Clinical Grade Human Pluripotent Stem Cell-Derived Engineered Skin Substitutes Promote Keratinocytes Wound Closure In Vitro.

Chronic wounds, such as leg ulcers associated with sickle cell disease, occur as a consequence of a prolonged inflammatory phase during the healing process. They are extremely hard to heal and persist as a significant health care problem due to the absence of effective treatment and the uprising number of patients. Indeed, there is a critical need to develop novel cell- and tissue-based therapies to treat these chronic wounds. Development in skin engineering leads to a small catalogue of available substitutes manufactured in Good Manufacturing Practices compliant (GMPc) conditions. Those substitutes are produced using primary cells that could limit their use due to restricted sourcing. Here, we propose GMPc protocols to produce functional populations of keratinocytes and fibroblasts derived from pluripotent stem cells to reconstruct the associated dermo-epidermal substitute with plasma-based fibrin matrix. In addition, this manufactured composite skin is biologically active and enhances in vitro wounding of keratinocytes. The proposed composite skin opens new perspectives for skin replacement using allogeneic substitute.

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.

SISTEMA: A large and standardized collection of transcriptome data sets for human pluripotent stem cell research.

Human pluripotent stem cells have ushered in an exciting new era for disease modeling, drug discovery, and cell therapy development. Continued progress toward realizing the potential of human pluripotent stem cells will be facilitated by robust data sets and complementary resources that are easily accessed and interrogated by the stem cell community. In this context, we present SISTEMA, a quality-controlled curated gene expression database, built on a valuable catalog of human pluripotent stem cell lines, and their derivatives for which transcriptomic analyses have been generated using a single experimental pipeline. SISTEMA functions as a one-step resource that will assist the stem cell community to easily evaluate the expression level for genes of interest, while comparing them across different hPSC lines, cell types, pathological conditions, or after pharmacological treatments.

Rescuing compounds for Lesch-Nyhan disease identified using stem cell-based phenotypic screening.

Lesch-Nyhan disease (LND) is a rare monogenic disease caused by deficiency of the salvage pathway enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT). LND is characterized by severe neuropsychiatric symptoms that currently cannot be treated. Predictive in vivo models are lacking for screening and evaluating candidate drugs because LND-associated neurological symptoms are not recapitulated in HGPRT-deficient animals. Here, we used human neural stem cells and neurons derived from induced pluripotent stem cells (iPSCs) of children affected with LND to identify neural phenotypes of interest associated with HGPRT deficiency to develop a target-agnostic-based drug screening system. We screened more than 3000 molecules and identified 6 pharmacological compounds, all possessing an adenosine moiety, that corrected HGPRT deficiency-associated neuronal phenotypes by promoting metabolism compensations in an HGPRT-independent manner. This included S-adenosylmethionine, a compound that had already been used as a compassionate approach to ease the neuropsychiatric symptoms in LND. Interestingly, these compounds compensate abnormal metabolism in a manner complementary to the gold standard allopurinol and can be provided to patients with LND via simple food supplementation. This experimental paradigm can be easily adapted to other metabolic disorders affecting normal brain development and functioning in the absence of a relevant animal model.

Clinical-grade production and safe delivery of human ESC derived RPE sheets in primates and rodents.

Age-related macular degeneration as well as some forms of Retinitis Pigmentosa (RP) are characterized by a retinal degeneration involving the retinal pigment epithelium (RPE). Various strategies were proposed to cure these disorders including the replacement of RPE cells using human pluripotent stem cells (hPSCs), an unlimited source material to generate in vitro RPE cells. The formulation strategy of the cell therapy (either a reconstructed sheet or a cell suspension) is crucial to achieve an efficient and long lasting therapeutic effect. We previously developed a hPSC-RPE sheet disposed on human amniotic membrane that sustained the vision of rodents with retinal degeneration compared to the same cells injected as a suspension. However, the transplantation strategy was difficult to implement in large animals. Herein we developed two medical devices for the preparation, conservation and implantation of the hPSC-RPE sheet in nonhuman primates. The surgery was safe and well tolerated during the 7-week follow up. The graft integrity was preserved in primates. Moreover, the hPSC-RPE sheet did not induce teratoma or grafted cell dispersion to other organs in rodent models. This work clears the way for the first cell therapy for RP patients carrying RPE gene mutations (LRAT, RPE65 and MERTK).

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.

Pluripotent Stem Cell-Based Drug Screening Reveals Cardiac Glycosides as Modulators of Myotonic Dystrophy Type 1.

There is currently no treatment for myotonic dystrophy type 1 (DM1), the most frequent myopathy of genetic origin. This progressive neuromuscular disease is caused by nuclear-retained RNAs containing expanded CUG repeats. These toxic RNAs alter the activities of RNA splicing factors, resulting in alternative splicing misregulation. By combining human mutated pluripotent stem cells and phenotypic drug screening, we revealed that cardiac glycosides act as modulators for both upstream nuclear aggregations of DMPK mRNAs and several downstream alternative mRNA splicing defects. However, these occurred at different drug concentration ranges. Similar biological effects were recorded in a DM1 mouse model. At the mechanistic level, we demonstrated that this effect was calcium dependent and was synergic with inhibition of the ERK pathway. These results further underscore the value of stem-cell-based assays for drug discovery in monogenic diseases.

Improved mobility with metformin in patients with myotonic dystrophy type 1: a randomized controlled trial.

Metformin, the well-known anti-diabetic drug, has been shown recently to improve the grip test performance of the DMSXL mouse model of myotonic dystrophy type 1. The drug may have positively affected muscle function via several molecular mechanisms, on RNA splicing, autophagia, insulin sensitivity or glycogen synthesis. Myotonic dystrophy remains essentially an unmet medical need. Since metformin benefits from a good toxicity profile, we investigated its potential for improving mobility in patients. Forty ambulatory adult patients were recruited consecutively at the neuromuscular reference centre of Henri-Mondor Hospital. Participants and investigators were all blinded to treatment until the end of the trial. Oral metformin or placebo was provided three times daily, with a dose-escalation period over 4 weeks up to 3 g/day, followed by 48 weeks at maximum dose. The primary outcome was the change in the distance walked during the 6-minute walk test, from baseline to the end of the study. Concomitant changes in muscle strength and effect on myotonia, gait variables, biological parameters and quality of life were explored. Patients randomized into two arms eventually revealed similar results in all physical measures and in the mean 6-minute walk test at baseline. For the 23/40 patients who fully completed the 1-year study, differences between the groups were statistically significant, with the treated group (n = 9) gaining a distance of 32.9 ± 32.7 m, while the placebo group (n = 14) gained 3.7 ± 32.4 m (P < 0.05). This improvement in mobility was associated with an increase in total mechanical power (P = 0.01), due to a concomitant increase in the cranial and antero-posterior directions suggesting an effect of the treatment on gait. Subanalysis revealed positive effects of metformin treatment on the 6-minute walk test at the first intermediate evaluation (after 16 weeks of treatment), quantitatively similar to those recorded at 1 year. In contrast, except for the expected limited weight loss associated to metformin treatment, there was no change in any of the other secondary endpoints, including myotonia and muscle strength. Patients in the treated group had a higher incidence of mild-to-moderate adverse effects, mostly gastrointestinal dysfunctions that required symptomatic treatment. Although results were statistically significant only for the per protocol population of patients and not in the intent-to-treat analysis, metformin at the maximal tolerated dose provided a promising effect on the mobility and gait abilities of myotonic patients. These encouraging results obtained in a small-scale monocentric phase II study call for replication in a well-powered multicentre phase III trial.

Differentiation of nonhuman primate pluripotent stem cells into functional keratinocytes.

BACKGROUND: Epidermal grafting using cells derived from pluripotent stem cells will change the face of this side of regenerative cutaneous medicine. To date, the safety of the graft would be the major unmet deal in order to implement long-term skin grafting. In this context, experiments on large animals appear unavoidable to assess this question and possible rejection. Cellular tools for large animal models should be constructed. METHODS: In this study, we generated monkey pluripotent stem cell-derived keratinocytes and evaluated their capacities to reconstruct an epidermis, in vitro as well as in vivo. RESULTS: Monkey pluripotent stem cells were differentiated efficiently into keratinocytes able to reconstruct fully epidermis presenting a low level of major histocompatibility complex class-I antigens, opening the way for autologous or allogeneic epidermal long-term grafting. CONCLUSIONS: Functional keratinocytes generated from nonhuman primate embryonic stem cells and induced pluripotent stem cells reproduce an in-vitro and in-vivo stratified epidermis. These monkey skin grafts will be considered to model autologous or allogeneic epidermal grafting using either embryonic stem cells or induced pluripotent stem cells. This graft model will allow us to further investigate the safety, efficacy and immunogenicity of nonhuman primate PSC-derived epidermis in the perspective of human skin cell therapy.

Human ESC-derived retinal epithelial cell sheets potentiate rescue of photoreceptor cell loss in rats with retinal degeneration.

Replacing defective retinal pigment epithelial (RPE) cells with those derived from human embryonic stem cells (hESCs) or human-induced pluripotent stem cells (hiPSCs) is a potential strategy for treating retinal degenerative diseases. Early clinical trials have demonstrated that hESC-derived or hiPSC-derived RPE cells can be delivered safely as a suspension to the human eye. The next step is transplantation of hESC/hiPSC-derived RPE cells as cell sheets that are more physiological. We have developed a tissue-engineered product consisting of hESC-derived RPE cells grown as sheets on human amniotic membrane as a biocompatible substrate. We established a surgical approach to engraft this tissue-engineered product into the subretinal space of the eyes of rats with photoreceptor cell loss. We show that transplantation of the hESC-RPE cell sheets grown on a human amniotic membrane scaffold resulted in rescue of photoreceptor cell death and improved visual acuity in rats with retinal degeneration compared to hESC-RPE cells injected as a cell suspension. These results suggest that tissue-engineered hESC-RPE cell sheets produced under good manufacturing practice conditions may be a useful approach for treating diseases of retinal degeneration.

Impedimetric real-time monitoring of neural pluripotent stem cell differentiation process on microelectrode arrays.

In today's neurodevelopment and -disease research, human neural stem/progenitor cell-derived networks represent the sole accessible in vitro model possessing a primary phenotype. However, cultivation and moreover, differentiation as well as maturation of human neural stem/progenitor cells are very complex and time-consuming processes. Therefore, techniques for the sensitive non-invasive, real-time monitoring of neuronal differentiation and maturation are highly demanded. Using impedance spectroscopy, the differentiation of several human neural stem/progenitor cell lines was analyzed in detail. After development of an optimum microelectrode array for reliable and sensitive long-term monitoring, distinct cell-dependent impedimetric parameters that could specifically be associated with the progress and quality of neuronal differentiation were identified. Cellular impedance changes correlated well with the temporal regulation of biomolecular progenitor versus mature neural marker expression as well as cellular structure changes accompanying neuronal differentiation. More strikingly, the capability of the impedimetric differentiation monitoring system for the use as a screening tool was demonstrated by applying compounds that are known to promote neuronal differentiation such as the γ-secretase inhibitor DAPT. The non-invasive impedance spectroscopy-based measurement system can be used for sensitive and quantitative monitoring of neuronal differentiation processes. Therefore, this technique could be a very useful tool for quality control of neuronal differentiation and moreover, for neurogenic compound identification and industrial high-content screening demands in the field of safety assessment as well as drug development.

Human Pluripotent Stem Cell-derived Cortical Neurons for High Throughput Medication Screening in Autism: A Proof of Concept Study in SHANK3 Haploinsufficiency Syndrome.

Autism spectrum disorders affect millions of individuals worldwide, but their heterogeneity complicates therapeutic intervention that is essentially symptomatic. A versatile yet relevant model to rationally screen among hundreds of therapeutic options would help improving clinical practice. Here we investigated whether neurons differentiated from pluripotent stem cells can provide such a tool using SHANK3 haploinsufficiency as a proof of principle. A library of compounds was screened for potential to increase SHANK3 mRNA content in neurons differentiated from control human embryonic stem cells. Using induced pluripotent stem cell technology, active compounds were then evaluated for efficacy in correcting dysfunctional networks of neurons differentiated from individuals with deleterious point mutations of SHANK3. Among 202 compounds tested, lithium and valproic acid showed the best efficacy at corrected SHANK3 haploinsufficiency associated phenotypes in cellulo. Lithium pharmacotherapy was subsequently provided to one patient and, after one year, an encouraging decrease in autism severity was observed. This demonstrated that pluripotent stem cell-derived neurons provide a novel cellular paradigm exploitable in the search for specific disease-modifying treatments.

PPARγ Is Activated during Congenital Cytomegalovirus Infection and Inhibits Neuronogenesis from Human Neural Stem Cells.

Congenital infection by human cytomegalovirus (HCMV) is a leading cause of permanent sequelae of the central nervous system, including sensorineural deafness, cerebral palsies or devastating neurodevelopmental abnormalities (0.1% of all births). To gain insight on the impact of HCMV on neuronal development, we used both neural stem cells from human embryonic stem cells (NSC) and brain sections from infected fetuses and investigated the outcomes of infection on Peroxisome Proliferator-Activated Receptor gamma (PPARγ), a transcription factor critical in the developing brain. We observed that HCMV infection dramatically impaired the rate of neuronogenesis and strongly increased PPARγ levels and activity. Consistent with these findings, levels of 9-hydroxyoctadecadienoic acid (9-HODE), a known PPARγ agonist, were significantly increased in infected NSCs. Likewise, exposure of uninfected NSCs to 9-HODE recapitulated the effect of infection on PPARγ activity. It also increased the rate of cells expressing the IE antigen in HCMV-infected NSCs. Further, we demonstrated that (1) pharmacological activation of ectopically expressed PPARγ was sufficient to induce impaired neuronogenesis of uninfected NSCs, (2) treatment of uninfected NSCs with 9-HODE impaired NSC differentiation and (3) treatment of HCMV-infected NSCs with the PPARγ inhibitor T0070907 restored a normal rate of differentiation. The role of PPARγ in the disease phenotype was strongly supported by the immunodetection of nuclear PPARγ in brain germinative zones of congenitally infected fetuses (N = 20), but not in control samples. Altogether, our findings reveal a key role for PPARγ in neurogenesis and in the pathophysiology of HCMV congenital infection. They also pave the way to the identification of PPARγ gene targets in the infected brain.

Metformin decreases progerin expression and alleviates pathological defects of Hutchinson-Gilford progeria syndrome cells.

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, LmnaG609G/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.

Dp412e: a novel human embryonic dystrophin isoform induced by BMP4 in early differentiated cells.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a devastating X-linked recessive genetic myopathy. DMD physiopathology is still not fully understood and a prenatal onset is suspected but difficult to address. METHODS: The bone morphogenetic protein 4 (BMP4) is a critical signaling molecule involved in mesoderm commitment. Human induced pluripotent stem cells (hiPSCs) from DMD and healthy individuals and human embryonic stem cells (hESCs) treated with BMP4 allowed us to model the early steps of myogenesis in normal and DMD contexts. RESULTS: Unexpectedly, 72h following BMP4 treatment, a new long DMD transcript was detected in all tested hiPSCs and hESCs, at levels similar to that found in adult skeletal muscle. This novel transcript named "Dp412e" has a specific untranslated first exon which is conserved only in a sub-group of anthropoids including human. The corresponding novel dystrophin protein of 412-kiloDalton (kDa), characterized by an N-terminal-truncated actin-binding domain, was detected in normal BMP4-treated hiPSCs/hESCs and in embryoid bodies. Finally, using a phosphorodiamidate morpholino oligomer (PMO) targeting the DMD exon 53, we demonstrated the feasibility of exon skipping validation with this BMP4-inducible hiPSCs model. CONCLUSIONS: In this study, the use of hiPSCs to analyze early phases of human development in normal and DMD contexts has led to the discovery of an embryonic 412 kDa dystrophin isoform. Deciphering the regulation process(es) and the function(s) associated to this new isoform can contribute to a better understanding of the DMD physiopathology and potential developmental defects. Moreover, the simple and robust BMP4-inducible model highlighted here, providing large amount of a long DMD transcript and the corresponding protein in only 3 days, is already well-adapted to high-throughput and high-content screening approaches. Therefore, availability of this powerful cell platform can accelerate the development, validation and improvement of DMD genetic therapies.