Synergistic Effect of miR-200 and Young Extracellular Matrix-based Bio-scaffolds to Reduce Signs of Aging in Senescent Fibroblasts

Aging is defined as a complex, multifaceted degenerative process that causes a gradual decline of physiological functions and a rising mortality risk with time. Stopping senescence or even rejuvenating the body represent one of the long-standing human dreams. Somatic cell nuclear transfer as well as cell reprogramming have suggested the possibility to slow or even reverse signs of aging. We exploited miR-200 family ability to induce a transient high plasticity state in human skin fibroblasts isolated from old individuals and we investigated whether this ameliorates cellular and physiological hallmarks of senescence. In addition, based on the assumption that extracellular matrix (ECM) provides biomechanical stimuli directly influencing cell behavior, we examine whether ECM-based bio-scaffolds, obtained from decellularized ovaries of young swine, stably maintain the rejuvenated phenotype acquired by cells after miR-200 exposure. The results show the existence of multiple factors that cooperate to control a unique program, driving the cell clock. In particular, miR-200 family directly regulates the molecular mechanisms erasing cell senescence. However, this effect is transient, reversible, and quickly lost. On the other hand, the use of an adequate young microenvironment stabilizes the miR-200-mediated rejuvenating effects, suggesting that synergistic interactions occur among molecular effectors and ECM-derived biomechanical stimuli. The model here described is a useful tool to better characterize these complex regulations and to finely dissect the multiple and concurring biochemical and biomechanical cues driving the cell biological clock. Graphical Abstract


Introduction
Advances in medical care, improvements in sanitation, and rising living standards contribute to increase life expectancy. Although this reflects positive human development, it also poses new important challenges that will aggravate in the years to come. Among them, aging is one of the most critical and emerging problems in public health, due to the exponential increase of elderly patients suffering with chronic ageonset diseases, often with multiple co-morbidities. Aging is indeed defined as a complex, multifaceted process characterized by a progressive accumulation of macroscopic and microscopic modifications that are accompanied by molecular and cellular damages, negatively affecting organ, tissue, cell, and subcellular organelle functions [1,2]. These senescence processes are influenced by environmental factors as well as by genetic and epigenetic dysregulations that induce a gradual organism decline [3]. In agreement with this, several studies have described a link between DNA methylation and aging [4][5][6][7], demonstrating that around 29% of the methylation sites in the genome is modified with increasing age, thus reflecting the natural development of age-related phenotypes and diseases, such as diabetes, autoimmune and cardiovascular disorders [8].
In the present work, we exploit miR-200 ability to induce a transient high plasticity state in cells isolated from old individuals. We investigate whether this is able to ameliorate cellular and physiological hallmarks of aging in senescent cells. We examine whether extracellular matrix (ECM)based scaffolds are able to boost and properly maintain the rejuvenated phenotype acquired by cells after miR-200 exposure. Specifically, we take advantage of acellular scaffold low immunogenicity, repopulating porcine decellularized scaffolds with cellular elements of human origin. In addition, we select the ovary based on its complex microanatomy and heterogenous architecture that may provide different mechanotransducive cues, possibly influencing cell proliferation and metabolism and translating physical forces into the activation of local intracellular pathways.

Materials and Methods
All reagents were purchased from Thermo Fisher Scientific unless otherwise indicated.

Ethics Statement
Human cells were purchased from the NIGMS Human Genetic Cell Repository at the Coriell Institute for Medical Research (USA). Ethical approval was not required for this study because it did not involve living animals. All the methods in our study were carried out in accordance with the approved guidelines.

Culture of Human Fibroblasts Obtained From Young and Aged Individuals
Skin fibroblast cell lines obtained from 29 to 32 years old donors (Young, n = 3) and from 82 to 92 years old individuals (Aged, n = 3) were purchased from the NIGMS Human Genetic Cell Repository at the Coriell Institute for Medical Research (USA). Cells were grown in fibroblast culture medium (FCM) consisting of Eagle's Minimum Essential Medium (MEM) supplemented with 15% Foetal Bovine Serum (FBS), 2mM glutamine (Sigma-Aldrich), 1% antibiotic/antimycotic solution (Sigma-Aldrich), and maintained in 5% CO 2 at 37 °C. All experiments were carried out in triplicates at least three times. 9 h and incubated with 2% deoxycholate (Sigma-Aldrich) for 12 h. At the end of the decellularization protocol, the ovarian bio-scaffold generated were extensively washed in DI-H 2 O for 6 h and sterilized with 70% ethanol and 2% antibiotic/antimycotic solution (Sigma-Aldrich) for 30 min. All the steps described were performed at room temperature using an orbital shaker at 300 rpm. A fragment from each bio-scaffold was collected and analysed for evaluating the efficacy of the decellularization process (data not shown).

Seeding and Culture of miR-200b/c Treated Fibroblasts Onto Young and Aged ECM-based Bio-scaffolds
miR-200b/c treated fibroblasts were seeded onto Young and Aged ECM-based bio-scaffolds (3D). Specifically, 15 Young and 15 Aged 7 mm x 1 mm fragments were cut from ovarian cortex and repopulated with 6.9 × 10 6 cells for each, in 300 µL of FCM and maintained in a 5% CO 2 incubator at 37 °C. Half medium volume was changed every other day. Cultures were arrested at day 2, 5, 7, and 10, and analysed as described below. 15 cortex fragments obtained from Young ovarian bio-scaffolds were repopulated with untreated Aged fibroblasts and used as controls.

Cell Growth Curve
Growth curve was assessed by plating 2.5 × 10 4 cells/cm 2 in 4-well multidishes (Nunc). Cell number was counted using Hycor KOVA TM Glasstic TM (Sentinel Diagnostic) and cell viability was determined by trypan blue dye exclusion assay (Sigma-Aldrich). Each time point was assessed in triplicate.

Cell Proliferation Index
Cell proliferation index was evaluated by proliferating cell nuclear antigen (PCNA) immuno-staining. Cells were fixed in methanol at -20 °C for 15 min, while paraffin-embedded tissues were treated with 10mM Sodium citrate solution (pH 6) containing 0,05% Tween-20 (Sigma-Aldrich) to unmask antigens. Samples were incubated in blocking solution containing 10% Goat Serum (Sigma-Aldrich) in PBS for 30 min. Primary antibody (1:200, Sigma-Aldrich) was incubated for 1 h, followed by a suitable secondary antibody exposure (1:250, Alexa Fluor™) for 1 h. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich). Samples were observed under the Eclipse TE200 microscope (Nikon). All steps were performed at room temperature, unless otherwise indicated. The number of immuno-positive cells was counted in 10 randomly selected fields at 100× total magnification. A minimum of 500 cells were scored in three independent replicates. The number of PCNA positive cells was expressed as a percentage of the total cell counted.

Reactive Oxygen Species (ROS) and β-galactosidase (β-GAL) Activity
ROS and β-GAL activities were analysed using human reactive oxygen species (MyBioSource, MBS166870) and Galactosidase beta ELISA kits (MyBioSource, MBS721441). Samples were sonicated at 20 kHz in ice for 20 min, homogenates were centrifuged at 3000 rpm for 20 min and supernatants were collected. Assays were carried out following the manufacturer's instructions and, at the end of the procedures, total ROS and β-GAL contents were quantified at 450 nm using a Multiskan FC. Standard curves were designed by plotting the absorbance means (y-axis) against the relative concentrations (x-axis) and the best fit line was determined by regression analyses. Absolute quantifications were then calculated.

Gene Expression Analysis
Quantitative PCR was performed using CFX96 Real-Time PCR detection system (Bio-Rad Laboratories). Total RNA was extracted using the TaqManGene Expression Cells to Ct kit (Applied Biosystems) and DNase I was added in lysis solutions at 1:100 concentration, as indicated by the manufacturer's instructions. Target genes were analysed using predesigned primers and probe sets from TaqManGene Expression Assays (Table 1). GAPDH and ACTB were used as reference genes. Gene expression levels were quantified with CFX Manager software (Bio-Rad Laboratories) and here reported with the highest expression set to 1 and the other relative to this.

DNA Quantification
Genomic DNA was extracted from fragments, ranging from 10 to 24 mg, with the PureLink® Genomic DNA Kit (Invitrogen), following the provider's instructions. DNA concentrations were measured with NanoDrop 8000 and normalized against the previously annotated fragment weights.

Cell Density
Cell number was counted in 15 tissue sections obtained from 3 Young (5 sections for each) and 3 Aged (5 sections for each) ECM-based bio-scaffolds repopulated with miR-200b/c treated fibroblasts as well as from 3 Young bio-scaffolds recellularized with untreated Aged cells (5 sections for each). 5-µm-thick sections were stained with DAPI and observed under an Eclipse E600 microscope (Nikon) equipped with a digital camera (Nikon). Pictures were acquired with NIS-Elements Software (Version 4.6; Nikon), using constant exposure parameters. Five randomly selected fields at 100X magnification were examined for each section and analysed using the Automated Cell Counter tool (ImageJ software version 1.53j). Briefly, 8-bit images were created and segmented with a thresholding algorithm to eliminate the background and to highlight areas occupied by the nuclei. Collected data were transformed in binary form. Nuclei were automatically counted using previously set size and circularity parameters. Cell density is expressed per mm 2 of tissue.

Statistical Analysis
Statistical analysis was performed using two-way ANOVA (SPSS 19.1; IBM). Data are presented as the mean ± the standard deviation (SD). Differences of p ≤ 0.05 were considered significant and are indicated with different superscripts.

miR-200b/c Exposure Induces High Plasticity and Erase Signs of Senescence in Fibroblasts Isolated From Aged Individuals
After miR-200b/c exposure, fibroblasts isolated from aged patients showed considerable phenotype changes. More in detail, the typical elongated shape, visible in untreated fibroblasts (T0, Fig. 1a), was replaced by a stem-cell-like morphology with cells smaller in size, granular and vacuolated cytoplasm, and larger nuclei. In addition, miR-2020b/c treated fibroblasts rearranged in a reticular pattern and tended to form distinguishable aggregates (Post miR-200b/c, Fig. 1a). Morphological changes were also accompanied by a significant increment in cell proliferation (Post miR-200b/c, Fig. 1c). In agreement with this, Post miR-200b/c aged cells showed a growth curve comparable to that of untreated fibroblasts isolated from young individuals (Young, Fig. 1c). PCNA immunocytochemical analysis confirmed these data ( Fig. 1d) and, in particular, its quantitative evaluation demonstrated a significantly higher PCNA positive cell rate after miR-200b/c exposure (Post miR-200b/c, Fig. 1f). These changes were paralleled by a significant decrease in β-galactosidase (β-GAL) activity (Post miR-200b/c, Fig. 1g) and reactive oxygen species (ROS) levels (Post miR-200b/c, Fig. 1h), with values post-treatment comparable to those young cells (Young, Fig. 1g and h).
Gene expression analysis were consistent with the morphological observations and indicated the onset of the pluripotency-related genes OCT4, NANOG, REX1, and SOX2 (Post miR200b/c, Fig. 2), which were originally undetectable in untreated fibroblasts (Aged, Young; Fig. 2). Furthermore, Post miR-200b/c significantly increased their expression of the cell proliferation marker MKI67 (Fig. 4), and the  (Fig. 3), picking to values comparable to young cells (Young; Fig. 3). Transcription levels of the reactive oxygen species modulator ROMO1 and of the senescencerelated markers, P53, P16 and P21, significantly decreased to levels distinctly of young cells (Young; Fig. 4).

miR-200b/c Treated Fibroblasts Cultured in 2D Systems Revert to Their Original Phenotype
Fibroblasts returned to FCM, after removal of miR-200b/c, progressively reverted to the original phenotype (Day 2, day 5 and day 7; Fig. 1a) and, by day 10, exhibited small central nuclei and an elongated spindle shape, comparable to those of untreated aged fibroblasts (T0, Fig. 1a). The morphological changes were also paralleled by a decrement in cell proliferation (Fig. 1c). This was also supported by PCNA positive cell rate values that slowly decreased, returning statistically comparable to those of aged fibroblasts by day 10 of culture ( Fig. 1d and f). Consistently, β-GAL activity (Fig. 1g) and ROS levels (Fig. 1h) gradually incremented during the culture period, up to day 10, when the values detected were statistically comparable to those observed in untreated aged cells (Aged).

miR-200b/c Treated Fibroblasts Repopulate 3D ECM-based Bio-scaffolds, but Maintain a Rejuvenated Phenotype Only on Young ECM
H & E (Fig. 5a) and DAPI staining (Fig. 5b) (Fig. 5c). Nevertheless, significant differences among the experimental groups were detected. Specifically, starting from day 2 of co-culture and onward, cell number was significantly higher in young decellularized bio-scaffolds repopulated with miR-200b/c exposed fibroblasts (miR-200b/c + Young scaffold) compared to miR-200b/c + Aged scaffold and the control group (w/o miR-200b/c + Young scaffold, Fig. 5c). These observations were also confirmed by DNA quantification (Fig. 5d).

Discussion
In the present study, we provide evidence that the use of miR-200b/c induces a transient high plasticity state and ameliorates cellular and physiological hallmarks of aging in senescent cells. In addition, we demonstrate that the rejuvenated phenotype is stably maintained when exposure to miR-200b/c is coupled with engrafting onto young ECMbased bio-scaffolds.
More in detail, exposure to miR-200b/c induces significant changes in fibroblasts isolated from aged patients that acquire morphological and molecular features distinctive of pluripotent cells. Fibroblast standard elongated shape is replaced by a round or oval one. Cells become smaller in size with granular, vacuolated cytoplasm and rearrange in distinguishable aggregates. All these aspects closely resemble those previously described for human ESCs [34] and iPSCs [35], which show a typical round morphology and form compact colonies, with distinct borders and welldefined edges [34][35][36][37][38]. In addition, senescent cells exposed to miR-200b/c display larger nuclei with less cytoplasm, an aspect usually correlated to the relaxed and accessible  [39][40][41][42]. All these observations suggest that the use of miR-200b/c may encourage the appearance of morphological properties previously described in both native and induced high plasticity cells, which is also supported by the molecular analyses performed, demonstrating the onset of the pluripotency-related genes, OCT4, NANOG, REX1, and SOX2, in response to miR-200b/c exposure. Overall, these data confirm and further expand previous studies indicating that overexpression of miR-200 supports Nanog active transcription and ESC self-renewal, while inhibiting embryoid body formation and repressing the expression of ectoderm, endoderm, and mesoderm markers [43,44].
Interestingly, the morphological changes described above are paralleled by significant reduction of aging hallmarks. In particular, miR-200b/c exposure causes an increment in cellular growth curve values, in proliferation rates and PCNA immune-positive cell number. These results suggest cell-cycle re-activation and the reestablishment of a robust cell division in senescent cells challenged with miR-200b/c. Consistent with this, recent pilot works described the possibility to achieve a young phenotype after restoration of a vigorous cell growth in non-dividing quiescent and senescent cells [45,46], pointing to a scenario where proliferation is an essential requirement for cellular rejuvenation [47].
Reduction of senescence-associated markers after miR-200b/c exposure is also demonstrated by a significant decrease in β-GAL and ROS activities and is in line with a recent study, showing miR-200 treatment ability to induce a decrement in β-GAL levels as well as in P53 and P21 gene transcription [48]. Consistent with this, our molecular data indicate that miR-200b/c treated cells display a downregulation of P53 and P21 as well as P16 genes. In addition, physiological signs of aging are damped by reducing the oxidative stress (ROMO1), promoting cell proliferation (MKI67) and increasing mitochondrial activity (TFAM, PDHA1, and COX4I1). Altogether these observations confirm the key role played by the miR-200 family, not only to promote and maintain high plasticity [29,43,49], but also to reduce signs of cellular senescence and to encourage the acquisition of a young phenotype. It is however important to note that when miR-200b/c are removed from cultures and cells are returned to FCM, they gradually revert to their original phenotype, exhibiting all morphological and molecular signs distinctive of aged cells by day 10 of culture. Specifically, cells increase their size, appear longer Insert scale bars: 50 μm. c Cell density analyses before cell seeding in aged (Pre-seeding aged scaffold, vertical stripped bars) and young scaffolds (Pre-seeding young scaffold, horizontal stripped bars), in untreated aged fibroblasts plated onto young 3D ECM-based bioscaffolds (w/o miR-200b/c + Young scaffold, dotted black bars) and in miR-200b/c treated aged fibroblasts plated onto aged (miR-200b/c + Aged scaffold, grey bars) or onto young 3D ECM-based bioscaffolds (Young scaffold) at day 2, 5, 7, and 10 of culture. Data are expressed as the mean. Error bars represent the standard error of the mean (SEM). Different superscripts indicate p < 0.05. d DNA quantification before cell seeding in aged (Pre-seeding aged scaffold, vertical stripped bars) and young scaffolds (Pre-seeding young scaffold, horizontal stripped bars), in untreated aged fibroblasts plated onto young 3D ECM-based bio-scaffolds (w/o miR-200b/c + Young scaffold, dotted black bars) and in miR-200b/c treated aged fibroblasts plated onto aged (miR-200b/c + Aged scaffold, grey bars) or onto young 3D ECM-based bio-scaffolds (Young scaffold) at day 2, 5, 7, and 10 of culture. Data are expressed as the mean. Error bars represent the standard error of the mean (SEM). Different superscripts indicate p < 0.05 with elongated shape, and decrease both their proliferation indexes and PCNA positive rates. The reacquisition of a senescent phenotype is also confirmed by an increased β-GAL and ROS activities and by changes in the transcription levels of MKI67, TFAM, PDHA1, COX4I1, ROMO1, P53, P16, and P21 genes, that returned statistically comparable to those of untreated aged cells. In our understanding, these results demonstrate not only an evident cell rejuvenation induced by miR-200b/c treatment, but also that this effect is transient and reversible.
In contrast, the rejuvenated state appears to be stably retained when cells are engrafted onto young 3D ECMbased bio-scaffolds. Indeed, following miR-200b/c removal, cells adhere and colonize the bio-scaffolds stably maintaining all the hallmarks typical of young cells, namely high number of PCNA positive cells, low values of β-GAL and ROS activities, high transcription levels for the genes involved in cell proliferation (MKI67), mitochondrial activity (TFAM, PDHA1, and COX4I1) and low transcription for the genes associated to cell senescence, such as ROMO1,

Conclusion
In conclusion, the data presented in this manuscript show that multiple factors cooperate to control a unique program, driving the cell clock. In particular, molecular mechanisms regulated by miR200 are directly involved in erasing cellular senescence, however, an adequate microenvironment is required to stabilize the rejuvenating effects, suggesting the involvement of synergistic interactions among molecular effectors and bio-scaffold-derived mechanical cues. It is clear that ovarian ECM creates a unique microenvironment deriving from the combination of its complex microanatomy and heterogenous architecture that may provide different mechanotransducive cues, influencing cell proliferation and function as well as resulting in the activation of local intracellular pathways. Altogether, the model here developed represents, in our understanding, a useful tool to better characterize these complex regulations and to allow a fine dissection of the multiple and concurring biochemical and biomechanical stimuli driving the process.