Emerging Cancer Immunotherapies: Cutting-Edge Advances and Innovations in Development.

The rise in biological therapies has revolutionized oncology, with immunotherapy leading the charge through breakthroughs such as CAR-T cell therapy for melanoma and B-ALL. Modified bispecific antibodies and CAR-T cells are being developed to enhance their effectiveness further. However, CAR-T cell therapy currently relies on a costly ex vivo manufacturing process, necessitating alternative strategies to overcome this bottleneck. Targeted in vivo viral transduction offers a promising avenue but remains under-optimized. Additionally, novel approaches are emerging, such as in vivo vaccine boosting of CAR-T cells to strengthen the immune response against tumors, and dendritic cell-based vaccines are under investigation. Beyond CAR-T cells, mRNA therapeutics represent another promising avenue. Targeted delivery of DNA/RNA using lipid nanoparticles (LNPs) shows potential, as LNPs can be directed to T cells. Moreover, CRISPR editing has demonstrated the ability to precisely edit the genome, enhancing the effector function and persistence of synthetic T cells. Enveloped delivery vehicles packaging Cas9 directed to modified T cells offer a virus-free method for safe and effective molecule release. While this platform still relies on ex vivo transduction, using cells from healthy donors or induced pluripotent stem cells can reduce costs, simplify manufacturing, and expand treatment to patients with low-quality T cells. The use of allogeneic CAR-T cells in cancer has gained attraction for its potential to lower costs and broaden accessibility. This review emphasizes critical strategies for improving the selectivity and efficacy of immunotherapies, paving the way for a more targeted and successful fight against cancer.

CD73-Adenosinergic Axis Mediates the Protective Effect of Extracellular Vesicles Derived from Mesenchymal Stromal Cells on Ischemic Renal Damage in a Rat Model of Donation after Circulatory Death.

We propose a new organ-conditioning strategy based on mesenchymal stromal cell (MSCs)/extracellular vesicle (EVs) delivery during hypothermic perfusion. MSCs/EVs marker CD73 is present on renal proximal tubular cells, and it protects against renal ischemia-reperfusion injury by converting adenosine monophosphate into adenosine (ADO). In this study, after checking if CD73-silenced EVs (EVsi) would impact in vitro tubular-cell proliferation, we perfused kidneys of a rat model of donation after circulatory death, with Belzer solution (BS) alone, BS supplemented with MSCs, EVs, or EVsi. The ADO and ATP levels were measured in the effluents and tissues. Global renal ischemic damage score (GRS), and tubular cell proliferation index (IPT) were evaluated in the tissue. EVsi did not induce cell proliferation in vitro. Ex vivo kidneys perfused with BS or BS + EVsi showed the worst GRS and higher effluent ADO levels than the MSC- and EV-perfused kidneys. In the EV-perfused kidneys, the tissue and effluent ATP levels and IPT were the highest, but not if CD73 was silenced. Tissue ATP content was positively correlated with tissue ADO content and negatively correlated with effluent ADO level in all groups. In conclusion, kidney conditioning with EVs protects against ischemic damage by activating the CD73/ADO system.

Mesenchymal Stem Cell-Derived Extracellular Vesicles Protect Human Corneal Endothelial Cells from Endoplasmic Reticulum Stress-Mediated Apoptosis.

Corneal endothelial dystrophy is a relevant cause of vision loss and corneal transplantation worldwide. In the present study, we analyzed the effect of mesenchymal stem cell (MSC)-derived extracellular vesicles (MSC-EVs) in an in vitro model of corneal dystrophy, characterized by endoplasmic reticulum stress. The effects of MSC-EVs were compared with those of serum-derived EVs, reported to display a pro-angiogenic activity. MSC-EVs were able to induce a significant down-regulation of the large majority of endoplasmic reticulum stress-related genes in human corneal endothelial cells after exposure to serum deprivation and tunicamycin. In parallel, they upregulated the Akt pathway and limited caspase-3 activation and apoptosis. At variance, the effect of the serum EVs was mainly limited to Akt phosphorylation, with minimal or absent effects on endoplasmic reticulum stress modulation and apoptosis prevention. The effects of MSC-EVs were correlated to the transfer of numerous endoplasmic reticulum (ER)-stress targeting miRNAs to corneal endothelial cells. These data suggest a potential therapeutic effect of MSC-EVs for corneal endothelial endoplasmic reticulum stress, a major player in corneal endothelial dystrophy.

Human Liver Stem Cells: A Liver-Derived Mesenchymal Stromal Cell-Like Population With Pro-regenerative Properties.

Human liver stem cells (HLSCs) were described for the first time in 2006 as a new stem cell population derived from healthy human livers. Like mesenchymal stromal cells, HLSCs exhibit multipotent and immunomodulatory properties. HLSCs can differentiate into several lineages under defined in vitro conditions, such as mature hepatocytes, osteocytes, endothelial cells, and islet-like cell organoids. Over the years, HLSCs have been shown to contribute to tissue repair and regeneration in different in vivo models, leading to more than five granted patents and over 15 peer reviewed scientific articles elucidating their potential therapeutic role in various experimental pathologies. In addition, HLSCs have recently completed a Phase 1 study evaluating their safety post intrahepatic injection in infants with inherited neonatal onset hyperammonemia. Even though a lot of progress has been made in understanding HLSCs over the past years, some important questions regarding the mechanisms of action remain to be elucidated. Among the mechanisms of interaction of HLSCs with their environment, a paracrine interface has emerged involving extracellular vesicles (EVs) as vehicles for transferring active biological materials. In our group, the EVs derived from HLSCs have been studied in vitro as well as in vivo. Our attention has mainly been focused on understanding the in vivo ability of HLSC-derived EVs as modulators of tissue regeneration, inflammation, fibrosis, and tumor growth. This review article aims to discuss in detail the role of HLSCs and HLSC-EVs in these processes and their possible future therapeutic applications.

Differential Therapeutic Effect of Extracellular Vesicles Derived by Bone Marrow and Adipose Mesenchymal Stem Cells on Wound Healing of Diabetic Ulcers and Correlation to Their Cargoes.

Extracellular vesicles (EVs) derived from mesenchymal stem cells isolated from both bone marrow (BMSCs) and adipose tissue (ADSCs) show potential therapeutic effects. These vesicles often show a similar beneficial effect on tissue regeneration, but in some contexts, they exert different biological properties. To date, a comparison of their molecular cargo that could explain the different biological effect is not available. Here, we demonstrated that ADSC-EVs, and not BMSC-EVs, promote wound healing on a murine model of diabetic wounds. Besides a general similarity, the bioinformatic analysis of their protein and miRNA cargo highlighted important differences between these two types of EVs. Molecules present exclusively in ADSC-EVs were highly correlated to angiogenesis, whereas those expressed in BMSC-EVs were preferentially involved in cellular proliferation. Finally, in vitro analysis confirmed that both ADSC and BMSC-EVs exploited beneficial effect on cells involved in skin wound healing such as fibroblasts, keratinocytes and endothelial cells, but through different cellular processes. Consistent with the bioinformatic analyses, BMSC-EVs were shown to mainly promote proliferation, whereas ADSC-EVs demonstrated a major effect on angiogenesis. Taken together, these results provide deeper comparative information on the cargo of ADSC-EVs and BMSC-EVs and the impact on regenerative processes essential for diabetic wound healing.

Coincubation as miR-Loading Strategy to Improve the Anti-Tumor Effect of Stem Cell-Derived EVs.

Extracellular vesicles are considered a novel therapeutic tool, due to their ability to transfer their cargoes to target cells. Different strategies to directly load extracellular vesicles with RNA species have been proposed. Electroporation has been used for the loading of non-active vesicles; however, the engineering of vesicles already carrying a therapeutically active cargo is still under investigation. Here, we set up a coincubation method to increase the anti-tumor effect of extracellular vesicles isolated from human liver stem cells (HLSC-EVs). Using the coincubation protocol, vesicles were loaded with the anti-tumor miRNA-145, and their effect was evaluated on renal cancer stem cell invasion. Loaded HLSC-EVs maintained their integrity and miR transfer ability. Loaded miR-145, but not miR-145 alone, was protected by RNAse digestion, possibly due to its binding to RNA-binding proteins on HLSC-EV surface, such as Annexin A2. Moreover, miR-145 coincubated HLSC-EVs were more effective in inhibiting the invasive properties of cancer stem cells, in comparison to naïve vesicles. The protocol reported here exploits a well described property of extracellular vesicles to bind nucleic acids on their surface and protect them from degradation, in order to obtain an effective miRNA loading, thus increasing the activity of therapeutically active naïve extracellular vesicles.

Extracellular Vesicles as Biomarkers of Acute Graft-vs.-Host Disease After Haploidentical Stem Cell Transplantation and Post-Transplant Cyclophosphamide.

Even with high-dose post-transplant cyclophosphamide (PT-Cy) which was initially introduced for graft-versus-host disease (GvHD) prevention in the setting of HLA-haploidentical transplantation, both acute and chronic GvHDs remain a major clinical challenge. Despite improvements in the understanding of the pathogenesis of both acute and chronic GvHDs, reliable biomarkers that predict their onset have yet to be identified. We recently studied the potential correlation between extracellular vesicles (EVs) and the onset of acute (a)GvHD in transplant recipients from related and unrelated donors. In the present study, we further investigated the role of the expression profile of membrane proteins and their microRNA (miRNA) cargo (miRNA100, miRNA155, and miRNA194) in predicting the onset of aGvHD in haploidentical transplant recipients with PT-Cy. Thirty-two consecutive patients were included. We evaluated the expression profile of EVs, by flow cytometry, and their miRNA cargo, by real-time PCR, at baseline, prior, and at different time points following transplant. Using logistic regression and Cox proportional hazard models, a significant association between expression profiles of antigens such as CD146, CD31, CD140a, CD120a, CD26, CD144, and CD30 on EVs, and their miRNA cargo with the onset of aGvHD was observed. Moreover, we also investigated a potential correlation between EV expression profile and cargo with plasma biomarkers (e.g., ST2, sTNFR1, and REG3a) that had been associated with aGVHD previously. This analysis showed that the combination of CD146, sTNFR1, and miR100 or miR194 strongly correlated with the onset of aGvHD (AUROC >0.975). A large prospective multicenter study is currently in progress to validate our findings.

The Inflammatory Cytokine IL-3 Hampers Cardioprotection Mediated by Endothelial Cell-Derived Extracellular Vesicles Possibly via Their Protein Cargo.

The biological relevance of extracellular vesicles (EV) released in an ischemia/reperfusion setting is still unclear. We hypothesized that the inflammatory microenvironment prevents cardioprotection mediated by endothelial cell (EC)-derived extracellular vesicles. The effects of naïve EC-derived EV (eEV) or eEV released in response to interleukin-3 (IL-3) (eEV-IL-3) were evaluated in cardiomyoblasts (H9c2) and rat hearts. In transwell assay, eEV protected the H9c2 exposed to hypoxia/reoxygenation (H/R) more efficiently than eEV-IL-3. Conversely, only eEV directly protected H9c2 cells to H/R-induced damage. Consistent with this latter observation, eEV, but not eEV-IL-3, exerted beneficial effects in the whole heart. Protein profiles of eEV and eEV-IL-3, established using label-free mass spectrometry, demonstrated that IL-3 drives changes in eEV-IL-3 protein cargo. Gene ontology analysis revealed that both eEV and eEV-IL-3 were equipped with full cardioprotective machinery, including the Nitric Oxide Signaling in the Cardiovascular System. eEV-IL-3 were also enriched in the endothelial-nitric oxide-synthase (eNOS)-antagonist caveolin-1 and proteins related to the inflammatory response. In vitro and ex vivo experiments demonstrated that a functional Mitogen-Activated Protein Kinase Kinase (MEK1/2)/eNOS/guanylyl-cyclase (GC) pathway is required for eEV-mediated cardioprotection. Consistently, eEV were found enriched in MEK1/2 and able to induce the expression of B-cell-lymphoma-2 (Bcl-2) and the phosphorylation of eNOS in vitro. We conclude that an inflammatory microenvironment containing IL-3 changes the eEV cargo and impairs eEV cardioprotective action.

Extracellular Vesicles After Allogeneic Hematopoietic Cell Transplantation: Emerging Role in Post-Transplant Complications.

Extracellular vesicles (EVs) play an important role in the cellular crosstalk by transferring bioactive molecules through biological barriers from a cell to another, thus influencing recipient cell functions and phenotype. Therefore, EVs are increasingly being explored as biomarkers of disease progression or response to therapy and as potential therapeutic agents in different contexts including in hematological malignancies. Recently, an EV role has emerged in allogeneic hematopoietic cell transplantation (allo-HCT) as well. Allogeneic hematopoietic cell transplantation often represents the only curative option in several hematological disorders, but it is associated with potentially life-threatening complications that can have a significant impact on clinical outcomes. The most common complications have been well-established and include graft-versus-host disease and infections. Furthermore, relapse remains an important cause of treatment failure. The aim of this review is to summarize the current knowledge, the potential applications, and clinical relevance of EVs in allo-HCT. Herein, we will mainly focus on the immune-modulating properties of EVs, in particular those derived from mesenchymal stromal cells, as potential therapeutic strategy to improve allo-HCT outcome. Moreover, we will briefly describe the main findings on EVs as biomarkers to monitor graft-versus-host disease onset and tumor relapse.

Extracellular vesicles from human liver stem cells inhibit renal cancer stem cell-derived tumor growth in vitro and in vivo.

Cancer stem cells (CSCs) are considered as responsible for initiation, maintenance and recurrence of solid tumors, thus representing the key for tumor eradication. The antitumor activity of extracellular vesicles (EVs) derived from different stem cell sources has been investigated with conflicting results. In our study, we evaluated, both in vitro and in vivo, the effect of EVs derived from human bone marrow mesenchymal stromal cells (MSCs) and from a population of human liver stem cells (HLSCs) of mesenchymal origin on renal CSCs. In vitro, both EV sources displayed pro-apoptotic, anti-proliferative and anti-invasive effects on renal CSCs, but not on differentiated tumor cells. Pre-treatment of renal CSCs with EVs, before subcutaneous injection in SCID mice, delayed tumor onset. We subsequently investigated the in vivo effect of MSC- and HLSC-EVs systemic administration on progression of CSC-generated renal tumors. Tumor bio-distribution analysis identified intravenous treatment as best route of administration. HLSC-EVs, but not MSC-EVs, significantly impaired subcutaneous tumor growth by reducing tumor vascularization and inducing tumor cell apoptosis. Moreover, intravenous treatment with HLSC-EVs improved metastasis-free survival. In EV treated tumor explants, we observed both the transfer and the induction of miR-145 and of miR-200 family members. In transfected CSCs, the same miRNAs affected cell growth, invasion and survival. In conclusion, our results showed a specific antitumor effect of HLSC-EVs on CSC-derived renal tumors in vivo, possibly ascribed to the transfer and induction of specific antitumor miRNAs. Our study provides further evidence for a possible clinical application of stem cell-EVs in tumor treatment.

Extracellular Vesicles Derived from Induced Pluripotent Stem Cells Promote Renoprotection in Acute Kidney Injury Model.

Induced pluripotent stem cells (iPSC) have been the focus of several studies due to their wide range of application, including in cellular therapy. The use of iPSC in regenerative medicine is limited by their tumorigenic potential. Extracellular vesicles (EV) derived from stem cells have been shown to support renal recovery after injury. However, no investigation has explored the potential of iPSC-EV in the treatment of kidney diseases. To evaluate this potential, we submitted renal tubule cells to hypoxia-reoxygenation injury, and we analyzed cell death rate and changes in functional mitochondria mass. An in vivo model of ischemia-reperfusion injury was used to evaluate morphological and functional alterations. Gene array profile was applied to investigate the mechanism involved in iPSC-EV effects. In addition, EV derived from adipose mesenchymal cells (ASC-EV) were also used to compare the potential of iPSC-EV in support of tissue recovery. The results showed that iPSC-EV were capable of reducing cell death and inflammatory response with similar efficacy than ASC-EV. Moreover, iPSC-EV protected functional mitochondria and regulated several genes associated with oxidative stress. Taken together, these results show that iPSC can be an alternative source of EV in the treatment of different aspects of kidney disease.

Extracellular Vesicles From Adipose Stem Cells Prevent Muscle Damage and Inflammation in a Mouse Model of Hind Limb Ischemia: Role of Neuregulin-1.

OBJECTIVES: Critical hindlimb ischemia is a severe consequence of peripheral artery disease. Surgical treatment does not prevent skeletal muscle impairment or improve long-term patient outcomes. The present study investigates the protective/regenerative potential and the mechanism of action of adipose stem cell-derived extracellular vesicles (ASC-EVs) in a mouse model of hindlimb ischemia. Approach and Results: We demonstrated that ASC-EVs exert a protective effect on muscle damage by acting both on tissue microvessels and muscle cells. The genes involved in muscle regeneration were up-regulated in the ischemic muscles of ASC-EV-treated animals. MyoD expression has also been confirmed in satellite cells. This was followed by a reduction in muscle function impairment in vivo. ASC-EVs drive myoblast proliferation and differentiation in the in vitro ischemia/reoxygenation model. Moreover, ASC-EVs have shown an anti-apoptotic effect both in vitro and in vivo. Transcriptomic analyses have revealed that ASC-EVs carry a variety of pro-angiogenic mRNAs, while proteomic analyses have demonstrated an enrichment of NRG1 (neuregulin 1). A NRG1 blocking antibody used in vivo demonstrated that NRG1 is relevant to ASC-EV-induced muscle protection, vascular growth, and recruitment of inflammatory cells. Finally, bioinformatic analyses on 18 molecules that were commonly detected in ASC-EVs, including mRNAs and proteins, confirmed the enrichment of pathways involved in vascular growth and muscle regeneration/protection. CONCLUSIONS: This study demonstrates that ASC-EVs display pro-angiogenic and skeletal muscle protective properties that are associated with their NRG1/mRNA cargo. We, therefore, propose that ASC-EVs are a useful tool for therapeutic angiogenesis and muscle protection.

Human Liver-Derived Stem Cells Improve Fibrosis and Inflammation Associated with Nonalcoholic Steatohepatitis.

Cell therapy may be regarded as a feasible alternative to whole organ transplantation to treat end-stage liver diseases. Human liver stem cells (HLSCs) are a population of cells easily obtainable and expandable from a human adult liver biopsy. HLSCs share with mesenchymal stromal cells the same phenotype, gene expression profile, and differentiation capabilities. In addition, HLSCs show a specific commitment to the hepatic phenotype. Injection of HLSCs into immunodeficient mice fed with a methionine-choline-deficient diet to induce nonalcoholic steatohepatitis ameliorates liver function and morphology. In particular, HLSC treatment induced a reduction of liver fibrosis and inflammation at morphological and molecular levels. Moreover, HLSCs were able to persist for up to 3 weeks after the injection. In conclusion, HLSCs have healing effects in a model of chronic liver disease.

Renal Regenerative Potential of Extracellular Vesicles Derived from miRNA-Engineered Mesenchymal Stromal Cells.

Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) possess pro-regenerative potential in different animal models with renal injury. EVs contain different molecules, including proteins, lipids and nucleic acids. Among the shuttled molecules, miRNAs have a relevant role in the pro-regenerative effects of EVs and are a promising target for therapeutic interventions. The aim of this study was to increase the content of specific miRNAs in EVs that are known to be involved in the pro-regenerative effect of EVs, and to assess the capacity of modified EVs to contribute to renal regeneration in in vivo models with acute kidney injuries. To this purpose, MSCs were transiently transfected with specific miRNA mimics by electroporation. Molecular analyses showed that, after transfection, MSCs and derived EVs were efficiently enriched in the selected miRNAs. In vitro and in vivo experiments indicated that EVs engineered with miRNAs maintained their pro-regenerative effects. Of relevance, engineered EVs were more effective than EVs derived from naïve MSCs when used at suboptimal doses. This suggests the potential use of a low amount of EVs (82.5 × 106) to obtain the renal regenerative effect.

Stem cell-derived extracellular vesicles inhibit and revert fibrosis progression in a mouse model of diabetic nephropathy.

Extracellular vesicles (EVs) that are derived from mesenchymal stromal cells (MSCs) have been shown to reprogram injured cells by activating regenerative processes. We herein investigate the potential therapeutic effect of EVs, shed by human bone marrow MSCs and by human liver stem-like cells (HLSCs), on the progression and reversion of fibrosis in a mouse model of diabetic nephropathy, as induced by streptozotocin. After the development of nephropathy, stem cell-derived EVs were administered weekly to diabetic mice for four weeks. The stem cell-derived EV treatment, but not the fibroblast EV treatment that was used as a control, significantly ameliorated functional parameters, such as albumin/creatinine excretion, plasma creatinine and blood urea nitrogen, which are altered in diabetic mice. Moreover, the renal fibrosis that develops during diabetic nephropathy progression was significantly inhibited in stem cell EV-treated animals. A correlation was found between the down regulation of several pro-fibrotic genes in renal tissues and the anti-fibrotic effect of HLSC and MSC EVs. A comparative analysis of HLSC and MSC EV miRNA content highlighted some common and some specific patterns of miRNAs that target predicted pro-fibrotic genes. In conclusion, stem cell-derived EVs inhibit fibrosis and prevent its progression in a model of diabetes-induced chronic kidney injury.

Extracellular vesicles from human liver stem cells inhibit tumor angiogenesis.

Human liver stem-like cells (HLSC) and derived extracellular vesicles (EVs) were previously shown to exhibit anti-tumor activity. In our study, we investigated whether HLSC-derived EVs (HLSC-EVs) were able to inhibit tumor angiogenesis in vitro and in vivo, in comparison with EVs derived from mesenchymal stem cells (MSC-EVs). The results obtained indicated that HLSC-EVs, but not MSC-EVs, inhibited the angiogenic properties of tumor-derived endothelial cells (TEC) both in vitro and in vivo in a model of subcutaneous implantation in Matrigel. Treatment of TEC with HLSC-EVs led to the down-regulation of pro-angiogenic genes. Since HLSC-EVs carry a specific set of microRNAs (miRNAs) that could target these genes, we investigated their potential role by transfecting TEC with HLSC-EV specific miRNAs. We observed that four miRNAs, namely miR-15a, miR-181b, miR-320c and miR-874, significantly inhibited the angiogenic properties of TEC in vitro, and decreased the expression of some predicted target genes (ITGB3, FGF1, EPHB4 and PLAU). In parallel, TEC treated with HLSC-EVs significantly enhanced expression of miR-15a, miR-181b, miR-320c and miR-874 associated with the down-regulation of FGF1 and PLAU. In summary, HLSC-EVs possess an anti-tumorigenic effect, based on their ability to inhibit tumor angiogenesis.

Islet-Like Structures Generated In Vitro from Adult Human Liver Stem Cells Revert Hyperglycemia in Diabetic SCID Mice.

A potential therapeutic strategy for diabetes is the transplantation of induced-insulin secreting cells. Based on the common embryonic origin of liver and pancreas, we studied the potential of adult human liver stem-like cells (HLSC) to generate in vitro insulin-producing 3D spheroid structures (HLSC-ILS). HLSC-ILS were generated by a one-step protocol based on charge dependent aggregation of HLSC induced by protamine. 3D aggregation promoted the spontaneous differentiation into cells expressing insulin and several key markers of pancreatic ß cells. HLSC-ILS showed endocrine granules similar to those seen in human ß cells. In static and dynamic in vitro conditions, such structures produced C-peptide after stimulation with high glucose. HLSC-ILS significantly reduced hyperglycemia and restored a normo-glycemic profile when implanted in streptozotocin-diabetic SCID mice. Diabetic mice expressed human C-peptide and very low or undetectable levels of murine C-peptide. Hyperglycemia and a diabetic profile were restored after HLSC-ISL explant. The gene expression profile of in vitro generated HLSC-ILS showed a differentiation from HLSC profile and an endocrine commitment with the enhanced expression of several markers of ß cell differentiation. The comparative analysis of gene expression profiles after 2 and 4 weeks of in vivo implantation showed a further ß-cell differentiation, with a genetic profile still immature but closer to that of human islets. In conclusion, protamine-induced spheroid aggregation of HLSC triggers a spontaneous differentiation to an endocrine phenotype. Although the in vitro differentiated HLSC-ILS were immature, they responded to high glucose with insulin secretion and in vivo reversed hyperglycemia in diabetic SCID mice.

Renal Regenerative Potential of Different Extracellular Vesicle Populations Derived from Bone Marrow Mesenchymal Stromal Cells.

Extracellular vesicles (EVs) derived from human bone marrow mesenchymal stromal cells (MSCs) promote the regeneration of kidneys in different animal models of acute kidney injury (AKI) in a manner comparable with the cells of origin. However, due to the heterogeneity observed in the EVs isolated from MSCs, it is unclear which population is responsible for the proregenerative effects. We therefore evaluated the effect of various EV populations separated by differential ultracentrifugation (10K population enriched with microvesicles and 100K population enriched with exosomes) on AKI recovery. Only the exosomal-enriched population induced an improvement of renal function and morphology comparable with that of the total EV population. Interestingly, the 100K EVs exerted a proproliferative effect on murine tubular epithelial cells, both in vitro and in vivo. Analysis of the molecular content from the different EV populations revealed a distinct profile. The 100K population, for instance, was enriched in specific mRNAs (CCNB1, CDK8, CDC6) reported to influence cell cycle entry and progression; miRNAs involved in regulating proliferative/antiapoptotic pathways and growth factors (hepatocyte growth factor and insulin-like growth factor-1) that could explain the effect of renal tubular cell proliferation. On the other hand, the EV population enriched in microvesicles (10K) was unable to induce renal regeneration and had a molecular profile with lower expression of proproliferative molecules. In conclusion, the different molecular composition of exosome- and microvesicle-enriched populations may explain the regenerative effect of EVs observed in AKI.

The effects of glomerular and tubular renal progenitors and derived extracellular vesicles on recovery from acute kidney injury.

BACKGROUND: Mesenchymal stromal cells (MSCs) and renal stem/progenitors improve the recovery of acute kidney injury (AKI) mainly through the release of paracrine mediators including the extracellular vesicles (EVs). Several studies have reported the existence of a resident population of MSCs within the glomeruli (Gl-MSCs). However, their contribution towards kidney repair still remains to be elucidated. The aim of the present study was to evaluate whether Gl-MSCs and Gl-MSC-EVs promote the recovery of AKI induced by ischemia-reperfusion injury (IRI) in SCID mice. Moreover, the effects of Gl-MSCs and Gl-MSC-EVs were compared with those of CD133+ progenitor cells isolated from human tubules of the renal cortical tissue (T-CD133+ cells) and their EVs (T-CD133+-EVs). METHODS: IRI was performed in mice by clamping the left renal pedicle for 35 minutes together with a right nephrectomy. Immediately after reperfusion, the animals were divided in different groups to be treated with: Gl-MSCs, T-CD133+ cells, Gl-MSC-EVs, T-CD133+-EVs or vehicle. To assess the role of vesicular RNA, EVs were either isolated by floating to avoid contamination of non-vesicles-associated RNA or treated with a high dose of RNase. Mice were sacrificed 48 hours after surgery. RESULTS: Gl-MSCs, and Gl-MSC-EVs both ameliorate kidney function and reduce the ischemic damage post IRI by activating tubular epithelial cell proliferation. Furthermore, T-CD133+ cells, but not their EVs, also significantly contributed to the renal recovery after IRI compared to the controls. Floating EVs were effective while RNase-inactivated EVs were ineffective. Analysis of the EV miRnome revealed that Gl-MSC-EVs selectively expressed a group of miRNAs, compared to EVs derived from fibroblasts, which were biologically ineffective in IRI. CONCLUSIONS: In this study, we demonstrate that Gl-MSCs may contribute in the recovery of mice with AKI induced by IRI primarily through the release of EVs.

Exosome and Microvesicle-Enriched Fractions Isolated from Mesenchymal Stem Cells by Gradient Separation Showed Different Molecular Signatures and Functions on Renal Tubular Epithelial Cells.

Several studies have suggested that extracellular vesicles (EVs) released from mesenchymal stem cells (MSCs) may mediate MSC paracrine action on kidney regeneration. This activity has been, at least in part, ascribed to the transfer of proteins/transcription factors and different RNA species. Information on the RNA/protein content of different MSC EV subpopulations and the correlation with their biological activity is currently incomplete. The aim of this study was to evaluate the molecular composition and the functional properties on renal target cells of MSC EV sub-populations separated by gradient floatation. The results demonstrated heterogeneity in quantity and composition of MSC EVs. Two peaks of diameter were observed (90-110 and 170-190 nm). The distribution of exosomal markers and miRNAs evaluated in the twelve gradient fractions showed an enrichment in fractions with a flotation density of 1.08-1.14 g/mL. Based on this observation, we evaluated the biological activity on renal cell proliferation and apoptosis resistance of low (CF1), medium (CF2) and high (CF3) floatation density fractions. EVs derived from all fractions, were internalized by renal cells, CF1 and CF2 but not CF3 fraction stimulated significant cell proliferation. CF2 also inhibited apoptosis on renal tubular cells submitted to ischemia-reperfusion injury. Comparative miRNomic and proteomic profiles reveal a cluster of miRNAs and proteins common to all three fractions and an enrichment of selected molecules related to renal regeneration in CF2 fraction. In conclusion, the CF2 fraction enriched in exosomal markers was the most active on renal tubular cell proliferation and protection from apoptosis.