Cellular Senescence and Senescence-Associated Secretory Phenotype Drive Multipotent Cardiac Stem/Progenitor Cell Dysfunction in Human Diabetic Cardiomyopathy Independently of Age

Background: Aging and Diabetes Mellitus (DM) independently and additively increase cardiovascular risk and a pathophysiological basis for this epidemiological link is currently the subject of intense investigations. Both aging and DM affect the biology and regenerative potential of tissue specic cardiac adult stem/progenitor cells (CSCs). In aged subjects over half of the CSCs are senescent with a senescence-associated secretory phenotype (SASP) that renders otherwise healthy CSCs to senescence, impairing their proliferative and differentiation potential. Although a link to organismal aging is clear, cells can undergo senescence, regardless of age. Yet, it is unclear whether the SASP is induced by Diabetes per se in CSCs and whether targeting senescent cells within the diabetic CSC compartment rescues their regenerative biology. Methods: In order to investigate the effects of DM on CSC senescence and to try separate those of aging from Diabetes on senescence events, we obtained peri-infarct/border zone biopsies from non-aged patients (50-64 years old) with DM type 2 (T2DM) and non-diabetic (NDM) patients with post-infarct cardiomyopathy undergoing surgical coronary revascularization. Results: Ischemic injury in DM is associated with a higher ROS production, as revealed by the exacerbated expression of 8-OH-deoxyguanosine, nitrotyrosine, and 4-hydroxinonenal targeting both cardiomyocytes as well as CSCs. The latter associated with an increased number of senescent and dysfunctional T2DM-hCSCs (isolated from atrial samples of T2DM and NDM patients with ischemic cardiomyopathy) identied by increased p16 INK4a positive cells, reduced telomerase activity and telomere length, reduced proliferation, clonogenesis/spherogenesis and myogenic differentiation when compared to NDM-hCSCs in vitro. Importantly, T2DM-hCSCs show a dened SASP, as demonstrated by the increased secretion of MMP-3, PAI1, IL-6, IL-8, IL-1β and Quantitative data are expressed as mean±SD. Binary data are reported by counts. *P<0.01 vs DM patients. Comparisons of the quantitative data have been made through use of Student's t test for independent samples. The χ2 test was used to compare binary data. T2DM indicates Type 2 Diabetes Mellitus; NDM non diabetes Mellitus; BMI body mass index; FBG, fasting blood glucose; LDL, low‐density lipoprotein; HDL, high‐density lipoprotein; TG, triglycerides; SDP,

cardiovascular diseases such as atherosclerosis and heart failure [1]. This epidemiological connection between aging, T2DM, and cardiovascular diseases postulates that there might be a pathophysiological link.
Aging has been associated with systemic in ammation and oxidative stress [2], which can be both a cause as well as a consequence of Diabetes [3]. Cellular senescence can be de ned as a permanent arrest of cellular growth and is a key feature of aging [4,5]. Cell senescence is also a cause and a consequence of Diabetes and plays an important role in its cardiovascular complications. Although senescent cells are classically reported as cell that irreversibly cease proliferation, they have the capacity to produce and secrete soluble factors that can in uence neighboring cells and tissues [6,7]. This feature of senescent cells to secrete these soluble factors has taken the name of senescence-associated secretory phenotype (SASP) [8]. Since chronic in ammation is an important pathophysiological factor of both aging and diabetes, the SASP has been pointed as the pathophysiological link between aging and diabetes in cardiovascular diseases [9].
Tissue-speci c adult stem cell senescence has emerged as an attractive theory for the decline in mammalian tissue and organ function during aging [10]. The mammalian heart, including the human, harbors a tissue speci c cardiac stem/progenitor cell (CSC) compartment [11][12][13][14][15] that undergoes senescence with age, which dictates a progressive and permanent dysfunction of more than half of these endogenous cells by 75 years of age [5,10,16,17]. The senescent CSCs exhibit a SASP that can negatively impact surrounding cells, causing otherwise healthy and cycling-competent CSCs to lose proliferative capacity and switch to a senescent phenotype. Nevertheless, even at the oldest age, it is still possible to retrieve a healthy, cycling-competent CSC fraction with an increased regenerative and reparative capacity [5,10,16]. Accordingly, experimental selective ablation of senescent CSCs either genetically or by a combination of senolytic drugs, fosters the expansion and functional regenerative recovery of the healthy aged CSCs [5,10,18].
Several reports have shown that Diabetes impairs the in vitro proliferative and differentiation potential of adult CSCs, further worsening their senescence phenotype even when compared with CSCs from nondiabetic ischemic patients [19][20][21]. Changes in chromatin conformation underlie the impaired proliferation, differentiation, and senescent behavior of diabetic CSC [22]. Yet, it is unclear whether the SASP is induced by Diabetes per se in CSCs and whether targeting senescent cells with the diabetic CSC compartment rescues CSC proliferation and differentiation defect in T2DM. On this premise here we show that the myocardium of T2DM patients, in a very narrow age window of 55-64 years, undergoing cardiac revascularization for ischemic heart disease, is characterized by an increased oxidative stress that affects the CSC compartment with an increased senescent phenotype when compared to myocardium and CSCs of age and sex matched NON-Diabetic Mellitus (NDM) patients. Importantly, Diabetic-Senescent CSCs have a SASP and senolityc treatment abrogate the senescent diabetic Diabetic-Senescent CSCs uncaging healthy CSCs to proliferate and differentiate normally.

Patients Cohort and Samples
Human myocardial samples were obtained from Type 2 diabetes mellitus (T2DM) and NON-Diabetic Mellitus (NDM) patients with post-infarct cardiomyopathy which undergoing surgical coronary revascularization. The samples were collected only when removal of tissue was required for surgical reasons. Collection of human tissues samples was approved by the local ethics committee at the University of Campania "L. Vanvitelli" of Naples. Before cardiac surgery, written informed consent was obtained from every patient. All patient data were kept anonymous. We included 10 T2DM and 6 NDM patients from which peri-infarct/border zone biopsies were obtained (Table 1). Freshly excised samples were formalin xed for immunohistochemistry analysis as described below. Furthermore, additional 6 T2DM and 6 NDM patients with similar characteristics were included (Table 1), from which atrial samples were obtained and processed for cell harvesting as below described.

Human CSCs Isolation
Human samples obtained from T2DM and NDM patients were stored in saline on ice until ready to process (~1hr). All steps were performed at 4°C unless stated otherwise. Brie y, cardiac tissue was minced then digested with collagenase II (0.3mg/ml; Worthington Laboratories) in Dulbecco's Modi ed Eagle's Medium (DMEM; Sigma-Aldrich) at 37°C in a series of sequential digestions for 3 minutes each.
Enzymatically released cells were ltered through a 40µm cell strainer (Becton Dickinson, BD) to eliminate the cardiomyocyte population and collected in enzyme quenching media (DMEM + 10% FBS). The isolated cardiac cells were collected by centrifugation at 400g for 10 min, resuspended in incubation media (PBS, 0.5% BSA, 2 mM EDTA). For the isolation of c-kit pos CD45 neg CD31 neg CSCs the MACS technology was used (Miltenyi Biotec). First, cardiomyocyte-depleted cardiac cells were negatively sorted for CD45 pos and CD31 pos cells by immunolabelling with anti-human CD45 and CD31 magnetic immunobeads (Miltenyi). The obtained CD45 neg CD31 neg population was then enriched for c-kit pos cardiac cells through incubation with anti-human CD117 immunobeads (Miltenyi) (1:10) and then positively sorted using MACS according to the manufacturer's instructions.

Histology and Immunohistochemistry
Tissue specimens were xed and embedded in para n for histochemical and immunohistochemical analysis. Human tissues were cut in sections of 5 µm. After dewaxing in xylene and rehydration in graded concentration of ethanol, when appropriate, Antigen Retrieval was performed by incubating section in 10 mM sodium citrate buffer (pH 6.0) at 98°C for 30 min. Non-speci c antibody binding was blocked by incubation with 10% normal donkey serum (Jackson ImmunoResearch) for 30 minutes at room temperature. Sections were stained for 1h at 37°C or overnight at 4°C and the following primary antibodies were used: c-kit (1:100 dilution, DAKO), c-TnI (1:200 dilution, Abcam), p16 INK4A (1:100 dilution, Santa Cruz Biotechnology), 8-OH-dG (1:100 dilution, Origene) and 3-NT (1:400 dilution, Merck Millipore). After washing in phosphate-buffered saline (PBS), sections were incubated with respective secondary antibodies (Jackson ImmunoResearch). Nuclear counterstains were performed by DAPI (4',6-diamidino-2phenylindole, Sigma) and sections were examined by confocal microscopy (LEICA TCS SP8). The number of positive cells was expressed as a percent fraction of the total cells number per mm 2 .
For immunohistochemical analysis of oxidative stress on tissue sections, after dewaxing, human sections were blocked with Dual Endogenous Enzyme Block for 10 min at room temperature. Then, the sections were stained with antibodies against 8-OH-dG (1:100 dilution, Origene), 3-NT (1:400 dilution, Merck Millipore) and 4-HNE antibody (1:50 dilution, Abcam) for 1h at 37°C. Positive reactions were visualized using a Labelled Polymer-HRP complex and 3,3'-diaminobenzidine tetrahydrochloride (DAB) chromogen using the EnVision+ Dual Link System-HRP (DAKO). Sections were then counterstained with hematoxylin, permanently mounted, and then examined with light microscopy (LEICA, DMI3000B). The morphometric analysis of immunohistochemistry was conducted using Image J.

FACS analysis
Cell analysis was performed on FACSCanto II (BD) with FlowJo software (TREE STAR) to identify the percentage of cardiac small cells expressing different cell-surface markers of interest at passage 2 (P2). A panel of different markers was used for immunophenotypic characterization of human ckit pos CD45 neg CD31 neg CSCs obtained from T2DM and NDM patients. Speci c antibodies used are listed in Table 2. Appropriate labelled isotype controls were used to de ne the speci c gates.
Proliferation, clonogenicity, cardiosphere formation and cardiomyocyte differentiation assays in vitro CSC proliferation was evaluated through BrdU incorporation and growth curve assay, at the indicated time points, on human CSCs. To assess the proliferative activity of freshly isolated ckit pos CD45 neg CD31 neg T2DM-hCSCs and NDM-hCSCs, BrdU 10 μM was administered in vitro. CSCs were plated at density of 1x10 3 in 24-well CELL-Start-coated dishes and then serum starved in 0% serum base medium. After 48 hrs, starvation medium was replaced by CSC growth medium and BrdU was added to the medium every 6hrs. The cells were xed at 24 hours and BrdU incorporation was assessed using the BrdU detection system kit (Roche) according to the manufacturer's instructions. Nuclei were counterstained with DAPI (Sigma-Aldrich). Cells were evaluated using a uorescent microscope (LEICA, DMI3000B). Accordingly, the number of BrdU pos cells was expressed as a percent fraction of the total cell nuclei.
Growth curve assay was archived by plating 5×10 3 cells in 24-well CELL-Start-coated dishes in CSC growth medium and then serum starved in 0% serum base medium. After 48 hrs, starvation medium was replaced by CSC growth medium and cells were then trypsinized and counted using trypan blue, 1:1 ratio, at the indicated time points.
Single cell cloning was employed through depositing of half c-kit pos CD45 neg CD31 neg T2DM-hCSCs and NDM-hCSCs into 96-well CELL-Start-coated Terasaki plates by serial dilution. Individual ckit pos CD45 neg CD31 neg T2DM-hCSCs and NDM-hCSCs were grown in CSC growth medium for 1-3 weeks when clones were identi ed and expanded. The clonogenicity of the c-kit pos CD45 neg CD31 neg T2DM-hCSCs and NDM-hCSCs was determined by counting the number of wells in each 96-well plate containing clones and expressed as a percentage. A total of 10 plates were analyzed.
For cardiosphere generation, 1x10 5 c-kit pos CD45 neg CD31 neg T2DM-hCSCs and NDM-hCSCs were placed in bacteriological dishes with CSC growth medium. Cardiospheres were counted per plate at 14 days and the number expressed as a percentage relative to the number of plated CSCs.
For speci c myogenic differentiation, c-kit pos CD45 neg CD31 neg T2DM-hCSCs and NDM-hCSCs-derivedcardiospheres were switched to StemPro®-34 SFM differentiation medium (a serum-free medium conditioned with StemPro®-Nutrient Supplement, Gibco, Life Technologies), Glutamine (2mM) and penicillin-streptomycin (1%, Life Technologies). For cardiomyocyte differentiation BMP4 (10ng/ml, Peprotech), Activin-A (50 ng/ml, Peprotech), β-FGF (10ng/ml, Peprotech), Wnt-11 (150ng/ml, R&D System) and Wnt-5a (150ng/ml, R&D System) were added to base differentiation medium. Then differentiating cardiospheres were pelleted and transferred to laminin coated dishes (1µg/ml) and Dkk-1 (150ng/ml, R&D System) was added to base differentiation medium until day 14. Cell differentiation was evaluated at 14 days. Immunocytochemistry A volume of 200 μl of a 0.15×10 6 cells/ml suspension of c-kit pos CD45 neg CD31 neg CSCs obtained from T2DM and NDM patients were directly loaded in cytofunnel and spin down at 800 rpm for 3 min onto poly-lysine-coated slides using a Shandon Cytospin 4 Cytocentrifuge (Thermo Fisher Scienti c). Slides were immediately xed using PFA 4% (Sigma-Aldrich). After xation, cells were allowed to air dry before proceeding with immunostaining. Slides were washed with PBS, and incubated in 0.1% Triton X-100:PBS at room temperature for 10 minutes. After washing with 0.1% tween:PBS and 30 minutes in 10% donkey serum, cells were incubated overnight at 4°C with primary antibodies against p16 INK4a (1:100 dilution, Santa Cruz Biotechnology), γ-H2AX (1:400 dilution, Cell Signaling) in 0.1% Tween:PBS. Slides were then washed and incubated with corresponding secondary antibodies (Jackson Immunoresearch) for 1 hour at 37°C. After washing, the nuclear DNA of the cells was counterstained with DAPI (Sigma-Aldrich) at 1µg/ml and mounted using Vectashield mounting media (Vector labs). Imagines were acquired using a confocal microscope (LEICA TCS SP8). The number of p16 INK4a positive and γ-H2AX positive cells was expressed as a percent fraction of the total CSC nuclei. Cell apoptosis were detected using the Terminal deoxynucleotidyl Transferase (TdT) assay (TUNEL assay, Merck Millipore) according to the manufacturer's instructions. Brie y, to perform TUNEL staining, cells were xed with paraformaldehyde 2% in PBS (pH 7.4) and permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate. Then cells were incubated with TUNEL reaction mixture (containing Terminal deoxynucleotidyl transferase enzyme + nucleotide mixture in reaction buffer) for 1 hour at 37°C in a humidi ed atmosphere in the dark. After washing, the nuclear DNA of the cells was counterstained with DAPI (Sigma-Aldrich) and the slides mounted using mounting media (Vector labs). Imagines were acquired using a confocal microscope (LEICA TCS SP8). The number of apoptotic cells was expressed as percentage of the total nuclei. SA-β-gal staining was performed using a SA-β-gal staining kit (Cell Signaling Technology) according to the manufacturer's instructions and protocols previously reported [23]. In brief, c-kit pos CD45 neg CD31 neg T2DM-hCSCs and NDM-hCSCs were grown in culture and then xed with 1X Fixative Solution for 10-15 min at room temperature. Following xation, cells were washed with PBS before being incubated in β-Galactosidase Staining Solution (pH 6.0) at 37°C in a dry incubator overnight. The enzymatic reaction was stopped by washing slides with ice-cold PBS and SA-β-gal staining was xed with ice-cold methanol for 30s before mounting/visualising. Senescent CSCs were identi ed as blue-stained cells using light microscopy. A minimum of 10 images were taken at x10 magni cation from random elds and the percentage of SA-β-gal cells were expressed as percentage of total nuclei.
To perform cTnI staining, human c-kit pos CD45 neg CD31 neg CSCs-derived cardiospheres were xed using PFA 4% (Sigma-Aldrich) after 14 days in differentiation media. After xation, cells were incubated with cTnI antibody (1:200, Abcam) for 2 hour at 37°C in a humidi ed atmosphere. The corresponding secondary antibody (Jackson Immunoresearch) was added for 1 hour at 37°C in the dark. The nuclear DNA of the cells was counterstained with DAPI (Sigma-Aldrich) and the slides mounted using mounting media (Vector labs). Imagines were acquired using a confocal microscope (LEICA TCS SP8).

Telomere length
Genomic DNA of human c-kit pos CD45 neg CD31 neg CSCs obtained from myocardial samples of T2DM and NDM patients was extracted using Quick-DNA Microprep Kit (Zymo Research). Telomere length was analyzed by using the Absolute Human Telomere Length Quanti cation qPCR Assay Kit (ScienCell Research Laboratories). Genomic DNA (10 ng) was ampli ed with the FastStart DNA Green Master (Roche Life Science) using a CFX384-Real-Time PCR System (Biorad) and data analysis was conducted according to manufacturer's instruction. For each genomic DNA samples, two different reaction were performed using two primer set: a Telomere primer set to recognize and amplify the telomere sequences and a single copy reference (SCR) primer set to normalize the data that recognizes and ampli es a 100 bp-long region on human chromosome 17, accordingly, a known telomere length as reference to calculate the telomere length of target samples. All reactions were run in triplicate. The average telomere length was calculated by following the manufacturer's instructions.

Telomerase Activity Quanti cation
Human c-kit pos CD45 neg CD31 neg CSCs obtained from myocardial samples of T2DM and NDM patients were processed according to the manufacturer's protocol using the Telomerase Activity Quanti cation qPCR Assay Kit (ScienCell Research Laboratories) [24]. Brie y, cell pellets were thawed in lysis reagent enables to release telomerases in the native state. Cell lysate samples were incubated with telomerase reaction buffer at 37°C for 3 hours. qRT-PCR was conducted with FastStart DNA Green Master (Roche Life Science) using a CFX384-Real-Time PCR System (Biorad) and data analysis was performed according to manufacturer's instruction. The telomere primers set recognizes and ampli es newly synthesized telomere sequences in the assay. All reactions were run in triplicate. Telomerase activity quanti cation was calculated by following the manufacturer's instructions.

Western Blot Analysis
Immunoblots were carried out using protein lysates obtained from c-kit pos CD45 neg CD31 neg T2DM-hCSCs and NDM-hCSCs. Aliquot equivalent of ~40 to 70 μg of proteins were separated on gradient (6-15%) SDS-polyacrylamide gels. After electrophoresis, proteins were transferred onto nitrocellulose lters, blocked with either 5% dry milk and incubated with Ab against p16 INK4A (1:1000, Santa Cruz Biotechnology) and GAPDH (1:1000, Santa Cruz Biotechnology). Proteins were detected by chemiluminescence using horseradish peroxidase-conjugated 2Abs and placing the nitrocellulose lters on a photographic lm. The acquisition was archived using Medical X-ray processor 2000 (CARESTREAM). Densitometry was obtained using ImageJ software. Immunoblots were performed in biological triplicates.
Senolytic drug treatment, viability in vitro c-kit pos CD45 neg CD31 neg T2DM-hCSCS were plated in 24-well CELL-Start-coated plates at 40% con uence and left for 72h. Then, the growing medium was replaced with new fresh medium in the absence or presence of a combination of two senolytics drug, Dasatinib (D, LC Laboratories) and Quercetin (Q, Sigma-Aldrich), respectively at doses 0.25μM and 10μM added for 6 hours. Then, the conditioned medium was replaced with complete fresh medium. After 48h the treatment D+Q was repeated as above.
For myogenic differentiation, T2DM-hCSCs were treated or untreated with D+Q in growth media as above. Then cells were placed in bacteriological dishes and the differentiation assay was performed as above described.

Statistical analysis
All data are presented in mean ± standard deviation. Data were analysed using t-test comparisons in GraphPad Prism version 8.0.0 for Windows, GraphPad Software, San Diego, California USA, www.graphpad.com. Differences of p<0,05 are considered statistically signi cant.

Increased Oxidative Stress in Diabetic Ischemic Cardiomyopathy
Diabetes is characterized by an enhanced oxygen toxicity [26]. ROS is the distal signal of the cascade of events triggered by Diabetes that leads to the initiation of the cell death pathway in the heart [27].
To assess the effects of Diabetes on ROS-mediated cytoplasmic and DNA damage, we obtained left ventricles (LV) samples from patients with Type 2 diabetes mellitus (T2DM) and NON-diabetic mellitus (NDM) patients with post-infarct cardiomyopathy undergoing surgical coronary revascularization. As inclusion criteria an age between 50-64 years was required in order to try separate the effects of aging from Diabetes on senescence. On this premise, we included 10 T2DM and 6 NDM patients from which peri-infarct/border zone biopsies were obtained (Table 1). These samples were analyzed for the presence of 8-OH-deoxyguanosine (8-OH-dG), Nitrotyrosine (3-NT), and 4-hydroxinonenal (4-HNE). Diabetes status resulted in an increase of myocardial cells, which stained positive for these markers of oxidative stress when compared to NDM patients ( Figure 1A-C). In particular, a higher percentage of both cardiomyocytes and CSC-enriched c-kit pos CD45 neg CD31 neg cardiac cells positive for 8-OH-dG and 3-NT were detected in T2DM as compared with NDM (Figure 2A,B). These data show that cardiac damage in Diabetes is associated with a higher ROS production that targets both muscle cells as well as progenitor cells.

Increased Oxidative Stress Associated with Increased Expression of Senescent Markers in hCSCs from
Diabetic Ischemic Cardiomyopathy Cellular senescence is a cell state triggered by stressful insults and certain physiological processes, characterized by a prolonged and generally irreversible cell-cycle arrest with secretory features, macromolecular damage, and altered metabolism. These mechanisms are generally interdependent as indeed oxygen toxicity and DNA damage alter telomeres, resulting in telomere shortening, cellular senescence, and cell dysfunction and/or death [28]. Although a link to organismal aging is clear, aging and senescence are not synonymous as, indeed, cells can undergo senescence, regardless of organismal age [29,30]. On the other hand, aging tissues experience a progressive decline in homeostatic and regenerative capacities, which has been attributed to senescence of their tissue-speci c stem cells [31]. On this premise, to assess whether the exaggerated ROS in Diabetes associated with senescence of tissue-resident CSCs, independently of age, we rst assessed the expression of p16 INK4a in CSCs-enriched c-kit pos CD45 neg CD31 neg cardiac cells in the myocardial sections from samples obtained from T2DM patients with ischemic cardiomyopathy as compared to NDM patients. p16 INK4a inactivates CDK4 and CDK6, which regulate cell-cycle progression in G1; p16 INK4a forms binary inhibitor-CDK complexes that are highly stable and cannot be dissociated by cyclins, and cells arrest permanently [32]. Therefore, p16 INK4a is widely used as senescent biomarker, despite not being su cient on its own to de ne/detect senescent cells [33][34][35][36].
The expression of p16 INK4a in CSCs-enriched c-kit pos CD45 neg CD31 neg cardiac cells markedly increased in myocardial samples from T2DM ischemic cardiomyopathy when compared to NDM counterparts ( Figure  2C), con rming that a higher oxidative stress couples with the induction of senescence in the cardiac progenitor pool.
To further assess senescence in the adult progenitor pool, we established cultures of human adult cardiac stem cells (hCSCs) from atrial specimens of T2DM (n=6) and NDM (n=6) patients undergoing to cardiac surgery (Table 1) using magnetic activated cell sorting (MACS) for CD45/CD31 negative sorting followed by c-kit positive sorting to isolate CSC-enriched c-kit pos CD45 neg CD31 neg cardiac cells [12,15,37].
By ow cytometry, the c-kit pos CD45 neg CD31 neg cardiac cells from T2DM (hereafter T2DM-hCSCs) showed a membrane phenotype typical of cardiac cells enriched with multipotent hCSCs ( Figure 3A) as we have previously shown [12], with no signi cant difference as compared to c-kit pos CD45 neg CD31 neg cardiac cells from NDM donors (hereafter NDM-hCSCs) (data not shown). Indeed, the hCSC-enriched c-kit pos CD45 neg CD31 neg cardiac cells at 14 days in culture after their primary harvest, expressed PDGFRα, CD166, CD105 and CD90, con rming the enrichment for the phenotypical identity of bona de CSCs ( Figure 3A) [37,38].
As rst assessment of replicative senescence in the isolate cells, we measured telomerase activity and telomeric length ( Figure 3B,C). T2DM-hCSCs showed a signi cant reduced telomerase activity which associated with a decreased average telomere length when compared to NDM-hCSCs ( Figure 3B,C). These data suggest that Diabetes determines exaggerated oxidative stress in hCSCs that is linked to replicative senescence as demonstrated by the appearance of p16 INK4a positive cells and reduced telomerase activity inducing telomere shortening.
CSCs from non-aged diabetic subjects show impaired cell growth and myogenic differentiation potential in vitro One common feature of senescent cells is an essentially irreversible cell-cycle arrest that can be an alarm response instigated by deleterious stimuli or aberrant proliferation [30]. The rst molecular feature associated with senescence is telomere shortening, a result of the DNA end-replication problem, during serial passages [28,39]. On the basis of the immunohistochemistry data showing increased p16 INK4a expression in myocardial sections and of the in vitro data demonstrating telomerase de cit with telomere length attrition, we evaluate replicative competence of T2DM-hCSCs. To this aim, freshly-isolated T2DM-hCSCs and NDM-hCSCs were plated for a week and then compared for their growth potential in vitro. T2DM-hCSCs show a signi cant decreased proliferation in vitro when compared to NDM-hCSCs, either assessed by growth curve kinetics over time as well as BrdU incorporation over 24 hours ( Figure 4A,B). To assess whether the proliferation defect in the expansion capacity of T2DM-hCSCs was coupled with a de cit in other 'stemness' capabilities, we tested these cells for their clonal ampli cation by depositing half-cell per well in a 96 wells plate and for their spheroid potential by plating them in bacteriological dishes [12]. Remarkably, at 14 days after cell deposition/plating in clonogenic or spherogenic medium, respectively, clonal e ciency was 2.5-fold lower and spherogenesis 2.5-fold lower in T2DM-hCSCs when compared to NDM-hCSCs ( Figure 4C,D).
The above data show that Diabetes increases senescence of hCSCs affecting their expansion, clonal and spheroid potential; we then tested the differentiation potential of these cells as it has been shown in different contexts that senescence and Diabetes both affect differentiation of tissue-dependent stem/progenitor cells [19][20][21]. Considering that myogenic differentiation is key to myocardial regeneration from endogenous progenitor cells, we evaluated cardiomyocyte differentiation of T2DM-hCSCs vs. NDM-hCSCs when grown in myogenic differentiation medium using the sphere-based myogenic assay that we previously established [38]. RT-PCR data show that T2DM-hCSCs upregulated signi cantly less than NDM-hCSCs the main cardiac transcription factors (GATA-4, NKX2.5 and MEF2C) and myocyte contractile genes (TNNT2, ACTC1, MYH6 and MYH7) during myogenic differentiation induction ( Figure 4K). Only 24±4% of T2DM-hCSCs, as compared to 52±11% of NDM-hCSCs, acquired the prototypical myocyte contractile marker cTnI by 4 weeks in culture, overall revealing a decreased capacity for myogenic differentiation ( Figure 4L).
Overall, these data show that Diabetes Mellitus hampers human CSC biology, inducing a variety of hallmarks of senescence that likely contribute to the de cit of their regenerative potential.
T2DM-hCSCs exhibit a de ned senescence-associated secretory phenotype (SASP) Senescent cells secrete a plethora of factors, including pro-in ammatory cytokines and chemokines, growth modulators, angiogenic factors, and matrix metalloproteinases (MMPs), collectively known as the senescent associated secretory phenotype (SASP) [8,39]. The SASP constitutes a hallmark of senescent cells and mediates many of their patho-physiological effects, such as reinforcing and spreading senescence in autocrine and paracrine fashions [40], activating immune responses [41], hampering tissue plasticity [42] and contributing to persistent chronic in ammation (known as in ammaging) [43]. Thus, the SASP explain some of the deleterious, pro-aging effects of senescent cells. On the other hand, all these patho-physiological mechanisms are equally active in Diabetes. Therefore, we evaluated whether increased senescence markers and functional defect associated also with SASP in T2DM-hCSCs.
We then treated for 7 days NDM-hCSCs with conditioned medium (CM) derived from T2DM-hCSCs (T2DM-CM) and measured cell proliferation and senescence of the treated NDM-hCSCs. As controls, NDM-hCSCs were treated either with normal growth media or CM from parallel cultures of NDM-hCSCs. Treatment of NDM-hCSCs with T2DM-CM resulted in a decreased proliferation (p < 0.05) ( Figure 5C,D) and an increased proportion of senescent p16 INK4a positive (p < 0.05), SA-β-gal-positive and γ-H2AX-positive hCSCs when compared to NDM-hCSCs in unconditioned medium (UM) or CM from NDM-hCSCs ( Figure 5E). These data show for the rst time that Diabetes induces SASP in hCSCs isolated from non-aged patients.
SASP appears to negatively affect neighbor hCSCs whereby senescence begets senescence, suggesting that targeting senescent cells could improve CSC dysfunction in Diabetes.
Senolytics rescue the regenerative de cit of diabetic hCSCs The above data show that T2DM-hCSCs harbor a high fraction of senescent cells that produce and secrete a detrimental SASP. Because senescent cells contribute to the outcome of a variety of cardiac disease, including age-related and -unrelated cardiac diseases like anthracycline cardiotoxicity [5,[44][45][46], much effort has been recently made to therapeutically target detrimental effects of cellular senescence.
In regard with the latter, senolytic drugs are agents that selectively induce apoptosis of senescent cells by overriding anti-apoptotic pathways in senescent cells [5]. One of the most well studied senolytics therapeutic approach is the combination of Dasatinib (D) and Quercetin (Q) [5], which we show can target senescent CSCs and improve cardiac function in cardiac aging in mice [5].
To that end, T2DM-hCSCs were plated in 24-well dishes at 40% con uence and left for 2 days. Then, cells were administered with a combination of D+Q at a dose of 0.25 μM D with 10 μM Q. Six hours later, D+Q conditioned medium was replaced with complete fresh medium. Untreated cells served as controls. Two days later, D+Q treatment was repeated as above. Two days after, T2DM-hCSCs were then analyzed for proliferation and markers of senescence, p16 INK4A , SA-β-gal and γ-H2AX. As shown in Figure 6, D+Q treatment removed from the cell culture the typical enlarged and attened cell morphology of senescent cells, consistent with the senolytics combination targeting and removing senescent cells ( Figure 6A).
Concurrently, D+Q increased T2DM-hCSCs proliferation as evaluated by growth curve kinetics when compared to untreated control T2DM-hCSCs ( Figure 6B). Accordingly, D+Q signi cantly reduced the number of SA-β-gal and γ-H2AX positive T2DM-hCSCs as well as the number of p16 INK4a senescent cells, which almost disappeared when compared to untreated T2DM-hCSCs ( Figure 6C-E). Furthermore, we determined whether D+Q would abrogate the SASP in T2DM-hCSCs. We found that the level of SASP factors secreted by T2DM-hCSCs was signi cantly reduced by the administration of D+Q combination ( Figure 6F).
Finally, we checked whether D+Q pre-treatment was able to rescue the altered myogenesis potential of diabetic hCSCs. To that end, T2DM-hCSCs were treated or untreated D+Q as above in growth media and then cells were plated in bacteriological dishes for sphere formation [12]. CSC-derived cardiospheres were then plated in laminin for additional 7 days in the stage speci c cardiomyogenic media [12]. Interestingly, we found that D+Q pre-treatment restored the myogenic capacity of T2DM-hCSCs as indeed this senolytic combination signi cantly increased myogenic transcription factor and myogenic contractile gene expression as compared to untreated cells in vitro ( Figure 6G). Accordingly, D+Q increased the number of T2DM-hCSC-derived cTnI pos cardiomyocytes after 14 days in differentiation media ( Figure 6H).
Overall, these ndings document that clearance of senescent cells using a combination of D+Q senolytics abrogates the SASP and restores a fully proliferative-and differentiation-competent hCSCs pool in T2DM.

Discussion
The main ndings emanating from this study are that: i) Ischemic damaged myocardial tissue of T2DM patients is characterized by an exaggerated oxidative stress targeting both cardiomyocytes and cardiac stem/progenitor cells (CSCs); ii) Increased oxidative stress in the myocardium of non-aged T2DM patients associates with an increased number of senescent and dysfucntional T2DM-hCSCs as shown by increased p16 INK4a expression, reduced telomerase activity and telomere length, reduced proliferation, clonogenesis/spherogenesis and myogenic differentiation; iii) T2DM-hCSCs from non-aged subjects show a senescence-associated secretory phenotype (SASP), as demonstrated by the increased secretion of several SASP factors, including MMP-3, PAI1, IL-6, IL-8, IL-1β and GM-CSF; iv) a combination of two senolytics, Dasatinib and Quercetin, clear senescent T2DM-hCSCs restoring expansion and myogenic differentiation capacities of the diabetic hCSC pool.
T2DM is a pre-conditioning and powerful driver of organismal aging [47]. The biological foundations of aging primarily involve cellular senescence and T2DM is plethoric in senescence-driving factors [47]. An intense debate has existed so far addressing whether senescence precedes or follows the onset of perpetual in ammation and insulin resistance (IR) in T2DM [48]. Independently from "who-precedes-who," diabetic patients experience an obvious accelerated aging process that increases their susceptibility to morbidity and earlier mortality [49]. Hence, diabetes-affected patients have a signi cantly shorter life expectancy than non-diabetic individuals, and cardiovascular disease accounts for a large part of the excess mortality [50]. Indeed, adults with T2DM have 2-4 times increased cardiovascular risk compared with adults without diabetes, and the risk rises with worsening glycemic control (T2DM) [1].
Adult tissue-speci c stem cells are multipotent cells that are considered a lifelong cellular reservoir to ensure the continuous generation, replacement, and restitution of multiple tissue lineages [51,52]. Therefore, adult stem cells play a vital role in preventing the aging of organs and tissues, and can delay aging. However, during aging, these cells also undergo some detrimental changes such as alterations in the microenvironment, a decline in the regenerative capacity, and loss of function so that aging has been linked to tissue-speci c adult stem cell exhaustion [31]. Converging evidence conclusively demonstrated how toxic high glucose load may be for survival, differentiation plasticity, and regenerative competence for different stem cells lineages [47,[53][54][55][56][57]. The self-sustaining vicious circle amalgamated by hyperglycemia/mitochondrial dysfunction/oxidative stress appears as a master driver to adult mesenchymal stem cell senescence [58]. Accordingly, the diabetic pro-oxidative environment is a major contributing factor for premature adult stem cell senescence and functional de cit [47,[58][59][60][61][62]. Therefore, Aging and T2DM drive adult stem cell senescence and regenerative de cit and conversely adult stem cell senescence and regenerative de cit drive the progressive pathology in aging and diabetes.
Like other tissue speci c adult stem/progenitor cells, also CSCs are not immortal [16]. They undergo cellular senescence characterized by increased ROS production and oxidative stress and loss of telomere/telomerase integrity in response to a variety of physiological and pathological demands with aging [16]. Nevertheless, the old myocardium preserves an endogenous functionally competent CSC cohort which appears to be resistant to the senescent phenotype occurring with aging [16]. The latter envisions the phenomenon of CSC ageing as a result of a stochastic and therefore reversible cell autonomous process, whereby targeting the senescent cells would bene t the recovery of a healthy CSC cohort [16]. Concurrently, T2DM impairs the in vitro proliferative and differentiation potential of human cardiac stem/progenitor cells, worsening their senescence phenotype when compared with CSCs from non-diabetic ischemic patients [22]. Additionally, miR-34a is signi cantly upregulated while SIRT1 is downregulated in adult CSCs harvested from T2DM patients, which is associated with a higher proapoptotic caspase-3/7 activity [63]. All the above studies were, however, conducted in hCSCs isolated from diabetic old patients (> 65 years), which could not clearly distinguish the role of age from DM on hCSC function. On the other hand, in an animal model of insulin-dependent DM in young mice, the myocardial accumulation of ROS drives CSC senescence through the expression of p53 and p16 INK4a proteins and telomere erosion [27]. p66 shc knockout inhibits CSC senescence and death, preventing the senescent phenotype and the development of cardiac failure by T2DM [27]. Additionally, Diabetes persistently decreases the ability of isolated CSCs from young male mice to proliferate, survive oxidative insults, and differentiate, which can be explained at least in part by an uncoupling of biosynthetic glucose metabolism pathways [64]. Diabetes suppresses CSC activation through a diminished SCF expression in the heart following permanent LAD ligation in young/adult rats [20]. Altogether, these data postulate the tantalizing hypothesis that the premature cellular senescence and ageing of resident CSCs underpins the development of diabetic heart disease [10].
In the present study we show premature aging in hCSCs isolated from non-aged (< 65 years old) patients with T2DM. Indeed, T2DM hCSCs harbor a signi cant fraction of senescent cells, with exaggerated ROS, DNA damage and telomere erosion. In particular, we provide evidence that independently from age, T2DM-hCSCs have a de ned pathologic SASP, which can drive senescence into healthy hCSCs to senescence. Furthermore, T2DM-hCSCs have a reduced capacity to differentiate into cardiomyocytes in vitro. The premature senescence is clearly central to the regenerative defect imposed by Diabetes on hCSCs as demonstrated by the elimination of senescent cells by senolytic treatment, a combination of Dasatinib and Quercetin, which rescued the expansion and myogenic capacity of T2DM-hCSCs. Therefore, this data suggests that eliminating senescent hCSCs by senolytic therapy in vivo could be a therapeutic strategy to prevent hCSC dysfunction even at early stages of Diabetic disease.
Although hampered by the di culty and limitation of working with samples of human tissue, which makes the data presented here mainly descriptive and falling short of being able to establish direct cause-effect relationship(s) (as it is also the case for most work based on primary human tissue), our ndings strongly point to Diabetes hampering human CSC biology, inducing a variety of hallmarks of senescence in non-aged subjects that contribute to the de cit of their regenerative potential. Diabetes induces a pathologic SASP in hCSCs that spread to otherwise healthy hCSCs, reducing the functional pool of hCSCs. This conclusion is reinforced by the evidence that clearance of senescent cells by senolytics abrogates the SASP and restores a fully proliferative-and differentiation-competent hCSC pool in T2DM. Nevertheless, this study cannot fully extrapolate the role of ischemic damage to the observed effects of Diabetes on hCSCs in vivo. Indeed, despite included patients were homogenous for number of vessel diseases and infarct area, Diabetes is known to determine more intense microvascular ischemia, which may have affected the status and function of hCSCs. On the other hand, isolated hCSCs were obtained from right atrial tissue which were not ischemic and therefore most likely the observed effects of Diabetes is independent form ischemia at least for the in vitro data. Yet, further work is needed to fully clarify this issue. Finally, the data of this study in human cells in vitro surely improve our understanding of the cellular mechanisms behind CSC dysfunction in T2DM, identifying senescent cells as potential drug targets and guiding the design of clinical test of candidate D + Q for the myocardial regenerative defect of the diabetic heart. However, human cells in vitro cannot fully recapitulate the human scenario whereby human organoids, stem cell-derived 3D culture systems, re-create the architecture and physiology of human organs in remarkable detail, providing unique opportunities for the study of human disease [65]. It will be therefore truly interesting to further evaluate the role of premature senescence of hCSCs in the context of diabetic human cardiac organoids in the next future [66].

Conclusions
DM hampers human CSC biology, inducing cellular senescence and senescence associate secretory phenotype (SASP) in non-aged subjects (< 65 years) that contribute to a signi cant de cit of their regenerative potential and in particular to their ability to differentiate in new cardiomyocytes. Clearance of senescent cells by senolytics abrogates the SASP and restores a fully proliferative-and differentiationcompetent hCSC pool in T2DM. Therefore, senolytics may represent a potential therapeutic approach to prevent or treat the reduced regenerative potential of the diabetic heart.

Declarations
Ethics approval and consent to participate: Ethics approval and consent to participate are both stated in the METHODS section above.
Consent for publication: Not Applicable.
Availability of data and materials: All data generated or analysed during this study are included in this published article