Directed conversion of Alzheimer's disease patient skin fibroblasts into functional neurons.

Directed conversion of mature human cells, as from fibroblasts to neurons, is of potential clinical utility for neurological disease modeling as well as cell therapeutics. Here, we describe the efficient generation of human-induced neuronal (hiN) cells from adult skin fibroblasts of unaffected individuals and Alzheimer's patients, using virally transduced transcription regulators and extrinsic support factors. hiN cells from unaffected individuals display morphological, electrophysiological, and gene expression profiles that typify glutamatergic forebrain neurons and are competent to integrate functionally into the rodent CNS. hiN cells from familial Alzheimer disease (FAD) patients with presenilin-1 or -2 mutations exhibit altered processing and localization of amyloid precursor protein (APP) and increased production of Aß, relative to the source patient fibroblasts or hiN cells from unaffected individuals. Together, our findings demonstrate directed conversion of human fibroblasts to a neuronal phenotype and reveal cell type-selective pathology in hiN cells derived from FAD patients.

Human iPSC-derived neurons reveal early developmental alteration of neurite outgrowth in the late-occurring neurodegenerative Wolfram syndrome.

Recent studies indicate that neurodegenerative processes that appear during childhood and adolescence in individuals with Wolfram syndrome (WS) occur in addition to early brain development alteration, which is clinically silent. Underlying pathological mechanisms are still unknown. We have used induced pluripotent stem cell-derived neural cells from individuals affected by WS in order to reveal their phenotypic and molecular correlates. We have observed that a subpopulation of Wolfram neurons displayed aberrant neurite outgrowth associated with altered expression of axon guidance genes. Selective inhibition of the ATF6α arm of the unfolded protein response prevented the altered phenotype, although acute endoplasmic reticulum stress response-which is activated in late Wolfram degenerative processes-was not detected. Among the drugs currently tried in individuals with WS, valproic acid was the one that prevented the pathological phenotypes. These results suggest that early defects in axon guidance may contribute to the loss of neurons in individuals with WS.

A general pattern of non-spiking neuron dynamics under the effect of potassium and calcium channel modifications.

Electrical activity of excitable cells results from ion exchanges through cell membranes, so that genetic or epigenetic changes in genes encoding ion channels are likely to affect neuronal electrical signaling throughout the brain. There is a large literature on the effect of variations in ion channels on the dynamics of spiking neurons that represent the main type of neurons found in the vertebrate nervous systems. Nevertheless, non-spiking neurons are also ubiquitous in many nervous tissues and play a critical role in the processing of some sensory systems. To our knowledge, however, how conductance variations affect the dynamics of non-spiking neurons has never been assessed. Based on experimental observations reported in the biological literature and on mathematical considerations, we first propose a phenotypic classification of non-spiking neurons. Then, we determine a general pattern of the phenotypic evolution of non-spiking neurons as a function of changes in calcium and potassium conductances. Furthermore, we study the homeostatic compensatory mechanisms of ion channels in a well-posed non-spiking retinal cone model. We show that there is a restricted range of ion conductance values for which the behavior and phenotype of the neuron are maintained. Finally, we discuss the implications of the phenotypic changes of individual cells at the level of neuronal network functioning of the C. elegans worm and the retina, which are two non-spiking nervous tissues composed of neurons with various phenotypes.

Conductance-Based Phenomenological Nonspiking Model: A Dimensionless and Simple Model That Reliably Predicts the Effects of Conductance Variations on Nonspiking Neuronal Dynamics.

The modeling of single neurons has proven to be an indispensable tool in deciphering the mechanisms underlying neural dynamics and signal processing. In that sense, two types of single-neuron models are extensively used: the conductance-based models (CBMs) and the so-called phenomenological models, which are often opposed in their objectives and their use. Indeed, the first type aims to describe the biophysical properties of the neuron cell membrane that underlie the evolution of its potential, while the second one describes the macroscopic behavior of the neuron without taking into account all of its underlying physiological processes. Therefore, CBMs are often used to study "low-level" functions of neural systems, while phenomenological models are limited to the description of "high-level" functions. In this letter, we develop a numerical procedure to endow a dimensionless and simple phenomenological nonspiking model with the capability to describe the effect of conductance variations on nonspiking neuronal dynamics with high accuracy. The procedure allows determining a relationship between the dimensionless parameters of the phenomenological model and the maximal conductances of CBMs. In this way, the simple model combines the biological plausibility of CBMs with the high computational efficiency of phenomenological models, and thus may serve as a building block for studying both high-level and low-level functions of nonspiking neural networks. We also demonstrate this capability in an abstract neural network inspired by the retina and C. elegans networks, two important nonspiking nervous tissues.

Distinctive Krebs cycle remodeling in iPSC-derived neural and mesenchymal stem cells.

Mitochondria play a vital role in proliferation and differentiation and their remodeling in the course of differentiation is related to the variable energy and metabolic needs of the cell. In this work, we show a distinctive mitochondrial remodeling in human induced pluripotent stem cells differentiated into neural or mesenchymal progenitors. While leading to upregulation of the citrate synthase-α-ketoglutarate dehydrogenase segment of the Krebs cycle and increased respiratory chain activities and respiration in the mesenchymal stem cells, the remodeling in the neural stem cells resulted in downregulation of α-ketoglutarate dehydrogenase, upregulation of isocitrate dehydrogenase 2 and the accumulation of α-ketoglutarate. The distinct, lineage-specific changes indicate an involvement of these Krebs cycle enzymes in cell differentiation.

Unsuspected task for an old team: succinate, fumarate and other Krebs cycle acids in metabolic remodeling.

Seventy years from the formalization of the Krebs cycle as the central metabolic turntable sustaining the cell respiratory process, key functions of several of its intermediates, especially succinate and fumarate, have been recently uncovered. The presumably immutable organization of the cycle has been challenged by a number of observations, and the variable subcellular location of a number of its constitutive protein components is now well recognized, although yet unexplained. Nonetheless, the most striking observations have been made in the recent period while investigating human diseases, especially a set of specific cancers, revealing the crucial role of Krebs cycle intermediates as factors affecting genes methylation and thus cell remodeling. We review here the recent advances and persisting incognita about the role of Krebs cycle acids in diverse aspects of cellular life and human pathology.

Cis-silencing of PIP5K1B evidenced in Friedreich's ataxia patient cells results in cytoskeleton anomalies.

Friedreich's ataxia (FRDA) is a progressive neurodegenerative disease characterized by ataxia, variously associating heart disease, diabetes mellitus and/or glucose intolerance. It results from intronic expansion of GAA triplet repeats at the FXN locus. Homozygous expansions cause silencing of the FXN gene and subsequent decreased expression of the encoded mitochondrial frataxin. Detailed analyses in fibroblasts and neuronal tissues from FRDA patients have revealed profound cytoskeleton anomalies. So far, however, the molecular mechanism underlying these cytoskeleton defects remains unknown. We show here that gene silencing spreads in cis over the PIP5K1B gene in cells from FRDA patients (circulating lymphocytes and primary fibroblasts), correlating with expanded GAA repeat size. PIP5K1B encodes phosphatidylinositol 4-phosphate 5-kinase ß type I (pip5k1ß), an enzyme functionally linked to actin cytoskeleton dynamics that phosphorylates phosphatidylinositol 4-phosphate [PI(4)P] to generate phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2]. Accordingly, loss of pip5k1ß function in FRDA cells was accompanied by decreased PI(4,5)P2 levels and was shown instrumental for destabilization of the actin network and delayed cell spreading. Knockdown of PIP5K1B in control fibroblasts using shRNA reproduced abnormal actin cytoskeleton remodeling, whereas over-expression of PIP5K1B, but not FXN, suppressed this phenotype in FRDA cells. In addition to provide new insights into the consequences of the FXN gene expansion, these findings raise the question whether PIP5K1B silencing may contribute to the variable manifestation of this complex disease.

Embryonic stem cells neural differentiation qualifies the role of Wnt/ß-Catenin signals in human telencephalic specification and regionalization.

Wnt-ligands are among key morphogens that mediate patterning of the anterior territories of the developing brain in mammals. We qualified the role of Wnt-signals in regional specification and subregional organization of the human telencephalon using human pluripotent stem cells (hPSCs). One step neural conversion of hPSCs using SMAD inhibitors leads to progenitors with a default rostral identity. It provides an ideal biological substrate for investigating the role of Wnt signaling in both anteroposterior and dorso-ventral processes. Challenging hPSC-neural derivatives with Wnt-antagonists, alone or combined with sonic hedgehog (Shh), we found that Wnt-inhibition promote both telencephalic specification and ventral patterning of telencephalic neural precursors in a dose-dependent manner. Using optimal Wnt-antagonist and Shh-agonist signals we produced human ventral-telencephalic precursors, committed to differentiation into striatal projection neurons both in vitro and in vivo after homotypic transplantation in quinolinate-lesioned rats. This study indicates that sequentially organized Wnt-signals play a key role in the development of human ventral telencephalic territories from which the striatum arise. In addition, the optimized production of hPSC-derived striatal cells described here offers a relevant biological resource for exploring and curing Huntington disease.

Depletion of WFS1 compromises mitochondrial function in hiPSC-derived neuronal models of Wolfram syndrome.

Mitochondrial dysfunction involving mitochondria-associated ER membrane (MAM) dysregulation is implicated in the pathogenesis of late-onset neurodegenerative diseases, but understanding is limited for rare early-onset conditions. Loss of the MAM-resident protein WFS1 causes Wolfram syndrome (WS), a rare early-onset neurodegenerative disease that has been linked to mitochondrial abnormalities. Here we demonstrate mitochondrial dysfunction in human induced pluripotent stem cell-derived neuronal cells of WS patients. VDAC1 is identified to interact with WFS1, whereas loss of this interaction in WS cells could compromise mitochondrial function. Restoring WFS1 levels in WS cells reinstates WFS1-VDAC1 interaction, which correlates with an increase in MAMs and mitochondrial network that could positively affect mitochondrial function. Genetic rescue by WFS1 overexpression or pharmacological agents modulating mitochondrial function improves the viability and bioenergetics of WS neurons. Our data implicate a role of WFS1 in regulating mitochondrial functionality and highlight a therapeutic intervention for WS and related rare diseases with mitochondrial defects.

Evolutionary forces shape the human RFPL1,2,3 genes toward a role in neocortex development.

The size and organization of the brain neocortex has dramatically changed during primate evolution. This is probably due to the emergence of novel genes after duplication events, evolutionary changes in gene expression, and/or acceleration in protein evolution. Here, we describe a human Ret finger protein-like (hRFPL)1,2,3 gene cluster on chromosome 22, which is transactivated by the corticogenic transcription factor Pax6. High hRFPL1,2,3 transcript levels were detected at the onset of neurogenesis in differentiating human embryonic stem cells and in the developing human neocortex, whereas the unique murine RFPL gene is expressed in liver but not in neural tissue. Study of the evolutionary history of the RFPL gene family revealed that the RFPL1,2,3 gene ancestor emerged after the Euarchonta-Glires split. Subsequent duplication events led to the presence of multiple RFPL1,2,3 genes in Catarrhini ( approximately 34 mya) resulting in an increase in gene copy number in the hominoid lineage. In Catarrhini, RFPL1,2,3 expression profile diverged toward the neocortex and cerebellum over the liver. Importantly, humans showed a striking increase in cortical RFPL1,2,3 expression in comparison to their cerebellum, and to chimpanzee and macaque neocortex. Acceleration in RFPL-protein evolution was also observed with signs of positive selection in the RFPL1,2,3 cluster and two neofunctionalization events (acquisition of a specific RFPL-Defining Motif in all RFPLs and of a N-terminal 29 amino-acid sequence in catarrhinian RFPL1,2,3). Thus, we propose that the recent emergence and multiplication of the RFPL1,2,3 genes contribute to changes in primate neocortex size and/or organization.

Striatal progenitors derived from human ES cells mature into DARPP32 neurons in vitro and in quinolinic acid-lesioned rats.

Substitutive cell therapy using fetal striatal grafts has demonstrated preliminary clinical success in patients with Huntington's disease, but the logistics required for accessing fetal cells preclude its extension to the relevant population of patients. Human embryonic stem (hES) cells theoretically meet this challenge, because they can be expanded indefinitely and differentiated into any cell type. We have designed an in vitro protocol combining substrates, media, and cytokines to push hES cells along the neural lineage, up to postmitotic neurons expressing striatal markers. The therapeutic potential of such hES-derived cells was further substantiated by their in vivo differentiation into striatal neurons following xenotransplantation into adult rats. Our results open the way toward hES cell therapy for Huntington's disease. Long-term proliferation of human neural progenitors leads, however, to xenograft overgrowth in the rat brain, suggesting that the path to the clinic requires a way to switch them off after grafting.

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.

Expression of miRNAs from the Imprinted DLK1/DIO3 Locus Signals the Osteogenic Potential of Human Pluripotent Stem Cells.

Substantial variations in differentiation properties have been reported among human pluripotent cell lines (hPSC), which could affect their utility and clinical safety. We characterized the variable osteogenic capacity observed between different human pluripotent stem cell lines. By focusing on the miRNA expression profile, we demonstrated that the osteogenic differentiation propensity of human pluripotent stem cell lines could be associated with the methylation status and the expression of miRNAs from the imprinted DLK1/DIO3 locus. More specifically, quantitative analysis of the expression of six different miRNAs of that locus prospectively identified human embryonic stem cells and human-induced pluripotent stem cells with differential osteogenic differentiation capacities. At the molecular and functional levels, we showed that these miRNAs modulated the expression of the activin receptor type 2B and the downstream signal transduction, which impacted osteogenesis. In conclusion, miRNAs of the imprinted DLK1/DIO3 locus appear to have both a predictive value and a functional impact in determining the osteogenic fate of human pluripotent stem cells.

Induced pluripotent stem cells-derived neurons from patients with Friedreich ataxia exhibit differential sensitivity to resveratrol and nicotinamide.

Translation of pharmacological results from in vitro cell testing to clinical trials is challenging. One of the causes that may underlie these discrepant results is the lack of the phenotypic or species-specific relevance of the tested cells; today, this lack of relevance may be reduced by relying on cells differentiated from human pluripotent stem cells. To analyse the benefits provided by this approach, we chose to focus on Friedreich ataxia, a neurodegenerative condition for which the recent clinical testing of two compounds was not successful. These compounds, namely, resveratrol and nicotinamide, were selected because they had been shown to stimulate the expression of frataxin in fibroblasts and lymphoblastoid cells. Our results indicated that these compounds failed to do so in iPSC-derived neurons generated from two patients with Friedreich ataxia. By comparing the effects of both molecules on different cell types that may be considered to be non-relevant for the disease, such as fibroblasts, or more relevant to the disease, such as neurons differentiated from iPSCs, a differential response was observed; this response suggests the importance of developing more predictive in vitro systems for drug discovery. Our results demonstrate the value of utilizing human iPSCs early in drug discovery to improve translational predictability.

Correction to: ZIKA virus elicits P53 activation and genotoxic stress in human neural progenitors similar to mutations involved in severe forms of genetic microcephaly.

The authors wish to point out that the name of the first author is appearing incorrectly on Pubmed: it should be El Ghouzzi V (and not Ghouzzi VE). In addition, the words "and p53" appear at the end of the title in the original publication ( https://www.nature.com/articles/cddis2016266 ) and in the previous erratum version ( https://www.nature.com/articles/cddis2016446 ). This is not correct.

ZIKA virus elicits P53 activation and genotoxic stress in human neural progenitors similar to mutations involved in severe forms of genetic microcephaly.

Epidemiological evidence from the current outbreak of Zika virus (ZIKV) and recent studies in animal models indicate a strong causal link between ZIKV and microcephaly. ZIKV infection induces cell-cycle arrest and apoptosis in proliferating neural progenitors. However, the mechanisms leading to these phenotypes are still largely obscure. In this report, we explored the possible similarities between transcriptional responses induced by ZIKV in human neural progenitors and those elicited by three different genetic mutations leading to severe forms of microcephaly in mice. We found that the strongest similarity between all these conditions is the activation of common P53 downstream genes. In agreement with these observations, we report that ZIKV infection increases total P53 levels and nuclear accumulation, as well as P53 Ser15 phosphorylation, correlated with genotoxic stress and apoptosis induction. Interestingly, increased P53 activation and apoptosis are induced not only in cells expressing high levels of viral antigens but also in cells showing low or undetectable levels of the same proteins. These results indicate that P53 activation is an early and specific event in ZIKV-infected cells, which could result from cell-autonomous and/or non-cell-autonomous mechanisms. Moreover, we highlight a small group of P53 effector proteins that could act as critical mediators, not only in ZIKV-induced microcephaly but also in many genetic microcephaly syndromes.

Methylation and transcripts expression at the imprinted GNAS locus in human embryonic and induced pluripotent stem cells and their derivatives.

Data from the literature indicate that genomic imprint marks are disturbed in human pluripotent stem cells (PSCs). GNAS is an imprinted locus that produces one biallelic (Gsα) and four monoallelic (NESP55, GNAS-AS1, XLsα, and A/B) transcripts due to differential methylation of their promoters (DMR). To document imprinting at the GNAS locus in PSCs, we studied GNAS locus DMR methylation and transcript (NESP55, XLsα, and A/B) expression in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) derived from two human fibroblasts and their progenies. Results showed that (1) methylation at the GNAS locus DMRs is DMR and cell line specific, (2) changes in allelic transcript expression can be independent of a change in allele-specific DNA methylation, and (3) interestingly, methylation at A/B DMR is correlated with A/B transcript expression. These results indicate that these models are valuable to study the mechanisms controlling GNAS methylation, factors involved in transcript expression, and possibly mechanisms involved in the pathophysiology of pseudohypoparathyroidism type 1B.

Improvement of culture conditions of human embryoid bodies using a controlled perfused and dialyzed bioreactor system.

In parallel to the active search for therapeutic and industrial applications of human embryonic stem cells (hESCs), designing automated means of producing those cells is a timely goal. Slow-turning lateral vessels (STLVs) with low shear stress have shown promise for expanding the cells at the embryoid body stage. We have improved this technology by developing two complementary systems, allowing continuous optimization of the culture conditions. First, perfused STLV bioreactors were set up, to provide continuous delivery of culture medium to the cells growing in the rotating chamber. This allowed the external control of the culture medium, and consequently optimized oxygenation, pH, nutrient supply, and waste elimination. Second, a dialysis chamber was adapted. This led to a further enhanced controlled environment and a decrease in the quantity of adjunct products (e.g., growth factors) necessary to the cells inside the bioreactor chamber. hESC aggregation and initial differentiation-taking neural induction as an example-were compared between the perfused and dialyzed STLV system and static cultures. Perfused and dialyzed STLV bioreactors promoted formation of embryoid bodies that were differentiated more rapidly and were homogeneously synchronized in a statistically significant manner.