Serum starvation affects mitochondrial metabolism of adipose-derived stem/stromal cells.

BACKGROUND AIMS: A large part of mesenchymal stromal cell (MSC) regenerative and immunomodulatory action is mediated by paracrine signaling. Hence, an increasing body of evidence acknowledges the potential of MSC secretome in a variety of preclinical and clinical scenarios. Mid-term serum deprivation is a common approach in the pipeline of MSC secretome production. Nevertheless, up to now, little is known about the impact of this procedure on the metabolic status of donor cells. METHODS: Here, through untargeted differential metabolomics, we revealed an impairment of mitochondrial metabolism in adipose-derived MSCs exposed for 72 h to serum deprivation. RESULTS: This evidence was further confirmed by the significant accumulation of reactive oxygen species and the reduction of succinate dehydrogenase activity. Probably as a repair mechanism, an upregulation of mitochondrial superoxide dismutase was also induced. CONCLUSIONS: Of note, the analysis of mitochondrial functionality indicated that, despite a significant reduction of basal respiration and ATP production, serum-starved MSCs still responded to changes in energy demand. This metabolic phenotype correlates with the obtained evidence of mitochondrial elongation and branching upon starvation.

Dynamics of Connexin 43 Down Modulation in Human Articular Chondrocytes Stimulated by Tumor Necrosis Factor Alpha.

Connexin 43 (Cx43) exerts pivotal functions in articular chondrocytes (CH). It is involved in the communication among cells and between cells and the extracellular environment, and it contributes to the maintenance of the correct cell phenotype. The pro-inflammatory cytokine TNFα induces a reduction in Cx43 expression in CH. Here, we studied the dynamics of this decrease in expression. We evaluated Cx43 protein and gene expression and the involvement of C-terminal domain (CTD) cleavage and proteasomal degradation. Treatments able to counteract TNFα action were also examined, together with Gap Junction (GJ) functionality and Cx43 localization. TNFα induced a significant reduction in Cx43 expression already at day 1, and the down modulation reached a peak at day 3 (-46%). The decrease was linked to neither gene expression modulation nor CTD cleavage. Differently, the proteasome inhibitor MG132 reverted TNFα effect, indicating the involvement of proteasomal degradation in Cx43 reduction. In addition, the co-treatment with the anabolic factor TGF-ß1 restored Cx43 levels. Cx43 decrease occurred both at the membrane level, where it partially influenced GJ communication, and in the nucleus. In conclusion, TNFα induced a rapid and lasting reduction in Cx43 expression mostly via the proteasome. The down modulation could be reverted by cartilage-protective factors such as MG132 and TGF-ß1. These findings suggest a possible involvement of Cx43 perturbation during joint inflammation.

Secretome of human adipose-derived mesenchymal stem cell relieves pain and neuroinflammation independently of the route of administration in experimental osteoarthritis.

OBJECTIVE: Treatment of pain associated with osteoarthritis (OA) is unsatisfactory and innovative approaches are needed. The secretome from human adipose-derived mesenchymal stem cells (hASC-Conditioned Medium, CM) has been successfully used to relieve painful symptoms in models of chronic pain. The aim of this study was to explore the efficacy of the hASC-CM to control pain and neuroinflammation in an animal model of OA. METHODS: OA was induced in mice by intra-articular monosodium-iodoacetate (MIA) injection. Thermal hyperalgesia and mechanical allodynia were assessed. Once hypersensitivity was established (7 days after MIA), hASC-CM was injected by IA, IPL and IV route and its effect monitored over time. Neuroinflammation in nerve, dorsal root ganglia and spinal cord was evaluated measuring proinflammatory markers and mediators by RT-qPCR. Protein content analysis of secretome by Mass Spectrometry was performed. RESULTS: A single injection with hASC-CM induced a fast and long lasting antihyperalgesic and antiallodynic effect. The IV route of administration appeared to be the most efficacious although all the treatments were effective. The effect on pain correlated with the ability of hASC-CM to reduce the neuroinflammatory condition in both the peripheral and central nervous system. Furthermore, the secretome analysis revealed 101 factors associated with immune regulation. CONCLUSION: We suggest that hASC-CM is a valid treatment option for controlling OA-related hypersensitivity, exerting a rapid and long lasting pain relief. The mechanisms underpinning its effects are likely linked to the positive modulation of neuroinflammation in peripheral and central nervous system that sustains peripheral and central sensitization.

Bioactive Lipids in MSCs Biology: State of the Art and Role in Inflammation.

Lipidomics is a lipid-targeted metabolomics approach that aims to the comprehensive analysis of lipids in biological systems in order to highlight the specific functions of lipid species in health and disease. Lipids play pivotal roles as they are major structural components of the cellular membranes and energy storage molecules but also, as most recently shown, they act as functional and regulatory components of intra- and intercellular signaling. Herein, emphasis is given to the recently highlighted roles of specific bioactive lipids species, as polyunsaturated fatty acids (PUFA)-derived mediators (generally known as eicosanoids), endocannabinoids (eCBs), and lysophospholipids (LPLs), and their involvement in the mesenchymal stem cells (MSCs)-related inflammatory scenario. Indeed, MSCs are a heterogenous population of multipotent cells that have attracted much attention for their potential in regulating inflammation, immunomodulatory capabilities, and reparative roles. The lipidomics of the inflammatory disease osteoarthritis (OA) and the influence of MSCs-derived lipids have also been addressed.

Genomic and transcriptomic characterisation of undifferentiated pleomorphic sarcoma of bone.

Undifferentiated pleomorphic sarcoma of bone (UPSb) is a rare primary bone sarcoma that lacks a specific line of differentiation. There is very little information about the genetic alterations leading to tumourigenesis or malignant transformation. Distinguishing between UPSb and other malignant bone sarcomas, including dedifferentiated chondrosarcoma and osteosarcoma, can be challenging due to overlapping features. To explore the genomic and transcriptomic landscape of UPSb tumours, whole-exome sequencing (WES) and RNA sequencing (RNA-Seq) were performed on UPSb tumours. All tumours lacked hotspot mutations in IDH1/2 132 or 172 codons, thereby excluding the diagnosis of dedifferentiated chondrosarcoma. Recurrent somatic mutations in TP53 were identified in four of 14 samples (29%). Moreover, recurrent mutations in histone chromatin remodelling genes, including H3F3A, ATRX and DOT1L, were identified in five of 14 samples (36%), highlighting the potential role of deregulated chromatin remodelling pathways in UPSb tumourigenesis. The majority of recurrent mutations in chromatin remodelling genes identified here are reported in COSMIC, including the H3F3A G34 and K36 hotspot residues. Copy number alteration analysis identified gains and losses in genes that have been previously altered in UPSb or UPS of soft tissue. Eight somatic gene fusions were identified by RNA-Seq, two of which, CLTC-VMP1 and FARP1-STK24, were reported previously in multiple cancers. Five gene fusions were genomically characterised. Hierarchical clustering analysis, using RNA-Seq data, distinctly clustered UPSb tumours from osteosarcoma and other sarcomas, thus molecularly distinguishing UPSb from other sarcomas. RNA-Seq expression profiling analysis and quantitative reverse transcription-polymerase chain reaction showed an elevated expression in FGF23, which can be a potential molecular biomarker for UPSb. To our knowledge, this study represents the first comprehensive WES and RNA-Seq analysis of UPSb tumours revealing novel protein-coding recurrent gene mutations, gene fusions and identifying a potential UPSb molecular biomarker, thereby broadening the understanding of the pathogenic mechanisms and highlighting the possibility of developing novel targeted therapeutics. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Genetic analyses of undifferentiated small round cell sarcoma identifies a novel sarcoma subtype with a recurrent CRTC1-SS18 gene fusion.

In recent years, undifferentiated small round cell sarcomas (USRCSs) have been divided into a variety of new, rare, sarcoma subtypes, including the group of Ewing-like sarcomas, which have the morphological appearance of Ewing sarcomas, but carry CIC-DUX4, BCOR-CCNB3 and other gene fusions different from the classic EWSR1-ETS gene fusion. Using high-throughput RNA-sequencing (RNA-seq) analyses, we identified a novel recurrent gene fusion, CRTC1-SS18, in two cases of USRCS that lacked any known translocation. RNA-seq results were confirmed by reverse transcription polymerase chain reaction, long-range polymerase chain reaction, and fluorescence in situ hybridization. In vitro, we showed that the cells expressing the gene fusion were morphologically distinct and had enhanced oncogenic potential as compared with control cells. Expression profile comparisons with tumours of other sarcoma subtypes demonstrated that both cases clustered close to EWSR1-CREB1-positive tumours. Moreover, these analyses indicated enhanced NTRK1 expression in CRTC1-SS18-positive tumours. We conclude that the novel gene fusion identified in this study adds a new subtype to the USRCSs with unique gene signatures, and may be of therapeutic relevance. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Nitrogen Containing Bisphosphonates Impair the Release of Bone Homeostasis Mediators and Matrix Production by Human Primary Pre-Osteoblasts.

Bisphosphonates (BPs) represent the first-line treatment for a wide array of bone disorders. Despite their well-known action on osteoclasts, the effects they induce on osteoblasts are still unclear. In order to shed light on this aspect we evaluated the impact of two nitrogen containing bisphosphonates, Alendronate (ALN) and Zoledronate (ZOL), on human primary pre-osteoblasts. At first, we showed an inhibitory effect on cell viability and alkaline phosphatase activity starting from µM concentrations of both drugs. In addition, an inhibitory trend on mineralized nodules deposition was observed. Then low doses of both ALN and ZOL rapidly increased the release of the pro-inflammatory mediators TNFα and IL-1ß, while increased DKK-1 and Sclerostin, both inhibitors of osteoblastogenesis. Finally, ALN and 10-7M ZOL decreased the expression of type I Collagen and Osteopontin, while both drugs slightly stimulated SPARC production. With these results, we would like to suggest a direct inhibitory action on bone-forming cells by nitrogen containing bisphosphonates.

17ß-estradiol differently affects osteogenic differentiation of mesenchymal stem/stromal cells from adipose tissue and bone marrow.

Adipose-derived and bone marrow stem/stromal cells (ASCs and BMSCs) have been often compared for their application in regenerative medicine, and several factors sustaining their differentiation and efficacy have been investigated. 17 ß-estradiol (E2) has been reported to influence some functions of progenitor cells. Here we studied the effects of 10 and 100nM E2 on ASC and BMSC vitality, proliferation and differentiation towards osteogenic and adipogenic lineages. E2 did not modulate ASC and BMSC vitality and growth rate, while the hormone produced a pro-adipogenic effect on both mesenchymal stem/stromal cells (MSCs). In particular, the synergy between 7-day pre-treatment and 100nM E2 led to the most evident result, increasing lipid vacuoles formation in ASCs and BMSCs of +44% and +82%, respectively. Despite the fact that E2 did not alter collagen deposition of osteo-induced MSCs, we observed a different modulation of ASC and BMSC alkaline phosphatase (ALP) activity. Indeed, this osteogenic marker was always enhanced by 17 ß-estradiol in BMSCs, and 7-day pre-treatment with 100nM E2 increased it of about 70%. In contrast, E2 weakened ASC osteogenic potential, reducing their ALP activity of about 20%, with the most evident effect on ASCs isolated from pre-menopausal women (-30%). Finally, we identified an estrogen receptor α (ERα) variant of about 37kDa expressed in both MSCs. Interestingly, adipogenic stimuli drastically reduced its expression, while osteogenic ones mildly increased this isoform in BMSCs only. In conclusion, E2 positively affected the adipogenic process of both MSCs while it favored osteogenic induction in BMSCs only, and both mesenchymal progenitors expressed a novel 37kDa ER-α variant whose expression was modulated during differentiation.

Hypoxia Promotes the Inflammatory Response and Stemness Features in Visceral Fat Stem Cells From Obese Subjects.

Low-grade chronic inflammation is a salient feature of obesity and many associated disorders. This condition frequently occurs in central obesity and is connected to alterations of the visceral adipose tissue (AT) microenvironment. Understanding how obesity is related to inflammation may allow the development of therapeutics aimed at improving metabolic parameters in obese patients. To achieve this aim, we compared the features of two subpopulations of adipose-derived stem cells (ASC) isolated from both subcutaneous and visceral AT of obese patients with the features of two subpopulations of ASC from the same isolation sites of non-obese individuals. In particular, the behavior of ASC of obese versus non-obese subjects during hypoxia, which occurs in obese AT and is an inducer of the inflammatory response, was evaluated. Obesity deeply influenced ASC from visceral AT (obV-ASC); these cells appeared to exhibit clearly distinguishable morphology and ultrastructure as well as reduced proliferation, clonogenicity and expression of stemness, differentiation and inflammation-related genes. These cells also exhibited a deregulated response to hypoxia, which induced strong tissue-specific NF-kB activation and an NF-kB-mediated increase in inflammatory and fibrogenic responses. Moreover, obV-ASC, which showed a less stem-like phenotype, recovered stemness features after hypoxia. Our findings demonstrated the peculiar behavior of obV-ASC, their influence on the obese visceral AT microenvironment and the therapeutic potential of NF-kB inhibitors. These novel findings suggest that the deregulated hyper-responsiveness to hypoxic stimulus of ASC from visceral AT of obese subjects may contribute via paracrine mechanisms to low-grade chronic inflammation, which has been implicated in obesity-related morbidity.

Effect of an Activated Platelet Concentrate on Differentiated Cells Involved in Tissue Healing.

Tissue healing is a complex process involving several players such as cells and growth factors released from platelets upon activation. Today, platelet concentrates (PCs) are used in many different medical fields including oral, orthopaedic, and reconstructive surgery since they allow growth factors delivery to the injured site, aiming at enhancing tissue regeneration. The purpose of this in vitro study was to evaluate the effect of the acellular plasma of an activated platelet concentrate obtained using a manual protocol, on the proliferation, and biological activity of differentiated cells involved in tissue healing. Human osteoblasts and dermal fibroblasts were grown in serum-free medium supplemented with PC derived from several donors. Human osteoblast and human dermal fibroblast proliferation was assessed by MTT test after 7 days and cells were count up to 12-day incubation. Human osteoblast osteo-differentiation was tested after 7 and 14-day incubation by alkaline phosphatase assay. The addition of PC to the culture medium caused an increased proliferation with respect to cells grown in standard condition. The results of the present study suggest that PC supports the proliferation of terminally differentiated cells involved in wound healing and tissue regeneration, confirming its beneficial clinical application in regenerative therapies.

Does Freeze-Thawing Influence the Effects of Platelet Concentrates? An In Vitro Study on Human Adipose-Derived Stem Cells.

Human adipose-derived stem cells (hASCs) have been proposed as a possible therapy for tissue regeneration in aesthetic, plastic, and reconstructive surgery. Today, platelet concentrates are used in a wide range of disciplines, but their storage has become a controversial aspect. The purpose of this in vitro study was to evaluate the effect of plasma rich in growth factors (PRGF), after a freeze-thawing cycle, on the proliferation and biological activity of progenitor cells involved in soft tissue healing. Different formulations of activated PRGF were added to hASCs cultured in serum-free medium. Cell proliferation was assessed by MTT test and cell count up to 7 and 12-day incubation. Osteo-differentiation ability of hASCs was also tested after 7 and 14-day incubation by alkaline phosphatase assay. The effects of 4 PRGF preparations (fresh/frozen and with/without platelets) were compared with corresponding formulations of plasma poor in growth factors and with standard medium. hASCs cultured in the presence of platelet concentrates increased proliferation rate with respect to cells grown in standard medium without significant differences among all the tested plasma formulations on cell viability up to 12 days of culture. PRGF activity is preserved after cryopreservation and platelet-rich preparations promoted osteo-differentiation of hASCs at day 7. In conclusion, PRGF supports the proliferation and the differentiation of progenitor cells in vitro also when applied after cryopreservation. Platelet concentrates, either alone or in combination with mesenchymal stem cells, might be a valuable tool in the field of tissue regeneration.

Boosting the therapeutic potential of cell secretome against osteoarthritis: Comparison of cytokine-based priming strategies.

The secretome, or conditioned medium (CM), from Mesenchymal Stem/stromal Cells (MSCs) has recently emerged as a promising cell-free therapeutic against osteoarthritis (OA), capable of promoting cartilage regeneration and immunoregulation. Priming MSCs with 10 ng/ml tumor necrosis factor α (TNFα) and/or 10 ng/ml interleukin 1ß (IL-1ß) aims at mimicking the pathological milieu of OA joints in order to target their secretion towards a pathology-tailored phenotype. Here we compare the composition of the CM obtained after 24 or 72 h from untreated and cytokine-treated adipose-derived MSCs (ASCs). The 72-hour double-primed CM presents a higher total protein yield, a larger number of extracellular vesicles, and a greater concentration of bioactive lipids, in particular sphingolipids, fatty acids, and eicosanoids. Moreover, the levels of several factors involved in immunomodulation and regeneration, such as TGF-ß1, PGE2, and CCL-2, are strongly upregulated. Additionally, the differential profiling of 80 bioactive molecules indicates that primed CM is enriched in immune cell chemotaxis and migration factors. Our results indicate that pre-conditioning ASCs with inflammatory cytokines can modulate the composition of their CM, promoting the release of factors with recognized anti-inflammatory, chondroprotective, and immunoregulatory properties.

FGF23 Expression Is a Promising Immunohistochemical Diagnostic Marker for Undifferentiated Pleomorphic Sarcoma of Bone (UPSb).

Background: Undifferentiated pleomorphic sarcoma of bone (UPSb) is a rare primary bone sarcoma that lacks a specific line of differentiation. Distinguishing between UPSb and other malignant bone sarcomas, including dedifferentiated chondrosarcoma and osteosarcoma, is challenging due to their overlapping features. We have previously identified that UPSb tumours have elevated mRNA levels of Fibroblast Growth Factor 23 (FGF23) transcripts compared to other sarcomas including osteosarcoma. In the present study, we evaluated the specificity and practicality of FGF23 immunoreactivity as a specific diagnostic tool to differentiate UPSb tumours from osteosarcomas and dedifferentiated chondrosarcomas. Methods: A total of 10 UPSb, 10 osteosarcoma, and 10 dedifferentiated chondrosarcoma cases (all high-grade), were retrieved and immunohistochemistry for FGF23 was performed. Results: FGF23 protein was expressed at high levels in 80-90% of undifferentiated pleomorphic sarcoma of the bone cases, whereas it was expressed at significantly lower levels in dedifferentiated chondrosarcoma and osteosarcoma cases. A semiquantitative analysis, considering the intensity of immunoreactivity, confirmed significantly elevated FGF23 expression levels in UPSb tissues compared to those observed in osteosarcoma and dedifferentiated chondrosarcoma tissues. Conclusions: The results we present here suggest that FGF23 immunohistochemistry may be a useful tool to aid in differentiating UPSb from morphologically similar malignant bone sarcomas, especially in situations where sampling is restricted and there is limited clinical information available.

Significant association between FGFR1 mutation frequency and age in central giant cell granuloma.

Central giant cell granulomas (CGCG) are rare intraosseous osteolytic lesions of uncertain aetiology. Despite the benign nature of this neoplasia, the lesions can rapidly grow and become large, painful, invasive, and destructive. The identification of molecular drivers could help in the selection of targeted therapies for specific cases. TRPV4, KRAS and FGFR1 mutations have been associated with these lesions but no correlation between the mutations and patient features was observed so far. In this study, we analysed 17 CGCG cases of an Italian cohort and identified an interesting and significant (p=0.0021) correlation between FGFR1 mutations and age. In detail, FGFR1 mutations were observed frequently and exclusively in CGCG from young (<18 years old) patients (4/5 lesions, 80%). Furthermore, the combination between ours and previously published data confirmed a significant difference in the frequency of FGFR1 mutations in CGCG from patients younger than 18 years at the time of diagnosis (9/23 lesions, 39%) when compared to older patients (1/31 lesions, 0.03%; p=0.0011), thus corroborating our observation in a cohort of 54 patients. FGFR1 variants in young CGCG patients could favour fast lesion growth, implying that they seek medical attention earlier. Our observation might help prioritise candidates for FGFR1 testing, thus opening treatment options with FGFR inhibitors.

Chemical and genetic blockade of HDACs enhances osteogenic differentiation of human adipose tissue-derived stem cells by oppositely affecting osteogenic and adipogenic transcription factors.

The human adipose-tissue derived stem/stromal cells (hASCs) are an interesting source for bone-tissue engineering applications. Our aim was to clarify in hASCs the role of acetylation in the control of Runt-related transcription factor 2 (Runx2) and Peroxisome proliferator activated receptor (PPAR) γ. These key osteogenic and adipogenic transcription factors are oppositely involved in osteo-differentiation. The hASCs, committed or not towards bone lineage with osteoinductive medium, were exposed to HDACs chemical blockade with Trichostatin A (TSA) or were genetically silenced for HDACs. Alkaline phosphatase (ALP) and collagen/calcium deposition, considered as early and late osteogenic markers, were evaluated concomitantly as index of osteo-differentiation. TSA pretreatment, useful experimental protocol to analyse pan-HDAC-chemical inhibition, and switch to osteogenic medium induced early-osteoblast maturation gene Runx2, while transiently decreased PPARγ and scarcely affected late-differentiation markers. Time-dependent effects were observed after knocking-down of HDAC1 and 3: Runx2 and ALP underwent early activation, followed by late-osteogenic markers increase and by PPARγ/ALP activity diminutions mostly after HDAC3 silencing. HDAC1 and 3 genetic blockade increased and decreased Runx2 and PPARγ target genes, respectively. Noteworthy, HDACs knocking-down favoured the commitment effect of osteogenic medium. Our results reveal a role for HDACs in orchestrating osteo-differentiation of hASCs at transcriptional level, and might provide new insights into the modulation of hASCs-based regenerative therapy.

Lipidomics of Cell Secretome Combined with the Study of Selected Bioactive Lipids in an In Vitro Model of Osteoarthritis.

Analytical advancements in lipidomics have enabled large-scale investigations of lipid biology. Herein, we focused on four bioactive lipid families, namely polyunsaturated fatty acids, eicosanoids, endocannabinoids, and N-acylethanolamines, and their involvement in the mesenchymal stem cells (MSC)-related inflammatory scenario. Since MSC secretome may represent a valid therapeutic alternative, here, the complete secretome and its vesicular component from adipose- and bone marrow-derived MSC and dermal fibroblasts were characterized by targeted mass spectrometry lipidomics. The 2-arachidonoylglycerol (2AG) and the palmitoylethanolamide (PEA), previously quantified in the MSC's secretome, were further investigated by assessing hypothetical effects in an in vitro model of osteoarthritis (OA) based on human primary articular chondrocytes (CH) stimulated with tumor necrosis factor alpha (TNFα). TNFα enhances the release of the inflammatory lipid prostaglandin E2 (PGE2), and an additional increment was observed when CH were treated with both TNFα and 2AG. In contrast, PEA downmodulates the PGE2 release to the levels of unstimulated CH suggesting a protective effect. TNFα also increases the expression of cyclooxygenase 2 (COX2), in particular when combined with 2AG, while PEA partly blunts TNFα-induced COX2 expression. In addition, TNFα-stimulated CH produce significantly higher levels of the inflammatory mediator nitric oxide (NO) both in the presence and in the absence of 2AG, and PEA was able to partially reduce NO release. Our results show a first partial lipidomic profile of MSC and DF secretome and suggest a possible implication of bioactive lipids in the OA scenario and in the future use of these cell-free products as innovative therapeutics.

Polythiophene-mediated light modulation of membrane potential and calcium signalling in human adipose-derived stem/stromal cells.

Recent progress in the fields of regenerative medicine and tissue engineering has been strongly fostered both by the investigation of crucial cues, able to trigger the regeneration of damaged tissues, and by the development of ad hoc functional materials, capable of selectively (re-)activating relevant physiological pathways. In parallel to the successful realization of biochemical cues and the optimization of delivery protocols, the use of biophysical stimuli has been emerging as an alternative, highly effective strategy. Techniques based on electrical, magnetic and mechanical stimulation have been reported to efficiently direct differentiation of stem cells and modulate cell physiology at different developmental stages. In this framework, the use of optical stimulation represents a valuable approach, possibly overcoming current limitations of chemical cues, like limited spatial and temporal resolution and poor control over the extracellular environment. Surprisingly, the effects of light on the physiological properties (light toxicity, cell membrane potential, and cell ionic trafficking) of undifferentiated cells, as well as on their differentiation pathways, were investigated to a very limited extent and rarely quantified in a systematic way. In this work, we aim at clarifying the effects of optical excitation on the physiological behaviour of undifferentiated human adipose-derived stem cells (hASC), cultured on top of a light-sensitive conjugated polymer, region-regular poly-3-hexyl-thiophene (P3HT). Interestingly, we observe statistically significant modulation of the cell membrane potential, as well as noticeable effects on intracellular calcium signalling, triggered by P3HT excitation upon green light stimuli. Possible mechanisms involved in the signal transduction pathways are considered and critically discussed. The capability to modulate the physiological response of hASC upon photoexcitation, in a highly controlled and selective manner, provides a promptly available and non invasive diagnostic tool, thus contributing to the understanding of the complex machinery behind stem cells and material interfaces. Moreover, it may open the route to novel techniques to drive the differentiation path with unprecedented versatility and operational easiness.

Human Osteochondral Explants as an Ex Vivo Model of Osteoarthritis for the Assessment of a Novel Class of Orthobiologics.

Osteoarthritis (OA) is a highly prevalent joint disease still lacking effective treatments. Its multifactorial etiology hampers the development of relevant preclinical models to evaluate innovative therapeutic solutions. In the last decade, the potential of Mesenchymal Stem Cell (MSC) secretome, or conditioned medium (CM), has emerged as an alternative to cell therapy. Here, we investigated the effects of the CM from adipose MSCs (ASCs), accounting for both soluble factors and extracellular vesicles, on human osteochondral explants. Biopsies, isolated from total knee replacement surgery, were cultured without additional treatment or with the CM from 106 ASCs, both in the absence and in the presence of 10 ng/mL TNFα. Tissue viability and several OA-related hallmarks were monitored at 1, 3 and 6 days. Specimen viability was maintained over culture. After 3 days, TNFα induced the enhancement of matrix metalloproteinase activity and glycosaminoglycan release, both efficiently counteracted by CM. The screening of inflammatory lipids, proteases and cytokines outlined interesting modulations, driving the attention to new players in the OA process. Here, we confirmed the promising beneficial action of ASC secretome in the OA context and profiled several bioactive factors involved in its progression, in the perspective of accelerating an answer to its unmet clinical needs.

Proteomic analysis of extracellular vesicles and conditioned medium from human adipose-derived stem/stromal cells and dermal fibroblasts.

Conditioned medium (CM) and extracellular vesicles (EV) from Adipose-derived Stem/stromal cells (ASC) and Dermal fibroblasts (DF) represent promising tools for therapeutic applications. Which one should be preferred is still under debate and no direct comparison of their proteome has been reported yet. Here, we apply quantitative proteomics to explore the protein composition of CM and EV from the two cell types. Data are available via ProteomeXchange (identifier PXD020219). We identified 1977 proteins by LC-MS/MS proteomic analysis. Unsupervised clustering analysis and PCA recognized CM and EV as separate groups. We identified 68 and 201 CM and EV specific factors. CM were enriched in proteins of endoplasmic reticulum, Golgi apparatus and lysosomes, whereas EV contained a large amount of GTPases, ribosome and translation factors. The analysis of ASC and DF secretomes revealed the presence of cell type-specific proteins. ASC-CM and -EV carried factors involved in ECM organization and immunological regulation, respectively. Conversely, DF-CM and -EV were enriched in epithelium development associated factors and -EV in Wnt signaling factors. In conclusion, this analysis provides evidence of a different protein composition between CM and EV and of the presence of cell type-specific bioactive mediators suggesting their specific future use as advanced therapy medicinal products. SIGNIFICANCE: The use of cell secretome presents several advantages over cell therapy such as the lower risks associated to the administration step and the avoidance of any potential risk of malignant transformation. The main secretome preparations consist in concentrated conditioned medium (CM) and extracellular vesicles (EV). Both of them showed well-documented therapeutic potentials. However, it is still not clear in which case it should be better to use one preparation over the other and an exhaustive comparison between their proteome has not been performed yet. The choice of the cell source is another relevant aspect that still needs to be addressed. In order to shed light on these questions we explored the protein composition of CM and EV obtained from Adipose-derived Stem/stromal Cells (ASC) and Dermal Fibroblasts (DF), by a comprehensive quantitative proteomics approach. The analysis showed a clear distinction between CM and EV proteome. CM were enriched in proteins of endoplasmic reticulum, Golgi apparatus and lysosomes, whereas EV contained a large amount of GTPases, ribosome and translation-related factors. Furthermore, the analysis of ASC and DF secretomes revealed specific biological processes for the different cell products. ASC secretome presented factors involved in ECM organization (hyaluronan and glycosaminoglycan metabolism) and immunological regulation (e.g. macrophage and IkB/NFkB signaling regulation), respectively. On the other hand, DF-CM and -EV were both enriched in epithelium development associated factors, whilst DF-CM in proteins involved in cellular processes regulation and -EV in Wnt signaling factors. In conclusion, our study shed a light on the different protein composition of CM and EV of two promising cell types, spanning from basic processes involved in secretion to specific pathways supporting their therapeutic potential and their possible future use as advanced therapy medicinal products.

Raman Fingerprint of Extracellular Vesicles and Conditioned Media for the Reproducibility Assessment of Cell-Free Therapeutics.

Extracellular Vesicles (EVs) and Conditioned Medium (CM) are promising cell-free approaches to repair damaged and diseased tissues for regenerative rehabilitation purposes. They both entail several advantages, mostly in terms of safety and handling, compared to the cell-based treatment. Despite the growing interest in both EVs and CM preparations, in the light of a clinical translation, a number of aspects still need to be addressed mainly because of limits in the reproducibility and reliability of the proposed protocols. Raman spectroscopy (RS) is a non-destructive vibrational investigation method that provides detailed information about the biochemical composition of a sample, with reported ability in bulk characterization of clusters of EVs from different cell types. In the present brief report, we acquired and compared the Raman spectra of the two most promising cell-free therapeutics, i.e., EVs and CM, derived from two cytotypes with a history in the field of regenerative medicine, adipose-derived mesenchymal stem/stromal cells (ASCs) and dermal fibroblasts (DFs). Our results show how RS can verify the reproducibility not only of EV isolation, but also of the whole CM, thus accounting for both the soluble and the vesicular components of cell secretion. RS can provide hints for the identification of the soluble factors that synergistically cooperate with EVs in the regenerative effect of CM. Still, we believe that the application of RS in the pipeline of cell-free products preparation for therapeutic purposes could help in accelerating translation to clinics and regulatory approval.