Chromatographic Scalable Method to Isolate Engineered Extracellular Vesicles Derived from Mesenchymal Stem Cells for the Treatment of Liver Fibrosis in Mice.

New therapeutic options for liver cirrhosis are needed. Mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) have emerged as a promising tool for delivering therapeutic factors in regenerative medicine. Our aim is to establish a new therapeutic tool that employs EVs derived from MSCs to deliver therapeutic factors for liver fibrosis. EVs were isolated from supernatants of adipose tissue MSCs, induced-pluripotent-stem-cell-derived MSCs, and umbilical cord perivascular cells (HUCPVC-EVs) by ion exchange chromatography (IEC). To produce engineered EVs, HUCPVCs were transduced with adenoviruses that code for insulin-like growth factor 1 (AdhIGF-I-HUCPVC-EVs) or green fluorescent protein. EVs were characterized by electron microscopy, flow cytometry, ELISA, and proteomic analysis. We evaluated EVs' antifibrotic effect in thioacetamide-induced liver fibrosis in mice and on hepatic stellate cells in vitro. We found that IEC-isolated HUCPVC-EVs have an analogous phenotype and antifibrotic activity to those isolated by ultracentrifugation. EVs derived from the three MSCs sources showed a similar phenotype and antifibrotic potential. EVs derived from AdhIGF-I-HUCPVC carried IGF-1 and showed a higher therapeutic effect in vitro and in vivo. Remarkably, proteomic analysis revealed that HUCPVC-EVs carry key proteins involved in their antifibrotic process. This scalable MSC-derived EV manufacturing strategy is a promising therapeutic tool for liver fibrosis.

Bioinformatic analysis of RHO family of GTPases identifies RAC1 pharmacological inhibition as a new therapeutic strategy for hepatocellular carcinoma.

OBJECTIVE: The RHO family of GTPases, particularly RAC1, has been linked with hepatocarcinogenesis, suggesting that their inhibition might be a rational therapeutic approach. We aimed to identify and target deregulated RHO family members in human hepatocellular carcinoma (HCC). DESIGN: We studied expression deregulation, clinical prognosis and transcription programmes relevant to HCC using public datasets. The therapeutic potential of RAC1 inhibitors in HCC was study in vitro and in vivo. RNA-Seq analysis and their correlation with the three different HCC datasets were used to characterise the underlying mechanism on RAC1 inhibition. The therapeutic effect of RAC1 inhibition on liver fibrosis was evaluated. RESULTS: Among the RHO family of GTPases we observed that RAC1 is upregulated, correlates with poor patient survival, and is strongly linked with a prooncogenic transcriptional programme. From a panel of novel RAC1 inhibitors studied, 1D-142 was able to induce apoptosis and cell cycle arrest in HCC cells, displaying a stronger effect in highly proliferative cells. Partial rescue of the RAC1-related oncogenic transcriptional programme was obtained on RAC1 inhibition by 1D-142 in HCC. Most importantly, the RAC1 inhibitor 1D-142 strongly reduce tumour growth and intrahepatic metastasis in HCC mice models. Additionally, 1D-142 decreases hepatic stellate cell activation and exerts an anti-fibrotic effect in vivo. CONCLUSIONS: The bioinformatics analysis of the HCC datasets, allows identifying RAC1 as a new therapeutic target for HCC. The targeted inhibition of RAC1 by 1D-142 resulted in a potent antitumoural effect in highly proliferative HCC established in fibrotic livers.

SPARC inhibition accelerates NAFLD-associated hepatocellular carcinoma development by dysregulating hepatic lipid metabolism.

BACKGROUND AND AIMS: Non-alcoholic fatty liver (NAFLD) and its more serious form non-alcoholic steatohepatitis increase risk of hepatocellular carcinoma (HCC). Lipid metabolic alterations and its role in HCC development remain unclear. SPARC (Secreted Protein, Acidic and Rich in Cysteine) is involved in lipid metabolism, NAFLD and diabetes, but the effects on hepatic lipid metabolism and HCC development is unknown. The aim of this study was to evaluate the role of SPARC in HCC development in the context of NAFLD. METHODS: Primary hepatocyte cultures from knockout (SPARC-/- ) or wild-type (SPARC+/+ ) mice, and HepG2 cells were used to assess the effects of free fatty acids on lipid accumulation, expression of lipogenic genes and de novo triglyceride (TG) synthesis. A NAFLD-HCC model was stabilized on SPARC-/- or SPARC+/+ mice. Correlations among SPARC, lipid metabolism-related gene expression patterns and clinical prognosis were studied using HCC gene expression dataset. RESULTS: SPARC-/- mice increases hepatic lipid deposits over time. Hepatocytes from SPARC-/- mice or inhibition of SPARC by an antisense adenovirus in HepG2 cells resulted in increased TG deposit, expression of lipid-related genes and nuclear translocation of SREBP1c. Human HCC database analysis revealed that SPARC negatively correlated with genes involved in lipid metabolism, and with poor survival. In NAFLD-HCC murine model, the absence of SPARC accelerates HCC development. RNA-seq study revealed that pathways related to lipid metabolism, cellular detoxification and proliferation were upregulated in SPARC-/- tumour-bearing mice. CONCLUSIONS: The absence of SPARC is associated with an altered hepatic lipid metabolism, and an accelerated NAFLD-related HCC development.

Human umbilical cord perivascular cells-derived extracellular vesicles mediate the transfer of IGF-I to the liver and ameliorate hepatic fibrogenesis in mice.

Extracellular vesicles (EVs) can mediate mesenchymal stromal cells (MSCs) paracrine effects. We aimed to evaluate the therapeutic potential of human umbilical cord perivascular cells (HUCPVCs) engineered to produce Insulin Growth Factor like-I (IGF-I) in experimental liver fibrosis and the role of EVs in this effect. HUCPVCs were engineered to produce human IGF-I (AdhIGF-I) or green fluorescence protein (AdGFP) using adenoviruses, and EVs were isolated from their conditioned medium (CM). In vitro effects of CM and EVs on hepatic stellate cells and hepatic macrophages were studied. Cells or EVs-based treatments were evaluated in thioacetamide-induced liver fibrosis in mice. The application of AdhIGF-I-HUCPVCs resulted in a further amelioration of liver fibrosis when compared to AdGFP-HUCPVCs and saline. Similarly, treatment with AdhIGF-I-HUCPVCs-derived EVs resulted in a reduction of collagen deposition and gene expression of the fibrogenic related molecules TGF-ß1, α-SMA, and COL1A2. In vitro incubation of hepatic stellate cells with EVs-AdhIGF-I-HUCPVCs significantly reduced activation of fibrogenic cells. In addition, EVs-AdhIGF-I-HUCPVCs trigger hepatic macrophages to switch their phenotype towards anti-inflammatory phagocytes, which might be involved in the antifibrotic effect. Consistently, high levels of IGF-I were observed within EVs-AdhIGF-I-HUCPVCs but not in controls EVs. Our results showed that hIGF-I carrying EVs could mediate the paracrine mechanism by which AdhIGF-I-HUCPVCs reduce liver fibrosis.

A comprehensive study of epigenetic alterations in hepatocellular carcinoma identifies potential therapeutic targets.

BACKGROUND & AIMS: A causal link has recently been established between epigenetic alterations and hepatocarcinogenesis, indicating that epigenetic inhibition may have therapeutic potential. We aimed to identify and target epigenetic modifiers that show molecular alterations in hepatocellular carcinoma (HCC). METHODS: We studied the molecular-clinical correlations of epigenetic modifiers including bromodomains, histone acetyltransferases, lysine methyltransferases and lysine demethylases in HCC using The Cancer Genome Atlas (TCGA) data of 365 patients with HCC. The therapeutic potential of epigenetic inhibitors was evaluated in vitro and in vivo. RNA sequencing analysis and its correlation with expression and clinical data in the TCGA dataset were used to identify expression programs normalized by Jumonji lysine demethylase (JmjC) inhibitors. RESULTS: Genetic alterations, aberrant expression, and correlation between tumor expression and poor patient prognosis of epigenetic enzymes are common events in HCC. Epigenetic inhibitors that target bromodomain (JQ-1), lysine methyltransferases (BIX-1294 and LLY-507) and JmjC lysine demethylases (JIB-04, GSK-J4 and SD-70) reduce HCC aggressiveness. The pan-JmjC inhibitor JIB-04 had a potent antitumor effect in tumor bearing mice. HCC cells treated with JmjC inhibitors showed overlapping changes in expression programs related with inhibition of cell proliferation and induction of cell death. JmjC inhibition reverses an aggressive HCC gene expression program that is also altered in patients with HCC. Several genes downregulated by JmjC inhibitors are highly expressed in tumor vs. non-tumor parenchyma, and their high expression correlates with a poor prognosis. We identified and validated a 4-gene expression prognostic signature consisting of CENPA, KIF20A, PLK1, and NCAPG. CONCLUSIONS: The epigenetic alterations identified in HCC can be used to predict prognosis and to define a subgroup of high-risk patients that would potentially benefit from JmjC inhibitor therapy. LAY SUMMARY: In this study, we found that mutations and changes in expression of epigenetic modifiers are common events in human hepatocellular carcinoma, leading to an aggressive gene expression program and poor clinical prognosis. The transcriptional program can be reversed by pharmacological inhibition of Jumonji enzymes. This inhibition blocks hepatocellular carcinoma progression, providing a novel potential therapeutic strategy.

4Mu Decreases CD47 Expression on Hepatic Cancer Stem Cells and Primes a Potent Antitumor T Cell Response Induced by Interleukin-12.

The tumor microenvironment (TME) represents a complex interplay between different cellular components, including tumor cells and cancer stem cells (CSCs), with the associated stroma; such interaction promotes tumor immune escape and sustains tumor growth. Several experimental approaches for cancer therapy are focused on TME remodeling, resulting in increased antitumor effects. We previously demonstrated that the hyaluronan synthesis inhibitor 4-methylumbelliferone (4Mu) decreases liver fibrosis and induces antitumor activity in hepatocellular carcinoma (HCC). In this work, 4Mu, in combination with an adenovirus encoding interleukin-12 genes (AdIL-12), elicited a potent antitumor effect and significantly prolonged animal survival (p < 0.05) in an orthotopic HCC model established in fibrotic livers. In assessing the presence of CSCs, we found reduced mRNA levels of CD133+, CD90+, EpCAM+, CD44+, and CD13+ CSC markers within HCC tumors (p < 0.01). Additionally, 4Mu downregulated the expression of the CSC marker CD47+ on HCC cells, promoted phagocytosis by antigen-presenting cells, and, combined with Ad-IL12, elicited a potent cytotoxic-specific T cell response. Finally, animal survival was increased when CD133low HCC cells, generated upon 4Mu treatment, were injected in a metastatic HCC model. In conclusion, the combined strategy ameliorates HCC aggressiveness by targeting CSCs and as a result of the induction of anticancer immunity.

Generation and characterization of human mesenchymal stem/stromal cells for cell therapy applications.

Stem Cell based-therapy is an active area of research in regenerative medicine. Mesenchymal stem/stromal cells (MSCs) are multipotent adult stem/progenitor cells, which could be easily expanded in vitro and have the ability to selectively migrate toward injured tissues, evade the immune system, and secrete trophic factors to support the repair of damaged tissues. The use of MSCs for cell and regenerative purposes has garnered the attention of scientists and clinicians. However, one of the most important issues before use MSCs in clinical practice is to standardize a number of aspects related to the source of MSCs, culture conditions, pre-condition protocols before transplantation, administration route, doses, or treatment duration. In this chapter, we described two standard protocols to isolate MSCs from bone marrow and umbilical cord connective tissue. In addition, basic characterization including immunophenotyping by flow cytometry and differentiation capability is also described.

IMT504 blocks allodynia in rats with spared nerve injury by promoting the migration of mesenchymal stem cells and by favoring an anti-inflammatory milieu at the injured nerve.

ABSTRACT: IMT504, a noncoding, non-CpG oligodeoxynucleotide, modulates pain-like behavior in rats undergoing peripheral nerve injury, through mechanisms that remain poorly characterized. Here, we chose the spared nerve injury model in rats to analyze the contribution of mesenchymal stem cells (MSCs) in the mechanisms of action of IMT504. We show that a single subcutaneous administration of IMT504 reverses mechanical and cold allodynia for at least 5 weeks posttreatment. This event correlated with long-lasting increases in the percentage of MSCs in peripheral blood and injured sciatic nerves, in a process seemingly influenced by modifications in the CXCL12-CXCR4 axis. Also, injured nerves presented with reduced tumor necrosis factor-α and interleukin-1ß and increased transforming growth factor-ß1 and interleukin-10 protein levels. In vitro analysis of IMT504-pretreated rat or human MSCs revealed internalized oligodeoxynucleotide and confirmed its promigratory effects. Moreover, IMT504-pretreatment induced transcript expression of Tgf-ß1 and Il-10 in MSCs; the increase in Il-10 becoming more robust after exposure to injured nerves. Ex vivo exposure of injured nerves to IMT504-pretreated MSCs confirmed the proinflammatory to anti-inflammatory switch observed in vivo. Interestingly, the sole exposure of injured nerves to IMT504 also resulted in downregulated Tnf-α and Il-1ß transcripts. Altogether, we reveal for the first time a direct association between the antiallodynic actions of IMT504, its promigratory and cytokine secretion modulating effects on MSCs, and further anti-inflammatory actions at injured nerves. The recapitulation of key outcomes in human MSCs supports the translational potential of IMT504 as a novel treatment for neuropathic pain with a unique mechanism of action involving the regulation of neuroimmune interactions.

4-methylumbelliferone-mediated polarization of M1 macrophages correlate with decreased hepatocellular carcinoma aggressiveness in mice.

Hepatocellular carcinoma (HCC) arises in the setting of advanced liver fibrosis, a dynamic and complex inflammatory disease. The tumor microenvironment (TME) is a mixture of cellular components including cancer cells, cancer stem cells (CSCs), tumor-associated macrophages (TAM), and dendritic cells (DCs), which might drive to tumor progression and resistance to therapies. In this work, we study the effects of 4-methylumbelliferone (4Mu) on TME and how this change could be exploited to promote a potent immune response against HCC. First, we observed that 4Mu therapy induced a switch of hepatic macrophages (Mϕ) towards an M1 type profile, and HCC cells (Hepa129 cells) exposed to conditioned medium (CM) derived from Mϕ treated with 4Mu showed reduced expression of several CSCs markers and aggressiveness. HCC cells incubated with CM derived from Mϕ treated with 4Mu grew in immunosuppressed mice while presented delayed tumor progression in immunocompetent mice. HCC cells treated with 4Mu were more susceptible to phagocytosis by DCs, and when DCs were pulsed with HCC cells previously treated with 4Mu displayed a potent antitumoral effect in therapeutic vaccination protocols. In conclusion, 4Mu has the ability to modulate TME into a less hostile milieu and to potentiate immunotherapeutic strategies against HCC.

[Mesenchymal stem/stromal cells. Its therapeutic potential in medicine]. / Células madre/estromales mesenquimales. Su potencial terapéutico en medicina.

Cell therapy and regenerative medicine are currently active areas for biomedical research. In most tissues, there are self-repair mechanisms carried out mainly by resident stem cells that can differentiate and replace dead cells or secrete trophic factors that stimulate the regenerative process. These mechanisms often fail in degenerative diseases; thus it is postulated that exogenous cell therapy can contribute to tissue regeneration and repair. Mesenchymal stem cells (MSCs) are multipotent adult stem/progenitor cells, which could be easily expanded in vitro and have the ability to selectively migrate toward injured tissues, evade the immune system recognition, and secrete trophic factors to support tissue repair. Furthermore, MSCs could be engineered for the delivery of therapeutic genes. The main sources for MSCs are bone marrow, adipose tissue, and umbilical cord. A number of pre-clinical and clinical studies have shown that MSCs therapy is safe for both autologous and allogeneic uses. This review summarizes information about the properties of MSCs and their therapeutic potential for a broad spectrum of diseases. We also present here the last data about clinical trials that position the use of MSCs as an interesting tool for tissue regeneration and the treatment of inflammatory diseases.

La terapia celular y la medicina regenerativa son áreas en gran desarrollo en la investigación biomédica. En la mayoría de los tejidos existen mecanismos de auto-reparación llevados a cabo, principalmente, por células madre o progenitoras residentes con capacidad para diferenciarse y reemplazar a las células dañadas o para secretar factores tróficos que induzcan el proceso regenerativo. Dado que estos mecanismos de reparación no siempre son suficientes, se postula que la terapia celular puede contribuir a la regeneración de los tejidos sometidos a injuria. Las células madre/estromales mesenquimales (MSCs, del inglés Mesenchymal Stem/Stromal Cells) son un tipo de progenitor adulto multipotente, que tienen la capacidad de expandirse in vitro con facilidad cuando son aisladas de su nicho in vivo, migrar selectivamente a los tejidos lesionados, modular y evadir el sistema inmunológico, y secretar factores tróficos que ayudan a la reparación tisular. Asimismo, la fácil manipulación ex vivo permitiría también usarlas como vehículos de genes terapéuticos. Las principales fuentes de obtención son la médula ósea, el tejido adiposo y cordón umbilical. Los numerosos estudios pre-clínicos y clínicos han demostrado que las MSCs parecieran ser seguras tanto para uso autólogo como alogénico. En este trabajo se resumen las propiedades de las MSCs y su potencial terapéutico para una amplia gama de enfermedades, también presentamos los distintos ensayos clínicos avanzados que las posicionan en el ámbito biomédico como una herramienta interesante para la regeneración de tejidos y el tratamiento de enfermedades inflamatorias.

Taking advantage of the potential of mesenchymal stromal cells in liver regeneration: Cells and extracellular vesicles as therapeutic strategies.

Cell-based therapies for acute and chronic liver diseases are under continuous progress. Mesenchymal stem/stromal cells (MSCs) are multipotent cells able to migrate selectively to damaged tissue and contribute to its healing and regeneration. The MSC pro-regenerative effect occurs due to their immunomodulatory capacity and their ability to produce factors that promote cell protection and survival. Likewise, it has been observed that part of their paracrine effect is mediated by MSC-derived extracellular vesicles (EVs). EVs contain proteins, lipids and nucleic acids (DNA, mRNA, miRNA, lncRNA) from the cell of origin, allowing for intercellular communication. Recently, different studies have demonstrated that MSC-derived EVs could reproduce, at least in part, the biological effects obtained by MSC-based therapies. Moreover, due to EVs' stability for long periods of time and easy isolation methods they have become a therapeutic option to MSCs treatments. This review summarizes the latest results achieved in clinical trials using MSCs as cell therapy for liver regeneration, the role of EVs in liver physiopathology and the potential of MSCderived EVs as intercellular mediators and therapeutic tools in liver diseases.

Células madre/estromales mesenquimales. Su potencial terapéutico en medicina / Mesenchymal stem/stromal cells. Their therapeutic potential in medicine

Resumen La terapia celular y la medicina regenerativa son áreas en gran desarrollo en la investigación biomédica. En la mayoría de los tejidos existen mecanismos de auto-reparación llevados a cabo, principalmente, por células madre o progenitoras residentes con capacidad para diferenciarse y reemplazar a las células dañadas o para secretar factores tróficos que induzcan el proceso regenerativo. Dado que estos mecanismos de reparación no siempre son suficientes, se postula que la terapia celular puede contribuir a la regeneración de los tejidos sometidos a injuria. Las células madre/estromales mesenquimales (MSCs, del inglés Mesenchymal Stem/Stromal Cells) son un tipo de progenitor adulto multipotente, que tienen la capacidad de expandirse in vitro con facilidad cuando son aisladas de su nicho in vivo, migrar selectivamente a los tejidos lesionados, modular y evadir el sistema inmunológico, y secretar factores tróficos que ayudan a la reparación tisular. Asimismo, la fácil manipulación ex vivo permitiría también usarlas como vehículos de genes terapéuticos. Las principales fuentes de obtención son la médula ósea, el tejido adiposo y cordón umbilical. Los numerosos estudios pre-clínicos y clínicos han demostrado que las MSCs parecieran ser seguras tanto para uso autólogo como alogénico. En este trabajo se resumen las propiedades de las MSCs y su potencial terapéutico para una amplia gama de enfermedades, también presentamos los distintos ensayos clínicos avanzados que las posicionan en el ámbito biomédico como una herramienta interesante para la regeneración de tejidos y el tratamiento de enfermedades inflamatorias.

Abstract Cell therapy and regenerative medicine are currently active areas for biomedical research. In most tissues, there are self-repair mechanisms carried out mainly by resident stem cells that can differentiate and replace dead cells or secrete trophic factors that stimulate the regenerative process. These mechanisms often fail in degenerative diseases; thus it is postulated that exogenous cell therapy can contribute to tissue regeneration and repair. Mesenchymal stem cells (MSCs) are multipotent adult stem/progenitor cells, which could be easily expanded in vitro and have the ability to selectively migrate toward injured tissues, evade the immune system recognition, and secrete trophic factors to support tissue repair. Furthermore, MSCs could be engineered for the delivery of therapeutic genes. The main sources for MSCs are bone marrow, adipose tissue, and umbilical cord. A number of pre-clinical and clinical studies have shown that MSCs therapy is safe for both autologous and allogeneic uses. This review summarizes information about the properties of MSCs and their therapeutic potential for a broad spectrum of diseases. We also present here the last data about clinical trials that position the use of MSCs as an interesting tool for tissue regeneration and the treatment of inflammatory diseases.