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. Grafted animals showed significantly better functional recovery than the control rats which received only contusion injury. Morphologically, the contusion cavity was significantly smaller, and the amount of spared tissue was significantly greater in grafted animals than in controls. Retrograde tracing studies showed a statistically significant increase in the number of FB-labeled neurons in different segments of the spinal cord, the brainstem and the sensorimotor cortex. The extent of functional improvement was inversely related to the amount of chondroitin-sulphate around the cavity and the astrocytic and microglial reactions in the injured segment. The grafts produced GDNF, IL-10 and MIP1-alpha for at least one week. These data suggest that grafted undifferentiated hiPSCs are able to induce morphological and functional recovery after spinal cord contusion injury.

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. NDRG1 and CTDP1 genes were screened only for founder mutations in Roma patients. Causative genetic mutations were identified in 67.2% of the CMT1 and in 33.6% of the CMT2 cases, which indicates an overall success rate of 59.9% in the study population. Considering all affected individuals, alterations were most frequently found in PMP22 (40.5%), followed by GJB1 (9.2%), MPZ (4.5%), MFN2 (2.5%), NDRG1 (1.5%), EGR2 (0.8%) and CTDP1 (0.8%). The phenotypic spectrum and the disease severity of the studied patients also varied broadly. Deafness and autoimmune disorders were more often associated with PMP22 duplication, while MFN2 and GJB1 mutations were frequently present with central nervous system abnormalities. Our study may be helpful in determining the strategy of genetic diagnostics in Hungarian CMT patients.

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. The iPSC line showed normal karyotype. The advantages of using RbiPSC are the easy access to primary material and the possibility to study the efficacy and safety of the iPSC-based therapies on a non-rodent animal model.

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. Furthermore, the iPSC line showed normal karyotype. Our model might offer a good platform to study the pathomechanism of ASD, also for drug testing, early biomarker discovery and gene therapy studies.

Generation of Mucopolysaccharidosis type II (MPS II) human induced pluripotent stem cell (iPSC) line from a 7-year-old male with pathogenic IDS mutation.

Peripheral blood was collected from a 7-year-old male patient with an X-linked recessive mutation of Iduronate 2-sulfatase (IDS) gene (NM_000202.7(IDS):c.182C>T) causing MPS II (OMIM 309900). Peripheral blood mononuclear cells (PBMCs) were reprogrammed by lentiviral delivery of a self-silencing hOKSM polycistronic vector. The pluripotency of the iPSC line was confirmed by the expression of pluripotency-associated markers and in vitro spontaneous differentiation towards the 3 germ layers. The iPSC line showed normal karyotype. The cell line offers a good platform to study MPS II pathophysiology, for drug testing, early biomarker discovery and gene therapy studies.

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). Peripheral blood mononuclear cells (PBMCs) were reprogrammed by lentiviral delivery of a self-silencing hOKSM polycistronic vector. The pluripotency of the iPSC line was confirmed by the expression of pluripotency-associated markers and in vitro spontaneous differentiation towards the 3 germ layers. The iPSC line showed normal karyotype. The cell line offers a good platform to study MPS II pathophysiology, for drug testing, early biomarker discovery and gene therapy studies.

Generation of Mucopolysaccharidosis type II (MPS II) human induced pluripotent stem cell (iPSC) line from a 1-year-old male with pathogenic IDS mutation.

Peripheral blood was collected from a 1-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). Peripheral blood mononuclear cells (PBMCs) were reprogrammed by lentiviral delivery of a self-silencing hOKSM polycistronic vector. The pluripotency of the iPSC line was confirmed by the expression of pluripotency-associated markers and in vitro spontaneous differentiation towards the 3 germ layers. The iPSC line showed normal karyotype. The cell line offers a good platform to study MPS II pathophysiology, for drug testing, early biomarker discovery and gene therapy studies.

Generation of human induced pluripotent stem cell (iPSC) line from an unaffected female carrier of Mucopolysaccharidosis type II (MPS II) disorder.

Peripheral blood was collected from a 39-year-old unaffected female carrier of an X-linked recessive mutation of Iduronate 2-sulfatase gene (NM_000202.7(IDS):c.85C>T) causing MPS II (OMIM 309900). Peripheral blood mononuclear cells (PBMCs) were reprogrammed by lentiviral delivery of a self-silencing hOKSM polycistronic vector. The pluripotency of iPSC line was confirmed by the expression of pluripotency-associated markers and in vitro spontaneous differentiation towards the 3 germ layers. The iPSC showed normal karyotype. The line offers a good platform to study MPS II pathophysiology, for drug testing, early biomarker discovery and gene therapy studies.

Establishment of EHMT1 mutant induced pluripotent stem cell (iPSC) line from a 11-year-old Kleefstra syndrome (KS) patient with autism and normal intellectual performance.

Peripheral blood was collected from a clinically characterized female Kleefstra syndrome patient with a heterozygous, de novo, premature termination codon (PTC) mutation (NM_024757.4(EHMT1):c.3413G>A; p.Trp1138Ter). Peripheral blood mononuclear cells (PBMCs) were reprogrammed with the human OSKM transcription factors using the Sendai-virus (SeV) delivery system. The pluripotency of transgene-free iPSC line was verified by the expression of pluripotency-associated markers and by in vitro spontaneous differentiation towards the 3 germ layers. Furthermore, the iPSC line showed normal karyotype. Our model might offer a good platform to study the pathomechanism of Kleefstra syndrome, also for drug testing, early biomarker discovery and gene therapy studies.

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. Furthermore, the iPSC line showed normal karyotype. Our model might offer a good platform to further study the pathomechanism of sporadic AD, to identify early biomarkers and also for drug testing and gene therapy studies.

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. Furthermore, the iPSC line showed normal karyotype. Our model might offer a good platform to further study the pathomechanism of sporadic AD, to identify early biomarkers and also for drug testing and gene therapy studies.

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. Expansion of NPCs in 3D culture methods also resulted in a more homogenous PAX6 expression when compared to 2D culture methods. Furthermore, the 3D propagation method for NPCs resulted in a significant higher expression of the astrocyte markers GFAP and aquaporin 4 (AQP4) in the differentiated cells. Thus, our 3D propagation method could constitute a useful tool to promote NPC homogeneity and also to increase the differentiation potential of iPSC towards astrocytes.

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. We determined the exon-intron boundaries and chromosomal localization of these genes using computational analysis. The sequences of mRNA and translated protein of the newly identified genes and those of POU5F1 were aligned to their mammalian orthologs to determine the degree of evolutionary conservation. Furthermore, the expression of these genes in embryonic and adult cells was studied at the mRNA and protein levels. We found the sequences and the expression pattern of these pluripotency-associated genes to be highly conserved between human and rabbit, indicating that the rabbit would be a valuable model for human preimplantation development. Implementing the newly identified genes either as biomarkers or as reprogramming factors might also pave the way towards the creation of stable pluripotent rabbit cell lines.

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. Control animals underwent ventral root avulsion and reimplantation, but did not receive iPSCs. The grafted iPSCs induced an improved reinnervation of the reimplanted ventral root by the host motoneurons as compared with the controls (number of retrogradely labeled motoneurons: 503 ± 38 [AR+iPSCs group] vs 48 ± 6 [controls, AR group]). Morphological reinnervation resulted in a functional recovery, i.e. the grafted animals exhibited more motor units in their reinnervated hind limb muscles, which produced a greater force than that in the controls (50 ± 2.1% vs 11.9 ± 4.2% maximal tetanic tension [% ratio of operated/intact side]). Grafting of undifferentiated iPSCs downregulated the astroglial activation within the L4 segment. The grafted cells differentiated into neurons and astrocytes in the injured cord. The grafted iPSCs, host neurons and glia were found to produce the cytokines and neurotrophic factors MIP-1a, IL-10, GDNF and NT-4. These findings suggest that, following ventral root avulsion injury, iPSCs are able to induce motoneuron survival and regeneration through combined neurotrophic and cytokine modulatory effects.

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. The effect of peptides was screened using a mouse embryonic stem cell line expressing luciferase dual reporter construct driven by promoters for neural tubulin and for elongation factor 1. Cell number was estimated by measuring total cellular DNA. We identified five peptides which enhanced activities of both promoters without relevant changes in cell number. We selected the two most potent peptides for further analysis: the NCAM-derived mimetic FGLL and the synthetic NCAM ligand, Plannexin. Both compounds induced phenotypic neuronal differentiation, as evidenced by increased neurite outgrowth. In summary, we used a simple, but sensitive screening approach to identify the neurogenic peptides. These peptides will not only provide new clues concerning pathways of neurogenesis, but they may also be interesting biotechnology tools for in vitro generation of neurons.

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. We performed the reprogramming procedure on embryonic fibroblasts from inbred (C57BL6) and outbred (ICR) mouse strains. The cells were treated with purified proteins four times, at 48-h intervals, and cultured on mitomycin C treated mouse embryonic fibroblast (MEF) cells in complete embryonic stem cell (ESC) medium until colonies formed. The iPSCs generated from the outbred fibroblasts exhibited similar morphology and growth properties to ESCs and were sustained in an undifferentiated state for more than 20 passages. The cells were checked for pluripotency-related markers (Oct4, Sox2, Klf4, cMyc, Nanog) by immunocytochemistry and by reverse transcription-polymerase chain reaction. The protein iPSCs (piPSCs) formed embryoid bodies and subsequently differentiated towards all three germ layer lineages. Importantly, the piPSCs could incorporate into the blastocyst and led to variable degrees of chimerism in newborn mice. These data show that recombinant purified cell-penetrating proteins are capable of reprogramming MEFs to iPSCs. We also demonstrated that the cells of the generated cell line satisfied all the requirements of bona fide mouse ESCs: form round colonies with defined boundaries; have a tendency to attach together with high nuclear/cytoplasmic ratio; express key pluripotency markers; and are capable of in vitro differentiation into ecto-, endo-, and mesoderm, and in vivo chimera formation.

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. Furthermore, unlike the alternative transposon piggyBac, SB has no SB-like elements within the human genome, minimising the possibility of mobilising endogenous transposon elements. All iPSC lines exhibited the expected characteristics of pluripotent human cells, including the ability to differentiate to derivatives of all three germ layers in vitro. Re-expression of the SB transposase in the iPSCs after reprogramming resulted in the mobilisation of some of the transposons. These results indicate that the SB transposon system is a useful addition to methods for generating human iPSCs, both for basic and applied biomedical research, and in the context of future therapeutic application.

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. Using 3 × 10(5) cells/ml, 10 rpm rotary speed and plating of EBs onto gelatin-coated surfaces three days after culture, were the best parameters for optimal size and EB quality on consequent cardiac differentiation. These optimized parameters enrich cardiac differentiation in ES cells when using the STLV method.

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. In contrast, lysosomal delivery of CD4 chimeras exposing K48-linked Ub chains was delayed, whereas delivery of monoubiquitinated CD4 chimeras was undetectable. Similar difference was observed in the lysosomal targeting of mono- versus polyubiquitinated invariant chain and CD4 ubiquitinated by the MARCH (membrane-associated RING-CH) IV Ub ligase. Consistent with this, Hrs (hepatocyte growth factor regulated tyrosine kinase phosphorylated substrate), an endosomal sorting adaptor, binds preferentially to K63-Ub chain and negligibly to mono-Ub. These results highlight the plasticity of Ub as a sorting signal and its recognition by the endosomal sorting machinery, and together with previous data, suggest a regulatory role for assembly and disassembly of Ub chains of specific topology in lysosomal cargo sorting.