was used to obtain adipose tissue samples from healthy subjects after their written informed consent. The

samples were washed with warm phosphate-buffered saline (PBS) to eliminate blood and oil. Next, the adipose tissue was digested using 0.025% type I collagenase (Worthington Biochemical

Corporation, Lakewood, NJ, USA) for 1 h at 37 °C. The lipoaspirate was centrifuged at 2000 _rpm_ for 5 min to separate the pellet containing ADSCs, and the pellet was resuspended in stromal

medium (DMEM-LG; Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA), 1% Penicillin–Streptomycin (Gibco, Carlsbad, CA, USA), and 1%

Glutamax (Gibco, Carlsbad, CA, USA). The cells were seeded at a density of 103 cells/cm2 and then incubated at 37 °C and 5% CO2. After 48 h, the nonadherent cells were removed, and the

remaining cells were maintained until they reached 80%–90% confluence, while the medium was changed every three days. ADSCs were harvested using 0.25% trypsin–EDTA (Gibco, Carlsbad, CA,

USA), and the cultures were either expanded or cryopreserved for future use. SURFACE MODIFICATION AND CELL CULTURE To determine the effects of adhesion molecule as coating materials for

ADSCs culture in vitro, the experiments were divided into three groups, ADSCs were cultured on uncoated surface as the control group (CTRL), coated surface with 5 µg/cm2 FN (Merck,

Darmstadt, Germany) and 0.5 µg/cm2 VN (Advanced BioMatrix, Carlsbad, CA, USA). In the FN-coated group, 1 mL FN was added to a 35 mm dish, followed by the removal of excess solution. Air

dried for at least 45 min at room temperature (RT) was performed. In the VN-coated group, 1 ml VN was added to 35 mm-dish, incubated for 1 h at RT, and then washed with PBS twice. To

investigate the cellular senescence, 1 × 105 ADSCs at the 2nd passage (P2) were seeded on uncoated (CTRL) and coated surface. ADSCs were cultured at 37 °C in 5% CO2 until the cells reached

the 5th passage (P5), 7th passage (P7), and 10th passage (P10). The biological activities of ADSCs under each condition were evaluated at P5, P7, and P10. The MDM2 inhibitor, Nutlin-3a

(Sigma-Aldrich, Taufkirchen, Germany), was dissolved in DMSO (PanReac Applichem, Darmstadt, Spain). Then 10 µM Nutlin-3a was added to the ADSCs culture at P10. This was followed by

incubation for 48 h before concluding the experiment. CELL ADHESION ASSAY 1 × 105 cells were seeded in a 24-well plate coated with FN and VN to evaluate the adhesion properties of ADSCs.

Here, ADSCs cultured on an uncoated surface were used as control. The seeded cells were incubated for 12 h in a humidified atmosphere containing 5% CO2 at 37 °C, and then the nonadherent

cells were carefully removed and discarded. The adherent cells were detached using 0.25% trypsin–EDTA (Sigma-Aldrich, St. Louis, MO, USA) and manually counted using a hemocytometer. The

percentage of cell adhesion was calculated using the following equation to assess cell adhesion rate: $${\text{Cell adhesion rate }}\left( \% \right) \, = \, \left( {{\text{Number of

adherent cells}}/{\text{Number of seeding cells}}} \right) \times {1}00.$$ PROLIFERATIVE ACTIVITY ADSCs were seeded on control, FN, and VN-coated dishes at a density of 1 × 105 cells to

investigate the impact of adhesion molecule-coated surfaces on cell growth. Upon ADSCs cultured reaching 90% confluence, the cells were detached by trypsinization, and their viability was

assessed using trypan blue staining. The proliferative activity of ADSCs was assessed by calculating the population doubling time (PDT) using the following expression: PDT = CT/PDN, where CT

denotes the culture time in hours. PDN denotes the number of population doublings and is calculated using the formula: PDN = [logNH − logNI]/log2, where NI and NH represent the initial cell

seeding number and the number of harvested cells, respectively. The PDT of ADSCs cultured on an uncoated surface was used as the control group for comparison. SENESCENCE-ASSOCIATED

Β-GALACTOSIDASE (SA-Β-GAL) STAINING SA-β-gal activity was assessed using a senescence cell staining kit (Cell Signaling Technology, Danvers, MA, USA) to investigate the impact of coated

surfaces of adhesion molecules on cellular senescence. Specifically, 3 × 104 ADSCs at P5, P7, and P10 were seeded in 35-mm culture dishes with coated surfaces using FN and VN. Then, the

cells were cultured in a humidified atmosphere containing 5% CO2 at 37 °C until the cultures reached 60% confluence. The cells were washed with PBS and then fixed for 15 min at RT before

being stained with SA-β-gal staining solution in a dry incubator at 37 °C for 16–18 h. The formation of a blue color, which represented senescent-positive cells, was observed using an

inverted microscope (Olympus, Shinjuku, Tokyo, Japan). The number of SA-β-gal-positive cells was counted and presented as the percentage of SA-β-gal activity using the following formula:

SA-β-gal activity (%) = (Number of SA-β-gal-positive cells/Number of total cells) × 100. RNA EXTRACTION AND QUANTITATIVE REAL-TIME PCR RNA was extracted using the phenol–chloroform procedure

from the P5, P7, and P10 of ADSCs grown under individual conditions in TRIzol reagent (Invitrogen, Waltham, MA, USA). The RNA concentration was measured using a nanodrop spectrometer

(Thermo Scientific, Waltham, MA, USA). cDNA was synthesized from 1 µg of RNA using iScript reverse transcription (Bio-Rad Laboratories, CA, USA), and the mRNA levels of _p16, p21, p53,

ITGA5, ITGAV, ITGB1, ITGB3_ and _ITGB5_ were examined using quantitative real-time PCR. Each cDNA sample was mixed with a PCR master mix containing target gene forward and reverse primers

(Table 1), nuclease-free water, and SYBR FAST qPCR master mix (KAPA Biosystem, Wilmington, MA, USA). Glyceraldehyde-phosphate dehydrogenase (_GAPDH_) was used as an internal reference

control to normalize the expression levels of the genes of interest. Finally, the difference in transcript levels of senescence-associated genes was calculated using the comparative CT

method (2−∆∆Ct) and presented as a relative gene expression to the control group. WESTERN BLOTTING ANALYSIS For immunoblotting, the protein from ADSCs culture in different coated surfaces

was dissolved in lysis buffer (Merck, Darmstadt, Germany) with protease inhibitors (Merck, Darmstadt, Germany), and the concentration was determined by Bicinchoninic acid (BCA) Protein Assay

Kit (Thermo Scientific, Waltham, MA, USA). The 20 µg of protein samples were separated on a 10% polyacrylamide gel and transferred to an Immobilon-P transfer membrane (Merck Millipore,

Burlington, MA, USA) using the Mini-Protean© system (Bio-Rad Laboratories, Feldkirchen, Germany). The membranes were blocked in 5% skimmed milk (Merck, Darmstadt, Germany) for 2 h and

incubated with the primary antibodies at 4 °C overnight. The primary antibodies used were AKT (#4691S), MDM2 (#86934S), p21 (#2947S), p53 (#2524S) (Cell Signaling Technology, Danvers, MA,

USA), and β-Actin (Merck, Darmstadt, Germany) as loading controls for normalization. In this process, the membranes were cut before hybridization with various antibodies, resulting in the

absence of images with adequate length. On the following day, the membranes were incubated using an ECL Prime Western Blotting Detection Reagent (GE Healthcare, Buckinghamshire, UK) at RT

for 2 h with secondary antibodies including anti-mouse IgG-HRP (#7074P2) and anti-rabbit IgG-HRP (#7076S) (Cell Signaling Technology, Danvers, MA, USA). The signal was then detected and

analyzed via the ChemiDoc™ MP Imaging System (Bio-Rad Laboratories, Los Angeles, CA, USA). IMMUNOFLUORESCENT STUDY ADSCs samples were fixed in warm 4% paraformaldehyde for 15 min at RT and

then washed three times with PBS. The samples were then permeabilized with 0.025% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) for 10 min and blocked in 5% bovine serum albumin (BSA)

(Sigma-Aldrich, St. Louis, MO, USA) in PBS for 1 h at RT. The samples were incubated overnight at 4 °C with primary antibodies, i.e., rabbit anti-HMGB1 (#3935S, 1:200) and rabbit IL-6

(#12912S, 1:200) (Cell Signaling Technology, Danvers, MA, USA), diluted in 1% BSA in PBS. The samples were then incubated with secondary antibody solutions, i.e., Alexa Fluor 488 goat

antirabbit (AB150077, 1:250) (Abcam, Cambridge, UK), for 45 min at RT. Finally, the samples were counterstained with Prolong Gold™ Antifade Reagent with DAPI (Thermo Scientific, Waltham, MA,

USA) for 24 h at RT in the dark. The samples were photographed using a fluorescence microscope (Olympus BX51, Shinjuku, Tokyo, Japan). STATISTICAL ANALYSIS The data are presented as mean ± 

standard deviation (SD) of at least three individual experiments (the Supplementary Figs. S1, and S3 are presented from two individual experiments). Statistical analysis was performed using

an ordinary one-way ANOVA of GraphPad Prism 9.0 software (GraphPad Software, San Diego, CA, USA). A _p_-value < 0.05 was considered statistically significant. STATEMENT Human adipose

tissue samples from healthy subject was approved by the Mahidol University Central Institutional Review Board and was performed in accordance with the Declaration of Helsinki (MU-CIRB

2018/202.1441) with the title “Study of Mesenchymal Stem Cells Senescence Derived Adipose Tissue”. There are no clinical trials or animal experiments in our research. All experiments were

performed in accordance with relevant guidelines and regulations. DATA AVAILABILITY The datasets generated during and/or analysed during the current study are available from the

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Invest._ 128, 1247–1254 (2018). Article  PubMed  PubMed Central  Google Scholar  Download references FUNDING This research was supported by Specific League Funds from Mahidol University.

AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Clinical Microscopy, Faculty of Medical Technology, Mahidol University, 999 Phutthamonthon Sai 4, Salaya, Phutthamonthon, Nakhon

Pathom, 73170, Thailand Patcharapa Tragoonlugkana & Aungkura Supokawej * Department of Surgery, Phramongkutklao Hospital and Phramongkutklao College of Medicine, Bangkok, 10400, Thailand

Chatchai Pruksapong * Department of Community Medical Technology, Faculty of Medical Technology, Mahidol University, Nakhon Pathom, 73170, Thailand Pawared Ontong * Department of Clinical

Microbiology and Applied Technology, Faculty of Medical Technology, Mahidol University, Nakhon Pathom, 73170, Thailand Witchayapon Kamprom Authors * Patcharapa Tragoonlugkana View author

publications You can also search for this author inPubMed Google Scholar * Chatchai Pruksapong View author publications You can also search for this author inPubMed Google Scholar * Pawared

Ontong View author publications You can also search for this author inPubMed Google Scholar * Witchayapon Kamprom View author publications You can also search for this author inPubMed Google

Scholar * Aungkura Supokawej View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS Conceptualization: AS; Methodology: PT, CP, PO, AS;

Investigation: PT, CP; Formal analysis PT, WK, AS; Visualization: PT, AS Writing – original draft preparation: PT, AS; Writing – review and editing: PT, PO, WK, AS; Supervision: AS.

CORRESPONDING AUTHOR Correspondence to Aungkura Supokawej. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION PUBLISHER'S NOTE

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Tragoonlugkana, P., Pruksapong, C., Ontong, P. _et al._ Fibronectin and vitronectin alleviate adipose-derived stem cells senescence during long-term culture through the AKT/MDM2/P53 pathway.

_Sci Rep_ 14, 14242 (2024). https://doi.org/10.1038/s41598-024-65339-z Download citation * Received: 19 February 2024 * Accepted: 19 June 2024 * Published: 20 June 2024 * DOI:

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currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing initiative KEYWORDS * Adipose-derived stem cells * Cell therapy * Good

manufacturing practice * Cell culture * Replicative senescence * Fibronectin * Vitronectin