Human pre-valvular endocardial cells derived from pluripotent stem cells recapitulate cardiac pathophysiological valvulogenesis.

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.

Mitral valve disease--morphology and mechanisms.

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.

Chromosome transplantation as a novel approach for correcting complex genomic disorders.

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.

Targeted Gene Correction in Osteopetrotic-Induced Pluripotent Stem Cells for the Generation of Functional Osteoclasts.

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.

MicroRNA-21 coordinates human multipotent cardiovascular progenitors therapeutic potential.

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.

The differentiation of cardiomyocytes from mouse embryonic stem cells is altered by dioxin.

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, ß-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.

Embryonic stem and haematopoietic progenitor cells resist to Aß oligomer toxicity and maintain the differentiation potency in culture.

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 Aß42 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.

Cardiac regeneration: still a 21st century challenge in search for cardiac progenitors from stem cells and embryos.

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.

Human embryonic and induced pluripotent stem cells in basic and clinical research in cardiology.

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.

A purified population of multipotent cardiovascular progenitors derived from primate pluripotent stem cells engrafts in postmyocardial infarcted nonhuman primates.

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.

Human embryonic stem cells and cardiac cell fate.

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.

Chromosome number variation in three mouse embryonic stem cell lines during culture.

Although mouse embryonic stem cell lines (mESCs) have been established since 1981, systematic studies about chromosomal changes during culture are lacking. In this study, we report the results of a cytogenetic analysis performed on three mESC lines (named UPV02, UPV06 and UPV08) cultured for a period of 3 months. At time intervals, the variation of the chromosome number together with the expression of markers of the undifferentiated status, i.e., OCT-4, SSEA-1, FOM-1 and alkaline phosphatase activity, were determined. The three mESC lines showed a progressive loss of euploid metaphases during the 3 months period of culture. Chromosome abnormalities were accumulated at the latest passages analysed. Metacentric chromosomes were the most frequent chromosome abnormality observed throughout the period of culture. Interestingly, in coincidence with, or few passages after, the drop of euploidy, the alkaline phosphatase activity was partially or totally lost, whereas the OCT-4, SSEA-1 and FOM-1 stem markers were always positive throughout the period of culture. Our results remark the necessity to perform the karyotype analysis during culture in order to develop new culture conditions to maintain the correct chromosome complement in long-term culture of mESC lines.

Expression of phase I and phase II genes in mouse embryonic stem cells cultured in the presence of 2,3,7,8-tetrachlorodibenzo-para-dioxin.

Embryonic stem (ES) cells have features that resemble the pluripotent cells of peri-implantation embryos and have been used as an in vitro model to assess the effects of test substances on these stages of development. Here, for the first time, we report on the effects of the xenobiotic 2,3,7,8-tetrachlorodibenzo-para-dioxin (TCDD) on mouse ES cells cultured with TCDD at concentrations ranging from 0.0001 to 100 nM for 15 min to 48 h. TCDD effects were determined by analysing the induction of Cyp1A1, Cyp1A2, Cyp1B1 (phase I) and Nqo1, Gsta1, Ugt1a6 (phase II) genes. Cyp1A1 was the phase I gene most rapidly induced (4 h at 1 nM); Cyp1B1 was induced at 48 h (1 nM), whereas Cyp1A2 expression was not affected. TCDD did not alter phase II gene expression, which remained at basal levels throughout the 48 h of culture. We studied more accurately the expression of Cyp1A1, the earliest gene to respond to the presence of TCDD. We found that: 1) Cyp1A1 gene induction is dependent on the duration of exposure (precisely it is first induced after 3 h of culture at 1 nM, the minimum effective-dose); 2) Cyp1A1 induction requires the continuous presence of TCDD, being interrupted 4 h after removal of the xenobiotic; and 3) induced expression of CYP1A1 protein is dependent on TCDD concentration, the higher the concentration the earlier the production of the enzyme. Furthermore, after 48 h of treatment, TCDD did not promote either apoptosis or changes to the differentiation status of the ES cells. These results are the first important step to investigate the effects of dioxin on the very early stages of mammalian development.

Embryonic stem cell differentiation studied by FT-IR spectroscopy.

We propose, here, an FT-IR method to monitor the spontaneous differentiation of murine embryonic stem (ES) cells in their early development. Principal component analysis and subsequent linear discriminant analysis enabled us to segregate stem cell spectra into separate clusters - corresponding to different differentiation times - and to identify the most significant spectral changes during differentiation. Between days 4 to 7 of differentiation, these spectral changes in the protein amide I band (1700-1600 cm(-1)) and in the nucleic acid absorption region (1050-850 cm(-1)) indicated that mRNA translation was taking place and that specific proteins were produced, reflecting the appearance of a new phenotype. The DNA/RNA hybrid bands (954 cm(-1) and 899 cm(-1)) were also observed, suggesting that the transcriptional switch of the genome started at this stage of differentiation. As confirmed by cytochemical assays, the FT-IR approach presented here allows to detect at molecular level the biological events of ES cell differentiation as they take place and to monitor in a rapid way the temporal evolution of the ES cell culture.

Stem cells.

The application of stem cells to regenerative medicine is one of the actual hot topics in biomedicine. This research could help the cure of a number of diseases that are affecting a large share of the population. Some good results in cell replacement have already been obtained (infarcted heart, diabetes, Parkinson disease), apart from those of more traditional applications like severe burns and blood tumors. We are now facing crucial questions in stem cell biology. One of the key questions is how a cell begins to proliferate or differentiate. Genome reprogramming, both following nuclear transfer and cytoplast action, will likely highlight some of the molecular mechanisms of cell differentiation and dedifferentiation. In turn, these clues should be useful to the production of populations of reprogrammed cells that could develop into tissues or, in the future, into proper organs. We will overview what stem cells are, what roles they play in normal developmental processes and how stem cells could have the potential to treat diseases.

Mouse fibroblasts are reprogrammed to Oct-4 and Rex-1 gene expression and alkaline phosphatase activity by embryonic stem cell extracts.

A recent remarkable study has shown that when mouse NIH-3T3 fibroblasts are exposed to an embryonic stem cell (ESC) extract, the majority of them expresses the Oct-4 gene, form ESC-like colonies, and embryoid-like bodies that differentiate into cells of the three germ layers. The use of cell extracts for inducing cell dedifferentiation could be a powerful system to obtain large quantities of pluripotent cells. It is thus of crucial importance that the robustness of this method of cell transdifferentiation is tested by other laboratories before it is advanced to a more ambitious use in cell therapy programs. We report here our experimental observations using the same reprogramming protocol on STO and NIH-3T3 mouse fibroblasts. Three are the main results: first, we confirmed an enduring reprogramming activity of the ESC extract, although on a much smaller number of cells that varies from approximately 0.003 to 0.04% of the total population of fibroblasts and with an effect limited to the induction of Oct-4 and Rex-1 gene expression and alkaline phosphatase activity. Second, the expression of OCT-4, SSEA-1, and Forssman antigen proteins was never detected. Third, our work has clearly demonstrated that ESCs may survive the procedure of extract preparation, may be source of contamination that is expanded in culture and give false positive results.