Acceleration of infectious disease drug discovery and development using a humanized model of drug metabolism

A key step in drug discovery, common to many disease areas, is preclinical demonstration of efficacy in a mouse model of disease. However, this demonstration and its translation to the clinic can be impeded by mouse-specific pathways of drug metabolism. Here we show that a mouse line extensively humanized for the cytochrome P450 gene superfamily (“8HUM”) can circumvent these problems. The pharmacokinetics, metabolite profiles and magnitude of drug-drug interactions of a test set of approved medicines were in much closer alignment with clinical observations than in wild-type mice. Infection with Mycobacterium tuberculosis, Leishmania donovani and Trypanosoma cruzi was well tolerated in 8HUM, permitting efficacy assessment. During such assessments, mouse-specific metabolic liabilities were bypassed while the impact of clinically relevant active metabolites and DDI on efficacy were well-captured. Removal of species differences in metabolism by replacement of wild-type mice with 8HUM therefore reduces compound attrition while improving clinical translation, accelerating drug discovery

Sertoli cells maintain leydig cell number and peritubular myoid cell activity in the adult mouse testis

The Sertoli cells are critical regulators of testis differentiation and development. In the adult, however, their known function is restricted largely to maintenance of spermatogenesis. To determine whether the Sertoli cells regulate other aspects of adult testis biology we have used a novel transgenic mouse model in which Amh-Cre induces expression of the receptor for Diphtheria toxin (iDTR) specifically within Sertoli cells. This causes controlled, cell-specific and acute ablation of the Sertoli cell population in the adult animal following Diphtheria toxin injection. Results show that Sertoli cell ablation leads to rapid loss of all germ cell populations. In addition, adult Leydig cell numbers decline by 75% with the remaining cells concentrated around the rete and in the sub-capsular region. In the absence of Sertoli cells, peritubular myoid cell activity is reduced but the cells retain an ability to exclude immune cells from the seminiferous tubules. These data demonstrate that, in addition to support of spermatogenesis, Sertoli cells are required in the adult testis both for retention of the normal adult Leydig cell population and for support of normal peritubular myoid cell function. This has implications for our understanding of male reproductive disorders and wider androgen-related conditions affecting male health

Bibliographie

Bibliographie. In: Paléorient, 1981, vol. 7, n°2. pp. 123-150

Biomaterials and Bioactive Molecules to Drive Differentiation in Striated Muscle Tissue Engineering.

Tissue engineering is an innovative, multidisciplinary approach which combines (bio)materials, cells and growth factors with the aim to obtain neo-organogenesis to repair or replenish damaged tissues and organs. The generation of engineered tissues and organs (e. g. skin and bladder) has entered into the clinical practice in response to the chronic lack of organ donors. In particular, for the skeletal and cardiac muscles the translational potential of tissue engineering approaches has clearly been shown, even though the construction of this tissue lags behind others given the hierarchical, highly organized architecture of striated muscles. Cardiovascular disease is the leading cause of death in the developed world, where the yearly incidence of Acute MI (AMI) is approx 2 million cases in Europe. Recovery from AMI and reperfusion is still less than ideal. Stem cell therapy may represent a valid treatment. However, delivery of stem cells alone to infarcted myocardium provides no structural support while the myocardium heals, and the injected stem cells do not properly integrate into the myocardium because they are not subjected to the mechanical forces that are known to drive myocardial cellular physiology. On the other hand, there are many clinical cases where the loss of skeletal muscle due to a traumatic injury, an aggressive tumour or prolonged denervation may be cured by the regeneration of this tissue. In vivo, stem or progenitor cells are sheltered in a specialized microenvironment (niche), which regulates their survival, proliferation and differentiation. The goal of this research topic is to highlight the available knowledge on biomaterials and bioactive molecules or a combination of them, which can be used successfully to differentiate stem or progenitor cells into beating cardiomyocytes or organized skeletal muscle in vivo. Innovations compared to the on-going trials may be: 1) the successful delivery of stem cells using sutural scaffolds instead of intracoronary or intramuscular injections; 2) protocols to use a limited number of autologous or allogeneic stem cells; 3) methods to drive their differentiation by modifying the chemical-physical properties of scaffolds or biomaterials, incorporating small molecules (i.e. miRNA) or growth factors; 4) methods to tailor the scaffolds to the elastic properties of the muscle; 5) studies which suggest how to realize scaffolds that optimize tissue functional integration, through the combination of the most up-to-date manufacturing technologies and use of bio-polymers with customized degradation properties

Muscle acellular scaffold as a biomaterial: effects on C2C12 cell differentiation and interaction with the murine host environment

International audienceThe extracellular matrix (ECM) of decellularized organs possesses the characteristics of the ideal tissue-engineering scaffold (i.e., histocompatibility, porosity, degradability, non-toxicity). We previously observed that the muscle acellular scaffold (MAS) is a pro-myogenic environment in vivo. In order to determine whether MAS, which is basically muscle ECM, behaves as a myogenic environment, regardless of its location, we analyzed MAS interaction with both muscle and non-muscle cells and tissues, to assess the effects of MAS on cell differentiation. Bone morphogenetic protein treatment of C2C12 cells cultured within MAS induced osteogenic differentiation in vitro, thus suggesting that MAS does not irreversibly commit cells to myogenesis. In vivo MAS supported formation of nascent muscle fibers when replacing a muscle (orthotopic position). However, heterotopically grafted MAS did not give rise to muscle fibers when transplanted within the renal capsule. Also, no muscle formation was observed when MAS was transplanted under the xiphoid process, in spite of the abundant presence of cells migrating along the laminin-based MAS structure. Taken together, our results suggest that MAS itself is not sufficient to induce myogenic differentiation. It is likely that the pro-myogenic environment of MAS is not strictly related to the intrinsic properties of the muscle scaffold (e.g., specific muscle ECM proteins). Indeed, it is more likely that myogenic stem cells colonizing MAS recognize a muscle environment that ultimately allows terminal myogenic differentiation. In conclusion, MAS may represent a suitable environment for muscle and non-muscle 3D constructs characterized by a highly organized structure whose relative stability promotes integration with the surrounding tissues. Our work highlights the plasticity of MAS, suggesting that it may be possible to consider MAS for a wider range of tissue engineering applications than the mere replacement of volumetric muscle loss

Autologous bone marrow stromal cells are promising candidates for cell therapy approaches to treat bone degeneration in sickle cell disease

Osteonecrosis of the femoral head is a frequent complication in adult patients with sickle cell disease (SCD). To delay hip arthroplasty, core decompression combined with concentrated total bone marrow (BM) treatment is currently performed in the early stages of the osteonecrosis. Cell therapy efficacy depends on the quantity of implanted BM stromal cells. For this reason, expanded bone marrow stromal cells (BMSCs, also known as bone marrow derived mesenchymal stem cells) can be used to improve osteonecrosis treatment in SCD patients. In this study, we quantitatively and qualitatively evaluated the function of BMSCs isolated from a large number of SCD patients with osteonecrosis (SCD-ON) compared with control groups (patients with osteonecrosis not related to SCD (ON) and normal donors (N)). BM total nuclear cells and colony-forming efficiency values (CFE) were significantly higher in SCD-ON patients than in age and sex-matched controls. The BMSCs from SCD-ON patients were similar to BMSCs from the control groups in terms of their phenotypic and functional properties. SCD-ON patients have a higher frequency of BMSCs that retain their bone regeneration potential. Our findings suggest that BMSCs isolated from SCD-ON patients can be used clinically in cell therapy approaches. This work provides important preclinical data that is necessary for the clinical application of expanded BMSCs in advanced therapies and medical products

Inferring Gene Networks in Bone Marrow Hematopoietic Stem Cell-Supporting Stromal Niche Populations

International audienceThe cardinal property of bone marrow (BM) stromal cells is their capacity to contribute to hematopoietic stem cell (HSC) niches by providing mediators assisting HSC functions. In this study we first contrasted transcriptomes of stromal cells at different developmental stages and then included large number of HSC-supportive and non-supportive samples. Application of a combination of algorithms, comprising one identifying reliable paths and potential causative relationships in complex systems, revealed gene networks characteristic of the BM stromal HSC-supportive capacity and of defined niche populations of perivascular cells, osteoblasts, and mesenchymal stromal cells. Inclusion of single-cell transcriptomes enabled establishing for the perivascular cell subset a partially oriented graph of direct gene-to-gene interactions. As proof of concept we showed that R-spondin-2, expressed by the perivascular subset, synergized with Kit ligand to amplify ex vivo hematopoietic precursors. This study by identifying classifiers and hubs constitutes a resource to unravel candidate BM stromal mediators

The cell adhesion molecule BT-IgSF is essential for a functional blood–testis barrier and male fertility in mice

The Ig-like cell adhesion molecule (IgCAM) brain- and testis-specific immunoglobulin superfamily (BT-IgSF) protein plays a major role in male fertility in mice. However, the molecular mechanism by which BT-IgSF supports fertility is unclear. Here, we found that it is localized in Sertoli cells at the blood-testis barrier (BTB) and at the apical ectoplasmic specialization. Absence of BT-IgSF in Sertoli cells in both global and conditional mouse mutants (i.e. AMHCre and Rosa26CreERT2 lines) resulted in male infertility, atrophic testes with vacuolation, azoospermia, and spermatogenesis arrest. Although transcripts of junctional proteins such as connexin43, ZO-1, occludin, and claudin11 were upregulated in the absence of BT-IgSF, the functional integrity of the BTB was impaired, as revealed by injection of a BTB-impermeable component into the testes under in vivo conditions. Disruption of the BTB coincided with mislocalization of connexin43, which was present throughout the seminiferous epithelium and not restricted to the BTB as in wildtype tissues, suggesting impaired cell-cell communication in the BT-IgSF-KO mice. Since EM images revealed a normal BTB structure between Sertoli cells in the BT-IgSF-KO mice, we conclude that infertility in these mice is most likely caused by a functionally impaired BTB. In summary, our results indicate that BT-IgSF is expressed at the BTB and is required for male fertility by supporting the functional integrity of the BTB