Transgene Excision Has No Impact on In Vivo Integration of Human iPS Derived Neural Precursors

The derivation of induced human pluripotent stem cells (hiPS) has generated significant enthusiasm particularly for the prospects of cell-based therapy. But there are concerns about the suitability of iPS cells for in vivo applications due in part to the introduction of potentially oncogenic transcription factors via viral vectors. Recently developed lentiviral vectors allow the excision of viral reprogramming factors and the development of transgene-free iPS lines. However it is unclear if reprogramming strategy has an impact on the differentiation potential and the in vivo behavior of hiPS progeny. Here we subject viral factor-free, c-myc-free and conventionally reprogrammed four-factor human iPS lines to a further challenge, by analyzing their differentiation potential along the 3 neural lineages and over extended periods of time in vitro, as well as by interrogating their ability to respond to local environmental cues by grafting into the striatum. We demonstrate similar and efficient differentiation into neurons, astrocytes and oligodendrocytes among all hiPS and human ES line controls. Upon intracranial grafting in the normal rat (Sprague Dawley), precursors derived from all hiPS lines exhibited good survival and response to environmental cues by integrating into the subventricular zone, acquiring phenotypes typical of type A, B or C cells and migrating along the rostral migratory stream into the olfactory bulb. There was no teratoma or other tumor formation 12 weeks after grafting in any of the 26 animals used in the study. Thus neither factor excision nor persistence of c-myc impact the behavior of hiPS lines in vivo.United States. National Institutes of HealthNew York State Stem Cell ScienceNational Institute of Neurological Disorders and Stroke (U.S.)Starr Foundation (Tri-Institutional Starr Stem Cell Scholars Fellowship

Differential Coupling of Self-Renewal Signaling Pathways in Murine Induced Pluripotent Stem Cells

The ability to reprogram somatic cells to induced pluripotent stem cells (iPSCs), exhibiting properties similar to those of embryonic stem cells (ESCs), has attracted much attention, with many studies focused on improving efficiency of derivation and unraveling the mechanisms of reprogramming. Despite this widespread interest, our knowledge of the molecular signaling pathways that are active in iPSCs and that play a role in controlling their fate have not been studied in detail. To address this shortfall, we have characterized the influence of different signals on the behavior of a model mouse iPSC line. We demonstrate significant responses of this iPSC line to the presence of serum, which leads to profoundly enhanced proliferation and, depending on the medium used, a reduction in the capacity of the iPSCs to self-renew. Surprisingly, this iPSC line was less sensitive to withdrawal of LIF compared to ESCs, exemplified by maintenance of expression of a Nanog-GFP reporter and enhanced self-renewal in the absence of LIF. While inhibition of phosphoinositide-3 kinase (PI3K) signaling decreased iPSC self-renewal, inhibition of Gsk-3 promoted it, even in the absence of LIF. High passages of this iPSC line displayed altered characteristics, including genetic instability and a reduced ability to self-renew. However, this second feature could be restored upon inhibition of Gsk-3. Collectively, our data suggest modulation of Gsk-3 activity plays a key role in the control of iPSC fate. We propose that more careful consideration should be given to characterization of the molecular pathways that control the fate of different iPSC lines, since perturbations from those observed in naïve pluripotent ESCs could render iPSCs and their derivatives susceptible to aberrant and potentially undesirable behaviors

Neurotrophic requirements of human motor neurons defined using amplified and purified stem-cell derived cultures

Neurotrophic requirements of human motor neurons defined using amplified and purified stem-cell derived culturesHuman motor neurons derived from embryonic and induced pluripotent stem cells (hESCs and hiPSCs) are a potentially important tool for studying motor neuron survival and pathological cell death. However, their basic survival requirements remain poorly characterized. Here, we sought to optimize a robust survival assay and characterize their response to different neurotrophic factors. First, to increase motor neuron yield, we screened a small-molecule collection and found that the Rho-associated kinase (ROCK) inhibitor Y-27632 enhances motor neuron progenitor proliferation up to 4-fold in hESC and hiPSC cultures. Next, we FACS-purified motor neurons expressing the Hb9::GFP reporter from Y-27632-amplified embryoid bodies and cultured them in the presence of mitotic inhibitors to eliminate dividing progenitors. Survival of these purified motor neurons in the absence of any other cell type was strongly dependent on neurotrophic support. GDNF, BDNF and CNTF all showed potent survival effects (EC(50) 1-2 pM). The number of surviving motor neurons was further enhanced in the presence of forskolin and IBMX, agents that increase endogenous cAMP levels. As a demonstration of the ability of the assay to detect novel neurotrophic agents, Y-27632 itself was found to support human motor neuron survival. Thus, purified human stem cell-derived motor neurons show survival requirements similar to those of primary rodent motor neurons and can be used for rigorous cell-based screening.This work was funded by Project A.L.S., P2ALS and NYSTEM grant number CO24415. The work of N.J.L. was supported by the Portuguese Foundation for Science and Technology SFRH/BD/33421/2008 and the Luso-American Development Foundation. B.J.-K. was supported by the National Institute of Neurological Disorders and Stroke (NINDS). L.R. was supported by the Swedish Brain Foundation/Hjarnfonden. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Inhibition of Apoptosis Blocks Human Motor Neuron Cell Death in a Stem Cell Model of Spinal Muscular Atrophy

Spinal muscular atrophy (SMA) is a genetic disorder caused by a deletion of the survival motor neuron 1 gene leading to motor neuron loss, muscle atrophy, paralysis, and death. We show here that induced pluripotent stem cell (iPSC) lines generated from two Type I SMA subjects–one produced with lentiviral constructs and the second using a virus-free plasmid–based approach–recapitulate the disease phenotype and generate significantly fewer motor neurons at later developmental time periods in culture compared to two separate control subject iPSC lines. During motor neuron development, both SMA lines showed an increase in Fas ligand-mediated apoptosis and increased caspase-8 and-3 activation. Importantly, this could be mitigated by addition of either a Fas blocking antibody or a caspase-3 inhibitor. Together, these data further validate this human stem cell model of SMA, suggesting that specific inhibitors of apoptotic pathways may be beneficial for patients

Parkinson's disease induced pluripotent stem cells with triplication of the α-synuclein locus

A major barrier to research on Parkinson's disease is inaccessibility of diseased tissue for study. One solution is to derive induced pluripotent stem cells from patients and differentiate them into neurons affected by disease. Triplication of SNCA, encoding α-synuclein, causes a fully penetrant, aggressive form of Parkinson's disease with dementia. α-Synuclein dysfunction is the critical pathogenic event in Parkinson's disease, multiple system atrophy and dementia with Lewy bodies. Here we produce multiple induced pluripotent stem cell lines from an SNCA triplication patient and an unaffected first-degree relative. When these cells are differentiated into midbrain dopaminergic neurons, those from the patient produce double the amount of α-synuclein protein as neurons from the unaffected relative, precisely recapitulating the cause of Parkinson's disease in these individuals. This model represents a new experimental system to identify compounds that reduce levels of α-synuclein, and to investigate the mechanistic basis of neurodegeneration caused by α-synuclein dysfunction

Modelling of the effect of ELMs on fuel retention at the bulk W divertor of JET

Effect of ELMs on fuel retention at the bulk W target of JET ITER-Like Wall was studied with multi-scale calculations. Plasma input parameters were taken from ELMy H-mode plasma experiment. The energetic intra-ELM fuel particles get implanted and create near-surface defects up to depths of few tens of nm, which act as the main fuel trapping sites during ELMs. Clustering of implantation-induced vacancies were found to take place. The incoming flux of inter-ELM plasma particles increases the different filling levels of trapped fuel in defects. The temperature increase of the W target during the pulse increases the fuel detrapping rate. The inter-ELM fuel particle flux refills the partially emptied trapping sites and fills new sites. This leads to a competing effect on the retention and release rates of the implanted particles. At high temperatures the main retention appeared in larger vacancy clusters due to increased clustering rate

The effect of beryllium oxide on retention in JET ITER-like wall tiles

Preliminary results investigating the microstructure, bonding and effect of beryllium oxide formation on retention in the JET ITER-like wall beryllium tiles, are presented. The tiles have been investigated by several techniques: Scanning Electron Microscopy (SEM) equipped with Energy Dispersive X-ray (EDX), Transmission Electron microscopy (TEM) equipped with EDX and Electron Energy Loss Spectroscopy (EELS), Raman Spectroscopy and Thermal Desorption Spectroscopy (TDS). This paper focuses on results from melted materials of the dump plate tiles in JET. From our results and the literature, it is concluded, beryllium can form micron deep oxide islands contrary to the nanometric oxides predicted under vacuum conditions. The deepest oxides analyzed were up to 2-micron thicknesses. The beryllium Deuteroxide (BeOxDy) bond was found with Raman Spectroscopy. Application of EELS confirmed the oxide presence and stoichiometry. Literature suggests these oxides form at temperatures greater than 700 °C where self-diffusion of beryllium ions through the surface oxide layer can occur. Further oxidation is made possible between oxygen plasma impurities and the beryllium ions now present at the wall surface. Under Ultra High Vacuum (UHV) nanometric Beryllium oxide layers are formed and passivate at room temperature. After continual cyclic heating (to the point of melt formation) in the presence of oxygen impurities from the plasma, oxide growth to the levels seen experimentally (approximately two microns) is proposed. This retention mechanism is not considered to contribute dramatically to overall retention in JET, due to low levels of melt formation. However, this mechanism, thought the result of operation environment and melt formation, could be of wider concern to ITER, dependent on wall temperatures