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

Hereditary erythrocytosis is a rare hematologic disorder characterized by an excess of red blood cell production. Here we describe a European collaborative study involving a collection of 2,160 patients with erythrocytosis sequenced in ten 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 hypoxia-inducible factor. We performed a comprehensive study to evaluate the causal role of the identified PHD2 variants: (i) in silico studies of localization, conservation, and deleterious effects; (ii) analysis of hematologic parameters of carriers identified in the UK Biobank; (iii) functional studies of the protein activity and stability; and (iv) a comprehensive study of PHD2 splicing. Altogether, these studies allowed the classification of 16 pathogenic or likely pathogenic mutants in a total of 48 patients and relatives. The in silico studies extended to the variants described in the literature showed that a minority of PHD2 variants can be classified as pathogenic (36/96), without any differences from the variants of unknown significance regarding the severity of the developed disease (hematologic parameters and complications). Here, we demonstrated the great value of federating laboratories working on such rare disorders in order to implement the criteria required for genetic classification, a strategy that should be extended to all hereditary hematologic diseases.

RRAD mutation causes electrical and cytoskeletal defects in cardiomyocytes derived from a familial case of Brugada syndrome.

AIMS: The Brugada syndrome (BrS) is an inherited cardiac disorder predisposing to ventricular arrhythmias. Despite considerable efforts, its genetic basis and cellular mechanisms remain largely unknown. The objective of this study was to identify a new susceptibility gene for BrS through familial investigation. METHODS AND RESULTS: Whole-exome sequencing performed in a three-generation pedigree with five affected members allowed the identification of one rare non-synonymous substitution (p.R211H) in RRAD, the gene encoding the RAD GTPase, carried by all affected members of the family. Three additional rare missense variants were found in 3/186 unrelated index cases. We detected higher levels of RRAD transcripts in subepicardium than in subendocardium in human heart, and in the right ventricle outflow tract compared to the other cardiac compartments in mice. The p.R211H variant was then subjected to electrophysiological and structural investigations in human cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs). Cardiomyocytes derived from induced pluripotent stem cells from two affected family members exhibited reduced action potential upstroke velocity, prolonged action potentials and increased incidence of early afterdepolarizations, with decreased Na+ peak current amplitude and increased Na+ persistent current amplitude, as well as abnormal distribution of actin and less focal adhesions, compared with intra-familial control iPSC-CMs Insertion of p.R211H-RRAD variant in control iPSCs by genome editing confirmed these results. In addition, iPSC-CMs from affected patients exhibited a decreased L-type Ca2+ current amplitude. CONCLUSION: This study identified a potential new BrS-susceptibility gene, RRAD. Cardiomyocytes derived from induced pluripotent stem cells expressing RRAD variant recapitulated single-cell electrophysiological features of BrS, including altered Na+ current, as well as cytoskeleton disturbances.

Generation of CD34+CD43+ Hematopoietic Progenitors to Induce Thymocytes from Human Pluripotent Stem Cells.

Immunotherapy using primary T cells has revolutionized medical care in some pathologies in recent years, but limitations associated to challenging cell genome edition, insufficient cell number production, the use of only autologous cells, and the lack of product standardization have limited its clinical use. The alternative use of T cells generated in vitro from human pluripotent stem cells (hPSCs) offers great advantages by providing a self-renewing source of T cells that can be readily genetically modified and facilitate the use of standardized universal off-the-shelf allogeneic cell products and rapid clinical access. However, despite their potential, a better understanding of the feasibility and functionality of T cells differentiated from hPSCs is necessary before moving into clinical settings. In this study, we generated human-induced pluripotent stem cells from T cells (T-iPSCs), allowing for the preservation of already recombined TCR, with the same properties as human embryonic stem cells (hESCs). Based on these cells, we differentiated, with high efficiency, hematopoietic progenitor stem cells (HPSCs) capable of self-renewal and differentiation into any cell blood type, in addition to DN3a thymic progenitors from several T-iPSC lines. In order to better comprehend the differentiation, we analyzed the transcriptomic profiles of the different cell types and demonstrated that HPSCs differentiated from hiPSCs had very similar profiles to cord blood hematopoietic stem cells (HSCs). Furthermore, differentiated T-cell progenitors had a similar profile to thymocytes at the DN3a stage of thymic lymphopoiesis. Therefore, utilizing this approach, we were able to regenerate precursors of therapeutic human T cells in order to potentially treat a wide range of diseases.

Generation of human induced pluripotent stem cell lines from three patients affected by Catecholaminergic Polymorphic ventricular tachycardia (CPVT) carrying heterozygous mutations in RYR2 gene.

Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) is an exercise and emotional stress-induced life-threatening inherited heart rhythm disorder, characterized by an abnormal cellular calcium homeostasis. Most reported cases have been linked to mutations in the gene encoding the type 2 ryanodine receptor gene, RYR2. We generated induced pluripotent stem cells (hiPSCs) from peripheral blood mononuclear cells (PBMC) from three CPVT-affected patients, two of them carrying p.R4959Q mutation and one carrying p.Y2476D mutation. These generated hiPSC lines are a useful model to study pathophysiological consequences of RYR2 dysfunction in humans and the molecular basis of CPVT.

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

AIMS: 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 (TF) 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 programme, 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 cardiac TF complex was newly identified, composed by IRX5 and GATA4, in which IRX5 potentiated GATA4-induction of SCN5A expression. CONCLUSION: 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.

Toward a better definition of hematopoietic progenitors suitable for B cell differentiation.

The success of inducing human pluripotent stem cells (hIPSC) offers new opportunities for cell-based therapy. Since B cells exert roles as effector and as regulator of immune responses in different clinical settings, we were interested in generating B cells from hIPSC. We differentiated human embryonic stem cells (hESC) and hIPSC into B cells onto OP9 and MS-5 stromal cells successively. We overcame issues in generating CD34+CD43+ hematopoietic progenitors with appropriate cytokine conditions and emphasized the difficulties to generate proper hematopoietic progenitors. We highlight CD31intCD45int phenotype as a possible marker of hematopoietic progenitors suitable for B cell differentiation. Defining precisely proper lymphoid progenitors will improve the study of their lineage commitment and the signals needed during the in vitro process.

Rapid and Reproducible Differentiation of Hematopoietic and T Cell Progenitors From Pluripotent Stem Cells.

Cell therapy using T cells has revolutionized medical care in recent years but limitations are associated with the difficulty of genome editing of the cells, the production of a sufficient number of cells and standardization of the product. Human pluripotent stem cells (hPSCs) can self-renew and differentiate into T cells to provide a standardized homogenous product of defined origin in indefinite quantity, therefore they are of great potential to alleviate limitations of therapeutic T cell production. The differentiation of hPSCs takes place in two steps: first the induction of hematopoietic stem/progenitor cells (HSPCs), then the induction of lymphopoiesis by Notch signaling. However, the differentiation of T cells from hPSCs can be difficult and lack reproducibility. One parameter that needs to be better assessed is the potential of DLL1 vs. DLL4 ligands of the Notch pathway to induce T cells. In addition, culture of hPSCs is labor-intensive and not compatible with GMP production, especially when they are cultured on feeder cells. Thus, the definition of a robust GMP-compatible differentiation protocol from hPSCs cultured in feeder-free conditions would increase the accessibility to off-the-shelf hematopoietic and T cell progenitors derived from hPSCs. In this article, we describe an efficient, rapid and reproducible protocol for the generation of hematopoietic and T cell progenitors in two steps: (1) generation of HSPCs from embryoid bodies (EB) in serum free medium and GMP-compatible feeder-free systems, (2) directed differentiation of hPSC-derived HSPCs into T-cell progenitors in the presence of bone marrow stromal cells expressing Notch-ligands OP9-DLL1 vs. OP9-DLL4.

A dominant vimentin variant causes a rare syndrome with premature aging.

Progeroid syndromes are a group of rare genetic disorders, which mimic natural aging. Unraveling the molecular defects in such conditions could impact our understanding of age-related syndromes such as Alzheimer's or cardiovascular diseases. Here we report a de novo heterozygous missense variant in the intermediate filament vimentin (c.1160 T > C; p.(Leu387Pro)) causing a multisystem disorder associated with frontonasal dysostosis and premature aging in a 39-year-old individual. Human vimentin p.(Leu387Pro) expression in zebrafish perturbed body fat distribution, and craniofacial and peripheral nervous system development. In addition, studies in patient-derived and transfected cells revealed that the variant affects vimentin turnover and its ability to form filaments in the absence of wild-type vimentin. Vimentin p.(Leu387Pro) expression diminished the amount of peripilin and reduced lipid accumulation in differentiating adipocytes, recapitulating key patient's features in vivo and in vitro. Our data highlight the function of vimentin during development and suggest its contribution to natural aging.

Induction of Human Trophoblast Stem Cells from Somatic Cells and Pluripotent Stem Cells.

Human 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.

Induced 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 mitochondrial 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.

Detection, isolation, and characterization of alpha-fetoprotein-specific T cell populations and clones using MHC class I multimer magnetic sorting.

alpha-fetoprotein (AFP) is a fetal protein specifically reexpressed in 50% of hepatocellular carcinomas. This protein could serve as a tumor-associated antigen for immunotherapy purpose. The aim of our work was to analyze the presence of AFP-specific T cell populations in peripheral blood mononuclear cells (PBMCs) from cirrhotic patients with or without hepatocellular carcinoma. Using peptide-major histocompatibility complex class I multimers, AFP-specific populations corresponding to 3 previously described human leukocyte antigen (HLA)-A*0201 major histocompatibility complex class I epitopes (AFP137, AFP158, and AFP325) were sorted magnetically from CD8 positive cells without prior stimulation with the target antigen. T cell populations specific for 1 peptide (AFP158) were frequent, whereas populations corresponding to peptide AFP137 were rare and absent for peptide AFP325. We also isolated and fully characterized T cell clones specific for AFP137 and AFP158 peptides. We show that these clones can be used to monitor dendritic cell loading with peptides and could be useful for future immunotherapy protocols.