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Hutchinson-Gilford progeria syndrome (HGPS) is an extremely rare genetic disorder associated with features of accelerated aging. HGPS is an autosomal dominant disease caused by a de novo mutation of LMNA gene, encoding A-type lamins, resulting in the truncated form of pre-lamin A called progerin. While asymptomatic at birth, patients develop symptoms within the first year of life when they begin to display accelerated aging and suffer from growth retardation, and severe cardiovascular complications including loss of vascular smooth muscle cells (VSMCs). Recent works reported the loss of VSMCs as a major factor triggering atherosclerosis in HGPS. Here, we investigated the mechanisms by which progerin expression leads to massive VSMCs loss. Using aorta tissue and primary cultures of murine VSMCs from a mouse model of HGPS, we showed increased VSMCs death associated with increased poly(ADP-Ribosyl)ation. Poly(ADP-Ribosyl)ation is recognized as a post-translational protein modification that coordinates the repair at DNA damage sites. Poly-ADP-ribose polymerase (PARP) catalyzes protein poly(ADP-Ribosyl)ation by utilizing nicotinamide adenine dinucleotide (NAD + ). Our results provided the first demonstration linking progerin accumulation, augmented poly(ADP-Ribosyl)ation and decreased nicotinamide adenine dinucleotide (NAD + ) level in VSMCs. Using high-throughput screening on VSMCs differentiated from iPSCs from HGPS patients, we identified a new compound, trifluridine able to increase NAD + levels through decrease of PARP-1 activity. Lastly, we demonstrate that trifluridine treatment in vivo was able to alleviate aortic VSMCs loss and clinical sign of progeria, suggesting a novel therapeutic approach of cardiovascular disease in progeria., (© 2024. The Author(s).)

Glycogen storage disease type III (GSDIII) is a rare metabolic disorder due to glycogen debranching enzyme (GDE) deficiency. Reduced GDE activity leads to pathological glycogen accumulation responsible for impaired hepatic metabolism and muscle weakness. To date, there is no curative treatment for GSDIII. We previously reported that 2 distinct dual AAV vectors encoding for GDE were needed to correct liver and muscle in a GSDIII mouse model. Here, we evaluated the efficacy of rapamycin in combination with AAV gene therapy. Simultaneous treatment with rapamycin and a potentially novel dual AAV vector expressing GDE in the liver and muscle resulted in a synergic effect demonstrated at biochemical and functional levels. Transcriptomic analysis confirmed synergy and suggested a putative mechanism based on the correction of lysosomal impairment. In GSDIII mice livers, dual AAV gene therapy combined with rapamycin reduced the effect of the immune response to AAV observed in this disease model. These data provide proof of concept of an approach exploiting the combination of gene therapy and rapamycin to improve efficacy and safety and to support clinical translation.

The design of human model systems is highly relevant to unveil the underlying mechanisms of aging and to provide insights on potential interventions to extend human health and life span. In this perspective, we explore the potential of 2D or 3D culture models comprising human induced pluripotent stem cells and transdifferentiated cells obtained from aged or age-related disorder-affected donors to enhance our understanding of human aging and to catalyze the discovery of anti-aging interventions., (© 2024. The Author(s).)

Glycogen storage disease type III (GSDIII) is a rare inborn error of metabolism affecting liver, skeletal muscle, and heart due to mutations of the AGL gene encoding for the glycogen debranching enzyme (GDE). No curative treatment exists for GSDIII. The 4.6 kb GDE cDNA represents the major technical challenge toward the development of a single recombinant adeno-associated virus-derived (rAAV-derived) vector gene therapy strategy. Using information on GDE structure and molecular modeling, we generated multiple truncated GDEs. Among them, an N-terminal-truncated mutant, ΔNter2-GDE, had a similar efficacy in vivo compared with the full-size enzyme. A rAAV vector expressing ΔNter2-GDE allowed significant glycogen reduction in heart and muscle of Agl-/- mice 3 months after i.v. injection, as well as normalization of histology features and restoration of muscle strength. Similarly, glycogen accumulation and histological features were corrected in a recently generated Agl-/- rat model. Finally, transduction with rAAV vectors encoding ΔNter2-GDE corrected glycogen accumulation in an in vitro human skeletal muscle cellular model of GSDIII. In conclusion, our results demonstrated the ability of a single rAAV vector expressing a functional mini-GDE transgene to correct the muscle and heart phenotype in multiple models of GSDIII, supporting its clinical translation to patients with GSDIII.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare and fatal genetic condition that arises from a single nucleotide alteration in the LMNA gene, leading to the production of a defective lamin A protein known as progerin. The accumulation of progerin accelerates the onset of a dramatic premature aging phenotype in children with HGPS, characterized by low body weight, lipodystrophy, metabolic dysfunction, skin, and musculoskeletal age-related dysfunctions. In most cases, these children die of age-related cardiovascular dysfunction by their early teenage years. The absence of effective treatments for HGPS underscores the critical need to explore novel safe therapeutic strategies. In this study, we show that treatment with the hormone ghrelin increases autophagy, decreases progerin levels, and alleviates other cellular hallmarks of premature aging in human HGPS fibroblasts. Additionally, using a HGPS mouse model (Lmna G609G/G609G mice), we demonstrate that ghrelin administration effectively rescues molecular and histopathological progeroid features, prevents progressive weight loss in later stages, reverses the lipodystrophic phenotype, and extends lifespan of these short-lived mice. Therefore, our findings uncover the potential of modulating ghrelin signaling offers new treatment targets and translational approaches that may improve outcomes and enhance the quality of life for patients with HGPS and other age-related pathologies., (© 2023 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

Glycogen storage disease type III (GSDIII) is an autosomal recessive disorder characterized by a deficiency of glycogen debranching enzyme (GDE) leading to cytosolic glycogen accumulation and inducing liver and muscle pathology. Skin fibroblasts from three GSDIII patients were reprogrammed into induced pluripotent stem cells (iPSCs) using non-integrated Sendai virus. All of the three lines exhibited normal morphology, expression of pluripotent markers, stable karyotype, potential of trilineage differentiation and absence of GDE expression, making them valuable tools for modeling GSDIII disease in vitro, studying pathological mechanisms and investigating potential treatments., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Introduction: Glycogen storage disease type III (GSDIII) is a rare genetic disease caused by mutations in the AGL gene encoding the glycogen debranching enzyme (GDE). The deficiency of this enzyme, involved in cytosolic glycogen degradation, leads to pathological glycogen accumulation in liver, skeletal muscles and heart. Although the disease manifests with hypoglycemia and liver metabolism impairment, the progressive myopathy is the major disease burden in adult GSDIII patients, without any curative treatment currently available. Methods: Here, we combined the self-renewal and differentiation capabilities of human induced pluripotent stem cells (hiPSCs) with cutting edge CRISPR/Cas9 gene editing technology to establish a stable AGL knockout cell line and to explore glycogen metabolism in GSDIII. Results: Following skeletal muscle cells differentiation of the edited and control hiPSC lines, our study reports that the insertion of a frameshift mutation in AGL gene results in the loss of GDE expression and persistent glycogen accumulation under glucose starvation conditions. Phenotypically, we demonstrated that the edited skeletal muscle cells faithfully recapitulate the phenotype of differentiated skeletal muscle cells of hiPSCs derived from a GSDIII patient. We also demonstrated that treatment with recombinant AAV vectors expressing the human GDE cleared the accumulated glycogen. Discussion: This study describes the first skeletal muscle cell model of GSDIII derived from hiPSCs and establishes a platform to study the mechanisms that contribute to muscle impairments in GSDIII and to assess the therapeutic potential of pharmacological inducers of glycogen degradation or gene therapy approaches., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Rossiaud, Fragner, Barbon, Gardin, Benabides, Pellier, Cosette, El Kassar, Giraud-Triboult, Nissan, Ronzitti and Hoch.)

Limb girdle muscular dystrophies (LGMD), caused by mutations in 29 different genes, are the fourth most prevalent group of genetic muscle diseases. Although the link between LGMD and its genetic origins has been determined, LGMD still represent an unmet medical need. Here, we describe a platform for modeling LGMD based on the use of human induced pluripotent stem cells (hiPSC). Thanks to the self-renewing and pluripotency properties of hiPSC, this platform provides a renewable and an alternative source of skeletal muscle cells (skMC) to primary, immortalized, or overexpressing cells. We report that skMC derived from hiPSC express the majority of the genes and proteins that cause LGMD. As a proof of concept, we demonstrate the importance of this cellular model for studying LGMDR9 by evaluating disease-specific phenotypes in skMC derived from hiPSC obtained from four patients.

Limb-girdle muscular dystrophy type R3 (LGMD R3) is a rare genetic disorder characterized by a progressive proximal muscle weakness and caused by mutations in the SGCA gene encoding alpha-sarcoglycan (α-SG). Here, we report the results of a mechanistic screening ascertaining the molecular mechanisms involved in the degradation of the most prevalent misfolded R77C-α-SG protein. We performed a combinatorial study to identify drugs potentializing the effect of a low dose of the proteasome inhibitor bortezomib on the R77C-α-SG degradation inhibition. Analysis of the screening associated to artificial intelligence-based predictive ADMET characterization of the hits led to identification of the HDAC inhibitor givinostat as potential therapeutical candidate. Functional characterization revealed that givinostat effect was related to autophagic pathway inhibition, unveiling new theories concerning degradation pathways of misfolded SG proteins. Beyond the identification of a new therapeutic option for LGMD R3 patients, our results shed light on the potential repurposing of givinostat for the treatment of other genetic diseases sharing similar protein degradation defects such as LGMD R5 and cystic fibrosis., Competing Interests: Authors NM, JD, MG, DP, and NS were employed by the company KANTIFY. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Hoch, Bourg, Degrugillier, Bruge, Benabides, Pellier, Tournois, Mahé, Maignan, Dawe, Georges, Papazian, Subramanian, Simon, Fanen, Delevoye, Richard and Nissan.)

What if the next generation of successful treatments was hidden in the current pharmacopoeia? Identifying new indications for existing drugs, also called the drug repurposing or drug rediscovery process, is a highly efficient and low-cost strategy. First reported almost a century ago, drug repurposing has emerged as a valuable therapeutic option for diseases that do not have specific treatments and rare diseases, in particular. This review focuses on Hutchinson-Gilford progeria syndrome (HGPS), a rare genetic disorder that induces accelerated and precocious aging, for which drug repurposing has led to the discovery of several potential treatments over the past decade., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

The vascular bioactivity/safety of nanomaterials is typically evaluated by animal testing, which is of low throughput and does not account for biological differences between animals and humans such as ageing, metabolism and disease profiles. The development of personalized human in vitro platforms to evaluate the interaction of nanomaterials with the vascular system would be important for both therapeutic and regenerative medicine. A library of 30 nanoparticle (NP) formulations, in use in imaging, antimicrobial and pharmaceutical applications, was evaluated in a reporter zebrafish model of vasculogenesis and then tested in personalized humanized models composed of human-induced pluripotent stem cell (hiPSC)-derived endothelial cells (ECs) with "young" and "aged" phenotypes in 3 vascular network formats: 2D (in polystyrene dish), 3D (in Matrigel) and in a blood vessel on a chip. As a proof of concept, vascular toxicity was used as the main readout. The results show that the toxicity profile of NPs to hiPSC-ECs was dependent on the "age" of the endothelial cells and vascular network format. hiPSC-ECs were less susceptible to the cytotoxicity effect of NPs when cultured in flow than in static conditions, the protective effect being mediated, at least in part, by glycocalyx. Overall, the results presented here highlight the relevance of in vitro hiPSC-derived vascular systems to screen vascular nanomaterial interactions.

Background: Severe ventricular rhythm disturbances are the hallmark of arrhythmogenic cardiomyopathy (ACM), and are often explained by structural conduction abnormalities. However, comprehensive investigations of ACM cell electrical instability are lacking. This study aimed to elucidate early electrical myogenic signature of ACM., Methods: We investigated a 41-year-old ACM patient with a missense mutation (c.394C>T) in the DSC2 gene, which encodes desmocollin 2. Pathogenicity of this variant was confirmed using a zebrafish DSC2 model system. Control and DSC2 patient-derived pluripotent stem cells were reprogrammed and differentiated into cardiomyocytes (hiPSC-CM) to examine the specific electromechanical phenotype and its modulation by antiarrhythmic drugs (AADs). Samples of the patient's heart and hiPSC-CM were examined to identify molecular and cellular alterations., Results: A shortened action potential duration was associated with reduced Ca 2+ current density and increased K + current density. This finding led to the elucidation of previously unknown abnormal repolarization dynamics in ACM patients. Moreover, the Ca 2+ mobilised during transients was decreased, and the Ca 2+ sparks frequency was increased. AAD testing revealed the following: (1) flecainide normalised Ca 2+ transients and significantly decreased Ca 2+ spark occurrence and (2) sotalol significantly lengthened the action potential and normalised the cells' contractile properties., Conclusions: Thorough analysis of hiPSC-CM derived from the DSC2 patient revealed abnormal repolarization dynamics, prompting the discovery of a short QT interval in some ACM patients. Overall, these results confirm a myogenic origin of ACM electrical instability and provide a rationale for prescribing class 1 and 3 AADs in ACM patients with increased ventricular repolarization reserve., (© 2021 The Authors. Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.)

Hutchinson-Gilford Progeria Syndrome (HGPS) is a premature aging disease in children that leads to early death. Smooth muscle cells (SMCs) are the most affected cells in HGPS individuals, although the reason for such vulnerability remains poorly understood. In this work, we develop a microfluidic chip formed by HGPS-SMCs generated from induced pluripotent stem cells (iPSCs), to study their vulnerability to flow shear stress. HGPS-iPSC SMCs cultured under arterial flow conditions detach from the chip after a few days of culture; this process is mediated by the upregulation of metalloprotease 13 (MMP13). Importantly, double-mutant Lmna G609G/G609G Mmp13 -/- mice or Lmna G609G/G609G Mmp13 +/+ mice treated with a MMP inhibitor show lower SMC loss in the aortic arch than controls. MMP13 upregulation appears to be mediated, at least in part, by the upregulation of glycocalyx. Our HGPS-SMCs chip represents a platform for developing treatments for HGPS individuals that may complement previous pre-clinical and clinical treatments.

Limb-girdle muscular dystrophy type 2D (LGMD2D) is characterized by a progressive proximal muscle weakness. LGMD2D is caused by mutations in the gene encoding α-sarcoglycan (α-SG), a dystrophin-associated glycoprotein that plays a key role in the maintenance of sarcolemma integrity in striated muscles. We report here on the development of a new in vitro high-throughput screening assay that allows the monitoring of the proper localization of the most prevalent mutant form of α-SG (R77C substitution). Using this assay, we screened a library of 2560 FDA-approved drugs and bioactive compounds and identified thiostrepton, a cyclic antibiotic, as a potential drug to repurpose for LGMD2D treatment. Characterization of the thiostrepton effect revealed a positive impact on R77C-α-SG and other missense mutant protein localization (R34H, I124T, V247M) in fibroblasts overexpressing these proteins. Finally, further investigations of the molecular mechanisms of action of the compound revealed an inhibition of the chymotrypsin-like activity of the proteasome 24 h after thiostrepton treatment and a synergistic effect with bortezomib, an FDA-approved proteasome inhibitor. This study reports on the first in vitro model for LGMD2D that is compatible with high-throughput screening and proposes a new therapeutic option for LGMD2D caused by missense mutations of α-SG.

Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare genetic disorder that leads to premature aging. In this study, we used induced pluripotent stem cells to investigate the hypopigmentation phenotypes observed in patients with progeria. Accordingly, two iPS cell lines were derived from cells from HGPS patients and differentiated into melanocytes. Measurements of melanin content revealed a lower synthesis of melanin in HGPS melanocytes as compared to non-pathologic cells. Analysis of the melanosome maturation process by electron microscopy revealed a lower percentage of mature, fully pigmented melanosomes. Finally, a functional rescue experiment revealed the direct role of progerin in the regulation of melanogenesis. Overall, these results report a new dysregulated pathway in HGPS and open up novel perspectives in the study of pigmentation phenotypes that are associated with normal and pathological aging.

Aging is characterized by a progressive accumulation of cellular damage, which leads to impaired function. Little is known whether substrates can influence cell aging. This is of utmost importance in the development of medical devices that are in contact with human tissue for long periods of time. To address this question, we have used an accelerated aging cell model derived from Hutchinson-Gilford Progeria Syndrome (HGPS) induced pluripotent stem cells (iPSCs). Our results show that HGPS-iPSC smooth muscle cells (SMCs) have an increased aging profile in substrates with specific micropatterns than in flat ones. This is characterized by an up-regulation in the expression of progerin, β-galactosidase, annexin 3 and 5, and caspase 9. Signs of cell aging are also observed in SMCs without HGPS cultured in substrates with specific microtopographies. It is further showed that specific micropatterned substrates induce cell aging by triggering a DNA damage program likely by the disruption between cyto- and nucleoskeleton.

Hutchinson-Gilford progeria syndrome (HGPS) is a lethal premature and accelerated aging disease caused by a de novo point mutation in LMNA encoding A-type lamins. Progerin, a truncated and toxic prelamin A issued from aberrant splicing, accumulates in HGPS cells' nuclei and is a hallmark of the disease. Small amounts of progerin are also produced during normal aging. We show that progerin is sequestered into abnormally shaped promyelocytic nuclear bodies, identified as novel biomarkers in late passage HGPS cell lines. We found that the proteasome inhibitor MG132 induces progerin degradation through macroautophagy and strongly reduces progerin production through downregulation of SRSF-1 and SRSF-5 accumulation, controlling prelamin A mRNA aberrant splicing. MG132 treatment improves cellular HGPS phenotypes. MG132 injection in skeletal muscle of Lmna G609G/G609G mice locally reduces SRSF-1 expression and progerin levels. Altogether, we demonstrate progerin reduction based on MG132 dual action and shed light on a promising class of molecules toward a potential therapy for children with HGPS., (© 2017 The Authors. Published under the terms of the CC BY 4.0 license.)

Organ-on-a-chip platforms seek to recapitulate the complex microenvironment of human organs using miniaturized microfluidic devices. Besides modeling healthy organs, these devices have been used to model diseases, yielding new insights into pathophysiology. Hutchinson-Gilford progeria syndrome (HGPS) is a premature aging disease showing accelerated vascular aging, leading to the death of patients due to cardiovascular diseases. HGPS targets primarily vascular cells, which reside in mechanically active tissues. Here, a progeria-on-a-chip model is developed and the effects of biomechanical strain are examined in the context of vascular aging and disease. Physiological strain induces a contractile phenotype in primary smooth muscle cells (SMCs), while a pathological strain induces a hypertensive phenotype similar to that of angiotensin II treatment. Interestingly, SMCs derived from human induced pluripotent stem cells of HGPS donors (HGPS iPS-SMCs), but not from healthy donors, show an exacerbated inflammatory response to strain. In particular, increased levels of inflammation markers as well as DNA damage are observed. Pharmacological intervention reverses the strain-induced damage by shifting gene expression profile away from inflammation. The progeria-on-a-chip is a relevant platform to study biomechanics in vascular biology, particularly in the setting of vascular disease and aging, while simultaneously facilitating the discovery of new drugs and/or therapeutic targets., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder that causes systemic accelerated aging in children. This syndrome is due to a mutation in the LMNA gene that leads to the production of a truncated and toxic form of lamin A called progerin. Because the balance between the A-type lamins is controlled by the RNA-binding protein SRSF1, we have hypothesized that its inhibition may have therapeutic effects for HGPS. For this purpose, we evaluated the antidiabetic drug metformin and demonstrated that 48 h treatment with 5 mmol/l metformin decreases SRSF1 and progerin expression in mesenchymal stem cells derived from HGPS induced pluripotent stem cells (HGPS MSCs). The effect of metformin on progerin was then confirmed in several in vitro models of HGPS, i.e., human primary HGPS fibroblasts, Lmna G609G/G609G mouse fibroblasts and healthy MSCs previously treated with a PMO (phosphorodiamidate morpholino oligonucleotide) that induces progerin. This was accompanied by an improvement in two in vitro phenotypes associated with the disease: nuclear shape abnormalities and premature osteoblastic differentiation of HGPS MSCs. Overall, these results suggest a novel approach towards therapeutics for HGPS that can be added to the currently assayed treatments that target other molecular defects associated with the disease., Competing Interests: The authors declare no conflict of interest.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare fatal genetic disorder that causes systemic accelerated aging in children. Thanks to the pluripotency and self-renewal properties of induced pluripotent stem cells (iPSC), HGPS iPSC-based modeling opens up the possibility of access to different relevant cell types for pharmacological approaches. In this study, 2800 small molecules were explored using high-throughput screening, looking for compounds that could potentially reduce the alkaline phosphatase activity of HGPS mesenchymal stem cells (MSCs) committed into osteogenic differentiation. Results revealed seven compounds that normalized the osteogenic differentiation process and, among these, all-trans retinoic acid and 13-cis-retinoic acid, that also decreased progerin expression. This study highlights the potential of high-throughput drug screening using HGPS iPS-derived cells, in order to find therapeutic compounds for HGPS and, potentially, for other aging-related disorders.

Major physiological changes are governed by alternative splicing of RNA, and its misregulation may lead to specific diseases. With the use of a genome-wide approach, we show here that this splicing step can be modified by medication and demonstrate the effects of the biguanide metformin, on alternative splicing. The mechanism of action involves AMPK activation and downregulation of the RBM3 RNA-binding protein. The effects of metformin treatment were tested on myotonic dystrophy type I (DM1), a multisystemic disease considered to be a spliceopathy. We show that this drug promotes a corrective effect on several splicing defects associated with DM1 in derivatives of human embryonic stem cells carrying the causal mutation of DM1 as well as in primary myoblasts derived from patients. The biological effects of metformin were shown to be compatible with typical therapeutic dosages in a clinical investigation involving diabetic patients. The drug appears to act as a modifier of alternative splicing of a subset of genes and may therefore have novel therapeutic potential for many more diseases besides those directly linked to defective alternative splicing.

Progeria, or Hutchinson-Gilford progeria syndrome (HGPS), is a rare, fatal genetic disease characterized by an appearance of accelerated aging in children. This syndrome is typically caused by mutations in codon 608 (p.G608G) of the LMNA, leading to the production of a mutated form of lamin A precursor called progerin. In HGPS, progerin accumulates in cells causing progressive molecular defects, including nuclear shape abnormalities, chromatin disorganization, damage to DNA and delays in cell proliferation. Here we report how, over the past five years, pluripotent stem cells have provided new insights into the study of HGPS and opened new original therapeutic perspectives to treat the disease., (Copyright © 2015 Elsevier B.V. All rights reserved.)

"Café-au-lait" macules (CALMs) and overall skin hyperpigmentation are early hallmarks of neurofibromatosis type 1 (NF1). One of the most frequent monogenic diseases, NF1 has subsequently been characterized with numerous benign Schwann cell-derived tumors. It is well established that neurofibromin, the NF1 gene product, is an antioncogene that down-regulates the RAS oncogene. In contrast, the molecular mechanisms associated with alteration of skin pigmentation have remained elusive. We have reassessed this issue by differentiating human embryonic stem cells into melanocytes. In the present study, we demonstrate that NF1 melanocytes reproduce the hyperpigmentation phenotype in vitro, and further characterize the link between loss of heterozygosity and the typical CALMs that appear over the general hyperpigmentation. Molecular mechanisms associated with these pathological phenotypes correlate with an increased activity of cAMP-mediated PKA and ERK1/2 signaling pathways, leading to overexpression of the transcription factor MITF and of the melanogenic enzymes tyrosinase and dopachrome tautomerase, all major players in melanogenesis. Finally, the hyperpigmentation phenotype can be rescued using specific inhibitors of these signaling pathways. These results open avenues for deciphering the pathological mechanisms involved in pigmentation diseases, and provide a robust assay for the development of new strategies for treating these diseases.

Hutchinson-Gilford progeria syndrome is a rare congenital disease characterized by premature aging in children. Identification of the mutation and related molecular mechanisms has rapidly led to independent clinical trials testing different marketed drugs with a preclinically documented impact on those mechanisms. However, the extensive functional effects of those drugs remain essentially unexplored. We have undertaken a systematic comparative study of the three main treatments currently administered or proposed to progeria-affected children, namely, a farnesyltransferase inhibitor, the combination of an aminobisphosphonate and a statin (zoledronate and pravastatin), and the macrolide antibiotic rapamycin. This work was based on the assumption that mesodermal stem cells, which are derived from Hutchinson-Gilford progeria syndrome-induced pluripotent stem cells expressing major defects associated with the disease, may be instrumental to revealing such effects. Whereas all three treatments significantly improved misshapen cell nuclei typically associated with progeria, differences were observed in terms of functional improvement in prelamin A farnesylation, progerin expression, defective cell proliferation, premature osteogenic differentiation, and ATP production. Finally, we have evaluated the effect of the different drug combinations on this cellular model. This study revealed no additional benefit compared with single-drug treatments, whereas a cytostatic effect equivalent to that of a farnesyltransferase inhibitor alone was systematically observed. Altogether, these results reveal the complexity of the modes of action of different drugs, even when they have been selected on the basis of a similar mechanistic hypothesis, and underscore the use of induced pluripotent stem cell derivatives as a critical and powerful tool for standardized, comparative pharmacological studies.

One puzzling observation in patients affected with Hutchinson-Gilford progeria syndrome (HGPS), who overall exhibit systemic and dramatic premature aging, is the absence of any conspicuous cognitive impairment. Recent studies based on induced pluripotent stem cells derived from HGPS patient cells have revealed a lack of expression in neural derivatives of lamin A, a major isoform of LMNA that is initially produced as a precursor called prelamin A. In HGPS, defective maturation of a mutated prelamin A induces the accumulation of toxic progerin in patient cells. Here, we show that a microRNA, miR-9, negatively controls lamin A and progerin expression in neural cells. This may bear major functional correlates, as alleviation of nuclear blebbing is observed in nonneural cells after miR-9 overexpression. Our results support the hypothesis, recently proposed from analyses in mice, that protection of neural cells from progerin accumulation in HGPS is due to the physiologically restricted expression of miR-9 to that cell lineage., (Copyright © 2012 The Authors. Published by Elsevier Inc. All rights reserved.)

The role of microRNAs (miRNAs) as coordinators of stem cell fate has emerged over the last decade. We have used human embryonic stem cells to identify miRNAs involved in neural lineage commitment induced by the inhibition of TGFβ-like molecule-mediated pathways. Among several candidate miRNAs expressed in the fetal brain, the two isoforms of miR-125 alone were detected in a time window compatible with a role in neural commitment in vitro. Functional analysis indicated that miR-125 isoforms were actively involved in the promotion of pluripotent cell conversion into SOX1-positive neural precursors. miR-125 promotes neural conversion by avoiding the persistence of non-differentiated stem cells and repressing alternative fate choices. This was associated with the regulation by miR-125 of SMAD4, a key regulator of pluripotent stem cell lineage commitment. Activation of miR-125 was directly responsive to the levels of TGFβ-like molecules, placing miR-125 at the core of mechanisms that lead to the irreversible neural lineage commitment of pluripotent stem cells in response to external stimuli.

Progeria, also known as HGPS (Hutchinson-Gilford progeria syndrome), is a rare fatal genetic disease characterized by an appearance of accelerated aging in children. This syndrome is typically caused by mutations in codon 608 (C1804T) of the gene encoding lamins A and C, LMNA, leading to the production of a truncated form of the protein called progerin. Owing to their unique potential to self-renew and to differentiate into any cell types of the organism, pluripotent stem cells offer a unique tool to study molecular and cellular mechanisms related to this global and systemic disease. Recent studies have exploited this potential by generating human induced pluripotent stem cells from HGPS patients' fibroblasts displaying several phenotypic defects characteristic of HGPS such as nuclear abnormalities, progerin expression, altered DNA-repair mechanisms and premature senescence. Altogether, these findings provide new insights on the use of pluripotent stem cells for pathological modelling and may open original therapeutic perspectives for diseases that lack pre-clinical in vitro human models, such as HGPS.

Melanocytes are essential for skin homeostasis and protection, and their defects in humans lead to a wide array of diseases that are potentially extremely severe. To date, the analysis of molecular mechanisms and the function of human melanocytes have been limited because of the difficulties in accessing large numbers of cells with the specific phenotypes. This issue can now be addressed via a differentiation protocol that allows melanocytes to be obtained from pluripotent stem cell lines, either induced or of embryonic origin, based on the use of moderate concentrations of a single cytokine, bone morphogenic protein 4. Human melanocytes derived from pluripotent stem cells exhibit all the characteristic features of their adult counterparts. This includes the enzymatic machinery required for the production and functional delivery of melanin to keratinocytes. Melanocytes also integrate appropriately into organotypic epidermis reconstructed in vitro. The availability of human cells committed to the melanocytic lineage in vitro will enable the investigation of those mechanisms that guide the developmental processes and will facilitate analysis of the molecular mechanisms responsible for genetic diseases. Access to an unlimited resource may also prove a vital tool for the treatment of hypopigmentation disorders when donors with matching haplotypes become available in clinically relevant banks of pluripotent stem cell lines.

The molecular mechanisms controlling the differentiation of human basal keratinocyte stem cells towards the epidermis are well characterized, whereas the earliest process leading to the specification of embryonic stem cells into keratinocytes is still not well understood. MicroRNAs are regulators of many cellular events, but evidence for microRNA acting on the differentiation of human embryonic stem cells into a specific lineage has been elusive. By using our recent protocol for obtaining functional keratinocytes from hESC, we attempted to analyze the role of microRNAs in the early stages of epidermal differentiation. Thus, we identified a set of 5 microRNAs, namely miR-200a, miR-200b, miR-203, miR-205 and miR-429, that are specifically overexpressed during the early stages of the differentiation process. Interestingly, our functional analyses revealed an instrumental role of miR-203, which had been previously shown to play a key role during the formation of the pluristratified epidermis by basal keratinocyte stem cells, in the early keratinocyte commitment. These results highlight the determinant and unique role of miR-203 during the entire process of epidermal development by extending its spectrum of action from the early commitment of embryonic stem cells to ultimate differentiation of the organ., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Although cell therapy has been clinically implemented for several decades, its use is hampered by the difficulty in supplying the amount of epidermal substitute needed to extend the application to all patients who may benefit from it. How human pluripotent stem cells may help meet this challenge is the topic of this review. After reporting on the main current applications and needs of skin grafting, we explore the potential of pluripotent stem cells--either of embryonic origin or produced by genetic reprogramming--to provide the needed clinical-grade keratinocytes, fulfilling industrial scale production, and quality standards. Immunogenicity is clearly an issue, although one may expect cells displaying characteristics of fetal or embryonic skin to have a much better tolerance than adult keratinocytes. The open possibility of a bank of pluripotent stem cell lines selected on the basis of interesting haplotypes may eventually provide a definitive answer. Actually, making the case for pluripotent stem cells in skin grafting goes well beyond that specific cell type. Most cell phenotypes that normally participate to the formation of dermis and epidermis can either already be obtained through in vitro differentiation from pluripotent stem cells or would likely migrate from the host into a graft. However, differentiation protocols for specialized glands and hair follicles remain to be designed. A future can be foreseen when reconstructive medicine will make use of composite grafts integrating several different cell types and biomaterials., (Copyright © 2011 AlphaMed Press.)

Mesenchymal stem cells (MSCs) are present in a wide variety of tissues during development of the human embryo starting as early as the first trimester. Gene expression profiling of these cells has focused primarily on the molecular signs characterizing their potential heterogeneity and their differentiation potential. In contrast, molecular mechanisms participating in the emergence of MSC identity in embryo are still poorly understood. In this study, human embryonic stem cells (hESs) were differentiated toward MSCs (ES-MSCs) to compare the genetic patterns between pluripotent hESs and multipotent MSCs by a large genomewide expression profiling of mRNAs and microRNAs (miRNAs). After whole genome differential transcriptomic analysis, a stringent protocol was used to search for genes differentially expressed between hESs and ES-MSCs, followed by several validation steps to identify the genes most specifically linked to the MSC phenotype. A network was obtained that encompassed 74 genes in 13 interconnected transcriptional systems that are likely to contribute to MSC identity. Pairs of negatively correlated miRNAs and mRNAs, which suggest miRNA-target relationships, were then extracted and validation was sought with the use of Pre-miRs. We report here that underexpression of miR-148a and miR-20b in ES-MSCs, compared with ESs, allows an increase in expression of the EPAS1 (Endothelial PAS domain 1) transcription factor that results in the expression of markers of the MSC phenotype specification.

Background: Cell therapy for large burns is dependent upon autologous epidermis reconstructed in vitro. However, the effectiveness of current procedures is limited by the delay needed to culture the patient's own keratinocytes. To assess whether the keratinocyte progeny of human embryonic stem cells (hESCs) could be used to form a temporary skin substitute for use in patients awaiting autologous grafts, we investigated the cells' capability of constructing a pluristratified epidermis., Methods: hESCs from lines H9 and SA01 were seeded at least in triplicate on fibroblast feeder cells for 40 days in a medium supplemented with bone morphogenetic protein 4 and ascorbic acid. Molecular characterisation of cell differentiation was done throughout the process by quantitative PCR, fluorescence-activated cell sorting, and immunocytochemical techniques. Keratinocyte molecular differentiation and functional capacity to construct a human epidermis were assessed in vitro and in vivo., Findings: From hESCs, we generated a homogeneous population of cells that showed phenotypic characteristics of basal keratinocytes. Expression levels of genes encoding keratin 14, keratin 5, integrin alpha6, integrin beta4, collagen VII, and laminin 5 in these cells were similar to those in basal keratinocytes. After seeding on an artificial matrix, keratinocytes derived from hESCs (K-hESCs) formed a pluristratified epidermis. Keratin-14 immunostaining was seen in the basal compartment, with keratin 10 present in layers overlying the basal layer. Involucrin and filaggrin, late markers of epidermal differentiation, were detected in the uppermost layers only. 12 weeks after grafting onto five immunodeficient mice, epidermis derived from K-hESCs had a structure consistent with that of mature human skin. Human involucrin was appropriately located in spinous and granular layers and few Ki67-positive cells were detected in the basal layer., Interpretation: hESCs can be differentiated into basal keratinocytes that are fully functional--ie, able to construct a pluristratified epidermis. This resource could be developed to provide temporary skin substitutes for patients awaiting autologous grafts., Funding: Institut National de la Santé et de la Recherche Médicale, University Evry Val d'Essonne, Association Française contre les Myopathies, Fondation René Touraine, and Genopole.

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.