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.

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.

Development of an Improved Guanidine-Based Rac1 Inhibitor with in†vivo Activity against Non-Small Cell Lung Cancer.

The Rho GTPase Rac1 is involved in the control of cytoskeleton reorganization and other fundamental cellular functions. Aberrant activity of Rac1 and its regulators is common in human cancer. In particular, deregulated expression/activity of Rac GEFs, responsible for Rac1 activation, has been associated to a metastatic phenotype and drug resistance. Thus, the development of novel Rac1-GEF interaction inhibitors is a promising strategy for finding new preclinical candidates. Here, we studied structure-activity relationships within a new family of N,N'-disubstituted guanidine as Rac1 inhibitors. We found that compound 1D-142, presents superior antiproliferative activity in human cancer cell lines and higher potency as Rac1-GEF interaction inhibitor in vitro than parental compounds. In addition, 1D-142 reduces Rac1-mediated TNFα-induced NF-κB nuclear translocation during cell proliferation and migration in NSCLC. Notably, 1D-142 allowed us to show for the first time the application of a Rac1 inhibitor in a lung cancer animal model.

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.

[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.

Contribution of neural crest and GLAST+ Wnt1+ bone marrow pericytes with liver fibrogenesis and/or regeneration.

BACKGROUND AND AIMS: Liver fibrosis results from cycles of liver damage and scar formation. We herein aimed at analysing neural crest cells and/or bone marrow stromal cells contribution to the liver. METHODS: Two liver fibrosis and one hepatectomy model were applied on double-transgenic loxP-Cre mouse lines. RESULTS: Increased numbers of glia with more complex processes were found in fibrotic livers. During embryonic development, only few cells were traced in the liver and bone marrow, in a minor fraction of mice of different neural crest reporter strains analysed: therefore, a neural crest origin of such cells is doubtful. In the fibrotic liver, a significantly higher incidence of endothelial cells and hepatocyte-like cells expressing the reporter gene Tomato were found in Wnt1-Cre-Tom and GLAST-CreERT2-Tom mice. Consistently, during early fibrogenesis stromal Wnt1-traced cells, with progenitor (CFU-F) properties, get likely mobilized to peripheral blood. Circulating adult Wnt1-traced cells are stromal cells and lack from the expression of other bone marrow and endothelial progenitor cells markers. Furthermore, in a 70% hepatectomy model GLAST+ Wnt1-traced pericytes were found to be mobilized from the bone marrow and the incidence of GLAST-traced hepatocyte-like cells was increased. Finally, GLAST-traced hepatocyte like-cells were found to maintain the expression of stromal markers. CONCLUSIONS: Our data suggest a gliosis process during liver fibrogenesis. While neural crest cells probably do not contribute with other liver cell types than glia, GLAST+ Wnt1-traced bone marrow pericytes are likely a source of endothelial and hepatocyte-like cells after liver injury and do not contribute to scarring.

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.

IL-8, GRO and MCP-1 produced by hepatocellular carcinoma microenvironment determine the migratory capacity of human bone marrow-derived mesenchymal stromal cells without affecting tumor aggressiveness.

New therapies are needed for advanced hepatocellular carcinoma (HCC) and the use of mesenchymal stromal cells (MSCs) carrying therapeutic genes is a promising strategy. HCC produce cytokines recruiting MSCs to the tumor milieu and modifying its biological properties. Our aim was to study changes generated on human MSCs exposed to conditioned media (CM) derived from human HCC fresh samples and xenografts. All CM shared similar cytokines expression pattern including CXCL1-2-3/GRO, CCL2/MCP-1 and CXCL8/IL-8 being the latter with the highest concentration. Neutralizing and knockdown experiments of CCL2/MCP-1, CXCL8/IL-8, CXCR1 and CXCR2 reduced in vitro MSC migration of ≥20%. Simultaneous CXCR1 and CXCR2 neutralization resulted in 50% of MSC migration inhibition. MSC stimulated with CM (sMSC) from HuH7 or HC-PT-5 showed a 2-fold increase of migration towards the CM compared with unstimulated MSC (usMSC). Gene expression profile of sMSC showed ~500 genes differentially expressed compared with usMSC, being 46 genes related with cell migration and invasion. sMSC increased fibroblasts and endothelial cells chemotaxis. Finally, sMSC with HuH7 CM and then inoculated in HCC tumor bearing-mice did not modify tumor growth. In this work we characterized factors produced by HCC responsible for the changes in MSC chemotactic capacity with would have an impact on therapeutic use of MSCs for human HCC.

Mesenchymal Stem/Stromal Cells in Liver Fibrosis: Recent Findings, Old/New Caveats and Future Perspectives.

Mesenchymal stem/stromal cells (MSCs) are progenitors which share plastic-adherence capacity and cell surface markers but have different properties according to their cell and tissue sources and to culture conditions applied. Many recent publications suggest that MSCs can differentiate into hepatic-like cells, which can be a consequence of either a positive selection of rare in vivo pluripotent cells or of the original plasticity of some cells contributing to MSC cultures. A possible role of MSCs in hereditary transmission of obesity and/or diabetes as well as properties of MSCs regarding immunomodulation, cell fusion and exosome release capacities are discussed according to recent literature. Limitations in methods used to track MSCs in vivo especially in the context of liver cirrhosis are addressed as well as strategies explored to enhance their migratory, survival and proliferation properties, which are known to be relevant for their future clinical use. Current knowledge regarding mechanisms involved in liver cirrhosis amelioration mediated by naïve and genetically modified MSCs as well as the effects of applying preconditioning and combined strategies to improve their therapeutic effects are evaluated. Finally, first reports of GMP guidelines and biosafety issues in MSCs applications are discussed.

Mesenchymal stromal cells engineered to produce IGF-I by recombinant adenovirus ameliorate liver fibrosis in mice.

Liver cirrhosis involves chronic wound healing and fibrotic processes. Mesenchymal stromal cells (MSCs) are multipotent adult progenitor cells that are used as vehicles of therapeutic genes. Insulin growth factor like-I (IGF-I) was shown to counteract liver fibrosis. We aimed at analyzing the effect of applying IGF-I overexpressing mouse bone marrow-derived MSCs on hepatic fibrosis. Fibrosis was induced by chronic thioacetamide application or bile duct ligation. MSCs engineered to produce green fluorescent protein (GFP) (AdGFP-MSCs) or IGF-I (AdIGF-I-MSCs) were applied systemically, and changes in collagen deposition and in the expression of key pro-fibrogenic and pro-regenerative genes/proteins were assessed. In addition, immunogenicity of transduced cells was analyzed. Liver fibrosis was further ameliorated after a single-dose application of AdIGF-I-MSCs when compared with AdGFP-MSCs and/or recombinant IGF-I treatments. Interestingly, an early and transitory upregulation in IGF-I and hepatocyte growth factor (HGF) mRNA expression was found in the liver of MSC-treated animals, which was more pronounced in AdIGF-I-MSCs condition. A reduction in hepatic stellate cell activation status was found after incubation with MSCs conditioned media. In addition, the AdIGF-I-MSCs cell-free supernatant induced the expression of IGF-I and HGF in primary cultured hepatocytes. From day 1 after transplantation, the proliferation marker proliferating cell nuclear antigen was upregulated in the liver of AdIGF-I-MSCs group, mainly in hepatocytes. MSCs were in vivo traced till day 14 after injection. In addition, multiple doses of Ad-IGF-I-MSCs likely suppressed antiviral immune response and it further reduced collagen deposition. Our results uncover early events that are likely involved in the anti-fibrogenic effect of genetically modified MSCs and overall would support the use of AdIGF-I-MSCs in treatment of liver fibrosis.

Lack of the matricellular protein SPARC (secreted protein, acidic and rich in cysteine) attenuates liver fibrogenesis in mice.

INTRODUCTION: Secreted Protein, Acidic and Rich in Cysteine (SPARC) is a matricellular protein involved in many biological processes and found over-expressed in cirrhotic livers. By mean of a genetic approach we herein provide evidence from different in vivo liver disease models suggesting a profibrogenic role for SPARC. METHODS: Two in vivo models of liver fibrosis, based on TAA administration and bile duct ligation, were developed on SPARC wild-type (SPARC(+/+)) and knock-out (SPARC(-/-)) mice. Hepatic SPARC expression was analyzed by qPCR. Fibrosis was assessed by Sirius Red staining, and the maturation state of collagen fibers was analyzed using polarized light. Necroinflammatory activity was evaluated by applying the Knodell score and liver inflammatory infiltration was characterized by immunohistochemistry. Hepatic stellate cell activation was assessed by α-SMA immunohistochemistry. In addition, pro-fibrogenic genes and inflammatory cytokines were measured by qPCR and/or ELISA. Liver gene expression profile was analyzed in SPARC(-/-) and SPARC(+/+) mice using Affymetrix Mouse Gene ST 1.0 array. RESULTS: SPARC expression was found induced in fibrotic livers of mouse and human. SPARC(-/-) mice showed a reduction in the degree of inflammation, mainly CD4+ cells, and fibrosis. Consistently, collagen deposits and mRNA expression levels were decreased in SPARC(-/-) mice when compared to SPARC(+/+) mice; in addition, MMP-2 expression was increased in SPARC(-/-) mice. A reduction in the number of activated myofibroblasts was observed. Moreover, TGF-ß1 expression levels were down-regulated in the liver as well as in the serum of TAA-treated knock-out animals. Ingenuity Pathway Analysis (IPA) analysis suggested several gene networks which might involve protective mechanisms of SPARC deficiency against liver fibrogenesis and a better established machinery to repair DNA and detoxify from external chemical stimuli. CONCLUSIONS: Overall our data suggest that SPARC plays a significant role in liver fibrogenesis. Interventions to inhibit SPARC expression are suggested as promising approaches for liver fibrosis treatment.