Daisuke Tsuji

Generation of human induced pluripotent stem cells from oral mucosa.

Induced pluripotent stem (iPS) cells are one of the most promising sources for cell therapy in regenerative medicine. Using a patients own genetically identical and histocompatible cells is the ideal way to practice personalized regenerative medicine. For personalized iPS cell therapy, the prerequisites for cell source preparation are a simple and safe procedure, no aesthetic or functional damage, and quick wound healing. Oral mucosa fibroblasts (OFs) may have high potential to fulfill these requirements. In this study, biopsy was performed in a dental chair; no significant incisional damage was recognized and rapid wound healing (within a week) was observed. We generated human iPS cells from the isolated OFs via the retroviral gene transfer of OCT4, SOX2, c-MYC, and KLF4. Reprogrammed cells showed ES-like morphology and expressed undifferentiated markers such as OCT4, NANOG, SSEA4, TRA-1-60, and TRA-1-81. Subsequent in vitro and in vivo analyses confirmed the pluripotency of resultant iPS cells, which matched the criteria for iPS cells. In addition, we found that the endogenous expression levels of c-MYC and KLF4 in OFs were similar to those in dermal fibroblasts. Taken together, we propose that OFs could be a practical source for preparing iPS cells to achieve personalized regenerative medicine in the near future.

Glycolysis Inhibition Inactivates ABC Transporters to Restore Drug Sensitivity in Malignant Cells

Cancer cells eventually acquire drug resistance largely via the aberrant expression of ATP-binding cassette (ABC) transporters, ATP-dependent efflux pumps. Because cancer cells produce ATP mostly through glycolysis, in the present study we explored the effects of inhibiting glycolysis on the ABC transporter function and drug sensitivity of malignant cells. Inhibition of glycolysis by 3-bromopyruvate (3BrPA) suppressed ATP production in malignant cells, and restored the retention of daunorubicin or mitoxantrone in ABC transporter-expressing, RPMI8226 (ABCG2), KG-1 (ABCB1) and HepG2 cells (ABCB1 and ABCG2). Interestingly, although side population (SP) cells isolated from RPMI8226 cells exhibited higher levels of glycolysis with an increased expression of genes involved in the glycolytic pathway, 3BrPA abolished Hoechst 33342 exclusion in SP cells. 3BrPA also disrupted clonogenic capacity in malignant cell lines including RPMI8226, KG-1, and HepG2. Furthermore, 3BrPA restored cytotoxic effects of daunorubicin and doxorubicin on KG-1 and RPMI8226 cells, and markedly suppressed subcutaneous tumor growth in combination with doxorubicin in RPMI8226-implanted mice. These results collectively suggest that the inhibition of glycolysis is able to overcome drug resistance in ABC transporter-expressing malignant cells through the inactivation of ABC transporters and impairment of SP cells with enhanced glycolysis as well as clonogenic cells.

Production of Recombinant β-Hexosaminidase A, a Potential Enzyme for Replacement Therapy for Tay-Sachs and Sandhoff Diseases, in the Methylotrophic Yeast Ogataea minuta

ABSTRACT Human β-hexosaminidase A (HexA) is a heterodimeric glycoprotein composed of α- and β-subunits that degrades GM2 gangliosides in lysosomes. GM2 gangliosidosis is a lysosomal storage disease in which an inherited deficiency of HexA causes the accumulation of GM2 gangliosides. In order to prepare a large amount of HexA for a treatment based on enzyme replacement therapy (ERT), recombinant HexA was produced in the methylotrophic yeast Ogataea minuta instead of in mammalian cells, which are commonly used to produce recombinant enzymes for ERT. The problem of antigenicity due to differences in N-glycan structures between mammalian and yeast glycoproteins was potentially resolved by using α-1,6-mannosyltransferase-deficient (och1Δ) yeast as the host. Genes encoding the α- and β-subunits of HexA were integrated into the yeast cell, and the heterodimer was expressed together with its isozymes HexS (αα) and HexB (ββ). A total of 57 mg of β-hexosaminidase isozymes, of which 13 mg was HexA (αβ), was produced per liter of medium. HexA was purified with immobilized metal affinity column for the His tag attached to the β-subunit. The purified HexA was treated with α-mannosidase to expose mannose-6-phosphate (M6P) residues on the N-glycans. The specific activities of HexA and M6P-exposed HexA (M6PHexA) for the artificial substrate 4MU-GlcNAc were 1.2 ± 0.1 and 1.7 ± 0.3 mmol/h/mg, respectively. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis pattern suggested a C-terminal truncation in the β-subunit of the recombinant protein. M6PHexA was incorporated dose dependently into GM2 gangliosidosis patient-derived fibroblasts via M6P receptors on the cell surface, and degradation of accumulated GM2 ganglioside was observed.

Highly Phosphomannosylated Enzyme Replacement Therapy for GM2 Gangliosidosis

Novel recombinant human lysosomal β‐hexosaminidase A (HexA) was developed for enzyme replacement therapy (ERT) for Tay‐Sachs and Sandhoff diseases, ie, autosomal recessive GM2 gangliosidoses, caused by HexA deficiency.

Therapeutic Potential of Intracerebroventricular Replacement of Modified Human β-Hexosaminidase B for GM2 Gangliosidosis

To develop a novel enzyme replacement therapy for neurodegenerative Tay-Sachs disease (TSD) and Sandhoff disease (SD), which are caused by deficiency of β-hexosaminidase (Hex) A, we designed a genetically engineered HEXB encoding the chimeric human β-subunit containing partial amino acid sequence of the α-subunit by structure-based homology modeling. We succeeded in producing the modified HexB by a Chinese hamster ovary (CHO) cell line stably expressing the chimeric HEXB, which can degrade artificial anionic substrates and GM2 ganglioside in vitro, and also retain the wild-type (WT) HexB-like thermostability in the presence of plasma. The modified HexB was efficiently incorporated via cation-independent mannose 6-phosphate receptor into fibroblasts derived from Tay-Sachs patients, and reduced the GM2 ganglioside accumulated in the cultured cells. Furthermore, intracerebroventricular administration of the modified HexB to Sandhoff mode mice restored the Hex activity in the brains, and reduced the GM2 ganglioside storage in the parenchyma. These results suggest that the intracerebroventricular enzyme replacement therapy involving the modified HexB should be more effective for Tay-Sachs and Sandhoff than that utilizing the HexA, especially as a low-antigenic enzyme replacement therapy for Tay-Sachs patients who have endogenous WT HexB.

Synthesis of a stimulus-responsive processing device and its application to a nucleocytoplasmic shuttle Peptide.

Stimulus-responsive processing (peptide bond cleavage) devices were developed. The processing reaction was triggered by stimulus-induced removal of a PG and the processing products were obtained in good purity. A photo-responsive processing device was successfully applied to develop a nucleocytoplasmic shuttle peptide. (F: fluorophore, NES: nuclear export signal. NLS: nuclear localization signal. PG: stimulusresponsive protective group)

Specific induction of macrophage inflammatory protein 1‐α in glial cells of Sandhoff disease model mice associated with accumulation of N‐acetylhexosaminyl glycoconjugates

Sandhoff disease is a lysosomal storage disease caused by simultaneous deficiencies of β‐hexosaminidase A (HexA; αβ) and B (HexB; ββ), due to a primary defect of the β‐subunit gene (HEXB) associated with excessive accumulation of GM2 ganglioside (GM2) and oligosaccharides with N‐acetylhexosamine residues at their non‐reducing termini, and with neurosomatic manifestations. To elucidate the neuroinflammatory mechanisms involved in its pathogenesis, we analyzed the expression of chemokines in Sandhoff disease model mice (SD mice) produced by disruption of the murine Hex β‐subunit gene allele (Hexb–/–). We demonstrated that chemokine macrophage inflammatory protein‐1 α (MIP‐1α) was induced in brain regions, including the cerebral cortex, brain stem and cerebellum, of SD mice from an early stage of the pathogenesis but not in other systemic organs. On the other hand, little changes in other chemokine mRNAs, including those of RANTES (regulated upon activation, normal T expressed and secreted), MCP‐1 (monocyte chemotactic protein‐1), SLC (secondary lymphoid‐tissue chemokine), fractalkine and SDF‐1 (stromal derived factor‐1), were detected. Significant up‐regulation of MIP‐1α mRNA and protein in the above‐mentioned brain regions was observed in parallel with the accumulation of natural substrates of HexA and HexB. Immunohistochemical analysis revealed that MIP‐1α‐immunoreactivity (IR) in the above‐mentioned brain regions of SD mice was co‐localized in Iba1‐IR‐positive microglial cells and partly in glial fibrillary acidic protein (GFAP)‐IR‐positive astrocytes, in which marked accumulation of N‐acetylglucosaminyl (GlcNAc)‐oligosaccharides was observed from the presymptomatic stage of the disease. In contrast, little MIP‐1α‐IR was observed in neurons in which GM2 accumulated predominantly. These results suggest that specific induction of MIP‐1α might coincide with the accumulation of GlcNAc‐oligosaccharides due to a HexB deficiency in resident microglia and astrocytes in the brains of SD mice causing their activation and acceleration of the progressive neurodegeneration in SD mice.

Chemical Synthesis of Biologically Active Monoglycosylated GM2‐Activator Protein Analogue Using N‐Sulfanylethylanilide Peptide

Going to SEA(lide): Total chemical synthesis of a 162-residue glycoprotein analogue of the monoglycosylated human GM2-activator protein (GM2AP) was achieved. Key steps were the use of N-sulfanylethylanilide (SEAlide) peptides in the kinetic chemical ligation synthesis of a large peptide fragment, and a convergent native chemical ligation for final fragment assembly.

Production of human β-hexosaminidase A with highly phosphorylated N-glycans by the overexpression of the Ogataea minuta MNN4 gene

Effective enzyme replacement therapy for lysosomal storage diseases requires a recombinant enzyme with highly phosphorylated N-glycans. Recombinant human beta-hexosaminidase A is a potentially therapeutic enzyme for GM2-gangliosidosis. Recombinant HexA has been produced by using the methylotrophic yeast Ogataea minuta as a host, and the purified enzyme was tested for its replacement effect on cultured fibroblasts derived from GM2-gangliosidosis patients. Although the therapeutic effect was observed, in order to obtain the higher therapeutic effect with a little dose as possible, increased phosphorylation of recombinant beta-hexosaminidase A N-glycans is suggested to be prerequisite. In the budding yeast Saccharomyces cerevisiae, the overexpression of MNN4, which encodes a positive regulator of mannosylphosphate transferase, led to increased mannosylphosphate contents. In the present study, we cloned OmMNN4, a homologous gene to ScMNN4, based on the genomic sequence of O. minuta. We overexpressed the cloned gene under the control of the alcohol oxidase promoter in a beta-hexosaminidase A-producing yeast strain. Structural analysis of pyridylamine-labeled N-glycans by high-performance liquid chromatography revealed that the overexpression of MNN4 caused a 3-fold increase in phosphorylated N-glycans of recombinant beta-hexosaminidase A. The recombinant enzyme prepared from strains overexpressing OmMNN4 was more effectively incorporated into cultured fibroblasts and neural cells, and it more rapidly degraded the accumulated GM2-ganglioside as compared to the control enzyme. These results suggest that beta-hexosaminidase A produced in a strain that overexpresses OmMNN4 will act as an effective enzyme for use in replacement therapy of GM2-gangliosidosis.

Metabolic correction in microglia derived from Sandhoff disease model mice

Sandhoff disease is an autosomal recessive lysosomal storage disease caused by a defect of the β‐subunit gene (HEXB) associated with simultaneous deficiencies of β‐hexosaminidase A (HexA; αβ) and B (HexB; ββ), and excessive accumulation of GM2 ganglioside (GM2) and oligosaccharides with N‐acetylglucosamine (GlcNAc) residues at their non‐reducing termini. Recent studies have shown the involvement of microglial activation in neuroinflammation and neurodegeneration of this disease. We isolated primary microglial cells from the neonatal brains of Sandhoff disease model mice (SD mice) produced by disruption of the murine Hex β‐subunit gene allele (Hexb–/–). The cells expressed microglial cell‐specific ionized calcium binding adaptor molecule 1 (Iba1)‐immunoreactivity (IR) and antigen recognized by Ricinus communis agglutinin lectin‐120 (RCA120), but not glial fibrillary acidic protein (GFAP)‐IR specific for astrocytes. They also demonstrated significant intracellular accumulation of GM2 and GlcNAc‐oligosaccharides. We produced a lentiviral vector encoding for the murine Hex β‐subunit and transduced it into the microglia from SD mice with the recombinant lentivirus, causing elimination of the intracellularly accumulated GM2 and GlcNAc‐oligosaccharides and secretion of Hex isozyme activities from the transduced SD microglial cells. Recomibinant HexA isozyme isolated from the conditioned medium of a Chinese hamster ovary (CHO) cell line simultaneously expressing the human HEXA (α‐subunit) and HEXB genes was also found to be incorporated into the SD microglia via cell surface cation‐independent mannose 6‐phosphate receptor and mannose receptor to degrade the intracellularly accumulated GM2 and GlcNAc‐oligosaccharides. These results suggest the therapeutic potential of recombinant lentivirus encoding the murine Hex β‐subunit and the human HexA isozyme (αβ heterodimer) for metabolic cross‐correction in microglial cells involved in progressive neurodegeneration in SD mice.