Wild-type ALK and activating ALK-R1275Q and ALK-F1174L mutations upregulate Myc and initiate tumor formation in murine neural crest progenitor cells.

The anaplastic lymphoma kinase (ALK) gene is overexpressed, mutated or amplified in most neuroblastoma (NB), a pediatric neural crest-derived embryonal tumor. The two most frequent mutations, ALK-F1174L and ALK-R1275Q, contribute to NB tumorigenesis in mouse models, and cooperate with MYCN in the oncogenic process. However, the precise role of activating ALK mutations or ALK-wt overexpression in NB tumor initiation needs further clarification. Human ALK-wt, ALK-F1174L, or ALK-R1275Q were stably expressed in murine neural crest progenitor cells (NCPC), MONC-1 or JoMa1, immortalized with v-Myc or Tamoxifen-inducible Myc-ERT, respectively. While orthotopic implantations of MONC- 1 parental cells in nude mice generated various tumor types, such as NB, osteo/ chondrosarcoma, and undifferentiated tumors, due to v-Myc oncogenic activity, MONC-1-ALK-F1174L cells only produced undifferentiated tumors. Furthermore, our data represent the first demonstration of ALK-wt transforming capacity, as ALK-wt expression in JoMa1 cells, likewise ALK-F1174L, or ALK-R1275Q, in absence of exogenous Myc-ERT activity, was sufficient to induce the formation of aggressive and undifferentiated neural crest cell-derived tumors, but not to drive NB development. Interestingly, JoMa1-ALK tumors and their derived cell lines upregulated Myc endogenous expression, resulting from ALK activation, and both ALK and Myc activities were necessary to confer tumorigenic properties on tumor-derived JoMa1 cells in vitro.

Neurogenic subventricular zone stem/progenitor cells are notch1-dependent in their active but not quiescent state.

The adult mammalian forebrain contains neural stem/progenitor cells (NSCs) that generate neurons throughout life. As in other somatic stem cell systems, NSCs are proposed to be predominantly quiescent and proliferate only sporadically to produce more committed progeny. However, quiescence has recently been shown not to be an essential criterion for stem cells. It is not known whether NSCs show differences in molecular dependence based on their proliferation state. The subventricular zone (SVZ) of the adult mouse brain has a remarkable capacity for repair by activation of NSCs. The molecular interplay controlling adult NSCs during neurogenesis or regeneration is not clear but resolving these interactions is critical in order to understand brain homeostasis and repair. Using conditional genetics and fate mapping, we show that Notch signaling is essential for neurogenesis in the SVZ. By mosaic analysis, we uncovered a surprising difference in Notch dependence between active neurogenic and regenerative NSCs. While both active and regenerative NSCs depend upon canonical Notch signaling, Notch1-deletion results in a selective loss of active NSCs (aNSCs). In sharp contrast, quiescent NSCs (qNSCs) remain after Notch1 ablation until induced during regeneration or aging, whereupon they become Notch1-dependent and fail to fully reinstate neurogenesis. Our results suggest that Notch1 is a key component of the adult SVZ niche, promoting maintenance of aNSCs, and that this function is compensated in qNSCs. Therefore, we confirm the importance of Notch signaling for maintaining NSCs and neurogenesis in the adult SVZ and reveal that NSCs display a selective reliance on Notch1 that may be dictated by mitotic state.

Progenitor cell therapy for heart disease.

Many cell types are currently being studied as potential sources of cardiomyocytes for cell transplantation therapy to repair and regenerate damaged myocardium. The question remains as to which progenitor cell represents the best candidate. Bone marrow-derived cells and endothelial progenitor cells have been tested in clinical studies. These cells are safe, but their cardiogenic potential is controversial. The functional benefits observed are probably due to enhanced angiogenesis, reduced ventricular remodeling, or to cytokine-mediated effects that promote the survival of endogenous cells. Human embryonic stem cells represent an unlimited source of cardiomyocytes due to their great differentiation potential, but each step of differentiation must be tightly controlled due to the high risk of teratoma formation. These cells, however, confront ethical barriers and there is a risk of graft rejection. These last two problems can be avoided by using induced pluripotent stem cells (iPS), which can be autologously derived, but the high risk of teratoma formation remains. Cardiac progenitor cells have the advantage of being cardiac committed, but important questions remain unanswered, such as what is the best marker to identify and isolate these cells? To date the different markers used to identify adult cardiac progenitor cells also recognize progenitor cells that are outside the heart. Thus, it cannot be determined whether the cardiac progenitor cells identified in the adult heart represent resident cells present since fetal life or extracardiac cells that colonized the heart after cardiac injury. Developmental studies have identified markers of multipotent progenitors, but it is unknown whether these markers are specific for adult progenitors when expressed in the adult myocardium. Cardiac regeneration is dependent on the stability of the cells transplanted into the host myocardium and on the electromechanical coupling with the endogenous cells. Finally, the promotion of endogenous regenerative processes by mobilizing endogenous progenitors represents a complementary approach to cell transplantation therapy.

TWEAK, via its receptor Fn14, is a novel regulator of mesenchymal progenitor cells and skeletal muscle regeneration.

Inflammation participates in tissue repair through multiple mechanisms including directly regulating the cell fate of resident progenitor cells critical for successful regeneration. Upon surveying target cell types of the TNF ligand TWEAK, we observed that TWEAK binds to all progenitor cells of the mesenchymal lineage and induces NF-kappaB activation and the expression of pro-survival, pro-proliferative and homing receptor genes in the mesenchymal stem cells, suggesting that this pro-inflammatory cytokine may play an important role in controlling progenitor cell biology. We explored this potential using both the established C2C12 cell line and primary mouse muscle myoblasts, and demonstrated that TWEAK promoted their proliferation and inhibited their terminal differentiation. By generating mice deficient in the TWEAK receptor Fn14, we further showed that Fn14-deficient primary myoblasts displayed significantly reduced proliferative capacity and altered myotube formation. Following cardiotoxin injection, a known trigger for satellite cell-driven skeletal muscle regeneration, Fn14-deficient mice exhibited reduced inflammatory response and delayed muscle fiber regeneration compared with wild-type mice. These results indicate that the TWEAK/Fn14 pathway is a novel regulator of skeletal muscle precursor cells and illustrate an important mechanism by which inflammatory cytokines influence tissue regeneration and repair. Coupled with our recent demonstration that TWEAK potentiates liver progenitor cell proliferation, the expression of Fn14 on all mesenchymal lineage progenitor cells supports a broad involvement of this pathway in other tissue injury and disease settings.

Extracellular vesicles from human cardiac progenitor cells inhibit cardiomyocyte apoptosis and improve cardiac function after myocardial infarction.

AIMS: Recent evidence suggests that cardiac progenitor cells (CPCs) may improve cardiac function after injury. The underlying mechanisms are indirect, but their mediators remain unidentified. Exosomes and other secreted membrane vesicles, hereafter collectively referred to as extracellular vesicles (EVs), act as paracrine signalling mediators. Here, we report that EVs secreted by human CPCs are crucial cardioprotective agents. METHODS AND RESULTS: CPCs were derived from atrial appendage explants from patients who underwent heart valve surgery. CPC-conditioned medium (CM) inhibited apoptosis in mouse HL-1 cardiomyocytic cells, while enhancing tube formation in human umbilical vein endothelial cells. These effects were abrogated by depleting CM of EVs. They were reproduced by EVs secreted by CPCs, but not by those secreted by human dermal fibroblasts. Transmission electron microscopy and nanoparticle tracking analysis showed most EVs to be 30-90 nm in diameter, the size of exosomes, although smaller and larger vesicles were also present. MicroRNAs most highly enriched in EVs secreted by CPCs compared with fibroblasts included miR-210, miR-132, and miR-146a-3p. miR-210 down-regulated its known targets, ephrin A3 and PTP1b, inhibiting apoptosis in cardiomyocytic cells. miR-132 down-regulated its target, RasGAP-p120, enhancing tube formation in endothelial cells. Infarcted hearts injected with EVs from CPCs, but not from fibroblasts, exhibited less cardiomyocyte apoptosis, enhanced angiogenesis, and improved LV ejection fraction (0.8 ± 6.8 vs. -21.3 ± 4.5%; P < 0.05) compared with those injected with control medium. CONCLUSION: EVs are the active component of the paracrine secretion by human CPCs. As a cell-free approach, EVs could circumvent many of the limitations of cell transplantation.

Ultrastructural evidence of exosome secretion by progenitor cells in adult mouse myocardium and adult human cardiospheres.

The demonstration of beneficial effects of cell therapy despite the persistence of only few transplanted cells in vivo suggests secreted factors may be the active component of this treatment. This so-called paracrine hypothesis is supported by observations that culture media conditioned by progenitor cells contain growth factors that mediate proangiogenic and cytoprotective effects. Cardiac progenitor cells in semi-suspension culture form spherical clusters (cardiospheres) that deliver paracrine signals to neighboring cells. A key component of paracrine secretion is exosomes, membrane vesicles that are stored intracellularly in endosomal compartments and are secreted when these structures fuse with the cell plasma membrane. Exosomes have been identified as the active component of proangiogenic effects of bone marrow CD34(+) stem cells in mice and the regenerative effects of embryonic mesenchymal stem cells in infarcted hearts in pigs and mice. Here, we provide electron microscopic evidence of exosome secretion by progenitor cells in mouse myocardium and human cardiospheres. Exosomes are emerging as an attractive vector of paracrine signals delivered by progenitor cells. They can be stored as an "off-the-shelf" product. As such, exosomes have the potential for circumventing many of the limitations of viable cells for therapeutic applications in regenerative medicine.

Isolation, culture and characterisation of human endothelial progenitor cells

Temps de parole: 30 minutes

Reticular dysgenesis-associated AK2 protects hematopoietic stem and progenitor cell development from oxidative stress.

Adenylate kinases (AKs) are phosphotransferases that regulate the cellular adenine nucleotide composition and play a critical role in the energy homeostasis of all tissues. The AK2 isoenzyme is expressed in the mitochondrial intermembrane space and is mutated in reticular dysgenesis (RD), a rare form of severe combined immunodeficiency (SCID) in humans. RD is characterized by a maturation arrest in the myeloid and lymphoid lineages, leading to early onset, recurrent, and overwhelming infections. To gain insight into the pathophysiology of RD, we studied the effects of AK2 deficiency using the zebrafish model and induced pluripotent stem cells (iPSCs) derived from fibroblasts of an RD patient. In zebrafish, Ak2 deficiency affected hematopoietic stem and progenitor cell (HSPC) development with increased oxidative stress and apoptosis. AK2-deficient iPSCs recapitulated the characteristic myeloid maturation arrest at the promyelocyte stage and demonstrated an increased AMP/ADP ratio, indicative of an energy-depleted adenine nucleotide profile. Antioxidant treatment rescued the hematopoietic phenotypes in vivo in ak2 mutant zebrafish and restored differentiation of AK2-deficient iPSCs into mature granulocytes. Our results link hematopoietic cell fate in AK2 deficiency to cellular energy depletion and increased oxidative stress. This points to the potential use of antioxidants as a supportive therapeutic modality for patients with RD.

Programming Hippocampal Neural Stem/Progenitor Cells into Oligodendrocytes Enhances Remyelination in the Adult Brain after Injury.

Demyelinating diseases are characterized by a loss of oligodendrocytes leading to axonal degeneration and impaired brain function. Current strategies used for the treatment of demyelinating disease such as multiple sclerosis largely rely on modulation of the immune system. Only limited treatment options are available for treating the later stages of the disease, and these treatments require regenerative therapies to ameliorate the consequences of oligodendrocyte loss and axonal impairment. Directed differentiation of adult hippocampal neural stem/progenitor cells (NSPCs) into oligodendrocytes may represent an endogenous source of glial cells for cell-replacement strategies aiming to treat demyelinating disease. Here, we show that Ascl1-mediated conversion of hippocampal NSPCs into mature oligodendrocytes enhances remyelination in a diphtheria-toxin (DT)-inducible, genetic model for demyelination. These findings highlight the potential of targeting hippocampal NSPCs for the treatment of demyelinated lesions in the adult brain.

Human Fetal Progenitor Tenocytes for Regenerative Medicine.

Tendon injuries are very frequent and affect a wide and heterogeneous population. Unfortunately, the healing process is long with outcomes that are not often satisfactory due to fibrotic tissue appearance, which leads to scar and adhesion development. Tissue engineering and cell therapies emerge as interesting alternatives to classical treatments. In this study, we evaluated human fetal progenitor tenocytes (hFPTs) as a potential cell source for treatment of tendon afflictions, as fetal cells are known to promote healing in a scarless regenerative process. hFPTs presented a rapid and stable growth up to passage 9, allowing to create a large cell bank for off-the-shelf availability. hFPTs showed a strong tenogenic phenotype with an excellent stability, even when placed in conditions normally inducing cells to differentiate. The karyotype also indicated a good stability up to passage 12, which is far beyond that necessary for clinical application (passage 6). When placed in coculture, hFPTs had the capacity to stimulate human adult tenocytes (hATs), which are responsible for the deposition of a new extracellular matrix during tendon healing. Finally, it was possible to distribute cells in porous or gel scaffolds with an excellent survival, thus permitting a large variety of applications (from simple injections to grafts acting as filling material). All of these results are encouraging in the development of an off-the-shelf cell source capable of stimulating tendon regeneration for the treatment of tendon injuries.

Cells with neuronal characteristics differentiate and persist for one year in rat optic nerve explants cultures.

Evidence concerning the presence or absence of common neuronglia lineages in the postnatal mammalian central nervous system is still a matter of speculation. We address this problem using optic nerve explants, which show an extremely long survival in culture. Morphological, immunocytochemical and immunochemical methods were applied. The results obtained from in vitro tissue were compared with optic nerves (ONs) and whole-brain samples from animals of different ages. Newborn rat ONs represented the starting material of our tissue culture; they are composed of unmyelinated axons, astrocytes and progenitor cells but devoid of neuronal cell bodies. At this age, Western blots of ONs were positively stained by neurofilament and synapsin I specific antibodies. These bands increased in intensity during postnatal in situ development. In explant cultures, the glia cells reach a stage of functional differentiation and they maintain, together with undifferentiated cells, a complex histotypic organization. After 6 days in vitro, neurofilaments and synapsin I could not be detected on immunoblots, indicating that 1) axonal degeneration was completed, and 2) neuronal somata were absent at the time. Surprisingly, after about 4-5 weeks in culture, a new cell type appeared, which showed characteristics typical of neurons. After 406 days in vitro, neurofilaments and synapsin I were unequivocally detectable on Western blots. Furthermore, both immunocytochemical staining and light and electron microscopic examinations corroborated the presence of this earlier-observed cell type. These in vitro results clearly show the high developmental plasticity of ON progenitor cells, even late in development. The existence of a common neuron-glia precursor, which never gives rise to neurons in situ, is suggested.

Soft tissue sarcomas with complex genomic profiles.

Soft tissue sarcomas (STS) with complex genomic profiles (50% of all STS) are predominantly composed of spindle cell/pleomorphic sarcomas, including leiomyosarcoma, myxofibrosarcoma, pleomorphic liposarcoma, pleomorphic rhabdomyosarcoma, malignant peripheral nerve sheath tumor, angiosarcoma, extraskeletal osteosarcoma, and spindle cell/pleomorphic unclassified sarcoma (previously called spindle cell/pleomorphic malignant fibrous histiocytoma). These neoplasms show, characteristically, gains and losses of numerous chromosomes or chromosome regions, as well as amplifications. Many of them share recurrent aberrations (e.g., gain of 5p13-p15) that seem to play a significant role in tumor progression and/or metastatic dissemination. In this paper, we review the cytogenetic, molecular genetic, and clinicopathologic characteristics of the most common STS displaying complex genomic profiles. Features of diagnostic or prognostic relevance will be discussed when needed.

Stem cell transplantation in brain tumors: a new field for molecular imaging?

Neural stem cells have been proposed as a new and promising treatment modality in various pathologies of the central nervous system, including malignant brain tumors. However, the underlying mechanism by which neural stem cells target tumor areas remains elusive. Monitoring of these cells is currently done by use of various modes of molecular imaging, such as optical imaging, magnetic resonance imaging and positron emission tomography, which is a novel technology for visualizing metabolism and signal transduction to gene expression. In this new context, the microenvironment of (malignant) brain tumors and the blood-brain barrier gains increased interest. The authors of this review give a unique overview of the current molecular-imaging techniques used in different therapeutic experimental brain tumor models in relation to neural stem cells. Such methods for molecular imaging of gene-engineered neural stem/progenitor cells are currently used to trace the location and temporal level of expression of therapeutic and endogenous genes in malignant brain tumors, closing the gap between in vitro and in vivo integrative biology of disease in neural stem cell transplantation.