Stem cell extracellular vesicles for neural regeneration

In the last decade, multipotent mesenchymal stromal cells (MSCs) demonstrated a significant therapeutic efficacy, particularly in cell therapy approaches aiming at tissue regeneration. MSCs exert their action via trophic support, induction of angiogenesis, immunomodulation and reduction of necrosis at affected tissues. Importantly, these regenerative and protective properties are largely associated to MSC secretome. Unfortunately, cell-based approaches not always meet the criteria for a smooth translation to the clinic. For instance, the use of stem cells in pathologies with a very short therapeutic window, such as few hours, is not compatible with the requested minimal criteria for MSC release, before administration to the patient. Notwithstanding, in the regenerative medicine field, the MSC mechanism of action paradigm was recently extended to include the action of extracellular vesicles (EVs), which are cytoplasm-containing cellular bodies secreted by a wide range of cell types. Intriguingly, many studies reported that EVs generated by MSCs are able to recapitulate the majority of the regenerative properties of parental MSCs. Starting from these premises, the objectives of the present doctoral research project were: to address EV-mediated cell-to-cell communication as novel MSC mechanism of action; to address reprogrammed MSC-EV generation; to define, for the first time in the literature, stem cell EV molecular content (e.g.: miRNome), comparing reprogrammed to non-reprogrammed MSC-EVs; to challenge stem cell-EV therapeutic potential in a model of acute tissue damage, as a proof-of-concept for feasibility and effectiveness of a stem cell-based albeit cell-free regenerative strategy. Intriguingly, EVs may be produced in a ready-to-use formulation, so that clinicians could use them as soon as a therapeutic need arises, also in the case of an urgent one. In this way, EV-shuttled MSC regenerative properties could exert beneficial effects also on pathologies currently lacking any cell therapy option. To develop this innovative therapeutic strategy, MSCs were isolated from different tissues and their biological properties were evaluated in order to choose the MSC source most suitable for the implementation of the project. Thus, both MSC transcriptome and immunophenotype were addressed. MSCs from adult sources (e.g.: bone marrow) showed senescence-related features in vitro, correlated to donor’s age in vivo. On the other hand, MSCs from perinatal tissues (e.g.: cord blood) showed a phenotype more similar to that of pericytes, which are the in vivo progenitors of MSCs. Therefore, cord blood was chosen as MSC source, also in the prospective of clinical translation, since public banking of cord blood units for clinical use already exists worldwide. Next, thanks to an extended analysis of the stromal populations present in cord blood, a MSC subpopulation showing higher proliferation properties and significantly longer telomeres was isolated. In addition, the standard cord blood MSC isolation protocol was improved, leading to an efficiency of 80%. Eventually, MSC secretome-associated anti-inflammatory and anti-apoptotic properties were observed in vitro and in vivo. In order to investigate if EVs contributed to MSC paracrine properties, MSC-EV secretion and regenerative properties were assessed. The MSC-EV therapeutic effectiveness was challenged in an in vitro model of acute tissue damage. Intriguingly, MSC-EVs could rescue damage-induced cell mortality, showing the same protective effect of parental MSCs. In spite of the use of a high proliferative cord blood MSC subpopulation, primary cultures still show a limited lifespan. In order to increase their replicative potential and to better exploit their EV production, induced cellular reprogramming was tested on MSCs as an alternative to traditional immortalization techniques. In this way, MSC-derived cell lines endowed with unlimited lifespan were generated, and permanent modification of their genome was avoided. The next step was to confirm the generation of EVs from reprogrammed MSCs, since reprogramming drastically changes cell identity. Furthermore, the EV miRNome load of reprogrammed and non-reprogrammed MSCs was addressed. Importantly, the majority of miRNAs were common between the two samples, indicating that reprogramming did not change the EV miRNA content. This result could have relevant consequences on the functional features of reprogrammed MSC-EVs, since EV-mediated miRNA transfer from donor to target cells was proposed as one of MSC mechanisms of action. In the last part of this doctoral research, stem cell (non-reprogrammed and reprogrammed MSCs)-EV therapeutic effectiveness was addressed and compared to that of parental MSCs. In order to do that, an organotypic ex vivo mouse model of brain ischemia was used. This model recapitulated the modulation of some ischemic damage-related parameters, including increased secretion of inflammatory cytokines, high tissue necrosis and the impairment of neuronal and astrocytic cell populations. Therefore, this model mimicked early phase events of brain ischemia, whose thrombolytic clinical treatment must be administered within 3-6 hours of first signs of ischemia. Notably, stem cell-EVs were tested for the first time in this pathological context to verify their potential role in tissue regeneration. Strikingly, stem cell-EV administration to affected tissues showed significant neuroprotective properties, which were comparable to those of parental MSCs. Importantly, the ischemic damage-related parameters previously described were rescued. In particular, inflammatory-associated parameters underwent the most statistically significant decrease, showing levels similar to or better than those of the uninjured brain tissue. This is of uttermost importance, considering that chronic inflammation is detrimental to tissue regeneration. To conclude, the results of the present PhD thesis confirmed the feasibility of stem cell EV-based therapies in regenerative medicine approaches. In the future, this innovative EV therapy may be applied to pathological contexts currently without a cell therapy option. In the framework of advanced therapy medicinal products, the new drug would be the EVs, rather than the parental stem cells. Finally, EVs could play the role of ready-to-use anti-inflammatory molecule carriers, in order to guarantee a rapid therapeutic action for the regeneration of injured tissues

A Chemically Defined Medium-Based Strategy to Efficiently Generate Clinically Relevant Cord Blood Mesenchymal Stromal Colonies.

During the last decade it has been demonstrated that mesenchymal progenitors are present and can be isolated also from cord blood (CB). Recently, we managed to set up a standard protocol allowing the isolation of mesenchymal stromal cells (MSCs) with high proliferative potential and multiple differentiation capabilities, whereas the generation rate of MSC-initiating colonies could still be further improved. Herein, we strikingly succeeded in defining some simple and basic culture conditions based on the use of a chemically defined medium that increased the colony isolation efficiency up to almost 80% of processed CB units. Importantly, this result was achieved irrespective of CB unit white blood cell content and time elapsed from delivery, two limiting parameters involved with processing CB units. Thus, this high efficiency is guaranteed without strict selection of the starting material. In addition, since we are profoundly concerned about how different culture conditions can influence cell behavior, we devoted part of this study to in-depth characterization of the established CB-MSC populations to confirm their stemness features in this novel isolation and culture system. Therefore, an extended study of their immunophenotype, including classical pericytic markers, and a detailed molecular analysis addressing telomere length and also stemness-related microRNA contribution were performed. In summary, we propose a straightforward, extremely efficient, and reliable approach to isolate and expand thoroughly characterized CB-MSCs, even when poor-quality CB units are the only available source, or there is no space for an isolation to fail

Carbon dots conjugated to SN38 for improved colorectal anticancer therapy

Irinotecan (CTP-11) is one of the standard therapies for colorectal cancer (CRC). CTP-11 is enzymatically con-verted to the hydrophobic 7-ethyl-10-hydroxycamptothecin (SN38), a one hundred-fold more active metabolite. Conjugation of hydrophobic anticancer drugs to nanomaterials is a strategy to improve their solubility, efficacy, and selectivity. Carbon dots (CDs) have garnered interest for their small sizes (<10 nm), low toxicity, high water solubility, and bright fluorescence. This paper describes the use of CDs to improve drug vehiculation, stability, and chemotherapeutic efficiency of SN38 through a direct intracellular uptake in CRC. The covalent conjugation of SN38 to CDs via a carbamate bond provides a CD-SN38 hybrid material for slow, sustained, and pH-responsive drug release. CD-SN38 successfully penetrates the CRC cells with a release in the nucleus affecting first the cell cycle and then the cytoskeleton. Moreover, CD-SN38 leads to a deregulation of the extracellular matrix (ECM), one of the major components of the cancer niche considered a possible target therapy for reducing the cancer progression. This work shows the combined therapeutic and imaging potential of CD-based hybrid materials for the treatment of CRC. Future efforts for targeted therapy of chronic diseases characterized by altered ECM deposition, such as chronic kidney disease and chronic allograft nephropathy in kidney transplant patients are envisaged

FOXP1 circular RNA sustains mesenchymal stem cell identity via microRNA inhibition

Stem cell identity and plasticity are controlled by master regulatory genes and complex circuits also involving non-coding RNAs. Circular RNAs (circRNAs) are a class of RNAs generated from protein-coding genes by backsplicing, resulting in stable RNA structures devoid of free 5' and 3' ends. Little is known of the mechanisms of action of circRNAs, let alone in stem cell biology. In this study, for the first time, we determined that a circRNA controls mesenchymal stem cell (MSC) identity and differentiation. High-throughput MSC expression profiling from different tissues revealed a large number of expressed circRNAs. Among those, circFOXP1 was enriched in MSCs compared to differentiated mesodermal derivatives. Silencing of circFOXP1 dramatically impaired MSC differentiation in culture and in vivo. Furthermore, we demonstrated a direct interaction between circFOXP1 and miR-17-3p/miR-127-5p, which results in the modulation of non-canonical Wnt and EGFR pathways. Finally, we addressed the interplay between canonical and non-canonical Wnt pathways. Reprogramming to pluripotency of MSCs reduced circFOXP1 and non-canonical Wnt, whereas canonical Wnt was boosted. The opposing effect was observed during generation of MSCs from human pluripotent stem cells. Our results provide unprecedented evidence for a regulatory role for circFOXP1 as a gatekeeper of pivotal stem cell molecular networks

A circular RNA map for human induced pluripotent stem cells of foetal origin

Background Adult skin fibroblasts represent the most common starting cell type used to generate human induced pluripotent stem cells (F-hiPSC) for clinical studies. Yet, a foetal source would offer unique advantages, primarily the absence of accumulated somatic mutations. Herein, we generated hiPSC from cord blood multipotent mesenchymal stromal cells (MSC-hiPSC) and compared them with F-hiPSC. Assessment of the full activation of the pluripotency gene regulatory network (PGRN) focused on circular RNA (circRNA), recently proposed to participate in the control of pluripotency. Methods Reprogramming was achieved by a footprint-free strategy. Self-renewal and pluripotency of cord blood MSC-hiPSC were investigated in vitro and in vivo, compared to parental MSC, to embryonic stem cells and to F-hiPSC. High-throughput array-based approaches and bioinformatics analyses were applied to address the PGRN. • View related content for this article Findings Cord blood MSC-hiPSC successfully acquired a complete pluripotent identity. Functional comparison with F-hiPSC showed no differences in terms of i) generation of mesenchymal-like derivatives, ii) their subsequent adipogenic, osteogenic and chondrogenic commitment, and iii) their hematopoietic support ability. At the transcriptional level, specific subsets of mRNA, miRNA and circRNA (n = 4,429) were evidenced, casting a further layer of complexity on the PGRN regulatory crosstalk. Interpretation A circRNA map of transcripts associated to naïve and primed pluripotency is provided for hiPSC of clinical-grade foetal origin, offering insights on still unreported regulatory circuits of the PGRN to consider for the optimization and development of efficient differentiation protocols for clinical translation

Large-scale production of extracellular vesicles: Report on the “massivEVs” ISEV workshop

Extracellular vesicles (EVs) large-scale production is a crucial point for the translation of EVs from discovery to application of EV-based products. In October 2021, the International Society for Extracellular Vesicles (ISEV), along with support by the FET-OPEN projects, “The Extracellular Vesicle Foundry” (evFOUNDRY) and “Extracellular vesicles from a natural source for tailor-made nanomaterials” (VES4US), organized a workshop entitled “massivEVs” to discuss the potential challenges for translation of EV-based products. This report gives an overview of the topics discussed during “massivEVs”, the most important points raised, and the points of consensus reached after discussion among academia and industry representatives. Overall, the review of the existing EV manufacturing, upscaling challenges and directions for their resolution highlighted in the workshop painted an optimistic future for the expanding EV field

Stem cell extracellular vesicles for neural regeneration

In the last decade, multipotent mesenchymal stromal cells (MSCs) demonstrated a significant therapeutic efficacy, particularly in cell therapy approaches aiming at tissue regeneration. MSCs exert their action via trophic support, induction of angiogenesis, immunomodulation and reduction of necrosis at affected tissues. Importantly, these regenerative and protective properties are largely associated to MSC secretome. Unfortunately, cell-based approaches not always meet the criteria for a smooth translation to the clinic. For instance, the use of stem cells in pathologies with a very short therapeutic window, such as few hours, is not compatible with the requested minimal criteria for MSC release, before administration to the patient. Notwithstanding, in the regenerative medicine field, the MSC mechanism of action paradigm was recently extended to include the action of extracellular vesicles (EVs), which are cytoplasm-containing cellular bodies secreted by a wide range of cell types. Intriguingly, many studies reported that EVs generated by MSCs are able to recapitulate the majority of the regenerative properties of parental MSCs. Starting from these premises, the objectives of the present doctoral research project were: to address EV-mediated cell-to-cell communication as novel MSC mechanism of action; to address reprogrammed MSC-EV generation; to define, for the first time in the literature, stem cell EV molecular content (e.g.: miRNome), comparing reprogrammed to non-reprogrammed MSC-EVs; to challenge stem cell-EV therapeutic potential in a model of acute tissue damage, as a proof-of-concept for feasibility and effectiveness of a stem cell-based albeit cell-free regenerative strategy. Intriguingly, EVs may be produced in a ready-to-use formulation, so that clinicians could use them as soon as a therapeutic need arises, also in the case of an urgent one. In this way, EV-shuttled MSC regenerative properties could exert beneficial effects also on pathologies currently lacking any cell therapy option. To develop this innovative therapeutic strategy, MSCs were isolated from different tissues and their biological properties were evaluated in order to choose the MSC source most suitable for the implementation of the project. Thus, both MSC transcriptome and immunophenotype were addressed. MSCs from adult sources (e.g.: bone marrow) showed senescence-related features in vitro, correlated to donor’s age in vivo. On the other hand, MSCs from perinatal tissues (e.g.: cord blood) showed a phenotype more similar to that of pericytes, which are the in vivo progenitors of MSCs. Therefore, cord blood was chosen as MSC source, also in the prospective of clinical translation, since public banking of cord blood units for clinical use already exists worldwide. Next, thanks to an extended analysis of the stromal populations present in cord blood, a MSC subpopulation showing higher proliferation properties and significantly longer telomeres was isolated. In addition, the standard cord blood MSC isolation protocol was improved, leading to an efficiency of 80%. Eventually, MSC secretome-associated anti-inflammatory and anti-apoptotic properties were observed in vitro and in vivo. In order to investigate if EVs contributed to MSC paracrine properties, MSC-EV secretion and regenerative properties were assessed. The MSC-EV therapeutic effectiveness was challenged in an in vitro model of acute tissue damage. Intriguingly, MSC-EVs could rescue damage-induced cell mortality, showing the same protective effect of parental MSCs. In spite of the use of a high proliferative cord blood MSC subpopulation, primary cultures still show a limited lifespan. In order to increase their replicative potential and to better exploit their EV production, induced cellular reprogramming was tested on MSCs as an alternative to traditional immortalization techniques. In this way, MSC-derived cell lines endowed with unlimited lifespan were generated, and permanent modification of their genome was avoided. The next step was to confirm the generation of EVs from reprogrammed MSCs, since reprogramming drastically changes cell identity. Furthermore, the EV miRNome load of reprogrammed and non-reprogrammed MSCs was addressed. Importantly, the majority of miRNAs were common between the two samples, indicating that reprogramming did not change the EV miRNA content. This result could have relevant consequences on the functional features of reprogrammed MSC-EVs, since EV-mediated miRNA transfer from donor to target cells was proposed as one of MSC mechanisms of action. In the last part of this doctoral research, stem cell (non-reprogrammed and reprogrammed MSCs)-EV therapeutic effectiveness was addressed and compared to that of parental MSCs. In order to do that, an organotypic ex vivo mouse model of brain ischemia was used. This model recapitulated the modulation of some ischemic damage-related parameters, including increased secretion of inflammatory cytokines, high tissue necrosis and the impairment of neuronal and astrocytic cell populations. Therefore, this model mimicked early phase events of brain ischemia, whose thrombolytic clinical treatment must be administered within 3-6 hours of first signs of ischemia. Notably, stem cell-EVs were tested for the first time in this pathological context to verify their potential role in tissue regeneration. Strikingly, stem cell-EV administration to affected tissues showed significant neuroprotective properties, which were comparable to those of parental MSCs. Importantly, the ischemic damage-related parameters previously described were rescued. In particular, inflammatory-associated parameters underwent the most statistically significant decrease, showing levels similar to or better than those of the uninjured brain tissue. This is of uttermost importance, considering that chronic inflammation is detrimental to tissue regeneration. To conclude, the results of the present PhD thesis confirmed the feasibility of stem cell EV-based therapies in regenerative medicine approaches. In the future, this innovative EV therapy may be applied to pathological contexts currently without a cell therapy option. In the framework of advanced therapy medicinal products, the new drug would be the EVs, rather than the parental stem cells. Finally, EVs could play the role of ready-to-use anti-inflammatory molecule carriers, in order to guarantee a rapid therapeutic action for the regeneration of injured tissues.Negli ultimi anni, le cellule stromali mesenchimali multipotenti umane (CSM) hanno mostrato una grande efficacia terapeutica, soprattutto in approcci di terapia cellulare aventi come obiettivo la rigenerazione tissutale. L’azione delle CSM avviene attraverso supporto trofico, induzione di angiogenesi, modulazione della risposta immunitaria e diminuzione della necrosi a livello dei tessuti colpiti. Inoltre, recente letteratura ha dimostrato che queste capacità rigenerative e protettive sono in larga parte associate al secretoma delle CSM. Purtroppo, gli approcci di terapia cellulare non sono sempre traslabili alla clinica. Ad esempio, l’utilizzo di cellule staminali in patologie caratterizzate da una finestra terapeutica molto stretta, dell’ordine di poche ore, non è compatibile con la necessità di scongelare e valutare i minimi standard di qualità delle CSM prima della somministrazione al paziente. Nonostante ciò, il paradigma del meccanismo d’azione delle CSM nel campo della medicina rigenerativa si è ulteriormente arricchito. Infatti, molti recenti studi hanno dimostrato che le vescicole extracellulari, ossia porzioni di citoplasma delimitate da membrana cellulare secrete dalle CSM, sono in grado di riprodurre la maggior parte delle proprietà rigenerative delle CSM stesse. Date queste premesse, gli obiettivi del progetto di ricerca del presente Dottorato sono stati i seguenti: indagare la comunicazione intercellulare tramite vescicole extracellulari quale innovativo meccanismo d’azione delle CSM; studiare la produzione di vescicole extracellulari da parte di CSM riprogrammate, e, per la prima volta in letteratura, definirne il contenuto molecolare (es.: miRNoma), a confronto con le CSM d’origine; testare il potenziale terapeutico di vescicole extracellulari da cellule staminali in un modello di danno tissutale acuto, come proof-of-concept della funzionalità di una strategia terapeutica cell-free. Infatti, le vescicole extracellulari potrebbero essere prodotte in formulazioni pronte all’uso, a immediata disposizione per ogni richiesta clinica, anche urgente. In questo modo le proprietà rigenerative delle CSM potrebbero essere veicolate dalle vescicole extracellulari anche in contesti patologici attualmente senza alcuna opzione di terapia cellulare. Per lo sviluppo di questa innovativa strategia terapeutica, CSM isolate da vari tessuti sono state caratterizzate e confrontate in base al loro trascrittoma e al loro immunofenotipo, allo scopo di valutarne le proprietà biologiche e quindi scegliere le CSM più adatte all’implementazione del progetto di Dottorato. Le CSM da tessuti adulti (e.g.: midollo osseo) hanno mostrato in vitro caratteristiche di senescenza correlate all’età del donatore in vivo. Al contrario, le CSM da tessuti perinatali (e.g.: sangue di cordone ombelicale) hanno mostrato un fenotipo più simile a quello dei periciti, ossia i progenitori delle CSM in vivo. Quindi, tenuto conto anche della traslabilità clinica, il sangue di cordone ombelicale è stato scelto come fonte di CSM, visto che la raccolta e la crioconservazione di unità di sangue placentare a fini terapeutici è già una realtà clinica. In seguito, un’analisi estesa delle popolazioni stromali presenti nel sangue di cordone ombelicale ha portato alla definizione di una sottopopolazione di CSM dotata di maggiori capacità proliferative e con una lunghezza del telomero significativamente più alta. Inoltre il protocollo standard di isolamento delle CSM da sangue di cordone ombelicale è stato migliorato, arrivando ad un’efficienza di circa 80%. Infine, le proprietà anti-infiammatorie e anti-apoptotiche del secretoma delle CSM sono state studiate sia in vitro che in vivo. Al fine di verificare se le vescicole extracellulari contribuissero alle proprietà paracrine delle CSM, se ne è caratterizzata la secrezione e se ne sono indagate le proprietà rigenerative. Una chiara efficacia terapeutica da parte delle vescicole extracellulari di CSM è stata dimostrata in un modello in vitro di danno tissutale acuto, in cui le vescicole extracellulari hanno eguagliato i risultati ottenuti con le CSM stesse. Nonostante l’utilizzo di CSM dalle elevate proprietà proliferative, la loro lifespan in coltura, in quanto cellule primarie, rimane limitata. Allo scopo di aumentarne il potenziale replicativo e di sfruttarne al meglio così la produzione di vescicole extracellulari, le CSM sono state sottoposte alla riprogrammazione cellulare indotta. In questo modo sono state generate linee cellulari derivate da CSM dal potenziale di crescita illimitato, evitando però di modificarne il genoma come nelle tradizionali tecniche di immortalizzazione. Siccome la riprogrammazione implica una modificazione radicale dell’identità della cellula d’origine, il passo successivo è stato quello di confermare la capacità di questa nuova popolazione di generare vescicole extracellulari, poi opportunamente caratterizzate. In particolare, il miRNoma delle vescicole extracellulari da cellule riprogrammate è stato oggetto di studio e di confronto con quello delle vescicole extracellulari delle CSM d’origine. Si è così potuto dimostrare che la maggior parte dei miRNA era presente nelle vescicole extracellulari sia prima che dopo la riprogrammazione. Ciò indica che il processo di riprogrammazione non ne ha alterato in modo sostanziale il contenuto. Questo potrebbe avere importanti ricadute sugli aspetti funzionali delle vescicole extracellulari da CSM riprogrammate. Infatti è stato ipotizzato che il trasferimento di miRNA specifici da cellule donatrici a cellule target mediato dalle vescicole extracellulari sia uno dei meccanismi d’azione delle CSM. Nell’ultima parte di questo progetto di Dottorato, l’utilità terapeutica delle vescicole extracellulari da cellule staminali (CSM e CSM riprogrammate) è stata confrontata con quella delle CSM d’origine. A tale scopo è stato utilizzato un modello ex vivo di ischemia cerebrale, in cui è stato osservato il movimento di alcuni parametri di danno ischemico acuto, tra cui un picco di produzione di citochine infiammatorie, una forte necrosi tissutale e una riduzione delle popolazioni cellulari neuronali e astrocitiche. Questo particolare modello mima infatti la fase acuta di questa condizione patologica, il cui trattamento a base di agenti trombolitici deve avvenire entro 3-6 ore dall’insorgenza dei primi sintomi. Quindi le vescicole extracellulari prodotte dalle CSM riprogrammate sono state testate per la prima volta in questo contesto patologico per verificare se potessero esercitare una funzione rigenerativa. La loro somministrazione al tessuto colpito dal danno ischemico ha generato uno spiccato effetto neuroprotettivo, pari a quello delle CSM d’origine, che ha riportato a valori simili a quelli del tessuto cerebrale non danneggiato i parametri di danno sopra descritti. Il risultato più interessante e statisticamente significativo è stato soprattutto a carico di quei parametri legati ai processi infiammatori, i quali sfavoriscono il recupero del danno tissutale. In conclusione, i risultati presentati in questa tesi di Dottorato confermano la possibilità di utilizzo di vescicole extracellulari secrete da cellule staminali in strategie di medicina rigenerativa. Questa innovativa extracellular vesicle therapy potrebbe in futuro essere applicata in contesti patologici per i quali ad oggi non è praticabile una terapia cellulare. A questo punto, nel quadro dei prodotti medicinali per le terapie avanzate, il “farmaco” non sarebbe più la cellula staminale, ma le rispettive vescicole extracellulari. Queste acquisirebbero così il ruolo di carrier di molecole antinfiammatorie, pronte all’uso e capaci di garantire un’azione terapeutica tempestiva per la rigenerazione di tessuti danneggiati

NG2 as an Identity and Quality Marker of Mesenchymal Stem Cell Extracellular Vesicles

The therapeutic potential of mesenchymal stem cell (MSC) extracellular vesicles (EV) is currently under investigation in many pathological contexts. Both adult and perinatal MSC are being considered as sources of EV. Herein, we address antigen expression of cord blood and bone marrow MSC and released EV to define an identity and quality parameter of MSC EV as a medicinal product in the context of clinical applications. The research focuses on EV-shuttled neural/glial antigen 2 (NG2), which has previously been detected as a promising surface marker to distinguish perinatal versus adult MSC. Indeed, NG2 was significantly more abundant in cord blood than bone marrow MSC and MSC EV. Ultracentrifuge-isolated EV were then challenged for their pro-angiogenic properties on an xCELLigence system as quality control. NG2+ cord blood MSC EV, but not bone marrow MSC EV, promote bFGF and PDGF-AA proliferative effect on endothelial cells. Likewise, they successfully rescue angiostatin-induced endothelial cell growth arrest. In both cases, the effects are NG2-dependent. These results point at NG2 as an identity and quality parameter for cord blood MSC EV, paving the way for their clinical translation

FGF23 and Fetuin-A Interaction and Mesenchymal Osteogenic Transformation

Recently, we found a strict bone association between Fibroblast growth factor 23 (FGF23) and Fetuin-A, both involved in cardiovascular and mineral bone disorders. In this study, an uninvestigated bone marrow positivity for both was found. Though the role of exogenous FGF23 on mesenchymal cells (MSCs) was reported, no information is as yet available on the possible production of this hormone by MSCs. To further analyze these uninvestigated aspects, we studied human primary cells and mouse and human cell lines by means of immunostaining, qRT-PCR, enzyme linked immunosorbent assays, chromatin immunoprecipitation, transfection, and a streamlined approach for the FGF23&#8315;Fetuin-A interaction called Duolink proximity ligation assay. Mesenchymal cells produce but do not secrete FGF23 and its expression increases during osteo-differentiation. Fibroblast growth factor 23 is also involved in the regulation of Fetuin-A by binding directly to the Fetuin-A promoter and then activating its transcription. Both FGF23 overexpression and addition induced an upregulation of Fetuin-A in the absence of osteo-inducer factors. Fibroblast growth factor 23 and Fetuin-A promoter were increased by osteo-inducer factors with this effect being abolished after FGF23 silencing. In conclusion, both FGF23 and Fetuin-A are present and strictly linked to each other in MSCs with FGF23 driving Fetuin-A production. This mechanism suggests a role for these two proteins in the osteoblast differentiation