Grafted human induced pluripotent stem cells improve the outcome of spinal cord injury: modulation of the lesion microenvironment.

Spinal cord injury results in irreversible tissue damage followed by a very limited recovery of function. In this study we investigated whether transplantation of undifferentiated human induced pluripotent stem cells (hiPSCs) into the injured rat spinal cord is able to induce morphological and functional improvement. hiPSCs were grafted intraspinally or intravenously one week after a thoracic (T11) spinal cord contusion injury performed in Fischer 344 rats.

Genotypic and phenotypic spectrum of the most common causative genes of Charcot-Marie-Tooth disease in Hungarian patients.

Charcot-Marie-Tooth neuropathy (CMT) is a genetically and clinically heterogeneous group of neuromuscular disorders with an overall prevalence of 1 per 2500. Here we report the first comprehensive genetic epidemiology study of Hungarian CMT patients. 409 CMT1 and 122 CMT2 patients were enrolled and genetic testing of PMP22, GJB1, MPZ, EGR2 and MFN2 genes were performed routinely.

Establishment of an induced pluripotent stem cell (iPSC) line from a 9-year old male with autism spectrum disorder (ASD).

Peripheral blood mononuclear cells (PBMCs) were collected from a clinically characterized patient with autism spectrum disorder (ASD). The PMBCs were reprogrammed with the human OSKM transcription factors using the Sendai-virus delivery system. The pluripotency of transgene-free iPSCs was verified by immunocytochemistry for pluripotency markers and by spontaneous in vitro differentiation towards the 3 germ layers.

Establishment of a rabbit induced pluripotent stem cell (RbiPSC) line using lentiviral delivery of human pluripotency factors.

Rabbit Embryonic Fibroblast (RbEF) cells (from Hycole hybrid rabbit foetus) were reprogrammed by lentiviral delivery of a self-silencing hOKSM polycistronic vector. The pluripotency of the newly generated RbiPSC was verified by the expression of pluripotency-associated markers and by in vitro spontaneous differentiation towards the 3 germ layers. Furthermore, the spontaneous differentiation potential of the iPSC was also tested in vivo by teratoma assay.

Generation of Mucopolysaccharidosis type II (MPS II) human induced pluripotent stem cell (iPSC) line from a 3-year-old male with pathogenic IDS mutation.

Peripheral blood was collected from a 3-year-old male patient with an X-linked recessive mutation of Iduronate 2-sulfatase (IDS) gene (NM_000202.7(IDS):c.85C>T) causing MPS II (OMIM 309900).

Establishment of induced pluripotent stem cell (iPSC) line from a 63-year old patient with late onset Alzheimer's disease (LOAD).

Peripheral blood mononuclear cells (PBMCs) were collected from a clinically characterised 63-year old woman with late onset Alzheimer's disease (LOAD). The PMBCs were reprogrammed with the human OSKM transcription factors using the Sendai-virus delivery system. The transgene-free iPSC showed pluripotency verified by immunocytochemistry for pluripotency markers and differentiated spontaneously towards the 3 germ layers in vitro.

Establishment of induced pluripotent stem cell (iPSC) line from a 57-year old patient with sporadic Alzheimer's disease.

Peripheral blood mononuclear cells (PBMCs) were collected from a clinically characterised 57-year old woman with sporadic Alzheimer's disease. The PMBCs were reprogrammed with the human OSKM transcription factors using the Sendai-virus delivery system. The transgene-free iPSC showed pluripotency verified by immunocytochemistry for pluripotency markers and differentiated spontaneously towards the 3 germ layers in vitro.

Neurosphere Based Differentiation of Human iPSC Improves Astrocyte Differentiation.

Neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (iPSCs) are traditionally maintained and proliferated utilizing two-dimensional (2D) adherent monolayer culture systems. However, NPCs cultured using this system hardly reflect the intrinsic spatial development of brain tissue. In this study, we determined that culturing iPSC-derived NPCs as three-dimensional (3D) floating neurospheres resulted in increased expression of the neural progenitor cell (NPC) markers, PAX6 and NESTIN.

Cloning and characterization of rabbit POU5F1, SOX2, KLF4, C-MYC and NANOG pluripotency-associated genes.

While the rabbit (Oryctolagus cuniculus) is an important research model for aspects of human development and disease that cannot be studied in rodents, the lack of data on the genetic regulation of rabbit preimplantation development is a limitation. To assist in the understanding of this process, our aim was to isolate and characterize genes necessary for the induction and maintenance of cellular pluripotency. We are the first to report the isolation of complete coding regions of rabbit SOX2, KLF4, C-MYC and NANOG, which encode transcription factors that play crucial regulatory roles during early mammalian embryonic development.

Grafted murine induced pluripotent stem cells prevent death of injured rat motoneurons otherwise destined to die.

Human plexus injuries often include the avulsion of one or more ventral roots, resulting in debilitating conditions. In this study the effects of undifferentiated murine iPSCs on damaged motoneurons were investigated following avulsion of the lumbar 4 (L4) ventral root, an injury known to induce the death of the majority of the affected motoneurons. Avulsion and reimplantation of the L4 ventral root (AR procedure) were accompanied by the transplantation of murine iPSCs into the injured spinal cord segment in rats.

Screening of bioactive peptides using an embryonic stem cell-based neurodifferentiation assay.

Differentiation of pluripotent stem cells, PSCs, towards neural lineages has attracted significant attention, given the potential use of such cells for in vitro studies and for regenerative medicine. The present experiments were designed to identify bioactive peptides which direct PSC differentiation towards neural cells. Fifteen peptides were designed based on NCAM, FGFR, and growth factors sequences.

Generation of mouse induced pluripotent stem cells by protein transduction.

Somatic cell reprogramming has generated enormous interest after the first report by Yamanaka and his coworkers in 2006 on the generation of induced pluripotent stem cells (iPSCs) from mouse fibroblasts. Here we report the generation of stable iPSCs from mouse fibroblasts by recombinant protein transduction (Klf4, Oct4, Sox2, and c-Myc), a procedure designed to circumvent the risks caused by integration of exogenous sequences in the target cell genome associated with gene delivery systems. The recombinant proteins were fused in the frame to the glutathione-S-transferase tag for affinity purification and to the transactivator transcription-nuclear localization signal polypeptide to facilitate membrane penetration and nuclear localization.

Generation of induced pluripotent stem cells from human foetal fibroblasts using the Sleeping Beauty transposon gene delivery system.

Transposon gene delivery systems offer an alternative, non-viral-based approach to generate induced pluripotent stem cells (iPSCs). Here we used the Sleeping Beauty (SB) transposon to generate four human iPSC lines from foetal fibroblasts. In contrast to other gene delivery systems, the SB transposon does not exhibit an integration bias towards particular genetic elements, thereby reducing the risk of insertional mutagenesis.

Enhanced cardiac differentiation of mouse embryonic stem cells by use of the slow-turning, lateral vessel (STLV) bioreactor.

Embryoid body (EB) formation is a common intermediate during in vitro differentiation of pluripotent stem cells into specialized cell types. We have optimized the slow-turning, lateral vessel (STLV) for large scale and homogenous EB production from mouse embryonic stem cells. The effects of inoculating different cell numbers, time of EB adherence to gelatin-coated dishes, and rotation speed for optimal EB formation and cardiac differentiation were investigated.

Plasticity of polyubiquitin recognition as lysosomal targeting signals by the endosomal sorting machinery.

Lysosomal targeting is fundamental for the regulated disposal of ubiquitinated membrane proteins from the cell surface. To elucidate ubiquitin (Ub) configurations that are necessary and sufficient as multivesicular body (MVB)/lysosomal-sorting motifs, the intraendosomal destination and transport kinetics of model transmembrane cargo molecules bearing monoubiquitinated, multi-monoubiquitinated, or polyubiquitinated cytoplasmic tails were determined. Monomeric CD4 chimeras with K63-linked poly-Ub chains and tetrameric CD4-mono-Ub chimeras were rapidly targeted to the lysosome.

Molecular basis of oligoubiquitin-dependent internalization of membrane proteins in Mammalian cells.

Ubiquitination induced down-regulation of cell surface proteins by internalization and lysosomal targeting plays a fundamental role in cell physiology and pathogenesis of diseases. The molecular basis of a single ubiquitin (Ub) as an autonomous endocytic signal, the widely accepted mechanism, however, remains elusive in higher eukaryotes. Using Ub containing reporter proteins without signalling abilities, we present evidence that only multiple Ub moieties, linked either covalently or assembled as oligomers with an intact interface for recognition by Ub-interacting motifs (UIMs), are recognized by the endocytic machinery in vivo and associate with a subset of Ub-binding clathrin adaptors in vitro.

Oligomerization state of the DNA fragmentation factor in normal and apoptotic cells.

The caspase-activated DNase (CAD) is the primary nuclease responsible for oligonucleosomal DNA fragmentation during apoptosis. The DNA fragmentation factor (DFF) is composed of the 40-kDa CAD (DFF40) in complex with its cognate 45-kDa inhibitor (inhibitor of CAD: ICAD or DFF45). The association of ICAD with CAD not only inhibits the DNase activity but is also essential for the co-translational folding of CAD.

Misfolding diverts CFTR from recycling to degradation: quality control at early endosomes.

To investigate the degradation mechanism of misfolded membrane proteins from the cell surface, we used mutant cystic fibrosis transmembrane conductance regulators (CFTRs) exhibiting conformational defects in post-Golgi compartments. Here, we show that the folding state of CFTR determines the post-endocytic trafficking of the channel. Although native CFTR recycled from early endosomes back to the cell surface, misfolding prevented recycling and facilitated lysosomal targeting by promoting the ubiquitination of the channel.