Acceptance of Murine Islet Allografts Without Immunosuppression in Inguinal Subcutaneous White Adipose Tissue Pretreated With bFGF.

Prevention of immune rejection without immunosuppression is the ultimate goal of transplant immunobiology. One way to achieve this in cellular transplantation, such as with islet transplantation, is to create a favorable local environment at the transplant site. In the current study, we found that C57BL/6 mice with streptozotocin-induced diabetes remained normoglycemic for >1 year after transplantation of BALB/c islets without immunosuppression when the inguinal subcutaneous white adipose tissue (ISWAT) was the site of transplantation and when the site was pretreated with basic fibroblast growth factor. Mechanistically, mesenchymal stem cells (MSCs) expanded in the ISWAT after the treatment was found to produce transforming growth factor-ß (TGF-ß), and prevention of islet allograft rejection could be achieved by cotransplantation with syngeneic MSCs isolated from the ISWAT after the treatment, which was abolished by anti-TGF-ß antibody treatment. Importantly, TGF-ß-producing cells remained present at the site of cotransplantation up to the end of observation period at 240 days after transplantation. These findings indicate that prevention of islet allograft rejection without immunosuppression is feasible with the use of syngeneic TGF-ß-producing MSCs expanded in the ISWAT after the treatment with bFGF, providing a novel strategy for prevention of islet allograft rejection without immunosuppression.

Dual-phase Au-Pt alloys free from magnetic susceptibility artifacts in magnetic resonance imaging.

PURPOSE: Magnetic resonance imaging (MRI) devices are frequently used in image-based diagnosis. In the case of large artifacts, which are generated in magnetic resonance (MR) images when magnetic materials, such as metals, are present in the body, these devices are less useful. This study aimed to develop a dual-phase Au-Pt alloy that does not generate artifacts in MR images and has high workability to prepare medical devices. MATERIALS AND METHODS: A processing method to produce a dual-phase Au-Pt alloy was established, and the magnetic susceptibility and artifacts of different alloy compositions were determined using a SQUID (superconducting quantum interference device) flux meter and a 1.5 T-MRI system. The crystallographic phases of the prepared alloy samples were identified using X-ray diffraction. Sample cross-sections were observed using a metallurgical microscope. Furthermore, a thinning test was conducted to examine alloy workability. RESULTS: Dual-phase Au-Pt alloys Au70Pt30 and Au67Pt33-the former heat-treated at 800 and 850 °C and the latter heat-treated at 900 °C-generated minimal artifacts when imaged in a 1.5 T-MRI system. Their volume magnetic susceptibility increased as the heat-treatment temperature decreased. The alloy surfaces were observed to be uniform. Moreover, the workability of the dual-phase alloy was considerably better than that of the single-phase alloy. CONCLUSION: Volume magnetic susceptibility could be controlled by changing the composition and processing temperature of the Au-Pt alloys. Dual-phase Au-Pt alloys those do not generate magnetic susceptibility artifacts in MRI images and have good workability could be prepared. The alloys are expected to be used in the preparation of various implantable medical devices.

A Novel Human Placental Barrier Model Based on Trophoblast Stem Cells Derived from Human Induced Pluripotent Stem Cells.

The placenta acts as an interface between the fetus and the expecting mother. Various drugs and environmental pollutants can pass through the human placental barrier and may harm the developing fetus. Currently available in vitro placental barrier models are often inadequate, because they are lacking the functional trophoblast cells. Therefore, we developed and characterized a new human placental model using trophoblast stem cells (TSCs) derived from human induced pluripotent stem cells. Umbilical vein endothelial cells, fibroblast, and TSCs were cocultured using micromesh cell culture technique. These cells formed a tight three-layered structure. This coculture model induced progressive fusion of TSCs and formed a syncytialized epithelium that resembles the in vivo syncytiotrophoblast. Our model allowed the cultured trophoblasts to form microvilli and to reconstitute expression and physiological localization of membrane transport proteins, such as transporter for ATP-binding cassette subfamily B member 1, ATP-binding cassette subfamily C member 3, and glucose transporter-1. Drug permeability assays were performed using five compounds. The results from the permeability assays were comparable to the ones obtained with ex vivo placental models. In conclusion, we developed a novel coculture model mimicking human placenta that provides a useful tool for the studies on transfer of substances between the mother and fetus. Impact statement Compared with the currently available in vitro placental barrier models, a novel three-dimensional coculture placental barrier model presented in this study morphologically and functionally modeled the true placental barrier. The use of human trophoblast stem cells from human induced pluripotent stem cells substantially improved the current model. The use of micromesh sheet as a bioscaffold facilitated the formation of a good multilayer structure, which is closer to the physical appearance of the placenta observed in human.

A comparative study of key physiological stem cell parameters between three human trophoblast cell lines.

Human trophoblast stem cells (TSCs) play a key role in the placenta. These cells are proliferative, undifferentiated, and can differentiate into mature trophoblast cell types. However, primary human TSCs are difficult to obtain. In our previous study, we established TSCs from human induced pluripotent stem cells (TShiPSC). Here, we aimed to characterize the identity of these TShiPSC cells by comparing them with BeWo choriocarcinoma cells and primary TSCs (CT cells). Compared with BeWo cells, CT and TShiPSC cells showed high secretion of human chorionic gonadotrophin (hCG) and syncytiotrophoblast differentiation ability. Global gene microarray analysis results showed that CT and TShiPSC cells, unlike BeWo cells, could be classified in the same group. Compared with BeWo cells, CT and TShiPSC cells showed high expression levels of TSC-specific genes and low expression of cancer adhesion and invasion genes. Analysis of placental barrier integrity showed that TShiPSC cells could form a good barrier. Prospective studies using TShiPSC cells hold great promise for elucidating the pathogenesis of infertility due to trophoblast defects.

Three-Dimensional Cell Sheet Construction Method with a Polyester Micromesh Sheet.

Cell sheet engineering has become important in a variety of fields, including regenerative medicine and transplantation. Our research group previously developed micromesh cultures that enable cells to form a cell sheet on a microstructured mesh sheet. Here, we present a more usable micromesh culture and devices that make it possible, aiming for widespread use. The devices are mainly constituted of a polyester micromesh sheet and three-dimensional (3D)-printed simple frames that fix the mesh sheet on it. Cells such as fibroblast Tig-1-20 cells, hepatoma HepG2 cells, or mesenchymal stem cells (MSC) were easily seeded on the polyester mesh sheet in the device and cultured for 16 days, which was followed by the formation of a 100-400-µm-thick cell sheet. The cell sheet was very robust, easy to handle, and could be readily removed from the device for subsequent analysis. Optical coherence tomography revealed the structure of the cell sheet as having the mesh sheet layer in the center of the cell sheet. Confocal microscopy demonstrated that Tig-1-20 cells in the cell sheet were aligned according to the shape of the mesh apertures, indicating that cell orientation can be controlled with this micromesh culture. As for another application, the device was used to construct a multilayered cell sheet that consists of three different types of cells. Furthermore, for mass production, the device frames were made using polyoxymethylene (POM) instead of 3D printing materials. Using the POM devices, a large MSC sheet for 10 cm dishes was successfully produced 7 days after cell seeding. This micromesh culture may become one of the useful cell sheet construction methods in future for medical and research fields. Impact statement Currently, cell sheets are constructed, for example, on a temperature-responsive polymer-coated dish or a porous membrane. These cell sheets are widely used but are not completely suitable in terms of robustness, ease of handling, cost, ease of microscopic cell observation, or nutrient supply. We previously reported that the micromesh culture can provide a three-dimensional (3D) cell sheet that has advantages for cell observation and nutrient supply. In this study, the micromesh culture was enhanced with a polyester micromesh sheet and a series of devices of polyoxymethylene, helping us to produce a robust, cost-effective, easily layered, and easy-to-use 3D cell sheet.

Compact fluidic system for functional assessment of pancreatic islets.

Transplantation of pancreatic islets is becoming a promising therapy for people with type I diabetes. In this study, we present a compact fluidic system that enables assessment of islet functionality ex vivo for efficient islet transplantation. The fluidic system includes a micromesh sheet-embedded chip. Islets can be loaded easily on the micromesh sheet and observed clearly by microscopy. Islets on the mesh sheet mainly remained in place during perfusion and did not get damaged by hydraulic pressure because of high porosity of the micromesh sheet. The fluidic system was assembled with a sample fraction chip of polydimethylsiloxane. The chip includes a channel and columns, both having surfaces that were super-hydrophilized so that solutions could flow smoothly within the chip by gravity. Using mouse pancreatic islets, a dynamic glucose-stimulated insulin secretion test was performed to examine the performance of the fluidic system. The system successfully analyzed levels and patterns of insulin secretion upon exposure of the islets to low- and high-glucose solutions in turns, thus demonstrating its capacity to assess islet functions more easily and cost-effectively.

Tuning intercellular adhesion with membrane-anchored oligonucleotides.

Adhesive interactions between cells play an integral role in development, differentiation and regeneration. Existing methods for controlling cell-cell cohesion and adhesion by manipulating protein expression are constrained by biological interdependencies, e.g. coupling of cadherins to actomyosin force-feedback mechanisms. We use oligonucleotides conjugated to PEGylated lipid anchors (ssDNAPEGDPPE) to introduce artificial cell-cell adhesion that is largely decoupled from the internal cytoskeleton. We describe cell-cell doublets with a mechanical model based on isotropic, elastic deformation of spheres to estimate the adhesion at the cell-cell interface. Physical manipulation of adhesion by modulating the PEG-lipid to ssDNAPEGDPPE ratio, and conversely treating with actin-depolymerizing cytochalasin D, resulted in decreases and increases in doublet contact area, respectively. Our data are relevant to the ongoing discussion over mechanisms of tissue surface tension and in agreement with models based on opposing cortical and cohesive forces. PEG-lipid modulation of doublet geometries resulted in a well-defined curve indicating continuity, enabling prescriptive calibration for controlling doublet geometry. Our study demonstrates tuning of basic doublet adhesion, laying the foundation for more complex multicellular adhesion control independent of protein expression.

Establishment of human trophoblast stem cells from human induced pluripotent stem cell-derived cystic cells under micromesh culture.

BACKGROUND: Trophoblasts as a specific cell lineage are crucial for the correct function of the placenta. Human trophoblast stem cells (hTSCs) are a proliferative population that can differentiate into syncytiotrophoblasts and extravillous cytotrophoblasts. Many studies have reported that chemical supplements induce the differentiation of trophoblasts from human induced pluripotent stem cells (hiPSCs). However, there have been no reports of the establishment of proliferative hTSCs from hiPSCs. Our previous report showed that culturing hiPSCs on micromesh as a bioscaffold induced cystic cells with trophoblast properties. Here, we aimed to establish hTSCs from hiPSCs. METHODS: We used the micromesh culture technique to induce hiPSC differentiation into trophoblast cysts. We then reseeded and purified cystic cells. RESULTS: The cells derived from the reseeded cysts were highly proliferative. Low expression levels of pluripotency genes and high expression levels of TSC-specific genes were detected in proliferative cells. The cells could be passaged, and further directional differentiation into syncytiotrophoblast- and extravillous cytotrophoblast-like cells was confirmed by marker expression and hormone secretion. CONCLUSIONS: We established hiPSC-derived hTSCs, which may be applicable for studying the functions of trophoblasts and the placenta. Our experimental system may provide useful tools for understanding the pathogenesis of infertility owing to trophoblast defects in the future.

Chemically defined conditions for long-term maintenance of pancreatic progenitors derived from human induced pluripotent stem cells.

Large numbers of hormone-releasing cells, approximately 109 endocrine cells, are required to treat type I diabetes patients by cell transplantation. The SOX9-positive pancreatic epithelium proliferates extensively during the early stages of pancreatic development. SOX9-positive pancreatic epithelium is thought to be an expandable cell source of ß cells for transplantation therapy. In this study, we attempted to expand pancreatic progenitors (PPs: PDX1+/SOX9+) derived from four human iPSC lines in three-dimensional (3D) culture using a chemically defined medium and examined the potential of the derived PPs to differentiate into ß-like cells. PPs from four human iPSC lines were maintained and effectively proliferated in a chemically defined medium containing epidermal growth factor and R-spondin-1, CHIR99021, fibroblast growth factor-7, and SB431542. PPs derived from one iPSC line can be expanded by more than 104-fold in chemically defined medium containing two of the fives, epidermal growth factor and R-spondin-1. The expanded PPs were also stable following cryopreservation. After freezing and thawing, the PPs proliferated without a decrease in the rate. PPs obtained after 50 days of culture successfully differentiated into insulin-positive ß-like cells, glucagon-positive α-like cells, and somatostatin-positive δ-like cells. The differentiation efficiency of expanded PPs was similar to that of PPs without expansion culture.

Induction of in Vivo Ectopic Hematopoiesis by a Three-Dimensional Structured Extracellular Matrix Derived from Decellularized Cancellous Bone.

An in vitro blood production system could be an alternative to blood donation. We constructed a hematopoietic microenvironment using decellularized cancellous bones (DCBs) as scaffolds to sustain hematopoietic stem cells and supporting cells. The subcutaneous implantation of DCBs into mice with or without human mesenchymal stem cells (hMSCs) revealed that regardless of the presence of hMSCs DCBs were recellularized by some host cells and induced hematopoiesis. The ability of DCB to promote hematopoiesis was investigated by focusing on the components and the structure of cancellous bone, specifically reticular and adipose tissues and trabecular bone. Two decellularization methods were used to prepare DCBs. The DCBs differed concerning reticular tissue and adipose tissue. DCBs with these tissues could be recellularized at the original cellular location. An implantation experiment with DCBs revealed that they were very favorable for the persistent homing of hematopoietic stem cells. In addition, DCBs promoted ectopic hematopoiesis. The findings indicate that reticular tissues are important in directing hematopoiesis of DCBs.

Preparation of Immunotolerant Space Under the Skin and Transplantation of Islets in the Space.

IMPACT STATEMENT: Although transplantation of islets of Langerhans has been accepted as a fundamental treatment for insulin-dependent diabetes mellitus (IDDM), several problems are still remaining in order that it becomes the standard medical treatment of IDDM patients. In this study, using diabetic rat models, a subcutaneous space surrounded with highly vascularized granulomatous tissue was formed by agarose-bFGF rod implantation. Allogeneic islets transplanted into the space could survive and release insulin for a long period under no immunosuppressive medication. In conjunction with sufficient supply of islets from iPS/ES cells, our method can make islet transplantation the standard medical treatment of IDDM patients.

Bioabsorbable device to prepare subcutaneous pockets for islet transplantation.

Small invasive transplantation of islets and long maintenance of the islet graft without immunosuppression has been studied for the treatment of type 1 diabetes. Clinically, subcutaneous pockets surrounding vascular-rich tissue are prepared for islet transplantation using a device made of the materials. Here, gelatin sheets were implanted into two dorsal subcutaneous sites in diabetic ACI rats, and a mixture of bFGF and sodium hyaluronate solution was injected around the gelatin sheets. A total of 1500 islets isolated from F344 rats were transplanted into each of the pockets 7 days after injection of the bFGF mixture. Nine of 10 diabetic ACI rats with allogeneic islet graft demonstrated long-term normoglycemia without administration of immunosuppressant. Gelatin sheets almost disappeared 67 days after implantation. Thus, subcutaneous immune-tolerant sites can be prepared using gelatin sheets and a sodium hyaluronate-bFGF mixture. Allogeneic islets transplanted into the sites can survive and control blood glucose levels for a long period, even without immunosuppression. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1107-1112, 2019.

Development of trophoblast cystic structures from human induced pluripotent stem cells in limited-area cell culture.

We developed a novel engineering technique to induce differentiation of human induced pluripotent stem cells (hiPSCs) into organoids mimicking the trophectoderm (TE). Here, hiPSCs were cultured on a limited area of 2-4 mm in diameter. After 15-20 days, spherical cysts appeared on the surface of the limited area. Secretion of human chorionic gonadotrophin (hCG) began to increase after ∼ 20 days and remained dramatically elevated over the next 20 days. Limited-area-cultured cysts exhibited expression of hCG, which was a result of epithelial differentiation. Low expression levels of pluripotent genes and high expression levels of trophoblast lineage-specific genes were detected in the cells of spherical cysts. Multinucleated syncytia trophoblast was observed in the reseeded cystic cells. We observed hiPSC-derived cysts that morphologically resembled trophectoderm in vivo. The limited-area cell culture induced a three-dimensional (3D) trophectoderm organoid, which has potential for use in the study of human trophoblast differentiation and placental morphogenesis.

Design of Bioartificial Pancreases From the Standpoint of Oxygen Supply.

A bioartificial pancreas (BAP), in which islets of Langerhans (islets) are enclosed in a semipermeable membrane, has been developed to realize islet transplantation without the use of immunosuppressive drugs. Although recent progress in induced pluripotent stem (iPS) and embryonic stem (ES) cells has attracted attention owing to the potential applications of these cells as insulin-releasing cells, concerns about the safety of implantation of these cells remain. The use of the BAP has the advantage of easy removal if insulin-releasing cells derived from iPS/ES cells undesirably proliferate and form tumors in the BAP. Oxygen supply is a crucial issue for cell survival in BAPs as insufficient oxygen supply causes central necrosis of cell aggregates. In this study, we derived several simple equations considering oxygen supply in BAPs in order to provide insights into the rational design of three different types of BAPs (spherical microcapsules, cylindrical capsules, and planar capsules). The equations give (i) the thickness of a capsule membrane leading to no central necrosis of encapsulated cell aggregates as a function of the original size of the cell aggregate; (ii) the oxygen concentration profiles in BAPs; (iii) the effects of encapsulation of a cell aggregate on insulin release; (iv) the amount of encapsulated cells required to normalize blood glucose levels of a patient; and (v) the total volumes and sizes of BAPs. As an example, we used the equations in order to design three different types of BAPs for subcutaneous implantation.

Long-term Functioning of Allogeneic Islets in Subcutaneous Tissue Pretreated With a Novel Cyclic Peptide Without Immunosuppressive Medication.

BACKGROUND: There exists a need for a minimally invasive method of islet transplantation without immunosuppressive drugs for the treatment of type 1 diabetes. METHODS: In diabetic August Copenhagen Irish rats, an agarose rod containing the cyclic oligopeptide SEK-1005 (agarose-SEK rod) was implanted at 2 dorsal subcutaneous sites. Then these rods were removed, and 1500 islets of Langerhans isolated from Fischer 344 rats were transplanted into each of the pockets. RESULTS: Ten days after implantation of agarose-SEK rods, vascularized pockets were present. Nonfasting blood glucose levels confirmed long-term survival of the allogeneic islet grafts, without immunosuppressive therapy, in 8 of 10 recipients. Flow cytometry and gene expression analyses were performed to investigate the mechanisms underlying graft acceptance. Agarose-SEK rod implantation led to the formation of granulomatous tissue containing regulatory T cells that suppressed immune reactions against the allogeneic islet grafts. CONCLUSIONS: These results indicate that the use of an agarose-SEK rod to prevascularize a subcutaneous site may be a useful method for achieving successful allogeneic islet transplantation without immunosuppression.

Evaluation of the Perceptual Characteristics of a Force Induced by Asymmetric Vibrations.

This paper describes the perceptual characteristics of a sense of a force induced by asymmetric vibration using a vibration speaker-type non-grounded haptic interface. We confirm that the vibration speaker generates a perceived force that pulls or pushes a user's hand in a particular direction when an asymmetric amplitude signal that is generated by inverting a part of a sine wave is input. In this paper, to verify the system with respect to various factors of force perception caused by asymmetric vibration, we conducted six experiments and the following results were obtained. (1) The force vector can be controlled by reversing the asymmetric waves. (2) By investigating the physical characteristics of the vibration, asymmetric vibration was confirmed. (3) The presentation of vibration in the shear direction on the finger pad is effective. (4) The point of subjective equality of the perceived force can be controlled by up to 0.43 N by changing the amplitude voltage of the input signals. (5) The minimum stimulation time required for force perception is 66.7 ms. (6) When the vibration is continuously presented for 40 to 50 s, the perceived force decreases because of adaptation. Hence, we confirmed that we can control both the direction and magnitude of the reaction force by changing the input signal of the vibration speaker.

Encountered-Type Haptic Interface for Representation of Shape and Rigidity of 3D Virtual Objects.

This paper describes the development of an encountered-type haptic interface that can generate the physical characteristics, such as shape and rigidity, of three-dimensional (3D) virtual objects using an array of newly developed non-expandable balloons. To alter the rigidity of each non-expandable balloon, the volume of air in it is controlled through a linear actuator and a pressure sensor based on Hooke's law. Furthermore, to change the volume of each balloon, its exposed surface area is controlled by using another linear actuator with a trumpet-shaped tube. A position control mechanism is constructed to display virtual objects using the balloons. The 3D position of each balloon is controlled using a flexible tube and a string. The performance of the system is tested and the results confirm the effectiveness of the proposed principle and interface.

Preparation of an Au-Pt alloy free from artifacts in magnetic resonance imaging.

PURPOSE: When magnetic resonance imaging (MRI) is performed on patients carrying metallic implants, artifacts can disturb the images around the implants, often making it difficult to interpret them appropriately. However, metallic materials are and will be indispensable as raw materials for medical devices because of their electric conductivity, visibility under X-ray fluoroscopy, and other favorable features. What is now desired is to develop a metallic material which causes no artifacts during MRI. MATERIALS AND METHODS: In the present study, we prepared a single-phase and homogeneous Au-Pt alloys (Au; diamagnetic metal, and Pt; paramagnetic metal) by the processing of thermal treatment. Volume magnetic susceptibility was measured with a SQUID Flux Meter and MRI artifact was evaluated using a 1.5-T scanner. RESULTS: After final thermal treatment, an entirely recrystallized homogeneous organization was noted. The Au-35Pt alloy was shown to have a volume magnetic susceptibility of -8.8ppm, causing almost free from artifacts during MRI. CONCLUSIONS: We thus prepared an Au-35Pt alloy which had a magnetic susceptibility very close to that of living tissue and caused much fewer artifacts during MRI. It is promising as a material for spinal cages, intracranial electrodes, cerebral aneurysm embolization coils, markers for MRI and so on.

Closed-channel culture system for efficient and reproducible differentiation of human pluripotent stem cells into islet cells.

Human pluripotent stem cells (hPSCs) are thought to be a promising cell-source solution for regenerative medicine due to their indefinite proliferative potential and ability to differentiate to functional somatic cells. However, issues remain with regard to achieving reproducible differentiation of cells with the required functionality for realizing human transplantation therapies and with regard to reducing the potential for bacterial or fungal contamination. To meet these needs, we have developed a closed-channel culture device and corresponding control system. Uniformly-sized spheroidal hPSCs aggregates were formed inside wells within a closed-channel and maintained continuously throughout the culture process. Functional islet-like endocrine cell aggregates were reproducibly induced following a 30-day differentiation protocol. Our system shows an easily scalable, novel method for inducing PSC differentiation with both purity and functionality.

Interaction of poly(ethylene glycol)-conjugated phospholipids with supported lipid membranes and their influence on protein adsorption.

We studied real-time interaction between poly(ethylene glycol)-conjugated phospholipids (PEG-lipids) and a supported lipid membrane by surface plasmon resonance (SPR) spectroscopy to understand dynamic behaviors of PEG-lipids on living cell membranes. Supported lipid membranes formed on a hydrophobic surface were employed as a model of living cell membrane. We prepared three kinds of PEG-lipids that carried alkyl chains of different lengths for SPR measurements and also performed fluorescence recovery after photobleaching (FRAP) to study the influence of acyl chain length on dynamics on the supported membrane. PEG-lipids were uniformly anchored to lipid membranes with high fluidity without clustering. Incorporation and dissociation rates of PEG-lipids into supported membranes strongly depended on the length of acyl chains; longer acyl chains reduced the incorporation rate and the dissociation rate of PEG-lipid. Furthermore, protein adsorption experiment with bovine serum albumin indicated that PEG modification prevented the adsorption of bovine serum albumin on such supported membrane.