Accumulated α-synuclein affects the progression of GM2 gangliosidoses.

The accumulation of α-synuclein (ASyn) has been observed in several lysosomal storage diseases (LSDs) but it remains unclear if ASyn accumulation contributes to LSD pathology. ASyn also accumulates in the neurons of Sandhoff disease (SD) patients and SD model mice (Hexb-/- ASyn+/+ mice). SD is a lysosomal storage disorder caused by the absence of a functional ß-subunit on the ß-hexosaminidase A and B enzymes, which leads to the accumulation of ganglioside in the central nervous system. Here, we explored the role of accumulated ASyn in the progression of Hexb-/- mice by creating a Hexb-/- ASyn-/- double-knockout mice. Our results show that Hexb-/- ASyn-/- mice demonstrated active microglia levels and less dopaminergic neuron loss, without altering the neuronal storage of ganglioside. The autophagy and ubiquitin proteasome pathways are defective in the neurons of Hexb-/- ASyn+/+ mice. In ultrastructural physiological studies, the mitochondria structures look degenerated and dysfunctional. As a result, expression of manganese superoxide dismutase 2 are reduced, and reactive oxygen species-mediated oxidative damage in the neurons of Hexb-/- ASyn+/+ mice. Interestingly, these dysfunctions improved in Hexb-/- ASyn-/- mice. But any clinical improvement were hardly observed in Hexb-/- ASyn-/- mice. Taken together, these findings suggest that ASyn accumulation plays an important role in the pathogenesis of neuropathy in SD and other LSDs, and is therefore a target for novel therapies.

In vivo maturation of human embryonic stem cell-derived teratoma over time.

Transformation of human embryonic stem cells (hESC) is of interest to scientists who use them as a raw material for cell-processed therapeutic products. However, the WHO and ICH guidelines provide only study design advice and general principles for tumorigenicity tests. In this study, we performed in vivo tumorigenicity tests (teratoma formation) and genome-wide sequencing analysis of undifferentiated hESCs i.e. SEES-1, -2 and -3 cells. We followed up with teratoma formation histopathologically after subcutaneous injection of SEES cells into immunodeficient mice in a qualitative manner and investigated the transforming potential of the teratomas. Maturity of SEES-teratomas perceptibly increased after long-term implantation, while areas of each tissue component remained unchanged. We found neither atypical cells/structures nor cancer in the teratomas even after long-term implantation. The teratomas generated by SEES cells matured histologically over time and did not increase in size. We also analyzed genomic structures and sequences of SEES cells during cultivation by SNP bead arrays and next-generation sequencing, respectively. The nucleotide substitution rate was 3.1 × 10-9, 4.0 × 10-9, and 4.6 × 10-9 per each division in SEES-1, SEES-2, and SEES-3 cells, respectively. Heterozygous single-nucleotide variations were detected, but no significant homologous mutations were found. Taken together, these results imply that SEES-1, -2, and -3 cells do not exhibit in vivo transformation and in vitro genomic instability.

Xenogeneic-free defined conditions for derivation and expansion of human embryonic stem cells with mesenchymal stem cells.

The potential applications of human embryonic stem cells (hESCs) in regenerative medicine and developmental research have made stem cell biology one of the most fascinating and rapidly expanding fields of biomedicine. The first clinical trial of hESCs in humans has begun, and the field of stem cell therapy has just entered a new era. Here, we report seven hESC lines (SEES-1, -2, -3, -4, -5, -6, and -7). Four of them were derived and maintained on irradiated human mesenchymal stem cells (hMSCs) grown in xenogeneic-free defined media and substrate. Xenogeneic-free hMSCs isolated from the subcutaneous tissue of extra fingers from individuals with polydactyly showed appropriate potentials as feeder layers in the pluripotency and growth of hESCs. In this report, we describe a comprehensive characterization of these newly derived SEES cell lines. In addition, we developed a scalable culture system for hESCs having high biological safety by using gamma-irradiated serum replacement and pharmaceutical-grade recombinant basic fibroblast growth factor (bFGF, also known as trafermin). This is first report describing the maintenance of hESC pluripotency using pharmaceutical-grade human recombinant bFGF (trafermin) and gamma-irradiated serum replacement. Our defined medium system provides a path to scalability in Good Manufacturing Practice (GMP) settings for the generation of clinically relevant cell types from pluripotent cells for therapeutic applications.

Dystrophin conferral using human endothelium expressing HLA-E in the non-immunosuppressive murine model of Duchenne muscular dystrophy.

Human leukocyte antigen (HLA)-E is a non-classical major histocompatibility complex class I (Ib) molecule, which plays an important role in immunosuppression. In this study, we investigated the immunomodulating effect of HLA-E in a xenogeneic system, using human placental artery-derived endothelial (hPAE) cells expressing HLA-E in a mouse model. In vitro cell lysis analysis by primed lymphocytes in combination with siRNA transfection showed that HLA-E is necessary for inhibition of the immune response. Similarly, in vivo cell implantation analysis with siRNA-mediated down-regulation of HLA-E demonstrates that HLA-E is involved in immunosuppression. As hPAE cells efficiently transdifferentiate into myoblasts/myocytes in vitro, we transplanted the cells into mdx mice, a model of Duchenne muscular dystrophy. hPAE cells conferred dystrophin to myocytes of the 'immunocompetent' mdx mice with extremely high efficiency. These findings suggest that HLA-E-expressing cells with a myogenic potential represent a promising source for cell-based therapy of patients with muscular dystrophy.

Lectin microarray analysis of pluripotent and multipotent stem cells.

Stem cells have a capability to self-renew and differentiate into multiple types of cells; specific markers are available to identify particular stem cells for developmental biology research. In this study, we aimed to define the status of somatic stem cells and the pluripotency of human embryonic stem (hES) and induced pluripotent stem (iPS) cells using a novel molecular methodology, lectin microarray analysis. Our lectin microarray analysis successfully categorized murine somatic stem cells into the appropriate groups of differentiation potency. We then classified hES and iPS cells by the same approach. Undifferentiated hES cells were clearly distinguished from differentiated hES cells after embryoid formation. The pair-wise comparison means based on 'false discovery rate' revealed that three lectins -Euonymus europaeus lectin (EEL), Maackia amurensis lectin (MAL) and Phaseolus vulgaris leucoagglutinin [PHA(L)]- generated maximal values to define undifferentiated and differentiated hES cells. Furthermore, to define a pluripotent stem cell state, we generated a discriminant for the undifferentiated state with pluripotency. The discriminant function based on lectin reactivities was highly accurate for judgment of stem cell pluripotency. These results suggest that glycomic analysis of stem cells leads to a novel comprehensive approach for quality control in cell-based therapy and regenerative medicine.

Thymic alterations in GM2 gangliosidoses model mice.

BACKGROUND: Sandhoff disease is a lysosomal storage disorder characterized by the absence of ß-hexosaminidase and storage of GM2 ganglioside and related glycolipids. We have previously found that the progressive neurologic disease induced in Hexb(-/-) mice, an animal model for Sandhoff disease, is associated with the production of pathogenic anti-glycolipid autoantibodies. METHODOLOGY/PRINCIPAL FINDINGS: In our current study, we report on the alterations in the thymus during the development of mild to severe progressive neurologic disease. The thymus from Hexb(-/-) mice of greater than 15 weeks of age showed a marked decrease in the percentage of immature CD4(+)/CD8(+) T cells and a significantly increased number of CD4(+)/CD8(-) T cells. During involution, the levels of both apoptotic thymic cells and IgG deposits to T cells were found to have increased, whilst swollen macrophages were prominently observed, particularly in the cortex. We employed cDNA microarray analysis to monitor gene expression during the involution process and found that genes associated with the immune responses were upregulated, particularly those expressed in macrophages. CXCL13 was one of these upregulated genes and is expressed specifically in the thymus. B1 cells were also found to have increased in the thy mus. It is significant that these alterations in the thymus were reduced in FcRγ additionally disrupted Hexb(-/-) mice. CONCLUSIONS/SIGNIFICANCE: These results suggest that the FcRγ chain may render the usually poorly immunogenic thymus into an organ prone to autoimmune responses, including the chemotaxis of B1 cells toward CXCL13.

A novel method for isolating podocytes using magnetic activated cell sorting.

BACKGROUND: A large body of accumulated data has now revealed that podocytes play a major role in the development of proteinuria. However, the mechanisms of podocyte injury, leading to foot process effacement and proteinuria, are still unclear partly due to the current lack of an appropriate strategy for preparing podocytes. In this study, we have developed a novel method of rapid isolation of podocytes from mice using magnetic activated cell sorting with an anti-nephrin antibody. METHODS: After endothelial cell depletion using anti-CD31 antibody, nephrin-positive cells were prepared from mouse kidneys using magnetic activated cell sorting with polyclonal rabbit anti-nephrin antibody. Purity of the positively sorted cells was determined by confocal microscopy and fluorescence-activated cell sorting (FACS) analysis. Expression profiles of podocyte-specific molecules in the sorted fractions were characterized by qualitative PCR and immunoblot analysis. RESULTS: Nephrin-positive cells, isolated from mouse kidneys within 6 h, showed dual positivity for synaptopodin and rabbit IgG on confocal microscopy. FACS analysis revealed that the purity of the positively sorted fractions was ∼75%. The nephrin-positive cells sorted by this approach showed a significantly higher expression of podocyte-specific molecules compared with nephrin-negative fractions. CONCLUSIONS: These data strongly suggest that our novel method for isolating podocytes has great utility for various downstream applications such as genomic analysis, proteomics and transcriptomics to elucidate molecular profiling of podocyte biology in vivo compared with conventional methods as our approach requires only several hours to complete and no tissue culture.

Neuronal and glial accumulation of alpha- and beta-synucleins in human lipidoses.

A number of the lysosomal storage diseases that have now been characterized are associated with intra-lysosomal accumulation of lipids, caused by defective lysosomal enzymes. We have previously reported neuronal accumulation of both alpha- and beta-synucleins in brain tissue of a GM2 gangliosidosis mouse model. Although alpha-synuclein has been implicated in several neurodegenerative disorders including Parkinson's disease, dementia with Lewy bodies and multiple system atrophy, its functions remain largely unclear. In our present study, we have examined a cohort of human lipidosis cases, including Sandhoff disease, Tay-Sachs disease, metachromatic leukodystrophy, beta-galactosialidosis and adrenoleukodystrophy, for the expression of alpha- and beta-synucleins and the associated lipid storage levels. The accumulation of alpha-synuclein was found in brain tissue in not only cases of lysosomal storage diseases, but also in instances of adrenoleukodystrophy, which is a peroxisomal disease. alpha-synuclein was detected in both neurons and glial cells of patients with these two disorders, although its distribution was found to be disease-dependent. In addition, alpha-synuclein-positive neurons were also found to be NeuN-positive, whereas NeuN-negative neurons did not show any accumulation of this protein. By comparison, the accumulation of beta-synuclein was detectable only in the pons of Sandhoff disease cases. This differential accumulation of alpha- and beta-synucleins in human lipidoses may be related to functional differences between these two proteins. In addition, the accumulation of alpha-synuclein may also be a condition that is common to lysosomal storage diseases and adrenoleukodystrophies that show an enhanced expression of this protein upon the elevation of stored lipids.

A catalytic subunit of cyclic AMP-dependent protein kinase, PKAC-1, regulates asexual differentiation in Neurospora crassa.

A cyclic AMP (cAMP)-dependent protein kinase pathway has been shown to regulate growth, morphogenesis and virulence in filamentous fungi. However, the precise mechanisms of regulation through the pathway remain poorly understood. In Neurospora crassa, the cr-1 adenylate cyclase mutant exhibits colonial growth with short aerial hyphae bearing conidia, and the mcb mutant, a mutant of the regulatory subunit of cAMP-dependent protein kinase (PKA), shows the loss of growth polarity at the restrictive temperature. In the present study, we isolated mutants of the catalytic subunit of the PKA gene pkac-1 through the process of repeat-induced point mutation (RIP). PKA activity of the mutants obtained through RIP was undetectable. The genome sequence predicts two distinct catalytic subunit genes of PKA, named pkac-1 (NCU06240.1, AAF75276) and pkac-2 (NCU00682.1), as is the case in most filamentous fungi. The results suggest that PKAC-1 works as the major PKA in N. crassa. The phenotype of the pkac-1 mutants included colonial growth, short aerial hyphae, premature conidiation on solid medium, inappropriate conidiation in submerged culture, and increased thermotolerance. This phenotype of pkac-1 mutants resembled to that of cr-1 mutants, except that the addition of cAMP did not rescue the abnormal morphology of pkac-1 mutants. The loss of growth polarity at the restrictive temperature in the mcb mutant was suppressed by pkac-1 mutation. These results suggest that the signal transduction pathway mediated by PKAC-1 plays an important role in regulation of aerial hyphae formation, conidiation, and hyphal growth with polarity.