Genetically modified mice have advanced our understanding of valve development and disease. Yet, human pathophysiological valvulogenesis remains poorly understood. Here we report that, by combining single cell sequencing and in vivo approaches, a population of human pre-valvular endocardial cells (HPVCs) can be derived from pluripotent stem cells. HPVCs express gene patterns conforming to the E9.0 mouse atrio-ventricular canal (AVC) endocardium signature. HPVCs treated with BMP2, cultured on mouse AVC cushions, or transplanted into the AVC of embryonic mouse hearts, undergo endothelial-to-mesenchymal transition and express markers of valve interstitial cells of different valvular layers, demonstrating cell specificity. Extending this model to patient-specific induced pluripotent stem cells recapitulates features of mitral valve prolapse and identified dysregulation of the SHH pathway. Concurrently increased ECM secretion can be rescued by SHH inhibition, thus providing a putative therapeutic target. In summary, we report a human cell model of valvulogenesis that faithfully recapitulates valve disease in a dish.

Genomic disorders resulting from large rearrangements of the genome remain an important unsolved issue in gene therapy. Chromosome transplantation, defined as the perfect replacement of an endogenous chromosome with a homologous one, has the potential of curing this kind of disorders. Here we report the first successful case of chromosome transplantation by replacement of an endogenous X chromosome carrying a mutation in the Hprt genewith a normal one in mouse embryonic stem cells (ESCs), correcting the genetic defect. The defect was also corrected by replacing the Y chromosome with an X chromosome. Chromosome transplanted clones maintained in vitro and in vivo features of stemness and contributed to chimera formation. Genome integrity was confirmed by cytogenetic and molecular genome analysis. The approach here proposed, with some modifications, might be used to cure various disorders due to other X chromosome aberrations in induced pluripotent stem (iPS) cells derived from affected patients.

Autosomal recessive osteopetrosis is a human bone disease mainly caused by TCIRG1 gene mutations that prevent osteoclasts resorbing activity, recapitulated by the oc/oc mouse model. Bone marrow transplantation is the only available treatment, limited by the need for a matched donor. The use of induced pluripotent stem cells (iPSCs) as an unlimited source of autologous cells to generate gene corrected osteoclasts might represent a powerful alternative. We generated iPSCs from oc/oc mice, corrected the mutation using a BAC carrying the entire Tcirg1 gene locus as a template for homologous recombination, and induced hematopoietic differentiation. Similarly to physiologic fetal hematopoiesis, iPSC-derived CD41+ cells gradually gave rise to CD45+ cells, which comprised both mature myeloid cells and high proliferative potential colony-forming cells. Finally, we differentiated the gene corrected iPSC-derived myeloid cells into osteoclasts with rescued bone resorbing activity. These results are promising for a future translation into the human clinical setting.

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Mitral valve disease is a frequent cause of heart failure and death. Emerging evidence indicates that the mitral valve is not a passive structure, but-even in adult life-remains dynamic and accessible for treatment. This concept motivates efforts to reduce the clinical progression of mitral valve disease through early detection and modification of underlying mechanisms. Discoveries of genetic mutations causing mitral valve elongation and prolapse have revealed that growth factor signalling and cell migration pathways are regulated by structural molecules in ways that can be modified to limit progression from developmental defects to valve degeneration with clinical complications. Mitral valve enlargement can determine left ventricular outflow tract obstruction in hypertrophic cardiomyopathy, and might be stimulated by potentially modifiable biological valvular-ventricular interactions. Mitral valve plasticity also allows adaptive growth in response to ventricular remodelling. However, adverse cellular and mechanobiological processes create relative leaflet deficiency in the ischaemic setting, leading to mitral regurgitation with increased heart failure and mortality. Our approach, which bridges clinicians and basic scientists, enables the correlation of observed disease with cellular and molecular mechanisms, leading to the discovery of new opportunities for improving the natural history of mitral valve disease.

Published clinical trials in patients with ischemic diseases show limited benefit of adult stem cell-based therapy, likely due to their restricted plasticity and commitment toward vascular cell lineage. We aim to uncover the potent regenerative ability of MesP1/stage-specific embryonic antigen 1 (SSEA-1)-expressing cardiovascular progenitors enriched from human embryonic stem cells (hESCs). Injection of only 10(4) hESC-derived SSEA-1(+) /MesP1(+) cells, or their progeny obtained after treatment with VEGF-A or PDGF-BB, was effective enough to enhance postischemic revascularization in immunodeficient mice with critical limb ischemia (CLI). However, the rate of incorporation of hESC-derived SSEA-1(+) /MesP1(+) cells and their derivatives in ischemic tissues was modest. Alternatively, these cells possessed a unique miR-21 signature that inhibited phosphotase and tensin homolog (PTEN) thereby activating HIF-1α and the systemic release of VEGF-A. Targeting miR-21 limited cell survival and inhibited their proangiogenic capacities both in the Matrigel model and in mice with CLI. We next assessed the impact of mR-21 in adult angiogenesis-promoting cells. We observed an impaired postischemic angiogenesis in miR-21-deficient mice. Notably, miR-21 was highly expressed in circulating and infiltrated monocytes where it targeted PTEN/HIF-1α/VEGF-A signaling and cell survival. As a result, miR-21-deficient mice displayed an impaired number of infiltrated monocytes and a defective angiogenic phenotype that could be partially restored by retransplantation of bone marrow-derived cells from wild-type littermates. hESC-derived SSEA-1(+) /MesP1(+) cells progenitor cells are powerful key integrators of therapeutic angiogenesis in ischemic milieu and miR-21 is instrumental in this process as well as in the orchestration of the biological activity of adult angiogenesis-promoting cells.

2,3,7,8-Tetrachlorodibenzo-para-dioxin (TCDD) causes abnormalities during heart development. Cardiomyocytes derived from embryonic stem (ES) cells are a robust model for the study of early cardiomyogenesis. Here, we evaluated the effects of TCDD at key stages during the differentiation of mouse ES cells into cardiomyocytes analysing: (i) the transcription of lineage differentiation (Brachyury, Nkx-2.5, Actc-1), cardiac-specific (Alpk3, cTnT, cTnI, cTnC) and detoxification phase I (Cyp1A1, Cyp1A2 and Cyp1B1) and phase II (Nqo1, Gsta1 and Ugt1a6) genes; (ii) the global gene expression; (iii) the ultrastructure of ES-derived cardiomyocytes; (iv) level of ATP production and (v) the immunolocalisation of sarcomeric α-actinin, β-myosin heavy chain and cTnT proteins. We show that TCDD affects the differentiation of ES cells into cardiomyocytes at several levels: (1) induces the expression of phase I genes; (2) down-regulates a group of heart-specific genes, some involved in the oxidative phosphorylation pathway; (3) reduces the efficiency of differentiation; (4) alters the arrangement of mitochondria, that show twisted and disrupted cristae, and of some sarcomeres, with misalignement or disarrangement of the myofibrillar organisation and (5) reduces ATP production. This study provides novel evidences that TCDD impairs cardiomyocyte differentiation. Sarcomeres and mitochondria could be a target for dioxin toxicity, their disruption representing a possible mechanism developing cardiac injury.

Regeneration of the heart after a stroke would be the best biologic response to restore its function. However, although this phenomenon occurs in primitive organisms, the regenerative potential is lost in mammals. Thus, the search for an appropriate cardiac progenitor with the potential to differentiate into a functional cardiomyocyte in vitro and in vivo has been the subject of intensive investigation. We summarize the cardiogenic transcriptional pathway that constitutes the molecular scaffold to drive pluripotent stem cells toward a cardiac progenitor fate. Then we overview the literature on derivation of cardiac progenitors from both embryos and stem cells.

Regenerative medicine deals with the possible use of stem cells to repair tissues damaged by aging and related diseases, including amyloidoses. In the latter case, the toxicity of the amyloid deposits can, in principle, question the possibility to graft specific tissues by undifferentiated cells. To assess whether stem cells are vulnerable to amyloid toxicity, we exposed, in culture, murine embryonic stem (ES) cells and haematopoietic progenitor (HP) cells to oligomers of the amyloidogenic peptide Aβ42 at concentrations previously shown to be cytotoxic to several other cell types. These stem cells did not display any sign of apoptosis and their survival, proliferation and differentiation were not affected by the oligomers although the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay revealed that ES, but not HP, cells displayed some impaired ability to reduce the tetrazole salts possibly as a result of transient oxidative stress. Our results support a remarkable resistance of the investigated stem cells against amyloids and hence their potential use in cell therapy of Alzheimer's disease and, possibly, other amyloid diseases.

Human Embryonic or pluripotent stem cells hold many promises in regenerative medicine. They also provide the scientific community with powerful models of early human development including cardiogenesis under normal or pathological (congenital and genetic diseases) situations. Furthermore their cardiac derivatives turn out to be very useful to study human cardiac electrophysiology, pharmacology or cardiac toxicology. The current overview provides the basic knowledge on developmental biology of the heart which can be applied to stem cell research to study early cardiogenesis. We summarize both the cardiogenic transcriptional network and the role of morphogens involved in early cardiogenesis. We review protocols of cardiac differentiation of pluripotent stem cells so far available. We finally discuss the translation of basic stem cell research into clinical applications.

As recently pointed out in the literature, Fourier transform infrared (FT-IR) spectroscopy is emerging as a powerful tool in stem cell research. In this work we characterized in situ by FT-IR microspectroscopy the differentiation of murine embryonic stem cells (ES) to monitor possible changes in the cell macromolecular content during the early stages of differentiation. Undifferentiated and differentiating cells at 4, 7, 9 and 14 days were measured. Data were analyzed by the principal component and subsequent linear discriminant analyses (PCA–LDA) that enabled us to segregate ES cell spectra into well separate clusters and to identify the most significant spectral changes. Important changes in the lipid (3050–2800 cm –1 ), protein (1700–1600 cm –1 ) and in the nucleic acid (1050–850 cm –1 ) absorption regions were observed between days 4 to 7 of differentiation, indicating the appearance – at day 7 – of the new phenotype into cardiomyocyte precursors. Also the presence of DNA/RNA hybrid bands (954 cm –1 and 899 cm –1 ) suggests that the transcriptional switch of the genome started at this stage of differentiation. Particularly noteworthy, we suggest that the 2936 cm –1 shoulder we observed could reflect methyl group vibrations thus allowing the detection of variations in methylation levels of the stem cell during differentiation. These infrared results were found to be in agreement with the biochemical characterization of these differentiating cells, underlying the great potential of FT-IR spectroscopy in stem cell research.

Cell therapy holds promise for tissue regeneration, including in individuals with advanced heart failure. However, treatment of heart disease with bone marrow cells and skeletal muscle progenitors has had only marginal positive benefits in clinical trials, perhaps because adult stem cells have limited plasticity. The identification, among human pluripotent stem cells, of early cardiovascular cell progenitors required for the development of the first cardiac lineage would shed light on human cardiogenesis and might pave the way for cell therapy for cardiac degenerative diseases. Here, we report the isolation of an early population of cardiovascular progenitors, characterized by expression of OCT4, stage-specific embryonic antigen 1 (SSEA-1), and mesoderm posterior 1 (MESP1), derived from human pluripotent stem cells treated with the cardiogenic morphogen BMP2. This progenitor population was multipotential and able to generate cardiomyocytes as well as smooth muscle and endothelial cells. When transplanted into the infarcted myocardium of immunosuppressed nonhuman primates, an SSEA-1(+) progenitor population derived from Rhesus embryonic stem cells differentiated into ventricular myocytes and reconstituted 20% of the scar tissue. Notably, primates transplanted with an unpurified population of cardiac-committed cells, which included SSEA-1(-) cells, developed teratomas in the scar tissue, whereas those transplanted with purified SSEA-1(+) cells did not. We therefore believe that the SSEA-1(+) progenitors that we have described here have the potential to be used in cardiac regenerative medicine.

Lionel Bonnevie, Patrick Bruneval, Chad Cowan, Julia Pouly, Shoukhrat Mitalipov, Elodie Gouadon, Patrice Binder, Albert Hagège, Michel Desnos, Jean-François Renaud, Philippe Menasché Michel Pucéat INSERM U633, Avenir program, Embryonic stem cells and cardiogenesis, 91058 Evry, France INSERM U633, University Paris Descartes, Paris, France 3 CNRS-UMR 8162, Université Paris-Sud, and Hôpital Marie Lannelongue, Le Plessis Robinson, France Institut de Pharmacologie Moléculaire et Cellulaire, CNRS, Nice, France Assistance Publique-Hôpitaux de Paris, Hôpital Européen Georges Pompidou, Department of Cardiology (AH and MD), Pathology (PB) and Cardiovascular Surgery (AB,JP,PM); Department of Cardiology, Hôpital d’Instruction des Armées Bégin, Saint-Mandé, France Stowers Medical Institute, Center for Regenerative Medicine and Technology, Cardiovascular Research Center, Boston, MA 8 Division of Reproductive Sciences, Oregon National Primate Research Center, Beaverton, OR, USA. 9 IMASSA Institut de médecine aérospatiale du service de santé des armées, Brétigny sur Orge, France. 10 University Montpellier II , France INSERM U970 To whom correspondence should be addressed: michel.puceat@inserm.fr (33) 1 60 87 89 23 FAX (33) 1 60 87 89 99 INSERM campus genopole 1, 4 rue Pierre Fontaine, EVRY 91058, France or philippe.menasche@egp.aphp.fr (33) 1 56 09 36 22 FAX (33) 1 56 09 32 61 Hopital European Georges Pompidou, 20 rue Leblanc 75015 Paris

As recently pointed out in the literature, Fourier transform infrared (FT-IR) spectroscopy is emerging as a powerful tool in stem cell research. In this work we characterizedin situby FT-IR microspectroscopy the differentiation of murine embryonic stem cells (ES) to monitor possible changes in the cell macromolecular content during the early stages of differentiation. Undifferentiated and differentiating cells at 4, 7, 9 and 14 days were measured. Data were analyzed by the principal component and subsequent linear discriminant analyses (PCA–LDA) that enabled us to segregate ES cell spectra into well separate clusters and to identify the most significant spectral changes. Important changes in the lipid (3050–2800 cm–1), protein (1700–1600 cm–1) and in the nucleic acid (1050–850 cm–1) absorption regions were observed between days 4 to 7 of differentiation, indicating the appearance – at day 7 – of the new phenotype into cardiomyocyte precursors. Also the presence of DNA/RNA hybrid bands (954 cm–1and 899 cm–1) suggests that the transcriptional switch of the genome started at this stage of differentiation. Particularly noteworthy, we suggest that the 2936 cm–1shoulder we observed could reflect methyl group vibrations thus allowing the detection of variations in methylation levels of the stem cell during differentiation. These infrared results were found to be in agreement with the biochemical characterization of these differentiating cells, underlying the great potential of FT-IR spectroscopy in stem cell research.

Human embryonic stem (HES) cells are pluripotent and give rise to any cell lineage. More specifically, how the first embryonic lineage (i.e., cardiac lineage) is acquired remains in many aspects questionable. Herein, we summarize the protocols that have been used to direct the fate of HES cells toward the cardiomyocytic lineage. We further discuss the regulation of transcriptional pathways underlying this process of differentiation. Finally, we propose perspectives of this research in the near future.