[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.
Full-text · Article · Nov 2015 · Scientific Reports
[Show abstract][Hide abstract] 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.
No preview · Article · May 2015 · Biotechnology and Bioengineering
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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.
No preview · Article · Nov 2014 · Acta Biomaterialia
[Show abstract][Hide abstract] 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.
No preview · Article · Oct 2014 · Biochimica et Biophysica Acta (BBA) - General Subjects
[Show abstract][Hide abstract] 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.
No preview · Article · Sep 2014 · Analytical Biochemistry
[Show abstract][Hide abstract] 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.
Preview · Article · Jun 2014 · American Journal of Transplantation
[Show abstract][Hide abstract] ABSTRACT: Aim. Shigyaku-san is known to alleviate the effects of experimental liver damage. The aim of this study was to clarify the effects of Shigyaku-san on liver function and regeneration after hepatectomy in rats. Methods. Rats were divided into three groups (control, Shigyaku-san, no-hepatectomy). Blood biochemistry was analyzed at 2, 6, 24, and 72 h postoperatively. The remnant liver, liverbody weight ratio and immunohistochemistry stains were evaluated before hepatectomy and at 24 and 72 h postoperatively. Serum IL-6 and TNF-α were measured before hepatectomy and 2 and 6 h postoperatively. Results. Serum ALT and T-Chol were significantly lower in the Shigyaku-san group than in the control group at 24 and 72 h postoperatively, and serum LDH was significantly lower 6 h postoperatively. Regenerated liver weight was significantly increased in the Shigyaku-san group compared with the control group at 72 h postoperatively. The proliferating cell nuclear antigen (PCNA) labeling index (LI) and liver-body weight ratio were significantly higher in the Shigyaku-san group than in the control group 24 and 72 h postoperatively. Ki67 was significantly higher in the Shigyaku-san group than in the control group 72 h postoperatively. Compared with the control group, serum IL-6 was significantly increased 2 h postoperatively, but significantly decreased 6 h postoperatively in the Shigyaku-san group. Conclusion. These data suggest that Shigyaku-san protects against liver damage and promotes liver regeneration after partial hepatectomy in rats.
[Show abstract][Hide abstract] 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.
No preview · Article · Jun 2014 · Tissue Engineering Part A
[Show abstract][Hide abstract] ABSTRACT: Here, we outline a method for surface-mediated transfection of small interfering RNA (siRNA) using electroporation. Cells are cultured on an electrode carrying siRNA. After the cells are adhered on the electrode surface, an electric pulse is applied to release siRNA from the surface and transfect it into the cells. A siRNA electroporation microarray is also presented for high-throughput screening of the functions of many siRNAs.
No preview · Article · Feb 2014 · Methods in molecular biology (Clifton, N.J.)
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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.
No preview · Article · Nov 2013 · Bioorganic & medicinal chemistry
[Show abstract][Hide abstract] ABSTRACT: Objectives: Recently, segmental lung resection has been increasingly performed. For primary lung cancer, we performed segmentectomy
in lung cancer patients with pure ground glass opacity (GGO) or more than 50% GGO and <2 cm diameter as a radical segmentectomy
(RS), and in cases with poor lung function, relapse, or high-risk conditions as a palliative segmentectomy (PS). In this study,
we compared RS with PS patients.
Methods: Of 147 patients who underwent segmentectomy (81 men, 66 women; mean age 65.3 ± 12.3 years) using our method (Iwata
et al. Interact Cardiovasc Thorac Surg 2013;16(4):423-5), video-assisted thoracoscopic (VATS) segmentectomy was performed
in 96 patients (66.7%). Segmentectomy was performed to remove a primary lung cancer in 83 patients. We performed RS in 33
patients and PS in 49. We performed hilar or mediastinal lymph node resection.
Results: The mean ages of patients undergoing RS and PS were 67.2 ± 9.8 and 71.4 ± 8.8 years (P = 0.047), respectively. The mean FDG-PET standardized uptake values were 1.18 ± 1.49, and 7.39 ± 7.4 (P = 0.000), respectively. The mean operative times were 287 ± 84 and 308 ± 113 min (P = 0.288), respectively. The mean blood loss values were 59 ± 52 and 179 ± 307 ml (P = 0.010), respectively. The mean duration of drainage was 2.8 ± 1.3 and 3.5 ± 2.3 days (P = 0.101), respectively. Postoperative complications related to the operative procedures occurred in four (12.1%) and 16 patients
(32.7%), P = 0.034, respectively. Five-year survival of stage I lung cancer (RS in 33 vs PS in 38 patients) was 100% (median observation
duration 32.7 months) and 65.8% (34.5 months), P = 0.006, respectively.
Conclusions: Our radical segmentectomy is feasible for stage I lung cancer with features of pure GGO or >50% GGO and <2 cm
No preview · Article · Sep 2013 · Interactive Cardiovascular and Thoracic Surgery
[Show abstract][Hide abstract] 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.