A. Vanelli

Culture Conditions and Signalling Networks Promoting the Establishment of Cell Lines from Parthenogenetic and Biparental Pig Embryos

The generation of porcine embryonic stem cells (pESC) would potentially have great impact in the biomedical field given the long-standing history of the pig as a prime animal model for pre-clinical biomedical applications. These cells would also be beneficial for the agricultural area, allowing efficient genetic engineering of this animal, to improve health and production traits. Despite numerous reports, no conclusive results have been obtained on the isolation and propagation of pESC lines and the establishment of pluripotent cells from the pig has remained an elusive goal. In the present study we performed a systematic analysis of different culture media for their ability to support the establishment of homogenous outgrowths from in vitro-produced embryos. Furthermore, we investigated which molecular networks are responsive to the factors contained in the most efficient media, since the identification of dominant signaling pathways that regulate porcine stem-cell pluripotency is likely to facilitate the generation of genuine pESC. Finally we compared IVF blastocysts versus parthenotes as a possible source for putative pESC in terms of blastocyst rate, resilience to immunosurgery procedures, ability to attach to the feeder, to generate outgrowths and to establish stable cell lines.

Large animal models for cardiac stem cell therapies

Cardiovascular disease is the leading cause of death in developed countries and is one of the leading causes of disease burden in developing countries. Therapies have markedly increased survival in several categories of patients, nonetheless mortality still remains high. For this reason high hopes are associated with recent developments in stem cell biology and regenerative medicine that promise to replace damaged or lost cardiac muscle with healthy tissue, and thus to dramatically improve the quality of life and survival in patients with various cardiomyopathies. Much of our insight into the molecular and cellular basis of cardiovascular biology comes from small animal models, particularly mice. However, significant differences exist with regard to several cardiac characteristics when mice are compared with humans. For this reason, large animal models like dog, sheep and pig have a well established role in cardiac research. A distinct characteristic of cardiac stem cells is that they can either be endogenous or derive from outside the heart itself; they can originate as the natural course of their differentiation programme (e.g., embryonic stem cells) or can be the result of specific inductive conditions (e.g., mesenchymal stem cells). In this review we will summarize the current knowledge on the kind of heart-related stem cells currently available in large animal species and their relevance to human studies as pre-clinical models.

Parthenogenesis in non-rodent species: developmental competence and differentiation plasticity

An oocyte can activate its developmental process without the intervention of the male counterpart. This form of reproduction, known as parthenogenesis, occurs spontaneously in a variety of lower organisms, but not in mammals. However, it must be noted that mammalian oocytes can be activated in vitro, mimicking the intracellular calcium wave induced by the spermatozoon at fertilization, which triggers cleavage divisions and embryonic development. The resultant parthenotes are not capable of developing to term and arrest their growth at different stages, depending on the species. It is believed that this arrest is due to genomic imprinting, which causes the repression of genes normally expressed by the paternal allele. Human parthenogenetic embryos have recently been proposed as an alternative, less controversial source of embryonic stem cell lines, based on their inherent inability to form a new individual. However many aspects related to the biology of parthenogenetic embryos and parthenogenetically derived cell lines still need to be elucidated. Limited information is available in particular on the consequences of the lack of centrioles and on the parthenotes ability to assemble a new embryonic centrosome in the absence of the sperm centriole. Indeed, in lower species, successful parthenogenesis largely depends upon the oocytes ability to regenerate complete and functional centrosomes in the absence of the material supplied by a male gamete, while the control of this event appears to be less stringent in mammalian cells. In an attempt to better elucidate some of these aspects, parthenogenetic cell lines, recently derived in our laboratory, have been characterized for their pluripotency. In vitro and in vivo differentiation plasticity have been assessed, demonstrating the ability of these cells to differentiate into cell types derived from the three germ layers. These results confirmed common features between uni- and bi-parental embryonic stem cells. However data obtained with parthenogenetic cells indicate the presence of an intrinsic deregulation of the mechanisms controlling proliferation vs. differentiation and suggest their uni-parental origin as a possible cause.

Isolation, Characterization and Differentiation Potential of Cardiac Progenitor Cells in Adult Pigs

IntroductionCardiovascular disease (CVD) remains as the first cause ofdeath worldwide [1, 2]. It is known that a rising heart failureincidence is associated with unhealthy life styles and in-creasing life expectance. Current therapies are typicallysymptomatic and, even though they provide some survivalbenefit, they cannot reverse the loss of contractile cardiactissue due to ischemic injury. For this reason, high expect-ances are associated to the recent developments in stem cellbiology and regenerative medicine that promise to replacedamaged or lost cardiac muscle with healthy tissue and thusto improve the quality of life and survival in patients withvarious cardiomyopathies [3].The recent identification of different classes of cardiacprogenitor cells suggests that the heart, classically considereda terminally differentiated, post-mitotic organ, may rathercontain a stem cell compartment, responsible for both tissueturn-over and regeneration, which follows acute or chronicdamage to the cardiac tissue [4–6]. Several groups havealready reported the isolation of different types of cardiacstem-like cells based on distinct cell surface markers, suchas c-kit, Isl-1 and Sca-1 [7–11] or the ability to generategenuine cardiomyocytes and integrate into heart tissue asinduced pluripotent stem cells [12], or also in relation to theircapacity to influence the neovascularization of the ischemictissue, as satellite cells [13]. These cells are able to restorecardiac function after ischemic injury, although with variableefficiency [7, 14]. Another type of cells associated withthe heart was mesoangioblast, a class of vessel-associatedstem cells that can differentiate into various mesoderm celltypes.Theywereoriginallydescribedinthemouseembryonicdorsal aorta [15] and later similar cells have been identifiedand characterized from postnatal small vessels of humanskeletal muscle [16] and mouse and human heart [4, 17].However, the experiments carried out until now werepredominantly performed on mice and human. This restrictssignificantly the possibility to apply the results obtained inpreclinical studies that cannot be performed using the hu-man as a model and, at the same time, are limited by theevident differences between mouse and humans, such astheir size, mice heart rate and their general anatomy [18,19]. In particular, human and mouse hearts diverge in thecoronary architecture, the variations of which are muchbigger in humans compared to mice. As a consequence,whereas the size and location of the ischemic area are fairlyconstant in the mouse, a much larger variation exists in thehuman [20]. Differences can also be appreciated at thecellular level, as indicated by the higher capillary densityand the larger cross sectional area of the myocytes in hu-man, in comparison to the mouse [21, 22]. Consequently,extrapolation of murine systems, particularly after inductionof cardiovascular stress, must be meticulously monitored,when applied clinically, because of the obvious differences