Lack of robust methods for establishment and expansion of pluripotent human embryonic stem (hES) cells still hampers development of cell therapy. Laminins (LN) are a family of highly cell-type specific basement membrane proteins important for cell adhesion, differentiation, migration and phenotype stability. Here we produce and isolate a human recombinant LN-521 isoform and develop a cell culture matrix containing LN-521 and E-cadherin, which both localize to stem cell niches in vivo. This matrix allows clonal derivation, clonal survival and long-term self-renewal of hES cells under completely chemically defined and xeno-free conditions without ROCK inhibitors. Neither LN-521 nor E-cadherin alone enable clonal survival of hES cells. The LN-521/E-cadherin matrix allows hES cell line derivation from blastocyst inner cell mass and single blastomere cells without a need to destroy the embryo. This method can facilitate the generation of hES cell lines for development of different cell types for regenerative medicine purposes.

The alginate hydrogel is an effective and promising injectable delivery system in a myocardial infarction model. Sequential growth factor delivery of VEGF-A(165) and PDGF-BB induces mature vessels and improves cardiac function more than each factor singly. This may indicate clinical utility.

Adult human mesenchymal stem cells did not induce xenoreactivity in vitro in previously unexposed immunocompetent Sprague-Dawley rats. However, although mesenchymal stem cells are transplantable across allogeneic barriers, transplant rejection can occur in a xenogenic model. When transplanted into an immunoincompetent host, adult human mesenchymal stem cells showed persistent engraftment.

A detailed analysis was performed of the immunomodulatory and proteomic profiles of mesenchymal stromal cells (MSCs) administered to two patients with refractory acute respiratory distress syndrome (ARDS). The respiratory, hemodynamic, and multiorgan failure resolved and multiple pulmonary and systemic inflammation markers decreased. These observations highlight the mechanistic information that can be gained from clinical experience and the value of correlating in vitro potency assessments with clinical effects.

Recent studies have suggested that human embryonic stem cells (HESC) are immune-privileged and may thereby circumvent rejection. The expression of immunologically active molecules was studied by DNA microarray analysis and by flow cytometry. HESC were transplanted into immunologically competent mice and traced by fluorescence in-situ hybridization (FISH) and immunohistochemistry. The ability of HESC to directly and indirectly induce immune responses in CD4+ T-cells from naive and transplanted mice was studied. Their ability to induce immune responses of human CD4+ T-cells, when cultured in the presence of dendritic cells (DC) syngeneic to responder T-cells, was also analysed. HESC demonstrated expression of HLA class I and HLA class II genes, but the cell surface expression of HLA class II molecules was low even after incubation with IFNgamma. In wild-type mice, HESC could be demonstrated by FISH until 3 days after transplantation and were surrounded by heavy infiltrates of T-cells and macrophages. HESC induced a similar immune response as human fibroblast cells (HFib) on naive and immunized T-cells, both directly and in the presence of syngeneic DC. A similar response was observed in the allogeneic setting. It is concluded that HESC are immunologically inert and do not inhibit immune responses during direct or indirect antigen presentation, and they were acutely rejected in a xenogeneic setting.

Artículo de publicación ISIRationale: The ability of a cell to independently regulate nuclear and cytosolic Ca2+ signaling is currently attributed to the differential distribution of inositol 1,4,5-trisphosphate receptor channel isoforms in the nucleoplasmic versus the endoplasmic reticulum. In cardiac myocytes, T-tubules confer the necessary compartmentation of Ca2+ signals, which allows sarcomere contraction in response to plasma membrane depolarization, but whether there is a similar structure tunneling extracellular stimulation to control nuclear Ca2+ signals locally has not been explored. Objective: To study the role of perinuclear sarcolemma in selective nuclear Ca2+ signaling. Methods and Results: We report here that insulin-like growth factor 1 triggers a fast and independent nuclear Ca2+ signal in neonatal rat cardiac myocytes, human embryonic cardiac myocytes, and adult rat cardiac myocytes. This fast and localized response is achieved by activation of insulin-like growth factor 1 receptor signaling complexes present in perinuclear invaginations of the plasma membrane. The perinuclear insulin-like growth factor 1 receptor pool connects extracellular stimulation to local activation of nuclear Ca2+ signaling and transcriptional upregulation through the perinuclear hydrolysis of phosphatidylinositol 4,5-biphosphate inositol 1,4,5-trisphosphate production, nuclear Ca2+ release, and activation of the transcription factor myocyte-enhancing factor 2C. Genetically engineered Ca2+ buffers—parvalbumin—with cytosolic or nuclear localization demonstrated that the nuclear Ca2+ handling system is physically and functionally segregated from the cytosolic Ca2+ signaling machinery. Conclusions: These data reveal the existence of an inositol 1,4,5-trisphosphate–dependent nuclear Ca2+ toolkit located in direct apposition to the cell surface, which allows the local control of rapid and independent activation of nuclear Ca2+ signaling in response to an extracellular ligan

The aim of this study was to longitudinally characterize the distribution of cells actively expressing the progenitor transcription factor islet-1 (Isl1+) during the embryonic life, the postnatal period, and adulthood. In this study, we have used direct immunohistochemical staining toward the protein Isl1 in a longitudinal rat model. Cells actively expressing Isl1 were traced in embryos from gestational day (GD) 11 until adulthood. In early cardiac development (GD 11), the Isl1+ progenitors were located in a greater abundance in the paracardiac regions, areas suggested to be the second heart field. To a lesser extent, Isl1+ cells were present within the bulbotruncal region and the truncus arteriosus. During the following days until GD 15, the Isl1+ cells were mainly observed at the proximal outflow tract (OFT) and at the inflow area of the right atrium. No Isl1+ cells were detected in the left ventricle. Compared with GD 11, more Isl1+ cells seemed to co-express cardiomyocyte markers and a minority of the Isl1+ cells was undifferentiated. Unexpectedly, only few undifferentiated Isl1+ cells were Ki67+ while a lot of TnT+ cardiomyocytes were proliferating in the ventricles. After birth, immature Isl1+ cells were still present in the OFT where they resided until adulthood. Our data suggest that during embryogenesis, Isl1+ cells migrate from extracardiac regions into the proximal part of the heart, proliferating and giving rise to cardioblasts. Unexpectedly, only a minority of the Isl1+ cells while a majority of ventricular cardiomyocytes were proliferating. The Isl1+ cell pool persists into adulthood, which might open up new strategies to repair damaged myocardium.

Mechanical isolation of the ICM proved to be an effective way to derive new hESC lines. The technique is fast, does not require any extra investment and the xeno-components of immunosurgery could be avoided.