We describe the derivation of pluripotent embryonic stem (ES) cells from human blastocysts. Two diploid ES cell lines have been cultivated in vitro for extended periods while maintaining expression of markers characteristic of pluripotent primate cells. Human ES cells express the transcription factor Oct-4, essential for development of pluripotential cells in the mouse. When grafted into SCID mice, both lines give rise to teratomas containing derivatives of all three embryonic germ layers. Both cell lines differentiate in vitro into extraembryonic and somatic cell lineages. Neural progenitor cells may be isolated from differentiating ES cell cultures and induced to form mature neurons. Embryonic stem cells provide a model to study early human embryology, an investigational tool for discovery of novel growth factors and medicines, and a potential source of cells for use in transplantation therapy.

Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro.

The earliest stages of development in most animals, including the few mammalian species that have been investigated, are regulated by maternally inherited information. Dependence on expression of the embryonic genome cannot be detected until the mid two-cell stage in the mouse, the four-cell stage in the pig (J. Osborn & C. Polge, personal communication), and the eight-cell stage in the sheep. Information about the timing of activation of the embryonic genome in the human is of relevance not only to the therapeutic practice of in vitro fertilization and embryo transfer (IVF), but more importantly for the successful development of techniques for the preimplantation diagnosis of certain inherited genetic diseases. We describe here changes in the pattern of polypeptides synthesized during the pre-implantation stages of human development, and demonstrate that some of the major qualitative changes which occur between the four- and eight-cell stages are dependent on transcription. In addition, it appears that cleavage is not sensitive to transcriptional inhibition until after the four-cell stage.

Human gene expression first occurs between the four- and eight-cell stages of preimplantation development

The derivation of neural progenitor cells from human embryonic stem (ES) cells is of value both in the study of early human neurogenesis and in the creation of an unlimited source of donor cells for neural transplantation therapy. Here we report the generation of enriched and expandable preparations of proliferating neural progenitors from human ES cells. The neural progenitors could differentiate in vitro into the three neural lineages--astrocytes, oligodendrocytes, and mature neurons. When human neural progenitors were transplanted into the ventricles of newborn mouse brains, they incorporated in large numbers into the host brain parenchyma, demonstrated widespread distribution, and differentiated into progeny of the three neural lineages. The transplanted cells migrated along established brain migratory tracks in the host brain and differentiated in a region-specific manner, indicating that they could respond to local cues and participate in the processes of host brain development. Our observations set the stage for future developments that may allow the use of human ES cells for the treatment of neurological disorders.

Neural progenitors from human embryonic stem cells.

Oxidative stress is involved in the aetiology of defective embryo development. Reactive oxygen species (ROS) may originate from embryo metabolism and/or embryo surroundings. Embryo metabolism generates ROS via several enzymatic mechanisms. The relative contribution of each source seems different depending on the species, the stage of development, and the culture conditions. Several exogenous factors and culture conditions can enhance the production of ROS by embryos. ROS can alter most types of cellular molecules, and also induce development block and retardation. Multiple mechanisms of embryo protection against ROS exist, and these have complementary actions. External protection, present in follicular and tubal fluids, mainly comprises non-enzymatic antioxidants such as hypotaurine, taurine and ascorbic acid. Internal protection mainly comprises antioxidant enzymes: superoxide dismutase, glutathione peroxidase and gamma-glutamylcysteine synthetase. Transcripts encoding for these enzymes are present in the oocyte, embryo and oviduct. It may be important that these transcripts are stored during oocyte maturation in order to allow the embryo to acquire the aptitude to develop. It is now common to add antioxidant compounds to culture media. Nevertheless, maintaining the pro-oxidant-antioxidant equilibrium in embryos through such supplementation is a complex problem. Further studies are necessary to limit oxidative stress during embryo culture.

Oxidative stress and protection against reactive oxygen species in the pre-implantation embryo and its surroundings

Pluripotent stem cells provide a potential solution to current epidemic rates of heart failure by providing human cardiomyocytes to support heart regeneration. Studies of human embryonic-stem-cell-derived cardiomyocytes (hESC-CMs) in small-animal models have shown favourable effects of this treatment. However, it remains unknown whether clinical-scale hESC-CM transplantation is feasible, safe or can provide sufficient myocardial regeneration. Here we show that hESC-CMs can be produced at a clinical scale (more than one billion cells per batch) and cryopreserved with good viability. Using a non-human primate model of myocardial ischaemia followed by reperfusion, we show that cryopreservation and intra-myocardial delivery of one billion hESC-CMs generates extensive remuscularization of the infarcted heart. The hESC-CMs showed progressive but incomplete maturation over a 3-month period. Grafts were perfused by host vasculature, and electromechanical junctions between graft and host myocytes were present within 2 weeks of engraftment. Importantly, grafts showed regular calcium transients that were synchronized to the host electrocardiogram, indicating electromechanical coupling. In contrast to small-animal models, non-fatal ventricular arrhythmias were observed in hESC-CM-engrafted primates. Thus, hESC-CMs can remuscularize substantial amounts of the infarcted monkey heart. Comparable remuscularization of a human heart should be possible, but potential arrhythmic complications need to be overcome.

/pdf/human-embryonic-stem-cell-derived-cardiomyocytes-regenerate-3ebyaku6ri.pdf

Human embryonic-stem-cell-derived cardiomyocytes regenerate non-human primate hearts

Ovarian failure is the most common cause of infertility. Although numerous strategies have been proposed, a definitive solution for recovering ovarian functions and restoring fertility is currently unavailable. One innovative alternative may be represented by the development of an “artificial ovary” that could be transplanted in patients for re-establishing reproductive activities. Here, we describe a novel approach for successful repopulation of decellularized ovarian bioscaffolds in vitro. Porcine whole ovaries were subjected to a decellularization protocol that removed the cell compartment, while maintaining the macrostructure and microstructure of the original tissue. The obtained bioscaffolds were then repopulated with porcine ovarian cells or with epigenetically erased porcine and human dermal fibroblasts. The results obtained demonstrated that the decellularized extracellular matrix (ECM)-based scaffold may constitute a suitable niche for ex vivo culture of ovarian cells. Furthermore, it was able to properly drive epigenetically erased cell differentiation, fate, and viability. Overall, the method described represents a powerful tool for the in vitro creation of a bioengineered ovary that may constitute a promising solution for hormone and fertility restoration. In addition, it allows for the creation of a suitable 3D platform with useful applications both in toxicological and transplantation studies.

Ovarian Decellularized Bioscaffolds Provide an Optimal Microenvironment for Cell Growth and Differentiation In Vitro

In the presence of different environmental cues that are able to trigger specific responses, a given genotype has the ability to originate a variety of different phenotypes. This property is defined as plasticity and allows cell fate definition and tissue specialization. Fundamental epigenetic mechanisms drive these modifications in gene expression and include DNA methylation, histone modifications, chromatin remodeling, and microRNAs. Understanding these mechanisms can provide powerful tools to switch cell phenotype and implement cell therapy. Environmentally influenced epigenetic changes have also been associated to many diseases such as cancer and neurodegenerative disorders, with patients that do not respond, or only poorly respond, to conventional therapy. It is clear that disorders based on an individual’s personal genomic/epigenomic profile can rarely be successfully treated with standard therapies due to genetic heterogeneity and epigenetic alterations and a personalized medicine approach is far more appropriate to manage these patients. We here discuss the recent advances in small molecule approaches for personalized medicine, drug targeting, and generation of new cells for medical application. We also provide prospective views of the possibility to directly convert one cell type into another, in a safe and robust way, for cell-based clinical trials and regenerative medicine.

/pdf/the-quest-for-an-effective-and-safe-personalized-cell-4tjlvy0lcs.pdf

The quest for an effective and safe personalized cell therapy using epigenetic tools

The effect of modifications of the extracellular matrix on the biosynthesis of glycosaminoglycans was investigated in human skin fibroblast cultures by studying UDPGDH activity in order to evaluate: a), the histoenzymological and biochemical modifications induced by chondroitinase ABC treatment (new experimental conditions were developed in order to obtain minimum cell damage); b), the reversibility of these modifications; c), the effect of growing the cells in the presence of chondroitinsulfate; d), the specificity of the modifications induced. The results demonstrated that our experimental conditions specifically affected intracellular UDPGDH activity. Chondroitinase ABC treatment induced a reversible increase of UDP-glucuronic acid synthesis. On the contrary, the presence of chondroitinsulfate in the growth medium completely inhibited UDPGDH activity.

The effect of extracellular matrix modifications on UDP-glucose dehydrogenase activity in cultured human skin fibroblasts.

Different cell types have been suggested as candidates for use in regenerative medicine. Embryonic pluripotent stem cells can give rise to all cells of the body and possess unlimited self-renewal potential. However, they are unstable, difficult to control and have a risk of neoplastic transformation. Adult stem cells are safe but have limited proliferation and differentiation abilities and are usually not within easy access. In recent years, induced pluripotent stem (iPS) cells have become a new promising tool in regenerative medicine. However, the use of transgene vectors, commonly required for the induction of iPS cells, seriously limits their use in therapy. The same problem arising from the use of retroviruses is associated with the use of cells obtained through transdifferentiation. Developing knowledge of the mechanisms controlling epigenetic regulation of cell fate has boosted the use of epigenetic modifiers that drive cells into a ‘highly permissive’ state. We recently set up a new strategy for the conversion of an adult mature cell into another cell type. We increased cell plasticity using 5-aza-cytidine and took advantage of a brief window of epigenetic instability to redirect cells to a different lineage. This approach is termed ‘epigenetic conversion’. It is a simple, direct and safe way to obtain both cells for therapy avoiding gene transfection and a stable pluripotent state.

/pdf/phenotype-switching-through-epigenetic-conversion-2gkiuvpb7k.pdf

Phenotype switching through epigenetic conversion

Due to logistical and economical reasons assisted reproduction of domestic animals has been based mostly on the use of oocytes isolated from ovaries collected at the slaughterhouse. In order to propagate valuable or rare genetic material, perform somatic cell nuclear transfer, generate genetically modified animals it was essential to obtain fully competent oocytes that would allow full term development of the in vitro produced embryos. Such demanding need has soon made clearly evident the crucial role played by oocyte quality, how easy it is to compromise its developmental potential and the fact that it is impossible to restore it once it has been lost. Almost three decades after the first bovine, sheep, goat, horse and pig in vitro generated offsprings were born, a large body of information has accumulated on the mechanisms regulating oocyte competence and on how the latter may be preserved during all the required manipulations. The amount of knowledge is far from being complete and many laboratories are actively working to further expand it. In this review we will highlight the aspects of the ongoing research in which we have been actively involved.

Fulvio Gandolfi

Papers

Ovarian Decellularized Bioscaffolds Provide an Optimal Microenvironment for Cell Growth and Differentiation In Vitro

The quest for an effective and safe personalized cell therapy using epigenetic tools

The effect of extracellular matrix modifications on UDP-glucose dehydrogenase activity in cultured human skin fibroblasts.

Phenotype switching through epigenetic conversion

020. in vitro maturation of farm animals oocytes: a useful tool for investigating the mechanisms leading to full term development