Maurizio Pesce

Information obtained mainly from in vitro culture studies and genetic analysis of mouse mutants White spotting and Steel indicate a pivotal role of growth factors in the development of mouse primordial germ cells (PGCs). While stem cell factor (SCF) and TGF beta 1 seem to have a role in PGC migration (as an adhesion factor and a chemoattractant, respectively), the former is certainly required for PGC survival in vitro and probably in vivo as well. Recent findings suggest that the mechanism by which SCF supports PGC survival is by preventing PGC apoptosis. A similar action appears to be exerted by leukemia inhibitory factor (LIF), a further growth factor influencing PGC growth in culture. PGC proliferation seems to be mainly induced by cAMP dependent mechanisms, but further investigations are needed to clarify the interrelationships among the different molecular pathways activated by SCF, LIF, cAMP and other putative PGC growth factors (i.e. bFGF). Stimulation of long-term proliferat...

The POU transcription factor Oct-4 is expressed in early mouse embryogenesis and in pluripotent ES and EC stem cell lines. After gastrulation in the embryo, Oct-4 expression is confined to the germline. The present study provides evidence that Oct-4 undergoes downregulation during oogenesis and spermatogenesis, coincident with entry into meiosis. Furthermore, analysis of maturation stages of oocytes showed that Oct-4 is upregulated de novo in the final stages of meiotic prophase I in female germ cells. These data suggest that Oct-4 downregulation in germ cells in both sexes might represent one of the molecular triggers involved in the commitment to meiosis. The upregulation of Oct-4 in oocytes at the completion of the prophase I of meiotic division further suggests a specific involvement of this transcription factor in oocyte growth or the acquisition of meiotic competence.

The aim of the present study was to develop and validate a good manufacturing practice (GMP) compliant procedure for the preparation of bone marrow (BM) derived CD133(+) cells for cardiovascular repair. Starting from available laboratory protocols to purify CD133(+) cells from human cord blood, we implemented these procedures in a GMP facility and applied quality control conditions defining purity, microbiological safety and vitality of CD133(+) cells. Validation of CD133(+) cells isolation and release process were performed according to a two-step experimental program comprising release quality checking (step 1) as well as 'proofs of principle' of their phenotypic integrity and biological function (step 2). This testing program was accomplished using in vitro culture assays and in vivo testing in an immunosuppressed mouse model of hindlimb ischemia. These criteria and procedures were successfully applied to GMP production of CD133(+) cells from the BM for an ongoing clinical trial of autologous stem cells administration into patients with ischemic cardiomyopathy. Our results show that GMP implementation of currently available protocols for CD133(+) cells selection is feasible and reproducible, and enables the production of cells having a full biological potential according to the most recent quality requirements by European Regulatory Agencies.

We report the case of a 60-year-old man with patent coronary by-pass grafts on the left anterior descending and circumflex coronary arteries, who experienced recurrent Canadian class IV angina refractory to medical and interventional treatments for a dominant right coronary artery occlusion. He underwent autologous PBSC transplant into the inferior ventricular wall through a minimally invasive approach as a stand-alone therapy, in an attempt to induce therapeutic angiogenesis. Six months after the operation, angina significantly ameliorated; scintigraphy and coronary angiography showed a marked improvement in perfusion of the target injection area. These benefits had persisted at 25 months after stem cell transplant.

The Oct-4 transcription factor is expressed in embryonic stem cells and germ cells of the mouse embryo. Cells that are driven into embryonic and extraembryonic somatic differentiation lose Oct-4 expression. Oct-4 is crucial for the maintenance of the totipotency of embryonic cells at early developmental stages as demonstrated recently by homologous recombination. Since during embryogenesis Oct-4 is restricted to germ cells, it is possible that it plays a key role for the germline lineage by preventing differentiation of these cells during gastrulation. Thus, Oct-4 appears to be a key regulator of totipotency during the life cycle of mammals.

The assessment of progenitor cell survival and efficacy after transplantation is one of the major challenges in cardiovascular cell therapy. Translation of currently used imaging techniques to patients is not immediate. Possible options include iron oxide particle loading into cells to be tracked using magnetic resonance (MR) and by MR-based water diffusion anisotropy analysis. The aim of the present study was to assess, using these techniques, the localization and survival of human 'early' endothelial progenitor cells (EPCs) and their effects on vascular and skeletal muscle regeneration in a mouse model of hind limb ischaemia. A paramagnetic iron oxide particle loading protocol of human peripheral blood-derived early EPCs was devised. The iron+ EPCs maintained their phenotype and in vitro functional activity. In addition, the presence of iron+ cells was observed by MR until 7 days after injection into a pharmacologically immunosuppressed mouse model of hind limb ischaemia. Immunohistochemistry with human major histocompatibility complex antibodies revealed the absence of human cells at 7 days post-ischaemia. EPC death was confirmed by staining of iron+ cells with an anti-mouse CD68 antibody and by qPCR performed on DNA extracted from injected ischaemic limbs, at different times following injection. Surprisingly, early EPC injection enhanced arteriogenesis but caused a significant increase in ischaemic tissue inflammation and a retarded muscle regeneration, as evidenced by water diffusion anisotropy analysis and histology. In line with recent reports, our results show that the use of iron-based contrast agents does not allow detection of long-term EPC engraftment into ischaemic tissues. They further show that early EPCs exert a potent arteriogenic effect on ischaemic tissues that is not dependent on their prolonged survival. Unexpectedly, injection of these cells elicited a long-term inflammatory response that reflected a delayed muscle healing process.

The vascular competence of human-derived hematopoietic progenitors for postnatal vascularization is still poorly characterized. It is unclear whether, in the absence of ischemia, hematopoietic progenitors participate in neovascularization and whether they play a role in new blood vessel formation by incorporating into developing vessels or by a paracrine action. In the present study, human cord blood-derived CD34(+) (hCD34(+)) cells were transplanted into pre- and postgastrulation zebrafish embryos and in an adult vascular regeneration model induced by caudal fin amputation. When injected before gastrulation, hCD34(+) cells cosegregated with the presumptive zebrafish hemangioblasts, characterized by Scl and Gata2 expression, in the anterior and posterior lateral mesoderm and were involved in early development of the embryonic vasculature. These morphogenetic events occurred without apparent lineage reprogramming, as shown by CD45 expression. When transplanted postgastrulation, hCD34(+) cells were recruited into developing vessels, where they exhibited a potent paracrine proangiogenic action. Finally, hCD34(+) cells rescued vascular defects induced by Vegf-c in vivo targeting and enhanced vascular repair in the zebrafish fin amputation model. These results indicate an unexpected developmental ability of human-derived hematopoietic progenitors and support the hypothesis of an evolutionary conservation of molecular pathways involved in endothelial progenitor differentiation in vivo.