Hiroo Iwata

Kyoto University, Kioto, Kyōto, Japan

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Publications (385)1271.08 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Pluripotent stem cells, such as embryonic stem cells and induced pluripotent stem (iPS) cells, are regarded as new sources for cell replacement therapy. These cells can unlimitedly expand under undifferentiated conditions and be differentiated into multiple cell types. Automated culture systems enable the large-scale production of cells. In addition to reducing the time and effort of researchers, an automated culture system improves the reproducibility of cell cultures. In the present study, we newly designed a fully automated cell culture system for human iPS maintenance. Using an automated culture system, hiPS cells maintained their undifferentiated state for 60 days. Automatically prepared hiPS cells had a potency of differentiation into three germ layer cells including dopaminergic neurons and pancreatic cells.
    Scientific Reports 11/2015; 5:16647. DOI:10.1038/srep16647 · 5.58 Impact Factor
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    ABSTRACT: Eight types of poly(ethylene glycol)-lipid(PEG-lipids) carrying different lipid tails were synthesized. These PEG-lipids were labeled with fluorescein isothiocyanate (FITC-PEG-lipids) to examine their interaction with cells and to quantitatively determine amounts of PEG-lipids bound on the cell surface. FITC-PEG-lipids spontaneously anchored to the cell membrane within 15min without loss of cell viability. The type of lipid had very little effect on the anchoring rates, while an increase in the hydrophobicity of the lipid portion of the PEG-lipids slowed their dissociation rates. Densities of FITC-PEG-lipids on the cell surface ranged from 1×10(-3) to 1×10(-2)molecules/nm(2), depending on the kinds of lipids employed. The relationship between the stability of the lipids on the cell membrane and the hydrophobicity of the lipid moieties will give a basis for the selection of a hydrophobic moiety in PEG-lipid conjugates for use in specific applications. Copyright © 2015 Elsevier B.V. All rights reserved.
    Colloids and surfaces B: Biointerfaces 08/2015; 135:765-773. DOI:10.1016/j.colsurfb.2015.08.014 · 4.15 Impact Factor
  • Yusuke Arima · Hiroo Iwata ·
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    ABSTRACT: We examined the effect of surface chemistry on adsorption of fibronectin (Fn) and vitronectin (Vn) and subsequent cell adhesion, employing self-assembled monolayers (SAMs) of alkanethiols carrying terminal methyl (CH3), hydroxyl groups (OH), carboxylic acid (COOH), and amine (NH2). More Fn and Vn adsorbed to COOH- and NH2-SAMs than to CH3- and OH-SAMs from a mixture with bovine serum albumin (BSA) and from 2% fetal bovine serum. Adhesion of human umbilical vein endothelial cells (HUVECs) on CH3- and OH-SAMs preadsorbed with Fn and BSA decreased with decreasing adsorbed Fn; however, HUVECs adhered to COOH- and NH2-SAMs even in the presence of BSA at 1000-fold more than Fn in a mixture because of the preferential adsorption of Fn and/or displacement of preadsorbed BSA with Fn and Vn in a serum-containing medium. SAMs coated with a mixture of Vn and BSA exhibited adhesion of HUVECs regardless of surface functional groups. A well-organized focal adhesion complex and actin stress fibers were observed only for COOH- and NH2-SAMs when SAMs were preadsorbed with Vn and BSA. These results suggest that COOH- and NH2-SAMs allow for both cell adhesion and cell spreading because of the high density of cell-binding domains derived from adsorbed Vn. Copyright © 2015. Published by Elsevier Ltd.
    Acta biomaterialia 08/2015; 26. DOI:10.1016/j.actbio.2015.08.033 · 6.03 Impact Factor
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    ABSTRACT: Single-stranded oligonucleotide-conjugated lipids (ssDNA-PEG-lipids) that associate with the cell membrane confer to the cell an artificial adhesive capability via sequence-specific hybridization to complementary oligonucleotides, forming bonds of double stranded oligonucleotides (dsDNA). Such artificial tethers permit surface patterning of cells or controlled formation of cellular aggregates. However, the hybridization responsible for tethering cells to surfaces or to other cells is not trivially reversed under physiological conditions. In this study, we approach the unbinding of tethered cells by cleaving dsDNA bonds with restriction endonuclease BamHI or digesting bonds with the nonspecific nuclease Benzonase. The procedure was applied to CCRF-CEM cells bearing dsDNA suspended in isolation, cells tethered to glass substrates, and cells aggregated heterotypically with other ssDNA-bearing cells. Cells liberated from surfaces with BamHI could be flushed from flow chambers and viably recovered while the majority of cells not bearing enzyme recognition sequences were retained on the surface, and DNA-tethered cells could be nonspecifically recovered viably from surfaces after Benzonase treatment. Heterotypic aggregates of cells joined by recognition sequence DNA could be dispersed with 10 min exposure to BamHI while undispersed cells heterotypically aggregated with a control sequence remained. Likewise, 10 min exposure to Benzonase was sufficient to disperse aggregates independently of sequence. The potential to undo artificially engineered DNA-mediated adhesion offers new possibilities in the controlled arrangement of cells relative to other cells and in the study of membrane biophysics. Copyright © 2015 Elsevier Ltd. All rights reserved.
    Biomaterials 06/2015; 53. DOI:10.1016/j.biomaterials.2015.02.059 · 8.56 Impact Factor
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    ABSTRACT: Neural stem cells (NSCs) demonstrate encouraging results in cell replacement therapy for neurodegenerative disorders and traumatic injury in the central nervous system. Monitor the survival and migration of transplanted cells would provide us important information concerning the performance and integration of the graft during the therapy time course. Magnetic resonance imaging (MRI) allow us to monitor the transplanted cells in a non-invasive way. The only requirement is to use an appropriate contrast agent to label the transplanted cells. Superparamagnetic iron oxide (SPIO) nanoparticles are one of the most commonly used contrast agent for MRI detection of transplanted cells. SPIO nanoparticles demonstrated to be suitable for labeling several types of cells including NSCs. However, the current methods for SPIO labeling are non-specific, depending mostly on electrostatic interactions, demanding relatively high SPIO concentration, and long incubation time, which can affect the viability of cells. In this study, we propose a specific and relatively fast method to label NSCs with SPIO nanoparticles via DNA hybridization. Two short single stranded DNAs (ssDNAs), oligo[dT]20 and oligo[dA]20 were conjugated with a lipid molecule and SPIO nanoparticle respectively. The labeling process comprises two simple steps; first the cells are modified to present oligo[dT]20 ssDNA on the cell surface, then the oligo[dA]20 ssDNA conjugated with SPIO nanoparticles are presented to the modified cells to allow the oligo[dT]20-oligo[dA]20 hybridization. The method showed to be non-toxic at concentrations up to 50 μg/mL oligo[dA]20-SPIO nanoparticles. Presence of SPIO nanoparticles at cell surface and cell cytoplasm was verified by transmission electron microscopy (TEM). SPIO labeling via DNA hybridization demonstrated to not interfere on NSCs proliferation, aggregates formation, and differentiation. NSCs labeled with SPIO nanoparticles via DNA hybridization system were successfully detected by MRI in vitro as well in vivo. Cells transplanted into the rat brain striatum could be detected by MRI scanning up to 1 month post-transplantation. Copyright © 2015 Elsevier Ltd. All rights reserved.
    Biomaterials 06/2015; 54. DOI:10.1016/j.biomaterials.2015.03.017 · 8.56 Impact Factor
  • Mitsue Komatsu · Shuhei Konagaya · Edgar Y Egawa · Hiroo Iwata ·
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    ABSTRACT: Pluripotent stem cells (embryonic stem/induced pluripotent stem cells) have been widely studied as a potential cell source for cell transplantation therapy of Parkinson's disease. However, some difficulties remain to be overcome. These include the need to prepare a large number of dopamine (DA) neurons for clinical use and to culture the cells for a long period to allow their functional maturation and the removal of undifferentiated cells. In this study, aggregates of DA neuron precursors were enclosed in alginate-Ca(2+) microbeads, and the encapsulated aggregates were cultured for 25days to induce cell maturation. More than 60% of cells in the aggregates differentiated into tyrosine hydroxylase-positive DA neurons. The aggregates could release DA at the same level as aggregates maintained on culture dishes without encapsulation. In addition, by exposure to a citrate solution, the alginate-Ca(2+) gel layer could be easily removed from aggregates without damaging the DA neurons. When the aggregates were transplanted into rat brain, viable cells were found in the graft at one week post-transplantation, with cells extending neurites into the host tissue. Cell aggregates encapsulated in alginate-Ca(2+) beads successfully differentiated into mature DA neurons. The alginate-Ca(2+) microbead is suitable for maintaining DA precursor aggregates for a long period to allow their functional maturation. Copyright © 2015. Published by Elsevier B.V.
    Biochimica et Biophysica Acta 05/2015; 1850(9). DOI:10.1016/j.bbagen.2015.04.011 · 4.66 Impact Factor
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    ABSTRACT: Neural progenitor cells derived from induced pluripotent stem cells have been considered as a potential source for cell-transplantation therapy of central nervous disorders. However, efficient methods to expand neural progenitor cells are further required for their clinical applications. In this study, a protein array was fabricated with nine extracellular matrices and used to screen substrates suitable for the expansion of neural progenitor cells derived from mouse induced pluripotent stem cells. The results showed that neural progenitor cells efficiently proliferated on substrates with immobilized laminin-1, laminin-5, or Matrigel. Based on this result, further attempts were made to develop clinically compliant substrates with immobilized polypeptides that mimic laminin-1, one of the most effective extracellular matrices as identified in the array-based screening. We used here recombinant DNA technology to prepare polypeptide containing the globular domain 3 of laminin-1 and immobilized it onto glass-based substrates. Our results showed that neural progenitor cells selectively proliferated on substrate with the immobilized polypeptide while maintaining their differentiated state. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    Biotechnology and Bioengineering 05/2015; 112(11). DOI:10.1002/bit.25636 · 4.13 Impact Factor
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    ABSTRACT: Transplantation of islets of Langerhans (islets) has been investigated in the clinic to treat patients with insulin-dependent diabetes mellitus. Islet grafts have been maintained by administering immunosuppressive drugs, which can lead to complications in the long term. Alternatives to immunosuppressive therapy are eagerly desired. In this study, we examined the transplantation of coaggregates of CD4CD25 regulatory T (Treg) cells. Coaggregates of Treg cells from C57BL/6 mice and islet cells from BALB/c mice were prepared on agarose hydrogel with small round-bottomed wells. Four hundred coaggregates were transplanted into the livers of streptozotocin-induced diabetic C57BL/6 mice without systemic immunosuppression. The Treg cells and islet cells were distributed randomly in the coaggregates. When 400 coaggregates were transplanted into 9 C57BL/6 mice via the portal vein, 6 of the 9 recipients demonstrated blood glucose less than 250 mg/dL for more than 100 days. A number of insulin-positive cells were observed in the livers at 120 days after transplantation. The Treg cells and islet cells were distributed randomly in the coaggregates. After intraportal transplantation of the coaggregates, Treg cells in the aggregates enabled the long-term survival of allogeneic islet cell grafts in the liver without the use of immunosuppressive drugs.
    Transplantation 02/2015; 99(5). DOI:10.1097/TP.0000000000000579 · 3.83 Impact Factor
  • Toshiki Matsui · Yusuke Arima · Naohiro Takemoto · Hiroo Iwata ·
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    ABSTRACT: Polylactic acid (PLA) is a candidate material to prepare scaffolds for 3-D tissue regeneration. However, cells do not adhere or proliferate well on the surface of PLA because it is hydrophobic. We report a simple and rapid method for inducing cell adhesion to PLA through DNA hybridization. Single-stranded DNA (ssDNA) conjugated to poly(ethylene glycol) (PEG) and to a terminal phospholipid (ssDNA-PEG-lipid) was used for cell surface modification. Through DNA hybridization, modified cells were able to attach to PLA surfaces modified with complementary sequence (ssDNA′). Different cell types can be attached to PLA fibers and films in a spatially controlled manner by using ssDNAs with different sequences. In addition, they proliferate well in a culture medium supplemented with fetal bovine serum. The coexisting modes of cell adhesion through DNA hybridization and natural cytoskeletal adhesion machinery revealed no serious effects on cell growth. The combination of a 3-D scaffold made of PLA and cell immobilization on the PLA scaffold through DNA hybridization will be useful for the preparation of 3-D tissue and organs.
    Acta Biomaterialia 11/2014; 13. DOI:10.1016/j.actbio.2014.11.011 · 6.03 Impact Factor
  • Shuhei Konagaya · Hiroo Iwata ·
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    ABSTRACT: Backgroud Dopamine neurons derived from induced pluripotent stem cells have been widely studied for the treatment of Parkinson's disease. However, various difficulties remain to be overcome, such as tumor formation, fragility of dopamine neurons, difficulty in handling large numbers of dopamine neurons, and immune reactions. In this study, human induced pluripotent stem cell-derived precursors of dopamine neurons were encapsulated in agarose microbeads. Dopamine neurons in microbeads could be handled without specific protocols, because the microbeads protected the fragile dopamine neurons from mechanical stress.
    Biochimica et Biophysica Acta (BBA) - General Subjects 10/2014; 1850(1). DOI:10.1016/j.bbagen.2014.09.025 · 4.38 Impact Factor
  • Mitsuaki Toda · Yusuke Arima · Hiromi Takiguchi · Hiroo Iwata ·
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    ABSTRACT: Surface plasmon field-enhanced fluorescence spectroscopy (SPFS) is a promising methodology for point-of-care (POC) testing. The SPFS devices which have been reported have been equipped with an angle rotating stage to adjust the surface plasmon resonance (SPR) angle. In a clinical setting, however, the SPR angle determination is a tedious and time consuming process. In this study, we employed an SPFS instrument with a convergent optical system that allows the omission of this procedure. We demonstrated that this instrumentation allowed the sensitive determination of low concentrations of alpha-fetoprotein in serum and reduced the variation effect caused by the protein concentrations in samples. The SPFS with a convergent optical system is suitable for POC testing.
    Analytical Biochemistry 09/2014; 467. DOI:10.1016/j.ab.2014.08.029 · 2.22 Impact Factor
  • Source
    N M Luan · H Iwata ·
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    ABSTRACT: Establishment of noninvasive and efficient islet transplantation site together with the avoidance of immunosuppressive drugs for islet engraftment is currently the two major tasks for islet transplantation approach to treat patients with type 1 diabetes. Here, we proposed a method to achieve long-term allogeneic islet graft function without immunosuppression after transplantation in subcutaneous sites. Two agarose rods with basic fibroblast growth factor and heparin were implanted for 1 week in dorsal subcutaneous sites in diabetic rats. After rod removal, 1500 islets were transplanted into the prevascularized pockets. Islets transplanted in prevascularized but not nontreated subcutaneous sites rapidly reverted hyperglycemia in all streptozotocin-induced diabetic rats. In contrast to transient normalization of blood glucose when allogeneic islets were transplanted into liver, allogeneic islets transplanted into this prevascularized subcutaneous site demonstrated long-term graft survival and function in all three rat strain combinations (Fisher 344 to ACI, Lewis to ACI and Fisher 344 to Wistar), evidenced by nonfasting blood glucose level, plasma insulin concentration, intraperitoneal glucose tolerance test and immunohistochemistry. These results indicated that a subcutaneous site prevascularized by this method is potentially a suitable site for successful allogeneic islet transplantation without immunosuppression.
    American Journal of Transplantation 06/2014; 14(7). DOI:10.1111/ajt.12739 · 5.68 Impact Factor
  • Ian Torao Hoffecker · Hiroo Iwata ·
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    ABSTRACT: Co-localization of islets with immuno-privileged cell types such as mesenchymal stromal cells (MSC's) is a potentially multifaceted and adaptive approach to islet protection. We attempted to co-localize MSC's with islets by creating single-celled suspensions of MSC's and cells from dissociated islets on top of arrays of round bottom wells. Segregation between islet-derived cells and MSC's was observed within 3 days. When ROCK inhibitor Y-27632-containing media was used during the preparation of MSC/islet co-aggregates, co-aggregates sorted into core-shell structures with islet-derived cells occupying the exterior while MSC's occupied the core. Immunostaining revealed that MSC-derived regions transition from expression of N-cadherin, vimentin, and CD44 to expression of E-cadherin, while pan-cadherin staining indicated re-allocation of cadherins to cell borders, and shear-based cohesion measurements pointed to increased cohesive strength. The switch suggests that MSC-islet cohesion improved due to the greater degree of cell-cell adhesive compatibility. Functional evaluation of MSC-islet co-aggregates confirmed normal insulin secretory function and partial suppression of anti-CD3-activated splenocyte proliferation. These findings demonstrate that manipulation of cell-cell interactions can be harnessed to control spheroid architecture in MSC-islet co-aggregates, and this study also provides the basis for future islet therapies.
    Tissue Engineering Part A 06/2014; 20(11). DOI:10.1089/ten.TEA.2013.0305 · 4.64 Impact Factor
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    ABSTRACT: Transplantation of islets of Langerhans (islets) was used to treat insulin-dependent diabetes mellitus. However, islet grafts must be maintained by administration of immunosuppressive drugs, which can lead to complications in the long term. An approach that avoids immunosuppressive drug use is desirable. Co-aggregates of Sertoli cells and islet cells from BALB/c mice that were prepared by the hanging drop method were transplanted into C57BL/6 mouse liver through the portal vein as in human clinical islet transplantation. The core part of the aggregates contained mainly Sertoli cells, and these cells were surrounded by islet cells. The co-aggregates retained the functions of both Sertoli and islet cells. When 800 co-aggregates were transplanted into seven C57BL/6 mice via the portal vein, six of seven recipient mice demonstrated quasi-normoglycemia for more than 100 days. The hanging drop method is suitable for preparing aggregates of Sertoli and islet cells for transplantation. Notably, transplantation of these allogeneic co-aggregates into mice with chemically induced diabetes via the portal vein resulted in long-term graft survival without systemic immunosuppression.
    Transplantation 12/2013; 97(3). DOI:10.1097/01.TP.0000438198.76531.03 · 3.83 Impact Factor
  • Sho Deno · Naohiro Takemoto · Hiroo Iwata ·
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    ABSTRACT: Ischemia-reperfusion damage is a problem in organ transplantation. Reactive oxygen species are produced in cells by blood-mediated reactions at the time of blood reperfusion. In this study, we developed a method to immobilize and internalize antioxidants in endothelial cells, using vitamin E-loaded liposomes. The liposomes loaded with vitamin E and human umbilical vein endothelial cells (HUVECs) were modified with poly(ethylene glycol)-phospholipid conjugates carrying 20-mer of deoxyadenylic acid (oligo(dA)20) and 20-mer of complementary deoxythymidylic acid (oligo(dT)20), respectively. The liposomes were effectively immobilized on HUVECs through DNA hybridization between oligo(dA)20 and oligo(dT)20. The liposomes loaded with vitamin E were gradually internalized into HUVECs. Then, the cells were treated with antimycin A to induce oxidative stress. We found the amount of reactive oxygen species was greatly reduced in HUVECs carrying vitamin E-loaded liposomes.
    Bioorganic & medicinal chemistry 11/2013; 22(1). DOI:10.1016/j.bmc.2013.11.023 · 2.79 Impact Factor
  • H. Iwata · K. Shirahashi · Y. Mizuno · M. Matsui · H. Takemura ·

    Interactive Cardiovascular and Thoracic Surgery 09/2013; 17(suppl 2):S89-S89. DOI:10.1093/icvts/ivt372.85 · 1.16 Impact Factor
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    ABSTRACT: A labeling method for islet cells with superparamagnetic iron oxide nanoparticles (SPIOs) based on DNA hybridization is proposed for monitoring of transplanted islets by magnetic resonance imaging (MRI). The surfaces of SPIOs were modified by via Michael reaction by reacting oligo-(deoxyadenylic acid)-bearing a terminal thiol group at the 5'-end ((dA)20-SH) with maleic acid functional groups on the SPIOs. The SPIOs were immobilized on islet cells which had been pretreated with oligo-(thymidylic acid)-poly(ethylene glycol)-phospholipid conjugates ((dT)20-PEG-DPPE) through DNA hybridization. Transmission electron microscopy observations revealed that SPIOs were initially anchored on the islet cell surfaces and subsequently transferred to endosomes or exfoliated with time. The SPIO-labeled islet cells could be clearly detected as dark spots by T2(∗)-weighted MR image, whereas non-labeled islet cells could not be detected.
    Bioorganic & medicinal chemistry 09/2013; 21(22). DOI:10.1016/j.bmc.2013.08.063 · 2.79 Impact Factor
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    ABSTRACT: Cell behavior can be manipulated by the topography of the culture surface. In this study, we examined the intercellular communication and osteogenic differentiation of mesenchymal stem cells (MSCs) grown on electrospun fibers with different orientations and densities. Human bone marrow-derived MSCs (hMSCs) were seeded on poly(ε-caprolactone) (PCL) electrospun scaffolds composed of aligned (1D) or cross-aligned (2D) fibers (1.0-1.2 µm diameter) with high, medium, or low fiber densities. It was found that cells preferred to adhere onto electrospun PCL fibers rather than on the flat substrate. The immunofluorescence staining showed that the expression of vinculin, a focal adhesion protein, was limited to the periphery and the two extremities of aligned cells on the edge of the fibers. Electron microscopy showed that cells extended their lamellipodia across the adjacent fibers and proliferated along the direction of fibers. Cells grown on 1D fibrous scaffolds at all fiber densities had an obvious alignment. On 2D fibers, a higher degree of cell alignment was observed at the higher fiber density. On 1D scaffolds, the gap junction intercellular communication (GJIC) quantified by the lucifer yellow dye transfer assay was significantly promoted in the aligned cells in the direction parallel to the fibers but was abolished in the direction perpendicular to the fibers. The expression of osteogenic marker genes (RUNX2, ALP, and OCN) was significantly enhanced in seven days by culture on 1D but not 2D fibers. It was thus proposed that the promoted osteogenic differentiation of hMSCs may be associated with the fiber-guided and directional induction of GJIC.
    Biomedical Materials 09/2013; 8(5):055002. DOI:10.1088/1748-6041/8/5/055002 · 3.70 Impact Factor
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    ABSTRACT: Poor viability of cells transplanted into the brain has been the critical problem associated with stem cell-based therapy for Parkinson's disease. To overcome this problem, a collagen hydrogel incorporating an integrin-binding protein complex was prepared and used as a carrier for neural stem cells. The protein complex consisted of two polypeptides containing the G3 domain of a laminin alpha-1 chain and the C-terminal oligopeptide of a laminin gamma-1 chain. These polypeptides were fused with alpha-helical segments which spontaneously formed a coiled-coil heterodimer and with the collagen-binding peptide that facilitated the binding of the heterodimer to collagen networks. In this study, neural stem cells stably expressing the enhanced green fluorescent protein (EGFP) were suspended in the hydrogel and transplanted into the striatum of healthy rats. The viability of transplanted cells was evaluated by histological analysis and quantitative reverse-transcriptase polymerase chain reaction for EGFP mRNA present in the tissue explants. Our results showed that the collagen hydrogel incorporating the integrin-binding protein complex serves to improve the viability of NSCs in the early stage after transplantation into the striatum.
    Bioconjugate Chemistry 08/2013; 24(11). DOI:10.1021/bc400005m · 4.51 Impact Factor
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    ABSTRACT: Human neural progenitor cells (hNPCs) are a potential source for cell transplantation therapy in central nervous disorders. Neurosphere culture, the standard method for obtaining hNPCs, suffers from several limitations including the heterogeneity of cells in a neurosphere and the limitation of growth rate due to the presence of differentiated cells in the neurospheres. To overcome these limitations, we developed culture substrates that enable the selective expansion of hNPCs in adherent culture. Epidermal growth factor and basic fibroblast growth factor were fused with hexahistidine (EGF-His and bFGF-His, respectively) and were immobilized alone or in combination onto Ni ion-bound glass through coordination. When hNPCs derived from human fetal brain were cultured on these substrates, adhesion and proliferation of hNPCs took place most efficiently on the substrate with both EGF-His and bFGF-His compared to substrates with either factor alone and to a control substrate without growth factors. The rate of cell proliferation was two-fold higher in the adherent culture on the substrate immobilized with both EGF-His and bFGF-His than in the standard neurosphere culture. A cell population obtained after 5 days of culture on the substrate contained nestin-expressing progenitors (>90%). We conclude that the culture substrate with co-immobilized EGF and bFGF is effective for the selective expansion of hNPCs.
    Biomaterials 05/2013; 34(25). DOI:10.1016/j.biomaterials.2013.04.041 · 8.56 Impact Factor

Publication Stats

7k Citations
1,271.08 Total Impact Points


  • 1977-2015
    • Kyoto University
      • • Institute for Frontier Medical Sciences
      • • Graduate School of Medicine / Faculty of Medicine
      • • Department of Neurosurgery
      • • Institute for Chemical Research
      • • Department of Polymer Chemistry
      Kioto, Kyōto, Japan
  • 2002-2009
    • Gifu University
      • • Department of General and Cardiothoracic Surgery
      • • Graduate School of Medicine
      Gihu, Gifu, Japan
    • Fukui University
      Hukui, Fukui, Japan
  • 2000-2003
    • Gifu University Hospital
      Gihu, Gifu, Japan
  • 1998
    • University of Alberta
      Edmonton, Alberta, Canada
  • 1986-1996
    • National Cerebral and Cardiovascular Center
      • Department of Cardiovascular Medicine
      Ōsaka, Ōsaka, Japan
  • 1984-1994
    • The Cardiovascular Institute
      Tōkyō, Japan
  • 1993
    • Hamamatsu Rosai Hospital
      Hamamatu, Shizuoka, Japan