Differentiation of pluripotent stem cells into melanocytes and potential applications : the example of pigmentation disorders due to neurofibromatosis type 1

La neurofibromatose de type 1 (NF1) constitue la maladie autosomique dominante la plus fréquente avec une incidence d’environ 1 naissance pour 3500. Les manifestations cliniques les plus courantes sont neurocutanées et se caractérisent par une hyperpigmentation générale diffuse avec une impression de peau brune, des tâches hyperpigmentées dites tâches café-au-lait, des tumeurs bénignes de la gaine des nerfs périphériques appelées neurofibromes et des troubles neurologiques. Le gène NF1, responsable de la maladie est un gène suppresseur de tumeur qui code pour la neurofibromine. Les mécanismes moléculaires associés à l’altération de la pigmentation chez ces patients restent jusqu’ici inconnus. Nous avons étudié cette question grâce à la mise en place au laboratoire d’un protocole de différenciation des cellules souches pluripotentes en mélanocytes et l’accès à deux lignées de cellules souches embryonnaires porteuses de la mutation causale de NF1 (hESC-NF1). Dans cette étude, nous avons montré qu’une perte d’expression de la neurofibromine dans les mélanocytes dérivés de hESC-NF1 reproduisait le phénotype d’hyperpigmentation généralisée et pouvait mimer la formation des tâches « café au lait ». L’analyse des mécanismes moléculaires associés à ce phénotype pathologique montre que les mélanocytes NF1 présentent une dérégulation des voies de signalisation AMPc et ERK conduisant à l’augmentation de la mélanogenèse. Le phénotype hyperpigmentaire des mélanocytes NF1 a pu être corrigé grâce à l’utilisation d’inhibiteurs pharmacologiques spécifiques de la voie AMPc, de la voie ERK et de la mélanogenèse. En parallèle, afin de pouvoir envisager une approche de thérapie cellulaire à partir des mélanocytes dérivés de cellules pluripotentes pour le traitement de maladies associées à une hypopigmentation d’origine génétiques ou non, un protocole de différenciation fondé sur une application séquentielle de cytokines permettant de diriger de manière spécifique les cellules souches pluripotentes en mélanocytes a été développé au laboratoire. Des mélanocytes fonctionnels ont été obtenus en 30 jours en engagement la différenciation des cellules souches vers la crête neurale afin d’induire l’émergence de précurseurs de mélanocytes par l’action de molécules telles que la BMP4, l’acide ascorbique, l’endotheline 3, le SCF et un activateur de WNT3a (CHIR99021L’ensemble de ce travail a permis de valider la pertinence de l’utilisation des cellules souches pluripotentes porteuses de maladie génétique ou pour le traitement de maladies hyper ou hypopigmentaire par des approches de modélisation pathologique et de criblage pharmacologique ou par des approches de thérapie cellulaire.Neurofibromatosis type 1 is one of the most common autosomal dominant diseases (1/3500). Café-au-lait” macules (CALMs); overall skin hyperpigmentation, neurofibromas (benign tumors resulting from Schwann cell proliferation) and deficits cognitive are the mains clinical manifestations. NF1 is caused by mutations in a tumor suppressor gene that encodes neurofibromin, a functional RAS-GTPase-activating protein (RAS-GAP). Neurofibromin down-regulates RAS signaling by accelerating the conversion of active RAS-GTP to inactive RAS-GDP. The resulting decreased expression of neurofibromin leads to activation of several important downstream signaling pathways, including MEK/MAPK, AKT/mTOR and cAMP-mediated protein kinase A pathways. In order to better understand molecular mechanisms linking neurofibromin and associated manifestations in the pathology, mouse models have been established. However, these models don’t reproduce systematically cutaneous manifestations associated with NF1. To overcome this issue, we take advantage that pluripotent stem cells possess the capacity of self-renew and are able to differentiate into a wide variety of cell types. A growing number of examples illustrate how such cells, retrieved from genetically selected donors carrying the causal mutation of a monogenic disorder, may reproduce disease-associated phenotypes. Two human embryonic stem cell lines derived from embryos characterized as mutant-gene carriers for NF1 during a pre-implantation diagnosis procedure were differentiated into a pure and proliferative melanocytes, in order to explore mechanisms associated with hyperpigmentation and “café-au-lait” macules associated with NF1. In this study, we demonstrated that NF1 melanocytes reproduce the hyperpigmentation phenotype in vitro, and further characterized the link between loss of heterozygosity and the typical “café au lait” macules that appear over the general hyperpigmentation. Molecular mechanisms associated with these pathological phenotypes correlate with an increased activity of cyclic AMP-mediated protein kinase A and ERK1/2 signaling pathways, leading to increase of melanogenesis. Finally, the hyperpigmentation phenotype could be rescued by using specific inhibitors of these signaling pathways. In parallel, to consider a cell therapy approach from melanocytes derived from pluripotent cells for the treatment of hypopigmentation disorders related to the death or dysfunction of melanocytes such as Griscelli or Vitiligo syndromes, a differentiation protocol based on sequential application of molecules to specifically induce the differentiation of pluripotent stem cells into melanocytes was developed in the laboratory. Functional melanocytes were obtained in 30 days by the commitment of pluripotent stem cells into melanocytes precursors via neural crest state by the action of molecules such as BMP4, ascorbic acid, endothelin 3, SCF and an activator of Wnt3a (CHIR99021). All of this work has validated the appropriateness of the use of pluripotent stem cells carring genetic mutations or not to treat hyper or hypopigmentation disease by identified pharmoclogical treatment or by a cell therapy approach

Differentiation of nonhuman primate pluripotent stem cells into functional keratinocytes

Abstract 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

Pathological modelling of pigmentation disorders associated with Hutchinson-Gilford Progeria Syndrome (HGPS) revealed an impaired melanogenesis pathway in iPS-derived melanocytes

International audienceHutchinson-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

Generating Functional and Highly Proliferative Melanocytes Derived from Human Pluripotent Stem Cells: A Promising Tool for Biotherapeutic Approaches to Treat Skin Pigmentation Disorders

Melanocytes are essential for skin homeostasis and protection, and their loss or misfunction leads to a wide spectrum of diseases. Cell therapy utilizing autologous melanocytes has been used for years as an adjunct treatment for hypopigmentary disorders such as vitiligo. However, these approaches are hindered by the poor proliferative capacity of melanocytes obtained from skin biopsies. Recent advances in the field of human pluripotent stem cells have fueled the prospect of generating melanocytes. Here, we have developed a well-characterized method to produce a pure and homogenous population of functional and proliferative melanocytes. The genetic stability and potential transformation of melanocytes from pluripotent stem cells have been evaluated over time during the in vitro culture process. Thanks to transcriptomic analysis, the molecular signatures all along the differentiation protocol have been characterized, providing a solid basis for standardizing the protocol. Altogether, our results promise meaningful, broadly applicable, and longer-lasting advances for pigmentation disorders and open perspectives for innovative biotherapies for pigment disorders

Late cardiac adverse events in patients with cancer treated with immune checkpoint inhibitors

International audienceBackground Immune checkpoint inhibitor (ICI)-associated early cardiac adverse events (CAEs), mostly acute and fulminant myocarditis, have been well characterized and mainly occur during the first 90 days after ICI therapy initiation. ICI-associated late CAEs (occurring after the first 90 days of treatment) have not yet been described.Methods First, we compared characteristics of a cohort involving early (defined as a CAE time to onset (TTO) of 90 days after ICI therapy initiation) ICI-associated CAE consecutive cases who were referred to three French cardio-oncology units. Second, ICI-associated CAE cases were searched in VigiBase, the WHO global individual case safety report database, and early and late ICI-associated CAEs were compared.Results In the cohort study, compared with early CAE cases (n=19, median TTO of 14 days), late ICI-associated CAE cases (n=19, median TTO of 304 days) exhibited significantly more left ventricular systolic dysfunction (LVSD) and heart failure (HF) and less frequent supraventricular arrhythmias. In VigiBase, compared with early cases (n=437, 73.3%, median TTO 21 days), the late ICI-associated CAE reports (n=159, 26.7%, median TTO 178 days) had significantly more frequent HF (21.1% vs 31.4%, respectively, p=0.01). Early and late ICI-associated CAE cases had similarly high mortality rates (40.0% vs 44.4% in the cohort and 30.0% vs 27.0% in VigiBase, respectively).Conclusions Late CAEs could occur with ICI therapy and were mainly revealed to be HF with LVSD.Trial registration numbersNCT03678337, NCT03882580, and NCT03492528

DataSheet1_Generation of heterozygous and homozygous NF1 lines from human-induced pluripotent stem cells using CRISPR/Cas9 to investigate bone defects associated with neurofibromatosis type 1.pdf

Neurofibromatosis type 1 (NF1) is one of the most common genetic disorders caused by heterozygous germline NF1 mutations. NF1 affects many systems, including the skeletal system. To date, no curative therapies are available for skeletal manifestations such as scoliosis and tibial dysplasia, mainly due to the lack of knowledge about the mechanisms that underlie this process. By using CRISPR/Cas9-mediated gene editing in human-induced pluripotent stem cells (hiPSCs) to minimize the variability due to genetic background and epigenetic factors, we generated isogenic heterozygous and homozygous NF1-deficient hiPSC lines to investigate the consequences of neurofibromin inactivation on osteoblastic differentiation. Here, we demonstrate that loss of one or both copies of NF1 does not alter the potential of isogenic hiPSCs to differentiate into mesenchymal stem cells (hiPSC-MSCs). However, NF1 (+/−) and NF1 (−/−) hiPSC-MSCs show a defect in osteogenic differentiation and mineralization. In addition, we show that a mono-allelic deletion in NF1 in an isogenic context is sufficient to impair cell differentiation into osteoblasts. Overall, this study highlights the relevance of generating isogenic lines, which may help in genotype–phenotype correlation and provide a human cellular model to understand the molecular mechanisms underlying NF1 and, thus, discover new therapeutic strategies.</p

A UV-Independent Topical Small-Molecule Approach for Melanin Production in Human Skin

SUMMARY The presence of dark melanin (eumelanin) within human epidermis represents one of the strongest predictors of low skin cancer risk. Topical rescue of eumelanin synthesis, previously achieved in “redhaired” Mc1r-deficient mice, demonstrated significant protection against UV damage. However, application of a topical strategy for human skin pigmentation has not been achieved, largely due to the greater barrier function of human epidermis. Salt-inducible kinase (SIK) has been demonstrated to regulate MITF, the master regulator of pigment gene expression, through its effects on CRTC and CREB activity. Here, we describe the development of small-molecule SIK inhibitors that were optimized for human skin penetration, resulting in MITF upregulation and induction of melanogenesis. When topically applied, pigment production was induced in Mc1r-deficient mice and normal human skin. These findings demonstrate a realistic pathway toward UV-independent topical modulation of human skin pigmentation, potentially impacting UV protection and skin cancer risk

A UV-Independent Topical Small-Molecule Approach for Melanin Production in Human Skin

SUMMARY The presence of dark melanin (eumelanin) within human epidermis represents one of the strongest predictors of low skin cancer risk. Topical rescue of eumelanin synthesis, previously achieved in “redhaired” Mc1r-deficient mice, demonstrated significant protection against UV damage. However, application of a topical strategy for human skin pigmentation has not been achieved, largely due to the greater barrier function of human epidermis. Salt-inducible kinase (SIK) has been demonstrated to regulate MITF, the master regulator of pigment gene expression, through its effects on CRTC and CREB activity. Here, we describe the development of small-molecule SIK inhibitors that were optimized for human skin penetration, resulting in MITF upregulation and induction of melanogenesis. When topically applied, pigment production was induced in Mc1r-deficient mice and normal human skin. These findings demonstrate a realistic pathway toward UV-independent topical modulation of human skin pigmentation, potentially impacting UV protection and skin cancer risk