NOTO Transcription Factor Directs Human Induced Pluripotent Stem Cell-Derived Mesendoderm Progenitors to a Notochordal Fate

International audienceThe founder cells of the Nucleus pulposus, the centre of the intervertebral disc, originate in the embryonic notochord. After birth, mature notochordal cells (NC) are identified as key regulators of disc homeostasis. Better understanding of their biology has great potential in delaying the onset of disc degeneration or as a regenerative-cell source for disc repair. Using human pluripotent stem cells, we developed a two-step method to generate a stable NC-like population with a distinct molecular signature. Time-course analysis of lineage-specific markers shows that WNT pathway activation and transfection of the notochord-related transcription factor NOTO are sufficient to induce high levels of mesendoderm progenitors and favour their commitment toward the notochordal lineage instead of paraxial and lateral mesodermal or endodermal lineages. This study results in the identification of NOTO-regulated genes including some that are found expressed in human healthy disc tissue and highlights NOTO function in coordinating the gene network to human notochord differentiation

TET3 controls the expression of the H3K27me3 demethylase Kdm6b during neural commitment

International audienceThe acquisition of cell identity is associated with developmentally regulated changes in the cellular histone methylation signatures. For instance, commitment to neural differentiation relies on the tightly controlled gain or loss of H3K27me3, a hallmark of polycomb-mediated transcriptional gene silencing, at specific gene sets. The KDM6B demethylase, which removes H3K27me3 marks at defined promoters and enhancers, is a key factor in neurogenesis. Therefore, to better understand the epigenetic regulation of neural fate acquisition, it is important to determine how Kdm6b expression is regulated. Here, we investigated the molecular mechanisms involved in the induction of Kdm6b expression upon neural commitment of mouse embryonic stem cells. We found that the increase in Kdm6b expression is linked to a rearrangement between two 3D configurations defined by the promoter contact with two different regions in the Kdm6b locus. This is associated with changes in 5-hydroxymethylcytosine (5hmC) levels at these two regions, and requires a functional ten-eleven-translocation (TET) 3 protein. Altogether, our data support a model whereby Kdm6b induction upon neural commitment relies on an intronic enhancer the activity of which is defined by its TET3-mediated 5-hmC level. This original observation reveals an unexpected interplay between the 5-hmC and H3K27me3 pathways during neural lineage commitment in mammals. It also questions to which extent KDM6B-mediated changes in H3K27me3 level account for the TET-mediated effects on gene expression

Generation of human induced pluripotent stem cell lines from four unrelated healthy control donors carrying European genetic background

International audienceFour human induced pluripotent stem cell (hiPSC) lines have been generated from healthy control European donors, and validated. This resource represents a useful tool for stem cell-based research, as references for developmental studies and disease modeling linked to any type of human tissue and organ, in an ethnical-, sex- and age-matched context. They providea reliable in-vitro model for single cell- and tissue-based investigations, and are also a valuable tool for genome editing-based studies

In vitro and in vivo models define a molecular signature reference for human embryonic notochordal cells

International audienceUnderstanding the emergence of human notochordal cells (NC) is essential for the development of regenerative approaches. We present a comprehensive investigation into the specification and generation of bona fide NC using a straightforward pluripotent stem cell (PSC)-based system benchmarked with human fetal notochord. By integrating in vitro and in vivo transcriptomic data at single-cell resolution, we establish an extended molecular signature and overcome the limitations associated with studying human notochordal lineage at early developmental stages. We show that TGF-β inhibition enhances the yield and homogeneity of notochordal lineage commitment in vitro. Furthermore, this study characterizes regulators of cell-fate decision and matrisome enriched in the notochordal niche. Importantly, we identify specific cell-surface markers opening avenues for differentiation refinement, NC purification, and functional studies. Altogether, this study provides a human notochord transcriptomic reference that will serve as a resource for notochord identification in human systems, diseased-tissues modeling, and facilitating future biomedical research

Human model of IRX5 mutations reveals key role for this transcription factor in ventricular conduction

International audienceAim: Several inherited arrhythmic diseases have been linked to single gene mutations in cardiac ion channels and interacting proteins. However, the mechanisms underlying most arrhythmias, are thought to involve altered regulation of the expression of multiple effectors. In this study, we aimed to examine the role of a transcription factor belonging to the Iroquois homeobox family, IRX5, in cardiac electrical function.Methods and results: Using human cardiac tissues, transcriptomic correlative analyses between IRX5 and genes involved in cardiac electrical activity showed that in human ventricular compartment, IRX5 expression strongly correlated to the expression of major actors of cardiac conduction, including the sodium channel, Nav1.5, and Connexin 40 (Cx40). We then generated human induced pluripotent stem cells (hiPSCs) derived from two Hamamy Syndrome-affected patients carrying distinct homozygous loss-of-function mutations in IRX5 gene. Cardiomyocytes derived from these hiPSCs showed impaired cardiac gene expression program, including misregulation in the control of Nav1.5 and Cx40 expression. In accordance with the prolonged QRS interval observed in Hamamy Syndrome patients, a slower ventricular action potential depolarization due to sodium current reduction was observed on electrophysiological analyses performed on patient-derived cardiomyocytes, confirming the functional role of IRX5 in electrical conduction. Finally, a novel cardiac transcription factor complex was identified, composed by IRX5 and GATA4, in which IRX5 potentiated GATA4-induction of SCN5A expression.Conclusions: Altogether, this work unveils a key role for IRX5 in the regulation of human ventricular depolarization and cardiac electrical conduction, providing therefore new insights into our understanding of cardiac diseases.Translational perspectives: Inherited cardiac arrhythmias account for about 20% of sudden cardiac deaths, of which a small portion are monogenic familial diseases with mutations in cardiac ion channels. However, pathogeny of inherited cardiac arrhythmias is increasingly thought to result from complex mechanisms involving altered regulation of multiple effectors expression. Taking advantage of cardiomyocytes derived from Hamamy syndrome patients, carrying loss-of-function mutations in IRX5 transcription factor, we uncovered an important role for IRX5 in the regulation of several major players of ventricular depolarization conduction and in arrhythmogenesis. Thus, this study supports systematic screening for genetic variants in IRX5 in inherited cardiac arrhythmias

A consistent arrhythmogenic trait in Brugada syndrome cellular phenotype

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Induction of human trophoblast stem cells from somatic cells and pluripotent stem cells

International audienceHuman trophoblast stem cells (hTSCs) derived from blastocysts and first-trimester cytotrophoblasts offer an unprecedented opportunity to study the placenta. However, access to human embryos and first-trimester placentas is limited, thus preventing the establishment of hTSCs from diverse genetic backgrounds associated with placental disorders. Here, we show that hTSCs can be generated from numerous genetic backgrounds using post-natal cells via two alternative methods: (1) somatic cell reprogramming of adult fibroblasts with OCT4, SOX2, KLF4, MYC (OSKM) and (2) cell fate conversion of naive and extended pluripotent stem cells. The resulting induced/converted hTSCs recapitulated hallmarks of hTSCs including long-term self-renewal, expression of specific transcription factors, transcriptomic signature, and the potential to differentiate into syncytiotrophoblast and extravillous trophoblast cells. We also clarified the developmental stage of hTSCs and show that these cells resemble day 8 cytotrophoblasts. Altogether, hTSC lines of diverse genetic origins open the possibility to model both placental development and diseases in a dish

Parallel derivation of isogenic human primed and naive induced pluripotent stem cells

International audienceInduced pluripotent stem cells (iPSCs) have considerably impacted human developmental biology and regenerative medicine, notably because they circumvent the use of cells of embryonic origin and offer the potential to generate patient-specific pluripotent stem cells. However, conventional reprogramming protocols produce developmentally advanced, or primed, human iPSCs (hiPSCs), restricting their use to post-implantation human development modeling. Hence, there is a need for hiPSCs resembling preimplantation naive epiblast. Here, we develop a method to generate naive hiPSCs directly from somatic cells, using OKMS overexpression and specific culture conditions, further enabling parallel generation of their isogenic primed counterparts. We benchmark naive hiPSCs against human preimplantation epiblast and reveal remarkable concordance in their transcriptome, dependency on mito-chondrial respiration and X-chromosome status. Collectively, our results are essential for the understanding of pluripotency regulation throughout preimplantation development and generate new opportunities for disease modeling and regenerative medicine

Characterization of genetic variants in the EGLN1/PHD2 gene identified in a European collection of patients with erythrocytosis

: Hereditary erythrocytosis (HE) is a rare hematological disorder characterized by an excess of red blood cell production. Here we describe a European collaborative study involving a collection of 2160 patients with erythrocytosis sequenced in 10 different laboratories. We focused our study on the EGLN1 gene and identified 39 germline missense variants including one gene deletion in 47 probands. EGLN1 encodes the PHD2 prolyl 4-hydroxylase, a major inhibitor of the Hypoxia-Inducible Factor. We performed a comprehensive study to evaluate the causal role of the identified PHD2 variants: in silico study of localization, conservation, and deleterious effects; analysis of hematological parameters of carriers identified in the UK Biobank; functional studies of the protein activity and stability; and comprehensive study of PHD2 splicing. Altogether, this study allowed the classification of 16 pathogenic or likely pathogenic mutants in a total of 48 patients and relatives. The in silico studies extented to the variants described in the literature showed that a minority of PHD2 variants can be classified as pathogenic (36/96), without any differences with the variants of unknown significance regarding the severity of the developed disease (hematological parameters and complications). Here, we demonstrated the great value of federating laboratories working on such rare pathology to implement the criteria required for genetic classification, a strategy that should be extended to all hereditary hematological diseases

A dominant vimentin variant causes a rare syndrome with premature aging

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