Generation of CD34 + CD43 + hematopoietic progenitors to induce thymocytes from human pluripotent stem cells

ABSTRACT 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 lack of product standardization have limited its uses in the clinic. 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 clinic access. However, despite their potential, the feasibility and functionality of T-cells differentiated from hPSCs needs better comprehension before moving to the clinic. In this study, we generated human induced pluripotent stem cells from T-cells (T-iPSCs) allowing 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, and then DN3a thymic progenitors from several T-iPSC lines. To better comprehend differentiation, we analyzed the transcriptomic profiles of the different cell types and demonstrated that HPSCs differentiated from hiPSCs had a very similar profile 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, with this approach, we were able to regenerate precursors of therapeutic human T cells to potentially treat a wide number of diseases

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

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

Regulatory B cell generation from human Induced Pluripotent Stem Cells with a repressible granzyme B expression

International audienceKidney transplanted patients with long term graft survival harbor a higher frequency of B lymphocytes with regulatory properties. These “regulatory” B lymphocytes prevent effector T cell proliferation through the Granzyme B (GzmB) molecule. Our aim was to generate regulatory B lymphocytes from human Induced Pluripotent Stem (hIPS) cells with a repressible GzmB expression as a tool to better understand their function and ultimately to translate them toward clinical use

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

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

A consistent arrhythmogenic trait in Brugada syndrome cellular phenotype

International audienc

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

International audienceAims : 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

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