In-Vitro Decidualization With Different Progesterone Concentration: Development of a Hormone-Responsive 3D Endometrial Tissue Using Rat Endometrial Tissues.

Infertility in women is associated with various uterine and ovarian disorders. Treatment strategies for infertility can range from medications to embryo implantation through assisted reproductive technology (ART). ART has enabled considerable progress; however, there is currently no treatment to replace the endometrium itself. Decidualization requires a complex interaction between endometrial tissue and estrogen and progesterone. We aimed to create a three-dimensional endometrial-like tissue model using in-vitro cell sheet engineering with rat endometrium, and culture cells at different progesterone concentrations to mimic local concentrations. Histological and morphological changes revealed that development of the endometrial-like tissue was not proportional to progesterone concentrations in terms of thickness, number of endometrial glands, or area fraction of intimal glands. These results suggest that decidualization may not be commensurate with the local endometrial progesterone concentration. Notably, the number of endometrial glands increased in the high concentration group and compaction occurred, indicating that the endometrial conditions in the high concentration group may be most conducive to increase pregnancy rates. These findings suggest that there may be an "optimal progesterone concentration" for decidualization, application of which may lead to new strategies for improving pregnancy rates in women with infertility.

The bioengineering of perfusable endocrine tissue with anastomosable blood vessels.

Organ transplantation is a definitive treatment for endocrine disorders, but donor shortages limit the use of this technique. The development of regenerative therapies would revolutionize the treatment of endocrine disorders. As is the case for harvested organs, the ideal bioengineered graft would comprise vascularized endocrine tissue, contain blood vessels that could be anastomosed to host vessels, have stable blood flow, and be suitable for transplantation into various sites. Here, we describe a transplantable endocrine tissue graft that was fabricated byex vivoperfusion of tricultured cell sheets (isletß-cells, vascular endothelial cells (vECs), and mesenchymal stem cells (MSCs)) on a vascularized tissue flap ofin vivoorigin. The present study has three key findings. First, mild hypothermic conditions enhanced the success ofex vivoperfusion culture. Specifically, graft construction failed at 37 °C but succeeded at 32 °C (mild hypothermia), and endocrine tissue fabricated under mild hypothermia contained aggregations of isletß-cells surrounded by dense vascular networks. Second, the construction of transplantable endocrine tissue byex vivoperfusion culture was better achieved using a vascular flap (VF) than a muscle flap. Third, the endocrine tissue construct generated using a VF could be transplanted into the rat by anastomosis of the graft artery and vein to host blood vessels, and the graft secreted insulin into the host's circulatory system for at least two weeks after transplantation. Endocrine tissues bioengineered using these techniques potentially could be used as novel endocrine therapies.

Tricultured Cell Sheets Develop into Functional Pancreatic Islet Tissue with a Vascular Network.

Methods to induce islet ß-cells from induced pluripotent stem cells or embryonic stem cells have been established. However, islet ß-cells are susceptible to apoptosis under hypoxic conditions, so the technique used to transplant ß-cells must maintain the viability of cells in vivo. This study describes the development of a tricultured cell sheet, which was made by coculturing islet ß-cells, vascular endothelial cells, and mesenchymal stem cells for 1 day. The islet ß-cells in the tricultured cell sheet self-organized into islet-like structures surrounded by a dense vascular network in vitro. Triple-layered tricultured cell sheets engrafted well after transplantation in vivo and developed into insulin-secreting tissue with abundant blood vessels and a high density of islet ß-cells. We anticipate that the tricultured cell sheet could be used as an in vitro pseudo-islet model for pharmaceutical testing and may have potential for development into transplantable grafts for use in regenerative medicine. Impact statement This research assessed whether tricultured cell sheets containing islet ß-cells, vascular endothelial cells, and mesenchymal stem cells were able to form islet tissue. There were two main findings. First, the islet ß-cells in the tricultured cell sheet self-organized into islet structures surrounded by a dense vascular network in vitro. Second, triple-layered tricultured sheets engrafted well onto rat muscle and developed into insulin-secreting tissue with an abundance of blood vessels. The tricultured cell sheet could be used as a pseudo-islet model for pharmaceutical testing and may have potential for development into a transplantable graft for application in the clinical setting.

Networked lymphatic endothelial cells in a transplanted cell sheet contribute to form functional lymphatic vessels.

This study evaluated whether cell sheets containing a network of lymphatic endothelial cells (LECs) promoted lymphangiogenesis after transplantation in vivo. Cell sheets with a LEC network were constructed by co-culturing LECs and adipose-derived stem cells (ASCs) on temperature-responsive culture dishes. A cell ratio of 3:2 (vs. 1:4) generated networks with more branches and longer branch lengths. LEC-derived lymphatic vessels were observed 2 weeks after transplantation of a three-layered cell sheet construct onto rat gluteal muscle. Lymphatic vessel number, diameter and depth were greatest for a construct comprising two ASC sheets stacked on a LEC/ASC (3:2 ratio) sheet. Transplantation of this construct in a rat model of femoral lymphangiectomy led to the formation of functional lymphatic vessels containing both transplanted and host LECs. Further development of this technique may lead to a new method of promoting lymphangiogenesis.

External pressure dynamics promote kidney viability and perfusate filtration during ex vivo kidney perfusion.

Normothermic machine perfusion (NMP) has not yet been established as a technique for preserving organs for a day. A key contributing factor to the same is that the perfusing solutions cannot circulate continuously and evenly in the organs. Here, we conceived a method of applying intermittent air pressure from outside the organ to assist its circulatory distribution during perfusion. We used a perfusion culture system while applying external pressure to culture rat kidneys and compared the circulatory distribution in the kidneys, changes in tissue morphology due to injury, and perfusate filtration. The intermittent pressurization (IMP) (-) group showed markedly poorer circulation on the upper side compared with that in the lower side, alongside histological damage. On the other hand, the IMP (+) group showed improved circulation in the upper side and had lesser histological damage. Furthermore, the IMP (+) group maintained the ability to filter perfusate for 24 h. In transplantation medicine and regenerative medicine research, this method has the potential to contribute to more efficient organ preservation and more functional tissue regeneration in the future.

Development of appropriate fatty acid formulations to raise the contractility of constructed myocardial tissues.

Introduction: Heart disease is a major cause of mortality worldwide, and the annual number of deaths due to heart disease has increased in recent years. Although heart failure is usually managed with medicines, the ultimate treatment for end-stage disease is heart transplantation or an artificial heart. However, the use of these surgical strategies is limited by issues such as thrombosis, rejection and donor shortages. Regenerative therapies, such as the transplantation of cultured cells and tissues constructed using tissue engineering techniques, are receiving great attention as possible alternative treatments for heart failure. Research is ongoing into the potential clinical use of cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs). However, the energy-producing capacity of cardiomyocytes maintained under previous culture conditions is lower than that of adult primary cardiomyocytes due to immaturity and a reliance on glucose metabolism. Therefore, the aims of this study were to compare the types of fatty acids metabolized between cardiomyocytes in culture and heart cells in vivo and investigate whether the addition of fatty acids to the culture medium affected energy production by cardiomyocytes. Methods: A fatty acid-containing medium was developed based on an analysis of fatty acid consumption by rat primary cardiomyocytes (rat-CMs), and the effects of this medium on adenosine triphosphate (ATP) production were investigated through bioluminescence imaging of luciferase-expressing rat-CMs. Next, the fatty acid content of the medium was further adjusted based on analyses of fatty acid utilization by porcine hearts and hiPSC-CMs. Oxygen consumption analyses were performed to explore whether the fatty acid-containing medium induced hiPSC-CMs to switch from anaerobic metabolism to aerobic metabolism. Furthermore, the effects of the medium on contractile force generated by hiPSC-CM-derived tissue were evaluated. Results: Rat serum, human serum and porcine plasma contained similar types of fatty acid (oleic acid, stearic acid, linoleic acid, palmitic acid and arachidonic acid). The types of fatty acid consumed were also similar between rat-CMs, hiPSC-CMs and porcine heart. The addition of fatty acids to the culture medium increased the bioluminescence of luciferase-expressing rat-CMs (an indirect measure of ATP level), oxygen consumption by hiPSC-CMs, and contractile force generated by cardiac tissues constructed from hiPSC-CMs. Conclusions: hiPSC-CMs metabolize similar types of fatty acid to those consumed by rat-CMs and porcine hearts. Furthermore, the addition of these fatty acids to the culture medium increased energy production by rat-CMs and hiPSC-CMs and enhanced the contractility of myocardial tissue generated from hiPSC-CMs. These findings suggest that the addition of fatty acids to the culture medium stimulates aerobic energy production by cardiomyocytes through ß-oxidation. Since cardiomyocytes cultured in standard media rely primarily on anaerobic glucose metabolism and remain in an immature state, further research is merited to establish whether the addition of fatty acids to the culture medium would improve the energy-producing capacity and maturity of hiPSC-CMs and cardiac tissue constructed from these cells. It is possible that optimizing the metabolism of cultured cardiomyocytes, which require high energy production to sustain their contractile function, will improve the properties of hiPSC-CM-derived tissue, allowing it to be better utilized for disease modeling, drug screening and regenerative therapies for heart failure.

In utero transplantation of myoblasts and adipose-derived mesenchymal stem cells to murine models of Duchenne muscular dystrophy does not lead to engraftment and frequently results in fetal death.

Introduction: Duchenne muscular dystrophy (DMD) is a progressive disease that leads to damage of muscle and myocardium due to genetic abnormalities in the dystrophin gene. In utero cell transplantation that might facilitate allogenic transplantation is worth considering to treat this disease. Methods: We performed allogeneic in utero transplantation of GFP-positive myoblasts and adipose-derived mesenchymal stem cells into murine DMD model animals. The transplantation route in this study was fetal intraperitoneal transplantation and transplacental transplantation. Transplanted animals were examined at 4-weeks old by immunofluorescence staining and RT-qPCR. Results: No GFP-positive cells were found by immunofluorescence staining of skeletal muscle and no GFP mRNA was detected by RT-qPCR in any animal, transplantation method and cell type. Compared with previous reports, myoblast transplantation exhibited an equivalent mortality rate, but adipose-derived stem cell (ASC) transplantation produced a higher mortality rate. Conclusions: In utero transplantation of myoblasts or ASCs to murine models of DMD does not lead to engraftment and, in ASC transplantation primarily, frequently results in fetal death.

Organ Bioluminescence Imaging Under Machine Perfusion Setting for Assessing Quality of Harvested Organ Preservation.

Determination of organ viability over a period of time is a key technology in the process of organ preservation. However, a robust methodology to address this issue has not been established. Luciferase-expressing organs enable the assessment of the variances in organ viability over time as well as the visualization of a damaged tissue region. Herein, we introduce the assessment method for organ viability in detail using luciferase-expressing organs harvested from transgenic Lewis rats (Luc-LEW Tg rats). We exemplify the femoral muscle pedicle flap for the methods of tissue preparation, of setting up the machine perfusion system, and of measuring emitted light to assess organ viability. This evaluation method would be applicable to other organ-preservation studies as an innovative tool for developing a profound understanding of organ preservation.

Laminin-221-derived recombinant fragment facilitates isolation of cultured skeletal myoblasts.

Introduction: Laminin is a major component of the basement membrane, containing multiple domains that bind integrin, collagen, nidogen, dystroglycan, and heparan sulfate. Laminin-221, expressed in skeletal and cardiac muscles, has strong affinity for the cell-surface receptor, integrin α7X2ß1. The E8 domain of laminin-221, which is essential for cell integrin binding, is commercially available as a purified recombinant protein fragment. In this study, recombinant E8 fragment was used to purify primary rodent myoblasts. We established a facile and inexpensive method for primary myoblast culture exploiting the high affinity binding of integrin α7X2ß1 to laminin-221. Methods: Total cell populations from dissociated muscle tissue were enzymatically digested and seeded onto laminin-221 E8 fragment-coated dishes. The culture medium containing non-adherent floating cells was removed after 2-hour culture at 37 °C. The adherent cells were subjected to immunofluorescence staining of desmin, differentiation experiments, and gene expression analysis. Results: The cells obtained were 70.3 ± 5.49% (n = 5) desmin positive in mouse and 67.7 ± 1.65% (n = 3) in rat. Immunofluorescent staining and gene expression analyses of cultured cells showed phenotypic traits of myoblasts. Conclusion: This study reports a novel facile method for primary culture of myoblasts obtained from mouse and rat skeletal muscle by exploiting the high affinity of integrin α7X2ß1 to laminin-221.

A novel alveolar epithelial cell sheet fabricated under feeder-free conditions for potential use in pulmonary regenerative therapy.

Introduction: Lung transplantation is the only effective treatment option for many patients with irreversible pulmonary injury, and the demand for lung transplantation is increasing worldwide and expected to continue to outstrip the number of available donors. Regenerative therapy with alveolar epithelial cells (AECs) holds promise as an alternative option to organ transplantation. AECs are usually co-cultured with mouse-derived 3T3 feeder cells, but the use of xenogeneic tissues for regenerative therapy raises safety concerns. Fabrication of AEC sheets under feeder-free conditions would avoid these safety issues. We describe a novel feeder-free method of fabricating AEC sheets that may be suitable for pulmonary regenerative therapy. Methods: Lung tissues excised from male outbred rats or transgenic rats expressing green fluorescent protein (GFP) were finely minced and dissociated with elastase. The isolated AECs were cultured under four different feeder-free conditions according to whether a rho kinase (ROCK) inhibitor was included in the low-calcium medium (LCM) and whether the tissue culture dish was coated with recombinant laminin-511 E8 fragment (rLN511E8). The expanded cells were cultured on temperature-responsive dishes and subsequently harvested as AEC sheets. Engraftment of GFP-AEC sheets after their transplantation onto a partially resected region of the left lung was assessed in athymic rats. Results: AECs proliferated and reached confluence when cultured in LCM containing a ROCK inhibitor on tissue culture dishes coated with rLN511E8. When both the ROCK inhibitor and rLN511E8-coated culture dish were used, the number of AECs obtained after 7 days of culture was significantly higher than that in the other three groups. Immunohistochemical analyses revealed that aquaporin-5, surfactant protein (SP)-A, SP-C, SP-D and Axin-2 were expressed by the cultured AECs. AEC sheets were harvested successfully from temperature-responsive culture dishes (by lowering the temperature) when the expanded AECs were cultured for 7 days in LCM + ROCK inhibitor and then for 3 days in LCM + ROCK inhibitor supplemented with 200 mg/L calcium chloride. The AEC sheets were firmly engrafted 7 days after transplantation onto the lung defect and expressed AEC marker proteins. Conclusions: AEC sheets fabricated under feeder-free conditions retained the features of AECs after transplantation onto the lung in vivo. Further improvement of this technique may allow the bioengineering of alveolar-like tissue for use in pulmonary regenerative therapy.

Bioartificial pulsatile cuffs fabricated from human induced pluripotent stem cell-derived cardiomyocytes using a pre-vascularization technique.

There is great interest in the development of techniques to bioengineer pulsatile myocardial tissue as a next-generation regenerative therapy for severe heart failure. However, creation of thick myocardial grafts for regenerative medicine requires the incorporation of blood vessels. In this study, we describe a new method of constructing a vascular network in vivo that allows the construction of thick human myocardial tissue from multi-layered cell sheets. A gelatin sheet pre-loaded with growth factors was transplanted onto the superficial femoral artery and vein of the rat. These structures were encapsulated together within an ethylene vinyl alcohol membrane and incubated in vivo for 3 weeks (with distal superficial femoral artery ligation after 2 weeks to promote blood flow to the vascular bed). Subsequently, six cardiomyocyte sheets were transplanted onto the vascular bed in two stages (three sheets, two times). Incubation of this construct for a further week generated vascularized human myocardial tissue with an independent circulation supplied by an artery and vein suitable for anastomosis to host vessels. Notably, laminating six cell sheets on the vascular bed in two stages rather than one allowed the creation of thicker myocardial tissue while suppressing tissue remodeling and fibrosis. Finally, the pulsatile myocardial tissue was shown to generate auxiliary pressure when wrapped around the common iliac artery of a rat. Further development of this technique might facilitate the generation of circulatory assist devices for patients with heart failure.

Aligned human induced pluripotent stem cell-derived cardiac tissue improves contractile properties through promoting unidirectional and synchronous cardiomyocyte contraction.

Alignment, as seen in the native myocardium, is crucial for the fabrication of functional cardiac tissue. However, it remains unclear whether the control of cardiomyocyte alignment influences cardiac function and the underlying mechanisms. We fabricated aligned human cardiac tissue using a micro-processed fibrin gel with inverted V-shaped ridges (MFG) and elucidated the effect of alignment control on contractile properties. When human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were seeded on MFG, hiPSC-CMs were aligned more uniformly than the control, and we succeeded in fabricating the aligned cardiac tissue. Assessing the contractile properties with the direct contractile measurement system, the contractile force, maximum contractile velocity, and relaxation velocity were significantly increased in aligned cardiac tissue compared with non-aligned cardiac tissue. However, gene expression profiles were not different between the two groups, suggesting that functional improvement of cardiac tissue through alignment control might not be dependent on cardiomyocyte maturation. Motion capture analysis revealed that the cardiomyocytes in the aligned cardiac tissues showed more unidirectional and synchronous contraction than the non-aligned cardiac tissues, indicating that cardiac tissue maturation involves electrical integration of cardiomyocytes. Herein, cardiomyocyte alignment control might improve the contractile properties of cardiac tissue through promoting unidirectional and synchronous cardiomyocyte contraction.

Perfusable vascular tree like construction in 3D cell-dense tissues using artificial vascular bed.

Perfusable vascular structures in cell-dense tissues are essential for fabricating functional three-dimensional (3D) tissues in vitro. However, it is challenging to introduce functional vascular networks observable as vascular tree, finely spaced at intervals of tens of micrometers as in living tissues, into a 3D cell-dense tissue. Herein, we propose a method for introducing numerous vascular networks that can be perfused with blood into 3D tissues constructed by cell sheet engineering. We devise an artificial vascular bed using a hydrogel that is barely deformed by cells, enabling perfusion of the culture medium directly beneath the cell sheets. Triple-layered cell sheets with an endothelial cell network prepared by fibroblast co-culture are transplanted onto the vascular bed and subjected to perfusion culture. We demonstrate that numerous vascular networks are formed with luminal structures in the cell sheets and can be perfused with India ink or blood after a five-day perfusion culture. Histological analysis also demonstrates that perfusable vascular structures are constructed at least 100 µm intervals uniformly and densely within the tissues. The results suggest that our perfusion culture method enhances vascularization within the 3D cell-dense tissues and enables the introduction of functional vasculature macroscopically observable as vascular tree in vitro. In conclusion, this technology can be used to fabricate functional tissues and organs for regenerative therapies and in vitro experimental models.

Continuous measurement of surface electrical potentials from transplanted cardiomyocyte tissue derived from human-induced pluripotent stem cells under physiological conditions in vivo.

Recording the electrical potentials of bioengineered cardiac tissue after transplantation would help to monitor the maturation of the tissue and detect adverse events such as arrhythmia. However, a few studies have reported the measurement of myocardial tissue potentials in vivo under physiological conditions. In this study, human-induced pluripotent stem cell-derived cardiomyocyte (hiPSCM) sheets were stacked and ectopically transplanted into the subcutaneous tissue of rats for culture in vivo. Three months after transplantation, a flexible nanomesh sensor was implanted onto the hiPSCM tissue to record its surface electrical potentials under physiological conditions, i.e., without the need for anesthetic agents that might adversely affect cardiomyocyte function. The nanomesh sensor was able to record electrical potentials in non-sedated, ambulating animals for up to 48 h. When compared with recordings made with conventional needle electrodes in anesthetized animals, the waveforms obtained with the nanomesh sensor showed less dispersion of waveform interval and waveform duration. However, waveform amplitude tended to show greater dispersion for the nanomesh sensor than for the needle electrodes, possibly due to motion artifacts produced by movements of the animal or local tissue changes in response to surgical implantation of the sensor. The implantable nanomesh sensor utilized in this study potentially could be used for long-term monitoring of bioengineered myocardial tissue in vivo under physiological conditions.

A novel method to align cells in a cardiac tissue-like construct fabricated by cell sheet-based tissue engineering.

Fabrication of cardiac tissue from human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) has received great interest, but a major challenge facing researchers is the alignment of cardiomyocytes in the same direction to optimize force generation. We have developed a novel method of fabricating a cardiac tissue-like construct with aligned cells based on the unidirectional stretching of an hiPS-CM sheet. A square cell sheet was harvested from a temperature-responsive culture dish and placed on a silicone surface, and an extending force was imposed on the silicone to stretch the cell sheet along one direction. To enable evaluation of cardiomyocyte morphology in vitro, a cell sheet was constructed by coculture of hiPS-CMs and human adipose-derived stem cells. In separate experiments, a stretched double-layered cell sheet constructed from hiPS-CMs alone was transplanted onto the muscle of an athymic rat, and its features were compared with those of a nonstretched (control) cell sheet. Immediately after stretching, the stretched cell sheet was significantly longer than the control cell sheet. Immunohistological analysis revealed that the cardiomyocytes showed unidirectional alignment in the stretched cell sheet but random directionality in the control cell sheet. Two weeks after transplantation, immunohistology demonstrated that the stretched cell sheet had retained the unidirectionality of its myocardial fibers and had an orientation intensity that was higher than that of the control cell sheet after transplantation or the stretched cell sheet before transplantation. Our technique provides a simple method of aligning an hiPS-CM-derived cardiac tissue-like construct without the use of a scaffold.

A novel rat model of inflammatory bowel disease developed using a device created with a 3D printer.

OBJECTIVE: Inflammatory bowel disease (IBD) is an intractable condition. Existing models of experimental IBD are limited by their inability to create consistent ulcers between animals. The aim of this study was to develop a novel model of experimental colitis with ulcers of reproducible size. DESIGN: We used a 3D printer to fabricate a novel device containing a small window (10 × 10 mm) that could be inserted rectally to facilitate the creation of a localized ulcer in the rat intestinal mucosa. The mucosa within the window of the device was exposed to 2,4,6-trinitrobenzene sulfonic acid (TNBS) to generate ulceration. We evaluated the effects of conventional drug therapies (mesalazine and prednisolone) and local transplantation of allogeneic adipose-derived mesenchymal stem cells (ASCs) on ulcer size (measured from photographic images using image analysis software) and degree of inflammation (assessed histologically). RESULTS: The novel method produced localized, circular or elliptical ulcers that were highly reproducible in terms of size and depth. The pathological characteristics of the lesions were similar to those reported previously for conventional models of TNBS-induced colitis that show greater variation in ulcer size. Ulcer area was significantly reduced by the administration of mesalazine or prednisolone as an enema or localized injection of ASCs. CONCLUSION: The new model of TNBS-induced colitis, made with the aid of a device fabricated by 3D printing, generated ulcers that were reproducible in size. We anticipate that our new model of colitis will provide more reliable measures of treatment effects and prove useful in future studies of IBD therapies.

Intermittent application of external positive pressure helps to preserve organ viability during ex vivo perfusion and culture.

The perfusion of medium through blood vessels allows the preservation of donor organs and culture of bioengineered organs. However, tissue damage due to inadequate perfusion remains a problem. We evaluated whether intermittent external pressurization would improve the perfusion and viability of organs in culture. A bioreactor system was used to perfuse and culture rat small intestine and femoral muscle preparations. Intermittent positive external pressure (10 mmHg) was applied for 20 s at intervals of 20 s. Intermittent pressurization resulted in uniform perfusion of small intestine preparations and minimal tissue damage after 20 h of perfusion, whereas non-pressurized (control) preparations exhibited significantly worse perfusion of the upper surface than the lower surface and histologic evidence of tissue damage. Longer term studies were undertaken in luciferase-expressing rat femoral muscle preparations. Compared with non-pressurized controls, intermittent pressurization led to better perfusion throughout the 14-day experimental period, improved organ viability as indicated by a higher bioluminescence intensity after perfusion with luciferin, and reduced levels of tissue necrosis with better preservation of vascular structures and skeletal muscle nuclei (histologic analyses). Therefore, intermittent application of external positive pressure improved the perfusion of small intestine and skeletal muscle preparations and enhanced tissue viability when compared with controls. We anticipate that this innovative perfusion technique could be used to improve the preservation of donor organs and culture of bioengineered organs.

Correction to: Intermittent application of external positive pressure helps to preserve organ viability during ex vivo perfusion and culture.

article Intermittent application.

Sivelestat sodium hydrate treatment for refractory Kawasaki disease.

BACKGROUND: There is still no definite treatment for refractory Kawasaki disease (KD). In this pilot study, we evaluated the safety and efficacy of a new protocol consisting of sivelestat sodium hydrate (SSH) combined with additional i.v. immunoglobulin (IVIG) for KD resistant to initial IVIG therapy. METHODS: This study is a prospective non-randomized, open-label and single-arm study undertaken in a population of refractory KD patients at Chiba University Hospital from December 2006 to March 2016. The subjects had KD resistant to initial IVIG (2 g/kg) and received SSH (0.2 mg/kg/h for 5 days) combined with additional IVIG (2 g/kg) as a second-line therapy. We evaluated the safety and efficacy of the treatment during the study period. RESULTS: Forty-six KD patients were enrolled in this study and no serious adverse event was noted. Of these, 45 patients were evaluated for the incidence of coronary artery lesions, which occurred in one patient (2.2%; 95% CI: 0.5-15.2). Twenty-eight (62.2%) responded promptly and were afebrile after the therapy. The median total duration of fever was 8 days (range, 6-28 days). CONCLUSIONS: Additional IVIG combined with SSH as a second-line therapy for KD refractory to initial IVIG therapy was safe and well tolerated and could be a promising option for severe KD. Further investigations are expected to clarify the safety and timing of SSH treatment for KD.

Allogeneic adipose-derived mesenchymal stem cell sheet that produces neurological improvement with angiogenesis and neurogenesis in a rat stroke model.

OBJECTIVE: Stem cell therapy is a promising strategy for the treatment of severe cerebral ischemia. However, targeting sufficient grafted cells to the affected area remains challenging. Choosing an adequate transplantation method for the CNS appears crucial for this therapy to become a clinical reality. The authors used a scaffold-free cell sheet as a translational intervention. This method involves the use of cell sheet layers and allows the transplantation of a large number of cells, locally and noninvasively. The authors evaluated the effectiveness of allogeneic adipose tissue-derived mesenchymal stem cell sheets in a rat model of stroke. METHODS: The animals, subjected to middle cerebral artery occlusion, were randomly divided in two groups: one in which a cell sheet was transplanted and the other in which a vehicle was used (n = 10/group). Over a period of 14 days after transplantation, the animals' behavior was evaluated, after which brain tissue samples were removed and fixed, and the extent of angiogenesis and infarct areas was evaluated histologically. RESULTS: Compared to the vehicle group, in the cell sheet group functional angiogenesis and neurogenesis were significantly increased, which resulted in behavioral improvement. Transplanted cells were identified within newly formed perivascular walls as pericytes, a proportion of which were functional. Newly formed blood vessels were found within the cell sheet that had anastomosed to the cerebral blood vessels in the host. CONCLUSIONS: The transplantation approach described here is expected to provide not only a paracrine effect but also a direct cell effect resulting in cell replacement that protects the damaged neurovascular unit. The behavioral improvement seen with this transplantation approach provides the basis for further research on cell sheet-based regenerative treatment as a translational treatment for patients with stroke.