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Abstract 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.

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.

This paper describes the development of a gait rehabilitation system with a locomotion interface (LI) for home-visit rehabilitation. For this purpose, the LI should be compact, small, and easy to move. The LI has two 2 degree-of-freedom (DOF) manipulators with footpads to move each foot along a trajectory. When the user stands on the footpads, the system can move his or her feet while the body remains stationary. The footpads can have various trajectories, which are prerecordings of the movements of healthy individuals walking on plane surfaces or slopes. The homes of stroke patients may have not only flat surfaces but also some slopes and staircases. The quadriceps femoris muscle is important for walking up and down slopes and staircases, and the eccentric and concentric contractions of this muscle are, in particular, difficult to train under normal circumstances. Therefore, we developed a graded-walking program for the system used in this study. Using this system, the user can undergo gait rehabilitation in their home, during visits by a physical therapist. An evaluation of the results of tests showed that the vastus medialis muscles of all the subjects were stimulated more than by walking on real slopes.

Human mesenchymal stem cells (hMSCs) are promising feeder cells for expanding hematopoietic stem cells (HSCs), but their potential is heterogeneous. We examined the hematopoiesis-supporting activity of hMSC at the clonal level in relation to the osteogenic potential and gene expression. Hematopoiesis-supporting activities of stably immortalized clonal hMSC lines were evaluated by the expansion of CD34 + CD38 - cells after 7-day coculture with human cord blood-derived CD34 + cells. Six of 16 clones expanded the numbers of CD34 + CD38 - cells >500-fold. These hematopoiesis-supportive clones also showed high gene expression of Jagged1, a Notch ligand, as well as high potential to deposit calcium after osteogenic induction. Thus, osteogenic hMSC clones may provide proper microenvironments for HSC expansion, ultimately conveying self-renewal signals to HSCs via the Notch pathway. However, they lost hematopoiesis-supporting activity after osteogenic differentiation. The hematopoiesis-supportive clones are potentially useful for hematopoietic microenvironment studies and as components of a coculture system for expansion of HSCs, free from contamination by xenogeneic pathogens.

The long-term durability of agarose microencapsulated islets against autoimmunity was evaluated in NOD mice. Islets were isolated from 6-8-week-old prediabetic male NOD mice and microencapsulated in 5% agarose hydrogel. Microencapsulated or nonencapsulated islets were transplanted into the omental pouch of spontaneously diabetic NOD mice. Although the diabetic NOD mice that received nonencapsulated islets experienced a temporary reversal of their hyperglycemic condition, all 10 of these mice returned to hyperglycemia within 3 weeks. In contrast, 9 of 10 mice transplanted with microencapsulated islets maintained normoglycemia for more than 100 days. Islet grafts were removed at 100, 150, 200, 300, and 400 days posttransplantation. A prompt return to hyperglycemia was observed in the mice after graft removal, indicating that the encapsulated islet grafts were responsible for maintaining euglycemia. Histological examination revealed viable islets in the capsules at all time points of graft removal. In addition, β-cells within the capsules remained well granulated as revealed by the immunohistochemical detection of insulin. No immune cells were detected inside the microcapsules and no morphological irregularities of the microcapsules were observed at any time point, suggesting that the microcapsules successfully protected the islets from cellular immunity. Sufficient vascularization was evident close to the microcapsules. Considerable numbers of islets showed central necrosis at 400 days posttransplantation, although the necrotic islets made up only a small percentage of the islet grafts. Islets with central necrosis also showed abundant insulin production throughout the entire islets, except for the necrotic part. These results demonstrate the long-term durability of agarose microcapsules against autoimmunity in a syngeneic islet transplantation model in NOD mice.

We present a simple method for neural cell fate specification directly from mouse embryonic stem cells (ES cells) in serum-free conditions in the absence of embryoid body formation. Dissociated ES cells were cultured in serum-free media supplemented with vitamin B 12 and heparin, but without any expensive cytokines. After 14 days in culture, β-tubulin type III (TuJ1) and tyrosine hydroxylase (TH)-positive colonies were detected by immunocytochemical examinations. In addition, specific gene analyses by RT-PCR demonstrated expression of an early central nerve system, mature neuron, and midbrain dopaminergic neuron-specific molecules (i.e., nestin, middle molecular mass neurofilament protein, Nurr1, and TH, respectively). Dopamine was also detected in the culture media by reverse-phase HPLC analysis. These facts indicate that addition of vitamin B 12 /heparin to serum-free culture media induced neurons from ES cells, which included cells that released dopamine. Other supplements, such as putrescine, biotin, and Fe2+, could not induce neurons from ES cells by themselves, but produced synergistic effects with vitamin B 12 /heparin. The rate of TuJ1+/TH+ colony formation was increased threefold and the amounts of dopamine released increased 1.5fold by the addition of a mixture of putrescine, biotin, and Fe2+ to vitamin B 12 /heparin culture media. Our method is a simple tool to differentiate ES cells to dopaminergic neurons for the preparation of dopamine-releasing cells for the cell transplantation therapy of Parkinson's disease. In addition, this method can facilitate the discovery of soluble factors and genes that can aid in the induction of the ES cell to its neural fate.

In a previous experiment, we demonstrated the anticomplementary efficacy of poly(stryrene sulfonic acid) (PSSa) and poly(2-acrylamido-2-methyl propane sulfonic acid) (PAMPS). The aim of this study was to examine their influence on the function of pancreatic islets in vitro. In this study, after culturing the rat islets with RPMI-1640 culture medium containing different concentrations of soluble PSSa or PAMPS for 24 h at 37°C, we performed morphological and functional examination of the rat islets. We found that the islets maintained their normal morphology regardless of whether they were in the PSSa or PAMPS groups when the concentrations of soluble PSSa or PAMPS in the media were below 1 g/dl. In the static incubation study, the islets cultured in the PAMPS groups showed significantly high insulin secretory response to glucose challenge but those in the PSSa groups lost the response when the concentrations of soluble PSSa or PAMPS in the media were below 1 g/dl. The PAMPS not only had strong anticomplementry effect, but also maintained the good insulin secretory capacity of the islets. These results indicated that PAMPS is a promising bioartificial material for future clinical application of biohybrid artificial pancreas preparation. It is well suitable for xenotransplantation experiments.

In this study, the insulin secretory characteristics of the microencapsulated hamster islets were studied during long-term culture. The hamster islets were encapsulated as single-layer agarose microbeads or three-layer agarose microbeads with agarose and agarose containing poly(styrene sulfonic acid) (PSSa), respectively. The influence of PSSa on the function of the rat islets microencapsulted in three-layer microbeads was primarily monitored. The aim of this study was to examine the influence of the PSSa on the in vitro function of the islets encapsulated in the agarose/PSSa microbeads compared with single-layer agarose microbeads during long-term culture. The microbeads were cultured for 30 days in medium of Eagle's MEM at 37°C in 5% CO 2 and 95% air. The basal insulin secretion into the culture medium was measured daily during the first 12 days and two times per week until 30 days. The microbeads were subjected to static incubation test on the 10th, 20th, and 30th day during culture. The basal insulin secretion level of the agarose/PSSa microbeads was significantly higher than that of single-layer agarose microbeads. The static incubation tests revealed a similar pattern of insulin secretion from both microbeads when they were exposed to high glucose challenge. In the static incubation test, both could significantly increase insulin release to more than 6.61 times (stimulation index) in response to high glucose stimulation and could significantly decrease when glucose concentration returned from high glucose to low glucose on the 10th, 20th, and 30th day of culture. This study demonstrated that the hamster islets enclosed in agarose/PSSa hydrogel not only continuously secreted basal amounts of insulin, but also maintained their response to high glucose stimulation similar to the agarose microbeads. The above results together with those of our previous in vivo study suggest that the three-layer microbeads (agarose/PSSa) are well suitable for xenotransplantation of islets for the clinical application.

The success rate of subcutaneous transplantation of pancreatic islets has been extremely low. Insufficient oxygen supply to the grafted islets is one possible major obstacle to the preservation of graft function. This study attempted to use basic fibroblast growth factor (bFGF) in subcutaneous transplantation to induce neovascularization and a sufficient blood flow around the space formed for grafted islets in the subcutaneous tissues. A bFGF-releasing device was designed enclosing bFGF in a polyethylene terephthalate mesh bag coated with polyvinylalcohol hydrogel. In the vascularized group (n = 5), two bFGF-releasing devices were implanted bilaterally into the subcutaneous tissue of the back of streptozotocin-induced diabetic Lewis rats. One week after implantation, isolated rat islets (5000) were syngeneically transplanted subcutaneously after the removal of the devices. In the control group (n = 5), no devices were implanted and the same number of rat islets was transplanted directly. One week after the implantation of the devices into the test animals, a thick, well-vascularized capsule was observed in the subcutaneous site. All vascularized recipient rats showed significant decreases in nonfasting blood glucose and maintained normoglycemia for more than 1 month after islet transplantation. However, in the control group, all rats failed to achieve normoglycemia after transplantation. This study provides evidence that the subcutaneous tissue is a promising site for pancreatic islet transplantation, offering convincing advantages in acceptability for diabetic recipients. Establishment of this subcutaneous islet transplantation technique will afford some new perspectives on successful clinical islet transplantation.

It is particularly difficult to isolate porcine islets (PI). Experience suggests that the success rate of porcine islet isolation (PII) is probably considerably influenced by the distension and digestion of the pancreas. In this study, we divided the digestion procedure into two stages and developed a new enzyme solution to improve both the distension and digestion procedures. As a result, we established a novel and stable method of large-scale adult porcine islet isolation (APII). The harvested pancreata of 2-year-old pigs weighing over 200 kg (n = 18) were distended by introducing our new enzyme solution gently and slowly through the pancreatic ducts. Two-stage digestion (cold, then warm) was then performed by first placing the distended pancreata on ice for 2 h to cause diffusion of the enzyme solution around the islets, and then by incubating the pancreata in a water bath at 37°C for 45 min without shaking. The islets were purified by a COBE 2991 cell processor on dextran T70 discontinuous density gradients. Histological study was performed on porcine pancreata sampled after 0, 15, 30, and 45 min of the second stage, and stained with H&E stain. Next, islet equivalent was calculated. Static incubation study was performed by stimulating the islets with 3.3 and 16.7 mM glucose in Krebs' Ringer bicarbonate buffer (KRBB) solution at 37°C for 1 h, and finally the insulin released was measured. The dilated acinar cells septa around the islets were observed at time 0. Destruction of the acinar cells around the islets by warm digestion was recognized at 15 and 30 min, and destroyed and separated acinar cells present around the islets at 45 min. During the entire course of the warm digestion, the islets remained intact. The number of isolated islets was 291,667 ± 240,452 IEQ/pancreas (n = 14) and 3,294 ± 2199 IEQ/g of pancreatic tissue. The purity of recovered porcine islets was over 90%. The concentration of the insulin secreted by 10,000 IEQ islets selected at random was 83.9 ± 13.4 μU/dish/h in response to 3.3 mM glucose and 104.1 ± 12.9 μU/dish/h in response to 16.7 mM glucose (n = 20). A success rate of approximately 80% was attained with APII. We demonstrated that this increase in the success rate was due to the improved distension and digestion provided by this method. This two-stage APII method with its new enzyme solution may facilitate the future use of porcine islets in clinical xenotransplantation trials.

To establish a large-scale isolation procedure for adult porcine islets usable as a donor source for xenotransplantation and as a model of human islet isolation, we improved several characteristics of the conventional isolation procedure. At a slaughterhouse we first selected a breeder pig over 1.5 years old (and over 200 kg in weight) with warm ischemic time (WIT) of 15 ± 2 minutes as nonheart-beating donors. Then, we made a special enzymic mixture that consisted of collagenase S-1 (260 U/mg, NittaZelatin, Japan), collagenase P (1.86 U/ml Lyo Boehringer-Mannheim, USA), DNase (Sigma, St. Louis, Mo), Disparse (NittaZelatin, Japan), and protease inhibitor (Sigma). Third, this mixture was injected very gently into the pancreatic duct at the time of pancreatic harvesting. To prevent overdigestion of the pancreas, the mixture was first cooled to less than 10°C. Fourth, during the warm digestion of pancreas, the pancreas with the enzymic mixture was quietly put in a water bath at 37°C without mechanical shaking. Fifth, we purified the islets with a COBE 2991 cell processor by the Dextran 70 gradient method, because Dextran 70 is very cheap and has the same purification effect as the Ficoll gradient. The results of 10 consecutive breeder porcine islet isolations are reported. The total yield of isolations of islets over 50 μm in the longest diameter after staining with Dithizone (DTZ) was 85,900 ± 19,954 islets, 291,667 ± 240,452 IEQ (2,900 ± 2,324 IEQ/g). The purity of the isolated islets was very high: 90.2 ± 3.8%. Glucose stimulation during in vitro incubation induced significant insulin release from isolated breeder porcine islets. In two of the diabetic rats receiving encapsulated islets grafts using a mesh-reinforced polyvinyl alcohol hydrogel bag (MRPB), a prominent reduction in serum glucose levels (less than 200 mg/dL) persisted for 13 and 19 days, respectively, after intraperitoneal xenotransplantation islets without immunosuppression. In conclusion, we succeeded in a more efficient and less-expensive isolation of a large amount of adult porcine islets from a nonheart-beating donor.

Isolation of islets of Langerhans (islets) has been performed by means of collagenase digestion of the pancreatic tissue combined with density gradient separation of islets from unwanted exocrine tissues. An enormous number of islets are necessary for clinical islet transplantation. The density gradient used for isolation of a large number of islets should satisfy several requirements in addition to those for the conventional density gradients, such as high viscosity for creating fine interfaces with a large area, easy sterilization, and low cost. This study is concerned with the development of a new density gradient made of low-molecular-weight gelatin. We isolated islets from the hamster pancreatic tissue using the gelatin density gradients. The yield and purity of islet and its insulin release function were compared with those of islets isolated using Ficoll and Ficoll-Conray density gradients that have been conventionally used. The new gelatin density gradient can separate islets from the unwanted exocrine tissue as effectively as the Ficoll density gradient and more effectively than the Ficoll-Conray density gradients. The islets collected using the gelatin gradient retain ability of insulin release increase in response to glucose stimulation, similar to those isolated by the Ficoll-Conray gradient and more than those collected by the Ficoll gradient. In addition, the gelatin effectively inhibited enzyme activities, that is, collagenase and proteolytic enzymes released from the exocrine tissue, and thus it can inhibit overdigestion of islets during their density gradient isolation. The gelatin gradient satisfies most of the additional requirements for islet isolation from the pancreatic tissue of large animals mentioned above. Although several factors, such as molecular weight of gelatin, osmolality of the gradient, and centrifugal conditions, still remain to be optimized, our results suggest that the gelatin gradient has potentiality to isolate islets from the pancreatic tissue of a large animal.

The permeation of components of complement and secreted peptides through polymer capsules (PM30, K6305, and K5708) were examined. To analyze permeability by complement, the degree of hemolysis of sensitized sheep erythrocytes (EA) (1 × 10 9 /ml) enclosed in each type of capsule was examined after 24-h incubation in culture medium containing 10% human serum. PM30 and K6305 prevented the permeation of complement well, while K5708 did not. EA suspended in alginate prevented hemolysis even in K5708. Peptide permeation through the capsules was assessed by measuring the concentration of ACTH secreted by proopiomelanocortin (POMC)-gene-transfected-Neuro2A in the culture medium on days 4, 7, 14, 21, and 28 after encapsulation. The ACTH levels in the culture medium remained high until day 28. Alginate appeared to prevent the secretion, because ACTH levels decreased in alginate-suspended cells after day 14. The PM30-K6305 double capsules containing cell lines, Neuro2A, BHK21 (hamster fibroblasts), L929 (mouse fibroblasts), and HF-SKFII (human fibroblasts) were transplanted into the cerebrospinal fluid (CSF) space of the monkeys in the lumber region. The morphological examination showed the partial survival of Neuro2A, and BHK21 and HF-SKFII, which were cells concordant with the monkeys. On the other hand, L929 cells, which were discordant with the monkeys, could not survive at all. Because these results suggest that the complement components penetrate the polymer capsules, concordant cells are preferable for xenografting with polymer capsules into the CSF space.

The biohybrid artificial pancreas is designed to enclose pancreatic endocrine tissues with a selectively permeable membrane that immunoisolates the graft from the host immune system, allowing those endocrine tissues to survive and control glucose metabolism for an extended period of time. The pancreatic B cell line MIN6 is established from a pancreas B cell tumor occurring in transgenic mice harboring the human insulin promoter gene connected to the SV40 T-antigen hybrid gene. It has been proven that glucose-stimulated insulin secretion in MIN6 cells retains a concentration-dependent response similar to that of normal islets. In this study, we performed the histological and functional examination of three-layer microbeads employing MIN6 cells after subcutaneous xenotransplantation to evaluate this device as bioartificial pancreas. MIN6 cells were microencapsulated in three-layer microbeads formulated with agarose, polystyrene sulfonic acid, polybrene, and carboxymethyl cellulose. Microbeads were xenogenically implanted in the subcutaneous tissue of the back of Lewis rats with streptozotocin-induced diabetes. One week after implantation, microbeads were retrieved and cultured for 24 h before the static incubation. There was no evidence of adhesion to the graft and the fibrosis in the transplantation site as determined by gross visual inspection. Microscopic examination demonstrated that retrieved microbeads maintained normal shape, containing intact MIN6 cells. Histological study showed that these MIN6 cells in the microbeads appeared to be viable without cellular infiltration within or around the microbeads. Immunohistochemical analysis of the microbeads clearly revealed the intense staining of insulin in the cytoplasm of encapsulated MIN6 cells. Insulin productivity of MIN6 cells in the microbeads is strongly suggested to be preserved. In response to 16.7 mM glucose stimulation, static incubation of microbeads 1 wk after implantation caused the 2.3 times increase in insulin secretion seen after 3.3 mM glucose stimulation (84.3 ± 10.0 vs. 37.4 ± 10.7 μU/3 × 10 6 cells/hr, n = 5 each, p < 0.01). This study demonstrates that three-layer microbeads encapsulating MIN6 cells retain excellent biocompatibility and maintain good insulin secretion even after subcutaneous xenotransplantation, suggesting the possible future clinical application of this unique bioartificial pancreas to subcutaneous xenotransplantation.

Troglitazone (CS-045), a thiazolidinedione derivative, is a new oral antidiabetic agent that enhances insulin sensitivity and improves insulin responsiveness. In this study we examined the effects of CS-045 on the survival of xenografted bioartificial pancreas. Isolated rat islets were microencapsulated with three-layer agarose microcapsules (polybrene, carboxymethyl cellulose, and an agarose-polystyrene sulfonic acid mixture). Diabetes was induced by intraperitoneal injection of streptozotocin 220 mg/kg. Recipient diabetic mice were separated into two groups. In the CS-045 treated group, the recipient mice were given feed mixed with CS-045 (0.2% w/w) starting from 1 wk before transplantation up to graft failure. The mice in the control group had feed without CS-045. Three hundred microencapsulated rat islets were xenotransplanted into the intraperitoneal cavity of each recipient mouse in both groups. One month after xenotransplantation, IVGTT was performed for all recipients. Xenotransplantation of 300 rat islets in microcapsules decreased the nonfasting blood glucose levels of both groups within 2 days. In the CS-045-treated group (n = 3), the normoglycemic period lasted for more than 1 mo without administration of immunosuppressive drugs (45 ± 4.3 days). However, in the control group (n = 4), the blood glucose levels of all recipients were already elevated on day 4. In the IVGTT study, the glucose assimilation was markedly and significantly better in the CS-045-treated group than in the control group (K = 1.7 ± 0.1 vs. 0.7 ± 0.28 respectively, p