Infrapatellar fat pad adipose-derived stem cells co-cultured with articular chondrocytes from osteoarthritis patients exhibit increased chondrogenic gene expression.

AIM: The variable results in clinical trials of adipose tissue-derived stem cells (ASCs) for chondral defects may be due to the different ex vivo culture conditions of the ASCs which are implanted to treat the lesions. We sought to determine the optimal in vitro chondrocyte co-culture condition that promotes infrapatellar fat pad-derived (IFPD) ASC chondrogenic gene expression in a novel co-culture combination. METHODS: In our study, we utilized an in vitro autologous co-culture of IFPD ASCs and articular chondrocytes derived from Kellgren-Lawrence Grade III/IV osteoarthritic human knee joints at ASC-to-chondrocyte seeding log ratios of 1:1, 10:1, and 100:1. Gene expression following in vitro co-culture was quantified by RT-qPCR with a panel comprising COL1A1, COL2A1, COL10A1, L-SOX5, SOX6, SOX9, ACAN, HSPG2, and COMP for chondrogenic gene expression. RESULTS: The chondrogenic gene expression profiles from co-cultures were greater than would be expected from an expression profile modeled from chondrocyte and ASC-only monocultures. Additionally, chondrogenic gene expression decreased with increasing ASC-to-chondrocyte seeding ratios. CONCLUSIONS: These findings provide insight into the mechanisms underlying clinical ASC therapies and signifies that IFPD ASCs pre-conditioned by chondrocyte co-culture may have improved chondrogenic potential for cartilage repair. This model can help further understand IFPD ASCs in chondral and osteochondral repair and the chondrogenic pathways involved. Video Abstract

Transcriptional and Epigenetic Consequences of DMSO Treatment on HepaRG Cells

Dimethyl sulfoxide (DMSO) is used to sustain or favor hepatocyte differentiation in vitro. Thus, DMSO is used in the differentiation protocol of the HepaRG cells that present the closest drug-metabolizing enzyme activities to primary human hepatocytes in culture. The aim of our study is to clarify its influence on liver-specific gene expression. For that purpose, we performed a large-scale analysis (gene expression and histone modification) to determine the global role of DMSO exposure during the differentiation process of the HepaRG cells. The addition of DMSO drives the upregulation of genes mainly regulated by PXR and PPARα whereas genes not affected by this addition are regulated by HNF1α, HNF4α, and PPARα. DMSO-differentiated-HepaRG cells show a differential expression for genes regulated by histone acetylation, while differentiated-HepaRG cells without DMSO show gene signatures associated with histone deacetylases. In addition, we observed an interplay between cytoskeleton organization and EMC remodeling with hepatocyte maturation

Biomarkers and new therapeutic approches targeting cancer stem cells in hepatocellular carcinoma

Le cancer primitif du foie, ou carcinome hépatocellulaire (CHC), est l’un des premiers cancers dans le monde chez l’homme adulte. L'identité des cellules à l'origine des CHC est controversée, toutefois l'existence de cellules souches/progénitrices (CS/P) dans certaines tumeurs a été rapportée. Ces tumeurs ont un phénotype très agressif, sont associées à un mauvais pronostic et ont tendance à échapper aux thérapies conventionnelles. Ainsi, des traitements ciblant spécifiquement les cellules souches cancéreuses pourraient constituer des compléments thérapeutiques efficaces aux traitements actuels. Pour cela, il est nécessaire de disposer de biomarqueurs afin d’identifier les sous-types de CHC dérivant de CS/P mais également de modèles cellulaires permettant de tester des molécules thérapeutiques. Les CS/P pourraient provenir soit du compartiment de cellules souches hépatiques soit de la dédifférenciation d’hépatocytes à l'intérieur de la tumeur. Aussi dans le cadre de cette thèse nous nous sommes attachés à étudier les mécanismes cellulaires et moléculaires impliqués 1) dans la retrodifférenciation des hépatocytes en CS/P et 2) dans le maintien des propriétés souches. Pour cette étude nous avons utilisé la lignée HepaRG issue d’un CHC sur infection virale C qui présente des caractéristiques de progéniteurs hépatiques se différenciant en hépatocytes et cholangiocytes. Dans la première partie de ce travail nous avons montré qu’un environnement inflammatoire favorise la retrodifferenciation d’hépatocytes HepaRG en CS/P. Alors que les voies activées par le TNF et l’IL-6 sont impliquées dans la perte du phénotype hépatocytaire, la voie TGF induit une transition épithélio-mésenchymateuse. Dans ces CS/P, nous observons l’enrichissement de signatures moléculaires associées à des souches embryonnaires et à des CHC avec mauvais pronostic. De plus, nous avons identifié plusieurs molécules pouvant inhiber ce processus de rétrodifférenciation des hépatocytes-HepaRG comme la trichostatine A. Dans une seconde partie nous avons montré que l’acquisition et le maintien des caractéristiques souches des cellules HepaRG étaient associés à une production de cytokines, à l’expression de métalloprotéinases impliquées dans les processus de migration et d’invasion, de facteur pro-angiogenique et à un changement dans l’activité mitochondriale conduisant à une reprogrammation métabolique. Les voies de signalisation impliquant l’angiopoïétine-like 4, PI3K et ERK régulent ces changements. De façon intéressante, nous montrons que le rétablissement de l’activité mitochondriale réduit la résistance des CS/P aux anticancéreux. Notre étude nous a permis d’acquérir une meilleure compréhension des voies de signalisation activées dans les cellules souches/progénitrices hépatiques cancéreuses et des mécanismes impliqués dans la rétro-différenciation des cellules matures tumorales, deux phénomènes probablement impliqués dans la récidive tumorale et la chimiorésistance.Biomarkers and new therapeutic approches targeting cancer stem cells in hepatocellular carcinom

The Unitary Micro-Immunotherapy Medicine Interferon-γ (4 CH) Displays Similar Immunostimulatory and Immunomodulatory Effects than Those of Biologically Active Human Interferon-γ on Various Cell Types

As a cytokine, gamma-interferon (IFN-γ) is considered a key player in the fine-tuned orchestration of immune responses. The extreme cellular sensitivity to cytokines is attested by the fact that very few of these bioactive molecules per cell are enough to trigger cellular functions. These findings can, at least partially, explain how/why homeopathically-prepared cytokines, and especially micro-immunotherapy (MI) medicines, are able to drive cellular responses. We focused our fundamental research on a unitary MI preparation of IFN-γ, specifically employed at 4 CH, manufactured and impregnated onto sucrose-lactose pillules as all other MI medicines. We assessed the IFN-γ concentration in the medium after dilution of the IFN-γ (4 CH)-bearing pillules and we evaluated in vitro drug responses in a wide range of immune cells, and in endothelial cells. Our results showed that IFN-γ (4 CH) stimulated the proliferation, the activation and the phagocytic capabilities of primary immune cells, as well as modulated their cytokine-secretion and immunity-related markers’ expression in a trend that is quite comparable with the well-recognized biological effects induced by IFN-γ. Altogether, these data provide novel and additional evidences on MI medicines, and specifically when active substances are prepared at 4 CH, thus suggesting the need for more investigations

The Micro-Immunotherapy Medicine 2LEID Exhibits an Immunostimulant Effect by Boosting Both Innate and Adaptive Immune Responses

This study aimed at evaluating the effects of the micro-immunotherapy medicine (MIM) 2LEID, both in vitro and in vivo, on several components of the innate and adaptive immune system. MIM increased the phagocytic activity of macrophages, and it augmented the expression of the activation markers CD69 and HLA-DR in NK cells and monocytes/macrophages, respectively. The effect of MIM was evaluated in a model of respiratory infection induced by influenza A virus administration to immunocompetent mice in which it was able to improve neutrophil recruitment within the lungs (p = 0.1051) and slightly increased the circulating levels of IgM (p = 0.1655). Furthermore, MIM stimulated the proliferation of CD3-primed T lymphocytes and decreased the secretion of the immunosuppressive cytokine IL-10 in CD14+-derived macrophages. Human umbilical vein endothelial cells were finally used to explore the effect of MIM on endothelial cells, in which it slightly increased the expression of immune-related markers such as HLA-I, CD137L, GITRL, PD-L1 and ICAM-1. In conclusion, the present study suggests that MIM might be a promising nonspecific (without antigen specificity) immunostimulant drug in preventing and early treating respiratory infections, but not only exclusively, as it would gently support several facets of the immune system and host defenses

Retrodifferentiation of human tumor hepatocytes to stem cells leads to metabolic reprogramming and chemoresistance

International audienceHuman hepatocellular carcinoma (HCC) heterogeneity promotes recurrence and therapeutic resistance. We recently demonstrated that inflammation favors hepatocyte retrodifferentiation into progenitor cells. Here, we identify the molecular effectors that induce metabolic reprogramming, chemoresistance, and invasiveness of retrodifferentiated HCC stem cells. Spheroid cultures of human HepaRG progenitors (HepaRG-Spheres), HBG-BC2, HepG2, and HuH7 cells and isolation of side population (SP) from HepaRG cells (HepaRG-SP) were analyzed by transcriptomics, signaling pathway analysis, and evaluation of chemotherapies. Gene expression profiling of HepaRG-SP and HepaRG-Spheres revealed enriched signatures related to cancer stem cells, metastasis, and recurrence and showed that HepaRG progenitors could retrodifferentiate into an immature state. The transcriptome from these stem cells matched that of proliferative bad outcome HCCs in a cohort of 457 patients. These HCC stem cells expressed high levels of cytokines triggering retrodifferentiation and displayed high migration and invasion potential. They also showed changes in mitochondrial activity with reduced membrane potential, low ATP production, and high lactate production. These changes were, in part, related to angiopoietin-like 4 (ANGPTL4)-induced upregulation of pyruvate dehydrogenase kinase 4 (PDK4), an inhibitor of mitochondrial pyruvate dehydrogenase. Upregulation of ANGPTL4 and PDK4 paralleled that of stem cells markers in human HCC specimens. Moreover, the PDK4 inhibitor dichloroacetate reversed chemoresistance to sorafenib or cisplatin in HCC stem cells derived from four HCC cell lines. In conclusion, retrodifferentiated cancer cells develop enhanced invasion and therapeutic resistance through ANGPTL4 and PDK4. Therefore, restoration of mitochondrial activity in combination with chemotherapy represents an attractive therapeutic approach in HCC

Inflammatory cytokines promote the retrodifferentiation of tumor-derived hepatocyte-like cells to progenitor cells.

International audienceHuman hepatocellular carcinoma (HCC) heterogeneity promotes recurrence and resistance to therapies. Recent studies have reported that HCC may be derived not only from adult hepatocytes and hepatoblasts but also hepatic stem/progenitors. In this context, HepaRG cells may represent a suitable cellular model to study stem/progenitor cancer cells and the retrodifferentiation of tumor-derived hepatocyte-like cells. Indeed, they differentiate into hepatocyte- and biliary-like cells. Moreover, tumor-derived HepaRG hepatocyte-like cells (HepaRG-tdHep) differentiate into both hepatocyte- and biliary-like cells through a hepatic progenitor. In this study we report the mechanisms and molecular effectors involved in the retrodifferentiation of HepaRG-tdHep into bipotent progenitors. Gene expression profiling was used to identify genomic changes during the retrodifferentiation of HepaRG-tdHep into progenitors. We demonstrated that gene expression signatures related to a poor-prognosis HCC subclass, proliferative progenitors, or embryonic stem cells were significantly enriched in HepaRG progenitors derived from HepaRG-tdHep. HepaRG-tdHep retrodifferentiation is mediated by crosstalk between transforming growth factor beta 1 (TGFβ1) and inflammatory cytokine pathways (e.g., tumor necrosis factor alpha [TNFα] and interleukin 6 [IL6]). Signatures related to TNFα, IL6, and TGFβ activation pathways are induced within the first hour of retrodifferentiation. Moreover, specific activation or inhibition of these signaling pathways allowed us to determine that TNFα and IL6 contribute to the loss of hepatic-specific marker expression and that TGFβ1 induces an epithelial-to-mesenchymal transition of HepaRG-tdHep. Interestingly, the retrodifferentiation process is blocked by the histone deacetylase inhibitor trichostatin A, opening new therapeutic opportunities. Cancer progenitor cells (or metastasis progenitors) may derive from tumor-derived hepatocyte-like cells in an inflammatory environment that is frequently associated with HCC

Transcriptional and epigenetic consequences of DMSO treatment on HepaRG cells

International audienceDimethyl sulfoxide (DMSO) is used to sustain or favor hepatocyte differentiation in vitro. Thus, DMSO is used in the differentiation protocol of the HepaRG cells that present the closest drug-metabolizing enzyme activities to primary human hepatocytes in culture. The aim of our study is to clarify its influence on liver-specific gene expression. For that purpose, we performed a large-scale analysis (gene expression and histone modification) to determine the global role of DMSO exposure during the differentiation process of the HepaRG cells. The addition of DMSO drives the upregulation of genes mainly regulated by PXR and PPAR alpha whereas genes not affected by this addition are regulated by HNF1 alpha, HNF4 alpha, and PPAR alpha. DMSO-differentiated-HepaRG cells show a differential expression for genes regulated by histone acetylation, while differentiated-HepaRG cells without DMSO show gene signatures associated with histone deacetylases. In addition, we observed an interplay between cytoskeleton organization and EMC remodeling with hepatocyte maturation