Jordi Barquinero

Flow Cytometry-Based Approach to ABCG2 Function Suggests That the Transporter Differentially Handles the Influx and Efflux of Drugs

To better characterize the function of the ABCG2 transporter in vitro, we generated three cell lines (MXRA, MXRG, and MXRT) stably expressing ABCG2 after transfection of wild‐type ABCG2 and two mutants (R482G and R482T), respectively.

Gene Therapy Using a Liver-targeted AAV Vector Restores Nucleoside and Nucleotide Homeostasis in a Murine Model of MNGIE

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder caused by mutations in TYMP, enconding thymidine phosphorylase (TP). TP deficiency results in systemic accumulation of thymidine and deoxyuridine, which interferes with mitochondrial DNA (mtDNA) replication and leads to mitochondrial dysfunction. To date, the only treatment available for MNGIE patients is allogeneic hematopoietic stem cell transplantation, which is associated with high morbidity and mortality. Here, we report that AAV2/8-mediated transfer of the human TYMP coding sequence (hcTYMP) under the control of a liver-specific promoter prevents the biochemical imbalances in a murine model of MNGIE. hcTYMP expression was restricted to liver, and a dose as low as 2 × 10(11) genome copies/kg led to a permanent reduction in systemic nucleoside levels to normal values in about 50% of treated mice. Higher doses resulted in reductions to normal or slightly below normal levels in virtually all mice treated. The nucleoside reduction achieved by this treatment prevented deoxycytidine triphosphate (dCTP) depletion, which is the limiting factor affecting mtDNA replication in this disease. These results demonstrate that the use of AAV to direct TYMP expression in liver is feasible as a potentially safe gene therapy strategy for MNGIE.

Identification of multipotent mesenchymal stromal cells in the reactive stroma of a prostate cancer xenograft by side population analysis.

Cancer stem cells are a distinct cellular population that is believed to be responsible for tumor initiation and maintenance. Recent data suggest that solid tumors also contain another type of stem cells, the mesenchymal stem cells or multipotent mesenchymal stromal cells (MSCs), which contribute to the formation of tumor-associated stroma. The Hoechst 33342 efflux assay has proved useful to identify a rare cellular fraction, named Side Population (SP), enriched in cells with stem-like properties. Using this assay, we identified SP cells in a prostate cancer xenograft containing human prostate cancer cells and mouse stromal cells. The SP isolation, subculture and sequential sorting allowed the generation of single-cell-derived clones of murine origin that were recognized as MSC by their morphology, plastic adherence, proliferative potential, adipogenic and osteogenic differentiation ability and immunophenotype (CD45(-), CD81(+) and Sca-1(+)). We also demonstrated that SP clonal cells secrete transforming growth factor beta1 (TGF-beta1) and that their inhibition reduces proliferation and accelerates differentiation. These results reveal the existence of SP cells in the stroma of a cancer xenograft, and provide evidence supporting their MSC nature and the role of TGF-beta1 in maintaining their proliferation and undifferentiated status. Our data also reveal the usefulness of the SP assay to identify and isolate MSC cells from carcinomas.

Tolerance Induction in Experimental Autoimmune Encephalomyelitis Using Non-myeloablative Hematopoietic Gene Therapy With Autoantigen

Experimental autoimmune encephalomyelitis (EAE) constitutes a paradigm of antigen (Ag)-specific T cell driven autoimmune diseases. In this study, we transferred bone marrow cells (BMCs) expressing an autoantigen (autoAg), the peptide 40-55 of the myelin oligodendrocytic glycoprotein (MOG(40-55)), to induce preventive and therapeutic immune tolerance in a murine EAE model. Transfer of BMC expressing MOG(40-55) (IiMOG-BMC) into partially myeloablated mice resulted in molecular chimerism and in robust protection from the experimental disease. In addition, in mice with established EAE, transfer of transduced BMC with or without partial myeloablation reduced the clinical and histopathological severity of the disease. In these experiments, improvement was observed even in the absence of engraftment of the transduced hematopoietic cells, probably rejected due to the previous immunization with the autoAg. Splenocytes from mice transplanted with IiMOG-BMC produced significantly higher amounts of interleukin (IL)-5 and IL-10 upon autoAg challenge than those of control animals, suggesting the participation of regulatory cells. Altogether, these results suggest that different tolerogenic mechanisms may be mediating the preventive and the therapeutic effects. In conclusion, this study demonstrates that a cell therapy using BMC expressing an autoAg can induce Ag-specific tolerance and ameliorate established EAE even in a nonmyeloablative setting.

Estrogen receptor beta displays cell cycle-dependent expression and regulates the G1 phase through a non-genomic mechanism in prostate carcinoma cells

Background: It is well known that estrogens regulate cell cycle progression, but the specific contributions and mechanisms of action of the estrogen receptor beta (ERβ) remain elusive. Methods: We have analyzed the levels of ERβ1 and ERβ2 throughout the cell cycle, as well as the mechanisms of action and the consequences of the over-expression of ERβ1 in the human prostate cancer LNCaP cell line. Results: Both ERβ1 mRNA and protein expression increased from the G1 to the S phase and decreased before entering the G2/M phase, whereas ERβ2 levels decreased during the S phase and increased in the G2/M phase. ERβ1 protein was detected in both the nuclear and non-nuclear fractions, and ERβ2 was found exclusively in the nucleus. Regarding the mechanisms of action, endogenous ERβ was able to activate transcription via ERE during the S phase in a ligand-dependent manner, whereas no changes in AP1 and NFκB transactivation were observed after exposure to estradiol or the specific inhibitor ICI 182,780. Over-expression of either wild type ERβ1 or ERβ1 mutated in the DNA-binding domain caused an arrest in early G1. This arrest was accompanied by the interaction of over-expressed ERβ1 with c-Jun N-terminal protein kinase 1 (JNK1) and a decrease in c-Jun phosphorylation and cyclin D1 expression. The administration of ICI impeded the JNK1–ERβ1 interaction, increased c-Jun phosphorylation and cyclin D1 expression and allowed the cells to progress to late G1, where they became arrested. Conclusions: Our results demonstrate that, in LNCaP prostate cancer cells, both ERβ isoforms are differentially expressed during the cell cycle and that ERβ regulates the G1 phase by a non-genomic mechanism.

Transgene Expression Levels Determine the Immunogenicity of Transduced Hematopoietic Grafts in Partially Myeloablated Mice

We investigated whether transgene expression levels influence the immunogenicity of transduced hematopoietic grafts upon transplantation into partially myeloablated mice. To this aim, bone marrow cells (BMCs) transduced with retroviral vectors driving green fluorescent protein (GFP) expression either at high (high-EGFP) or low levels (low-EGFP) were transplanted into congenic recipients conditioned with sublethal doses of total body irradiation (TBI) or busulfan. Virtually all recipients showed evidence of donor engraftment 4 weeks after transplantation. However, as opposed to recipients receiving low-EGFP transduced grafts, the risk of rejecting the EGFP(+) cells by 30 days after transplantation was significantly higher in mice conditioned with busulfan and receiving high-EGFP transduced grafts. Anti-EGFP cellular immune responses were demonstrated in high-EGFP-treated mice conditioned with busulfan by interferon-gamma (IFN-gamma), enzyme-linked immunospot assay (ELISPOT), and cytotoxic T lymphocyte (CTL) assays, in contrast to that observed in mice transplanted with low-EGFP BMC. These results show for the first time that transgene expression levels can be critical for the immunogenicity of gene-modified hematopoietic grafts, especially in immunocompetent or in partially immunosuppressed recipients. These results have profound implications in vector choice and in the design of gene therapy (GT) protocols.

Flow cytometry of the Side Population: Tips & tricks

Background: The Side Population (SP) has become an important hallmark for the definition of the stem cell compartment, especially in the detection of these cells and in their physical isolation by fluorescence-activated cell sorting (FACS). SP cells are CD34neg and were discovered using ultraviolet excitation based on the efflux of Hoechst 33342 (Ho342). Although the method works as originally described, we believe that this method is difficult for most investigators. First, because the ability to discriminate SP cells is based on the differential retention of Ho342 during a functional assay; second, because of the difficulties in setting the right experimental and acquisition conditions; and third, because the analysis of the acquired data requires an extensive expertise on flow cytometry to accurately detect the SP events. Methods: First of all and mainly for the SP application, the laser beam paths were exhaustively checked to ensure the lowest coefficients of variation. Blood suspensions were prepared by erythrocyte lysis with ammonium chloride and hematopoietic cells were labeled with Ho342. Results: The Ho342 concentration and the staining procedure are critical for the optimal resolution of the SP cells. Although UV laser alignment is very important to resolve the dim tail that outlines the SP, the problem with Ho342 excitation is not the Hoechst Blue emission, but rather the Hoechst Reds (because of the weak emission). Conclusions: Each laboratory must establish its own expected ranges based on its instrument and results may vary slightly due to instrument differences such as the narrowness of the band pass filters, laser power, laser emission wavelength, nozzle type, differential of pressure, light collection system (cuvette versus jet-in-air) and beam shaping optics.

Myeloid-Derived Suppressor Cells are Generated during Retroviral Transduction of Murine Bone Marrow:

Previous work by our group showed that transferring bone marrow cells transduced with an autoantigen into nonmyeloablated mice with experimental autoimmune encephalomyelitis induced immune tolerance and improved symptoms of the disease. Because this effect occurred in the absence of molecular chimerism, we hypothesized that the cells responsible did not have repopulating ability and that they were not mediating central but peripheral tolerance mechanisms. In the present study, we analyzed the immunophenotype of the cells that are generated in the transduction cultures and we evaluated the immunosuppressive activity of the main cell subpopulations produced. We show that both granulocytic (CD11b+ Gr-1hi) and monocytic (CD11b+Gr-1lo) myeloid-derived suppressor cells (G- and M-MDSCs, respectively) are generated during standard 4-day γ-retroviral transduction cultures (representing about 25% and 40% of the total cell output, respectively) and that the effectively transduced cells largely consist of these two cell types. A third cell population representing about 15% of the transduced cells did not express CD45 or hematopoietic lineage markers and expressed mesenchymal stromal cell markers. Transduced total bone marrow cells and sorted M-MDSCs expressed arginase and inducible nitric oxide synthase activities, produced reactive oxygen species, and inhibited antigen-induced T-cell proliferation in vitro. Transgene-expressing MDSCs could be exploited therapeutically to induce tolerance in autoimmune diseases and in gene therapy protocols.

A reproducible method for the isolation and expansion of ovine mesenchymal stromal cells from bone marrow for use in regenerative medicine preclinical studies

The use of multipotent mesenchymal stromal cells (MSCs) as candidate medicines for treating a variety of pathologies is based on their qualities as either progenitors for the regeneration of damaged tissue or producers of a number of molecules with pharmacological properties. Preclinical product development programmes include the use of well characterized cell populations for proof of efficacy and safety studies before testing in humans. In the field of orthopaedics, an increasing number of translational studies use sheep as an in vivo test system because of the similarities with humans in size and musculoskeletal architecture. However, robust and reproducible methods for the isolation, expansion, manipulation and characterization of ovine MSCs have not yet been standardised. The present study describes a method for isolation and expansion of fibroblastic‐like, adherent ovine MSCs that express CD44, CD90, CD140a, CD105 and CD166, and display trilineage differentiation potential. The 3‐week bioprocess proposed here typically yielded cell densities of 1.4 × 104 MSCs/cm2 at passage 2, with an expansion factor of 37.8 and approximately eight cumulative population doublings. The osteogenic potential of MSCs derived following this methodology was further evaluated in vivo in a translational model of osteonecrosis of the femoral head, in which the persistence of grafted cells in the host tissue and their lineage commitment into osteoblasts and osteocytes was demonstrated by tracking enhanced green fluorescent protein‐labelled cells. Copyright

Expression of microRNA‐155 in inflammatory cells modulates liver injury

MicroRNA 155 (miR‐155) is involved in immune and inflammatory diseases and is associated with liver fibrosis and steatohepatitis. However, the mechanisms involved in miR‐155 regulation of liver injury are largely unknown. The role of miR‐155 in acute liver injury was assessed in wild‐type (WT), miR‐155−/−, and miR‐155−/− mice transplanted with WT bone marrow. Additionally, miR‐155 expression was evaluated in liver tissue and peripheral blood mononuclear cells of patients with autoimmune hepatitis. Concanavalin A, but not acetaminophen, treatment increased the expression of miR‐155 in liver tissue of WT mice. Concanavalin A induced increases in cell death, liver aminotransferases, and expression of proinflammatory cytokines (chemokine [C‐X‐C motif] ligands 1, 5, 9, 10, and 11; chemokine [C‐C motif] ligands 2 and 20; and intercellular cell adhesion molecule 1) in miR‐155−/− compared to WT mice. Importantly, these animals showed a significant decrease in cluster of differentiation 4–positive/chemokine (C‐X‐C motif) receptor 3–positive and forkhead box p3–positive cell recruitment but no changes in other inflammatory cell populations. Mechanistically, miR‐155‐deficient regulatory T cells showed increased SH2 domain–containing inositol 5‐phosphatase 1 expression, a known target of miR‐155. Inhibition of SH2 domain–containing inositol 5‐phosphatase 1 in miR‐155−/− mice restored forkhead box p3 recruitment and reduced liver cytokine expression. Transplantation of bone marrow from WT animals into miR‐155−/− mice partially reversed the effect of concanavalin A on miR‐155−/− mice as assessed by proinflammatory cytokines and cell death protein expression. Patients with autoimmune hepatitis showed a marked increase in miR‐155 expression in the liver but reduced expression of miR‐155 in peripheral blood mononuclear cells. Conclusion: miR‐155 expression is altered in both liver tissue and circulating inflammatory cells during liver injury, thus regulating inflammatory cell recruitment and liver damage; these results suggest that maintaining miR‐155 expression in inflammatory cells might be a potential strategy to modulate liver injury. (Hepatology 2018).