Generation of highly pure pluripotent stem cell-derived myogenic progenitor cells and myotubes.

Driving efficient and pure skeletal muscle cell differentiation from pluripotent stem cells (PSCs) has been challenging. Here, we report an optimized protocol that generates skeletal muscle progenitor cells with high efficiency and purity in a short period of time. Human induced PSCs (hiPSCs) and murine embryonic stem cells (mESCs) were specified into the mesodermal myogenic fate using distinct and species-specific protocols. We used a specific maturation medium to promote the terminal differentiation of both human and mouse myoblast populations, and generated myotubes associated with a large pool of cell-cycle arrested PAX7+ cells. We also show that myotube maturation is modulated by dish-coating properties, cell density, and percentage of myogenic progenitor cells. Given the high efficiency in the generation of myogenic progenitors and differentiated myofibers, this protocol provides an attractive strategy for tissue engineering, modeling of muscle dystrophies, and evaluation of new therapeutic approaches in vitro.

Two induced pluripotent stem cell (iPSC) lines derived from patients affected by Waardenburg syndrome type 1 retain potential to activate neural crest markers.

Waardenburg syndrome type 1 (WS1), a rare genetic disease characterized by pigmentation defects and mild craniofacial anomalies often associated with congenital deafness is caused by heterozygous mutations in the PAX3 gene (2q36.1). We have generated two induced pluripotent stem cell lines (PCli029-A and PCli031-A) from two patients from the same family both carrying the same heterozygous deletion in PAX3 exon 1 (c.-70_85 + 366del). These cells are pluripotent as they can differentiate into ectoderm, mesoderm and endoderm. They also can activate the early neural crest marker SNAI2. These cells will be useful for studying the human neural crest-derived pigment cells.

A high-throughput screening platform for pigment regulating agents using pluripotent stem cell-derived melanocytes.

In this study, we describe a simple and straightforward assay using induced pluripotent stem cell-derived melanocytes and high-throughput flow cytometry, to identify the effect induced by pigment regulating agents on melanin content. The assay is based on the correlation between forward light-scatter characteristics and melanin content, with pigmented cells displaying high light absorption/low forward light scatter, while the opposite is true for lowly pigmented melanocytes, as a result of genetic background or chemical treatments. Orthogonal validation is then performed by regular melanin quantification. Such approach was validated using a set of 80 small molecules and yielded a confirmed hit. The assay described in this study may prove a useful tool to identify modulators of melanogenesis in human melanocytes.

Beneficial Effect of Human Induced Pluripotent Stem Cell-Derived Neural Precursors in Spinal Cord Injury Repair.

Despite advances in our understanding and research of induced pluripotent stem cells (iPSCs), their use in clinical practice is still limited due to lack of preclinical experiments. Neural precursors (NPs) derived from a clone of human iPSCs (IMR90) were used to treat a rat spinal cord lesion 1 week after induction. Functional recovery was evaluated using the BBB, beam walking, rotarod, and plantar tests. Lesion morphology, endogenous axonal sprouting, graft survival, and iPSC-NP differentiation were analyzed immunohistochemically. Quantitative polymerase chain reaction (qPCR) was used to evaluate the effect of transplanted iPSC-NPs on endogenous regenerative processes and also to monitor their behavior after transplantation. Human iPSC-NPs robustly survived in the lesion, migrated, and partially filled the lesion cavity during the entire period of observation. Transplanted animals displayed significant motor improvement already from the second week after the transplantation of iPSC-NPs. qPCR revealed the increased expression of human neurotrophins 8 weeks after transplantation. Simultaneously, the white and gray matter were spared in the host tissue. The grafted cells were immunohistochemically positive for doublecortin, MAP2, ßIII-tubulin, GFAP, and CNPase 8 weeks after transplantation. Human iPSC-NPs further matured, and 17 weeks after transplantation differentiated toward interneurons, dopaminergic neurons, serotoninergic neurons, and ChAT-positive motoneurons. Human iPSC-NPs possess neurotrophic properties that are associated with significant early functional improvement and the sparing of spinal cord tissue. Their ability to differentiate into tissue-specific neurons leads to the long-term restoration of the lesioned tissue, making the cells a promising candidate for future cell-based therapy of SCI.

Genetically-modified human pluripotent stem cells: new hopes for the understanding and the treatment of neurological diseases?

The fundamental inaccessibility of the human neural cell types affected by neurological disorders prevents their isolation for in vitro studies of disease mechanisms or for drug screening efforts. Pluripotent stem cells represent a new interesting way to generate models of human neurological disorders, explore the physiopathological mechanisms and develop new therapeutic strategies. Disease-specific human embryonic stem cells were the first source of material to be used to study certain disease states. The recent demonstration that human somatic cells, such as fibroblasts or blood cells, can be genetically converted to induced pluripotent stem cells (hiPSCs) together with the continuous improvement of methods to differentiate these cells into disease-affected neuronal subtypes opens new perspectives to model and understand a large number of human pathologies. This review focuses on the opportunities concerning the use disease-specific human pluripotent stem cells as well as the different challenges that still need to be overcome. We also discuss the recent improvements in the genetic manipulation of human pluripotent stem cells and the consequences of these on disease modeling and drug screening for neurological diseases.

Neuronal differentiation of human iPS-cells in a rat cortical primary culture.

We tested the neuronal differentiation of human iPS-cells under in vitro conditions. For this purpose we pre-differentiated human (h) iPS-cells into neural stem cells and co-cultivated them with a cortical primary culture from embryonic rats. After 2 days of co-cultivation a certain number of hiPS-cells exhibited a clear neuronal morphology combined with expression of betaIII-tubulin and doublecortin. In addition, we found hiPS-cells without neuronal differentiation and cells already expressing betaIII-tubulin but not having yet distinctive axonal and dendritic processes. Human neuronal progenitors, starting neuronal differentiation, were contacted by both neuronal processes from rat neurons and oligodendrocytes, indicating a possible instructive influence by the primary culture on human cells. After 7 days of co-cultivation, however, we observed a complete degeneration of human iPS-derived cells and phagocytosis by microglial cells. Immunocytochemical stainings surprisingly revealed that microglial cells of the cortical primary culture express both CD8 and T-cell receptors.

Human induced pluripotent stem cells improve stroke outcome and reduce secondary degeneration in the recipient brain.

Human induced pluripotent stem cells (hiPSCs) are a most appealing source for cell replacement therapy in acute brain lesions. We evaluated the potential of hiPSC therapy in stroke by transplanting hiPSC-derived neural progenitor cells (NPCs) into the postischemic striatum. Grafts received host tyrosine hydroxylase-positive afferents and contained developing interneurons and homotopic GABAergic medium spiny neurons that, with time, sent axons to the host substantia nigra. Grafting reversed stroke-induced somatosensory and motor deficits. Grafting also protected the host substantia nigra from the atrophy that follows disruption of reciprocal striatonigral connections. Graft innervation by tyrosine hydoxylase fibers, substantia nigra protection, and somatosensory functional recovery were early events, temporally dissociated from the slow maturation of GABAergic neurons in the grafts and innervation of substantia nigra. This suggests that grafted hiPSC-NPCs initially exert trophic effects on host brain structures, which precede integration and potential pathway reconstruction. We believe that transplantation of NPCs derived from hiPSCs can provide useful interventions to limit the functional consequences of stroke through both neuroprotective effects and reconstruction of impaired pathways.

Regenerative medicine for stroke - are we there yet?

With the development of stem cell (SC) biology, cell-based therapy has become a highly challenging field for experimental and clinical research. Among neurological disorders, stroke has pioneered the clinical application of SC. Safety concerns have prevailed for pilot clinical studies and important preclinical work is ongoing to help SC therapy reach the level of generalization. Stroke is classically divided into an acute, a subacute and a chronic phase. Each phase is defined by a complex array of events with overlapping and distinct kinetics that lead to both rapid tissue degeneration and long-lasting remodeling. Each SC type possesses intrinsic properties - transposed via cell-autonomous and non-cell-autonomous signaling - that would more specifically address some of these events. A better definition of what is expected from SC therapy in stroke might help assign SC sources to the acute or chronic phases and possibly optimize their use in the clinic.

Intracerebral transplantation for neurological disorders. Lessons from developmental, experimental, and clinical studies.

The use of human pluripotent stem cells (PSCs) for cell therapy faces a number of challenges that are progressively answered by results from clinical trials and experimental research. Among these is the control of differentiation before transplantation and the prediction of cell fate after administration into the human brain, two aspects that condition both the safety and efficacy of the approach. For neurological disorders, this includes two steps: firstly, the identification of the optimal maturation stage for transplantation along the continuum that transforms PSCs into fully differentiated neural cell types, together with the derivation of robust protocols for large-scale production of biological products, and, secondly, the understanding of the effects of environmental cues and their possible interference with transplanted cells commitment. This review will firstly summarize our knowledge on developmental processes that have been applied to achieve robust in vitro differentiation of PSCs into neural progenitors. In a second part, we summarize results from experimental and clinical transplantation studies that help understanding the dialogue that establishes between transplanted cells and their host brain.

The postischemic environment differentially impacts teratoma or tumor formation after transplantation of human embryonic stem cell-derived neural progenitors.

BACKGROUND AND PURPOSE: Risk of tumorigenesis is a major obstacle to human embryonic and induced pluripotent stem cell therapy. Likely linked to the stage of differentiation of the cells at the time of implantation, formation of teratoma/tumors can also be influenced by factors released by the host tissue. We have analyzed the relative effects of the stage of differentiation and the postischemic environment on the formation of adverse structures by transplanted human embryonic stem cell-derived neural progenitors. METHODS: Four differentiation stages were identified on the basis of quantitative polymerase chain reaction expression of pluripotency, proliferation, and differentiation markers. Neural progenitors were transplanted at these 4 stages into rats with no, small, or large middle cerebral artery occlusion lesions. The fate of each transplant was compared with their pretransplantation status 1 to 4 months posttransplantation. RESULTS: The influence of the postischemic environment was limited to graft survival and occurrence of nonneuroectodermal structures after transplantation of very immature neural progenitors. Both effects were lost with differentiation. We identified a particular stage of differentiation characterized in vitro by a rebound of proliferative activity that produced highly proliferative grafts susceptible to threaten surrounding host tissues. CONCLUSIONS: The effects of the ischemic environment on the formation of teratoma by transplanted human embryonic stem cell-derived neural progenitors are limited to early differentiation stages that will likely not be used for stem cell therapy. In contrast, hyperproliferation observed at later stages of differentiation corresponds to an intrinsic activity that should be monitored to avoid tumorigenesis.

[Cerebral ischaemia: tomorrow's therapeutic tracks]. / Ischémie cérébrale : les pistes thérapeutiques de demain.

Thrombolysis remains the main therapeutic strategy used in stroke, but with a limited use to only a part of stroke patients. A neuroprotective approach would be necessary with a double objective : (1) to serve as an add-on treatment with thrombolysis to improve safety and increase therapeutic window ; (2) to limit infarct area by delaying neuronal death. While numerous molecules failed in clinical trials in stroke, pharmacological development is on-going with pleiotropic drugs targeting both neuronal and vascular parts of neurovascular unit. Another approach targets the functional rehabilitation and the neurorepair using pharmacological ways or cell therapy.

[Neuroplasticity: from physiological adaptation to the concept of therapeutic plasticity]. / Neuroplasticité : de l'adaptation physiologique au concept de plasticité thérapeutique.

There is considerable evidence that the human brain maintains the ability to reorganize itself throughout life, an ability known as neuroplasticity. Initially demonstrated in physiological situations, neuroplasticity includes, and relies on, a number of adaptive mechanisms that include not only phenotypic modifications of neurons or synaptic reorganisation but also major modifications of brain circuitry after insults. Recently, the presence of neurogenic zones in the adult brain has unveiled a new aspect of brain plasticity that, together with emerging stem cell therapy, opens the possibility to take advantage of these natural reminders of the developmental period to repair lesioned tissues, a concept known as "therapeutic plasticity".

Cardioprotection against myocardial infarction with PTD-BIR3/RING, a XIAP mimicking protein.

The purpose of the present study was to investigate the potential cardioprotective effects of an original approach based on the properties of the X chromosome-linked Inhibitor of Apoptosis (XIAP), the most effective endogenous inhibitor of apoptosis. For this purpose, the C-terminal part of XIAP (BIR3 and RING domains) was fused to the protein transduction domain (PTD) of the HIV1 transactivator of transcription, which confers to fused protein the ability to cross cell membranes. This protein, so-called PTD-BIR3/RING, was administered intravenously in C57BL/6J mice subjected to 30 min coronary artery occlusion and 24 h of reperfusion. Administration of PTD-BIR3/RING at 5 min before and 30 min after the onset of reperfusion reduced infarct size vs control (23+/-2% vs 41+/-4% and 27+/-4% vs 41+/-3%, respectively, p<0.05). Similar reduction in infarct size was observed when PTD-BIR3/RING was administered prior to ischemia (28+/-1% vs 44+/-3%). In addition to inhibition of caspase-3 and -9 activities, PTD-BIR3/RING induced an inhibition of caspase-8 and several other actors of the apoptotic pathways. In conclusion, this study demonstrates that the administration of PTD-BIR3/RING reduces myocardial infarct size even when injected during reperfusion through interruption of caspase activation by pharmacologically mimicking endogenous XIAP.

Role for glutathione in the hyposensitivity of LPS-pretreated mice to LPS anorexia.

To study the role of the redox state regulator glutathione (GSH) in bacterial lipopolysaccharide (LPS)-induced anorexia we measured total reduced GSH (trGSH) in liver, serum and brain in response to intraperitoneal (ip) lipopolysaccharide (LPS, 4 microg/mouse) injection in LPS-naïve and LPS-pretreated (4 microg/mouse given 3 days earlier) mice. LPS reduced food intake in LPS-naïve mice and LPS pretreatment attenuated this effect. LPS decreased trGSH at 24 hours after injection in LPS-naïve mice but 4 days later trGSH levels were upregulated in brain and liver, and this was associated with a significant attenuation of LPS-induced anorexia. In addition, LPS increased mitochondrial GSH levels in brain and liver at 4 days after injection. Pharmacological GSH depletion with diethylmaleate and L-buthionine sulfoximine in LPS-pretreated mice ablated the hyposensitivity to the anorexic effect of LPS. Together, these findings suggest a prominent role for GSH and its intracellular repartition in LPS anorexia.

Role for nerve growth factor in the in vivo regulation of glutathione in response to LPS in mice.

Since the redox state regulator glutathione (GSH), which influences lipopolysaccharide (LPS) anorexia, may be controlled by cytokines, we studied the roles of tumour necrosis factor-alpha (TNFalpha) and nerve growth factor (NGF) in the GSH response to intraperitoneal (ip) LPS injection in mice. Basal NGF and total reduced GSH (trGSH) levels were up-regulated in brain and liver of TNFalpha-knock-out (KO) mice, and this was associated with attenuated LPS anorexia. The increases in NGF and trGSH presumably contributed to the attenuated anorexia in response to LPS because transgenic mice over-expressing NGF (NGF-tg mice) also had increased trGSH levels and displayed attenuated anorexia compared to the corresponding wild type (WT) mice. Attenuated LPS anorexia in NGF-tg mice was accompanied by reduced serum TNFalpha and IFNgamma levels compared to WT mice. In response to a second injection of LPS, NGF and trGSH levels, but not TNFalpha levels changed. This suggests that in vivo tissue trGSH changes following LPS in LPS-naïve or LPS-pretreated mice are regulated by NGF rather than TNFalpha. The finding that genetic TNFalpha deficiency did not inhibit the acute trGSH response to LPS supports this interpretation. In sum, the results indicate i) that a decrease or increase in NGF is accompanied by a decrease or increase in trGSH levels and ii) that elevated NGF and/or trGSH levels attenuate some of the responses to LPS such as anorexia and cytokine production.

Neuroprotection: Present and future.

The treatment of brain diseases (regardless of the latter's neurological or psychiatric expression) is based on either preventive, symptomatic or etiopathogenic approaches. The frequent observation of neuronal death during brain disease initially prompted researchers to favour neuroprotection for the etiopathogenic approach. The repeated failure to develop reliable neuroprotective agents has prompted emergence of the concept of "disease modifyer". The disease modifyer concept (based essentially on clinical endpoints) enables us to envisage the broader application of etiopathogenic treatments by freeing ourselves of the need to demonstrate a cellular mechanism of action. The formalization of disease modification prompts several lines of thought, which are developed in the present article.

PTD-XIAP protects against cerebral ischemia by anti-apoptotic and transcriptional regulatory mechanisms.

Caspases play a major role in the infarction process that follows occlusion of cerebral arteries and are important targets for stroke therapy. We have generated three fusion proteins that link various domains of the X chromosome-linked inhibitor of apoptosis (XIAP), a potent caspase inhibitor, to the protein transduction domain (PTD) of HIV-1/Tat, and have tested their efficacy after distal occlusion of the middle cerebral artery (dMCAO) in mice. PTD-XIAP failed to accumulate in brain structures after intravenous (iv) delivery, but properly transduced cortical cells when applied topically. Shorter constructs efficiently targeted the lesion after iv delivery. All proteins retained their caspase inhibitory activity and significantly reduced infarct volumes. PTD-XIAP reversed long-term impairments in the water maze test. Sequential activation of transcription factors was observed, suggesting that the effects of XIAP are mediated by both direct inhibition of apoptotic mechanisms and secondary regulation of transcription factors involved in neuronal survival.

Neuroprotection and neurodegenerative diseases: from biology to clinical practice.

Neurodegenerative diseases and, in particular, Alzheimer disease, are characterized by progressive neuronal loss correlated in time with the symptoms of the disease considered. Whereas the symptoms of those incapacitating diseases are beginning to be managed with a relative efficacy, the ultimate objective of therapy nonetheless remains preventing cell (neuronal and/or astrocytic) death in a neurocytoprotective approach. In biologic terms, in the light of progress at basic research level, three strategies may be envisaged: (1) antagonizing the cytotoxic causal events (excess intracellular calcium, accumulation of abnormal proteins, excitotoxic effects of amino acids, oxidative stress, processes related to inflammation, etc.); (2) stimulating the endogenous protective processes (anti-free radical or DNA repair systems, production of neurotrophic factors, potential cytoprotective action of steroids, etc.); (3) promoting damaged structure repair strategies (grafts) or deep brain or cortical neurostimulation with a view to triggering (beyond the symptomatic actions) potential 'protective' cell mechanisms. The clinical transition of the various strategies whose efficacy is being tested in animal and/or cell models, experimental analogs of the diseases, and thus the objective demonstration in humans of pharmacological and/or surgical neurocytoprotection, is currently the subject of considerable methodological debate (What are the right psychometric assessment criteria? What are the most pertinent laboratory or neuroradiological markers, etc.?). A number of clinical trials have been completed or are ongoing with drugs that are reputed to be neuroprotective. Thus, elements of the response are beginning to be generated with a view to determining whether it will soon be possible to effectively slow or even stop the neurodegenerative process whose etiology, in most cases, remains obscure.