RESUMEN
The number of patients with diabetes is on an increasing trend, thus leading to the belief that diabetes will be the largest medical problem of the 21st century. Islet transplantation can improve glycometabolic control in patients with type 1 diabetes. We studied the viability of Rho-associated protein kinase (ROCK) inhibitor Y-27632 in a culture system in vitro on freshly isolated rat islets. Islet isolation was conducted on a Lewis rat, and studies of culture solutions were split into two groups, one group using ROCK inhibitor Y-27632, and another without. On the seventh day of culture, we evaluated the differences for the cell morphology, viability, and insulin secretion. The Y-27632 group maintained form better than the group without Y-27632. With strong expression of Bcl-2 observed with the Y-27632 group, and expression suppressed with Bax, inhibition of apoptosis by Y-27632 was confirmed. The Y-27632 group predominantly secreted insulin. For islet transplantation, Y-27632 inhibited cell apoptosis in a graft and was also effective in promoting insulin secretion. We were able to confirm effective morphological and functional culture maintenance by separating islets from a rat and adding ROCK inhibitor Y-27632 to the medium.
RESUMEN
We created canine models of type 1 diabetes that were suitable for the assessment of cell therapies, such as islet transplantation and bioartificial pancreas, with low-dose streptozotocin (STZ) injection and partial pancreatectomy. In our model, a 50% pancreatectomy was performed with general anesthesia, followed by systemic injection of 35 mg/kg STZ into a vein of the foreleg. Four weeks after the administration of STZ, the fasting blood glucose level of our model dogs was found to be over 200 mg/dl twice on different days, and we could not detect any canine insulin by the intravenous glucose tolerance test (IVGTT). We therefore diagnosed the dogs to have induced diabetes. Some studies have reported high-dose STZ to be very toxic for both the kidney and liver, and therefore a lower dose is desirable to induce diabetic models without any associated kidney or liver damage. We think that the combination of a partial pancreatectomy can thus make it possible to reduce the dose of STZ, and it is therefore useful for the creation of type 1 diabetes models. We believe that our model is a safe and reliable model for type 1 diabetes in canines to assess the efficacy of pancreas-targeted cell therapies.
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BACKGROUND: Bioartificial liver systems, designed to support patients with liver failure, are composed of bioreactors and functional hepatocytes. Immunological rejection of the embedded hepatocytes by the host immune system is a serious concern that crucially degrades the performance of the device. Induced pluripotent stem (iPS) cells are considered a desirable source for bioartificial liver systems, because patient-derived iPS cells are free from immunological rejection. The purpose of this paper was to test the feasibility of a bioartificial liver system with iPS cell-derived hepatocyte-like cells. METHODS: Mouse iPS cells were differentiated into hepatocyte-like cells by a multi-step differentiation protocol via embryoid bodies and definitive endoderm. Differentiation of iPS cells was evaluated by morphology, PCR assay, and functional assays. iPS cell-derived hepatocyte-like cells were cultured in a bioreactor module with a pore size of 0.2 µm for 7 days. The amount of albumin secreted into the circulating medium was analyzed by ELISA. Additionally, after a 7-day culture in a bioreactor module, cells were observed by a scanning electron microscope. RESULTS: At the final stage of the differentiation program, iPS cells changed their morphology to a polygonal shape with two nucleoli and enriched cytoplasmic granules. Transmission electron microscope analysis revealed their polygonal shape, glycogen deposition in the cytoplasm, microvilli on their surfaces, and a duct-like arrangement. PCR analysis showed increased expression of albumin mRNA over the course of the differentiation program. Albumin and urea production was also observed. iPS-Heps culture in bioreactor modules showed the accumulation of albumin in the medium for up to 7 days. Scanning electron microscopy revealed the attachment of cell clusters to the hollow fibers of the module. These results indicated that iPS cells were differentiated into hepatocyte-like cells after culture for 7 days in a bioreactor module with a pore size of 0.2 µm. CONCLUSION: We consider the combination of a bioreactor module with a 0.2-µm pore membrane and embedded hepatocytes differentiated from iPS cells to be a promising option for bioartificial liver systems. This paper provides the basic concept and preliminary data for an iPS cell-oriented bioartificial liver system.PACS code: 87. Biological and medical physics, 87.85.-d Biomedical engineering, 87.85.Lf Tissue engineering, 87.85.Tu Modeling biomedical systems.
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Técnicas de Cultivo de Célula/instrumentación , Hepatocitos/citología , Hepatocitos/fisiología , Hígado Artificial , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/fisiología , Andamios del Tejido , Animales , Diferenciación Celular , Línea Celular , Diseño de Equipo , Análisis de Falla de Equipo , Ratones , Técnicas de Cultivo de Órganos/instrumentación , Proyectos PilotoRESUMEN
Cryopreservation could be a possible means of addressing the shortage of islets of Langerhans. We investigated the effects of EDT324 solution on the vitrification of isolated rat islets of Langerhans. Rat pancreatic islets were cryopreserved in 10% DMSO by a slow-rate freezing method or were cryopreserved in EDT324 solution by vitrification. The cryopreserved islets were compared in terms of viability, stimulation index and metabolic function after transplantation. After cryopreservation, the viability and stimulation of islets stored in EDT324 were 92.4% and 6.4, respectively, and were higher than islets stored by slow freezing (72.5% and 1.5, respectively). Streptozotocin-induced diabetic rats were transplanted with islets cryopreserved in EDT324, which corrected diabetes and achieved euglycemia within 2 days after transplantation. These results indicate that EDT324 allows successful cryopreservation of rat islets for long-term storage as an alternative solution to traditionally used solutions, such as 10% DMSO. Transplantation of cryopreserved islets into diabetic rats can achieve euglycemia.
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Criopreservación/métodos , Trasplante de Islotes Pancreáticos/métodos , Islotes Pancreáticos/cirugía , Vitrificación , Animales , Diabetes Mellitus Experimental/cirugía , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Masculino , Ratas , Ratas Endogámicas LewRESUMEN
Definitive endoderm (DE) derived from stem cells holds potential to differentiate into hepatocytes. Stem cell therapy using those cells has potential for a treatment of liver disease. To date, various ways of inducing hepatocytes from embryonic stem (ES) cells have been reported by researchers. However, it has not been proved enough that induced pluripotent stem (iPS) cells behave in the same manner as ES cells in endoderm differentiation. The purpose of this study was to establish an efficient method to induce DE from iPS cells, through comparatively analyzing the efficacy of endoderm formation from mouse ES cells. Furthermore, the efficiency of a serum-free medium in the differentiation into DE was investigated. Mouse ES cells and iPS cells were floated in culture medium for 2 or 5 days and embryoid bodies (EB) were formed. Subsequently, DE was induced with 100 ng/ml activin A and 100 ng/ml basic fibroblast growth factor (bFGF). RT-PCR and real-time PCR analyses were carried out at each step to determine the gene expression of EB markers. The difference in cellular proliferation between serum-containing and serum-free media was examined by an MTS assay in EB and DE induction. iPS cells showed the paralleled mRNA expression to ES cells in each step of differentiation into EB, but the levels of expression of Sox17 and Foxa2 were relatively higher in ES cell-derived DE, whereas Cxcr4 expression was higher in iPS cell-derived DE. The utilization of serum-free medium for iPS cells showed significantly favorable cellular proliferation during EB formation and subsequent DE induction. Forming EB for 5 days and subsequently DE induction with activin A and bFGF with serum-free medium was an appropriate protocol in iPS cells. This may represent an important step for generating hepatocytes from iPS cells for the development of cell therapy.
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Células Madre Embrionarias/citología , Endodermo/embriología , Células Madre Pluripotentes Inducidas/citología , Animales , Bioensayo , Proliferación Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Medios de Cultivo/farmacología , Cuerpos Embrioides/citología , Cuerpos Embrioides/efectos de los fármacos , Cuerpos Embrioides/metabolismo , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/metabolismo , Endodermo/citología , Endodermo/efectos de los fármacos , Endodermo/metabolismo , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Induced pluripotent stem (iPS) cells are pluripotent and are able to unlimitedly proliferate in vitro. This technical breakthrough in creating iPS cells from somatic cells has noteworthy implications for overcoming the immunological rejection and the ethical issues associated with the derivation of embryonic stem cells from embryos. In the current work, we present an efficient hepatic differentiation of mouse iPS cells in vitro. iPS cells were cultured free floating to induce the formation of embryoid bodies (EB) for 5 days. EB were transferred to a gelatin-coated plate and treated with 100 ng/ml activin A and 100 ng/ml basic fibroblast growth factor (bFGF) for 3 days to induce definitive endoderm. Cells were further cultured for 8 days with 100 ng/ml hepatocyte growth factor (HGF) to generate hepatocytes. Characterization was performed by RT-PCR assay. Functional analysis for albumin secretion and ammonia removal was also carried out. iPS cell-derived hepatocyte-like cells (iPS-Heps) were obtained at the end of the differentiation program. Expression levels of a gestational hepatocyte gene and lineage-specific hepatic genes intensified in iPS-Heps. The production of albumin increased in a time-dependent manner. iPS-Heps were capable of metabolizing ammonia. We present here instant hepatic differentiation of mouse iPS cells using combined 3-day treatments of activin A and bFGF with subsequent 8-day HGF. Our study will be an important step to generate hepatocytes from human iPS cells as a new source for liver-targeted cell therapies.
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Diferenciación Celular , Células Madre Pluripotentes Inducidas/citología , Hígado/citología , Activinas/farmacología , Albúminas/genética , Albúminas/metabolismo , Amoníaco/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Línea Celular , Forma de la Célula/efectos de los fármacos , Células Cultivadas , Factor 2 de Crecimiento de Fibroblastos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Factor de Crecimiento de Hepatocito/farmacología , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , RatonesRESUMEN
Maintenance of freshly isolated porcine liver cells in vitro is limited for a short period of time. Therefore, establishment of easy handling cell lines is extremely important for in vitro study for liver cells and their possible utilization for cell differentiation and growth of stem cells. Porcine liver cells were transduced with a retroviral vector SSR#69 expressing SV40T, one of SSR#69-immortalized porcine liver cell lines, JSNK-1, was established and characterized. Morphology of JSNK-1 cells was spindle shaped. When the cells became confluent, JSNK-1 cells revealed hills-and-valleys pattern. In the presence of vitamin A, JSNK-1 cells showed big droplets inside the cytoplasm, which were positive with PAS staining. JSNK-1 cells showed the gene expression of collagen type 1α1, collagen type 1α2, FLT-1, ß-actin, and SV40T. Immunostaining study revealed that JSNK-1 cells produced collagen, vimentin, and α-smooth muscle actin. JSNK-1 cells possessed the characteristics of the liver stellate cells. JSNK-1 cells produced hepatocyte growth factor (HGF) in a time-dependent manner. When cocultured with iPS cells towards the hepatic differentiation, JSNK-1 cells facilitated their hepatic differentiation in terms of albumin production. In conclusion, JSNK-1 cells would be valuable in the study of liver stellate cell pathophysiology and contribute to the optimization of hepatic differentiation of iPS cells.
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Antígenos Transformadores de Poliomavirus/metabolismo , Técnicas de Cultivo de Célula/métodos , Línea Celular Transformada/citología , Hígado/citología , Retroviridae/genética , Transducción Genética , Albúminas/biosíntesis , Animales , Bioensayo , Biomarcadores/metabolismo , Línea Celular Transformada/ultraestructura , Proliferación Celular , Forma de la Célula , Técnicas de Cocultivo , Colágeno/metabolismo , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica , Células Estrelladas Hepáticas/citología , Células Estrelladas Hepáticas/metabolismo , Factor de Crecimiento de Hepatocito/biosíntesis , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Sus scrofa , Vimentina/metabolismoRESUMEN
Worldwide, colorectal cancer is the third most common type of cancer affecting both sexes. It has been proposed that a small subset of cancer cells (cancer stem cells) within each tumor is able to initiate tumor growth. In 2007, two research groups simultaneously identified a colon cancer stem cell population in human tumors by the use of CD133 expression. In the present study, we used a human colon cancer cell line, SW620, to analyze the cancer stem cell-like characteristics of CD133(+) cells in vitro and in vivo. In vitro, CD133(+) SW620 cells had a higher proliferative capacity, were more irradiation- and chemotherapy-resistant, and had a higher expression of ß-catenin compared with CD133(-) cells. Injections of either CD133(+) or CD133(-) cells into the skin or rectal mucosa of NOD/SCID mice led to tumors; however, injection of CD133(+) cells resulted in the formation of larger tumors. Tumors derived from injections of CD133(-) cells did not contain any CD133(+) cells, whereas tumors derived from injections of CD133(+) cells did contain CD133(+) cells, suggesting self-renewing capability. However, the proportion of CD133(+) cells in the newly formed tumors in vivo was lower than the proportion of CD133(+) cells in vitro. In conclusion, the human colon cancer cell line, SW620, contains both CD133(+) and CD133(-) phenotypes, and the CD133(+) phenotype has characteristics consistent with those of cancer stem cells.
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Antígenos CD/metabolismo , Neoplasias del Colon/patología , Glicoproteínas/metabolismo , Péptidos/metabolismo , Antígeno AC133 , Animales , Antígenos CD/genética , Línea Celular Tumoral , Proliferación Celular , Neoplasias del Colon/genética , Resistencia a Antineoplásicos/genética , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica , Glicoproteínas/genética , Humanos , Ratones , Ratones SCID , Trasplante de Neoplasias , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Péptidos/genética , Tejido Subcutáneo/patologíaRESUMEN
It has been proposed in human colorectal cancers (CRC) a minority subset of cancer cells within tumors able to initiate tumor growth, defined as cancer stem cells (CSC). Solid human primary colonic and its ovarian metastatic cancer tissues were collected from fresh surgical samples and subsequent xenografts were established in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. The resulting tumors were disaggregated into single-cell suspensions and a CD133(-) cell line (NANK) was newly established and analyzed by flow cytometry. Surface markers of progenitor cells were immunophenotypically analyzed, and expression of stem cell and cancer-related genes was characterized. Secreted angiogenesis-associated molecules were investigated by proteomic array technology. Finally, different numbers of NANK were implanted and their tumor-initiating properties were investigated in NOD/SCID mice. Intraperitoneal injection of NANK in NOD/SCID mice induced tumors with developing progressive peritoneal dissemination and ascites. NANK cells maintained a differentiated phenotype and reproduced the full morphologic and phenotypic heterogeneity of their parental lesions. Noticeably, NANK lacked the expression of conventional CSC markers CD133 and CD44, self-renewal genes Oct-4 and Nanog, but showed the expression of an important gastrointestinal development marker CDX-2 and BMI-1 that is essential in regulating the proliferative activity of normal and leukemic stem cells. In addition, NANK secreted high amounts of important angiogenic cytokines. These results provide a novel and extensive model in human CSC for studying the generation and maintenance of phenotypic heterogeneity in CRC.
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Antígenos CD/metabolismo , Línea Celular Tumoral/patología , Separación Celular/métodos , Neoplasias del Colon/irrigación sanguínea , Neoplasias del Colon/patología , Glicoproteínas/metabolismo , Neovascularización Patológica/patología , Péptidos/metabolismo , Antígeno AC133 , Adulto , Animales , Biomarcadores de Tumor/metabolismo , Proliferación Celular , Citocinas/biosíntesis , Femenino , Humanos , Péptidos y Proteínas de Señalización Intercelular/biosíntesis , Ratones , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Neoplasias Ováricas/patología , Neoplasias Ováricas/secundario , Fenotipo , Factores de Tiempo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Liver diseases still have a high mortality even though liver transplantation has become a standard treatment. Currently, hepatocyte transplantation has been proposed as another promising strategy. One limitation is the availability of human livers as a source of hepatocytes. Because of an unlimited supply, the use of porcine hepatocytes might address this problem. Regardless of the source, once isolated hepatocytes lose specific functionality due to the loss of the natural microenvironment. For this reason, we tested the ability of a self-assembling peptide nanofiber (SAPNF) to provide a provisional three-dimensional (3D) support to interact with cells to control their function in vivo. Isolated porcine hepatocytes were embedded in SAPNF, or collagen type I and transplanted by direct injection into the splenic pulp of SCID mice suffering from acute liver failure (ALF) by 90% hepatectomy. SAPNF porcine hepatocyte transplantation produced engraftment that was far superior to that obtained using collagen and prolonged the survival of mice with ALF, in contrast with controls. An ultrastructural evaluation using transmission electron microscopy indicated extensive cell-cell communication and preservation of hepatocyte architecture. The transplanted SAPNF hepatocytes showed higher expression of albumin and PAS and lower apoptotic events assessed by TUNEL staining. Hepatocytes culture in a truly 3D network allows in vivo maintaining of differentiated functions, and once transplanted between widely divergent species can function to correct acute liver failure in mice and prolong their survival.
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Hepatocitos/trasplante , Fallo Hepático Agudo/terapia , Trasplante Heterólogo , Animales , Apoptosis/efectos de los fármacos , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Hepatectomía , Hepatocitos/citología , Hepatocitos/metabolismo , Hepatocitos/ultraestructura , Ratones , Ratones SCID , Nanofibras/química , Péptidos/farmacología , Regeneración/efectos de los fármacos , Bazo/efectos de los fármacos , Bazo/patología , Análisis de Supervivencia , Sus scrofaRESUMEN
The development of a bioartificial pancreas (BAP) with immunoisolating fashion has been gaining attention as a new method for treating diabetes. We have been proceeding with the development of a bag-type BAP that can be easily implanted and that allows for the optional injection or rejection of cells at any time. If fibrosis develops around a BAP device, then the permeability of substances transmitted through a semipermeable membrane will decrease, thereby reducing the reactivity with glucose, so it is necessary for the material of the device to have an excellent histocompatibility. Furthermore, in order to improve the efficacy of BAP treatment, it is important to maintain an environment of ample blood flow around the device. We have created a bag-type device for BAP that is 20 x 20 mm in size and comprises two layers of membranes. We have used an EVAL membrane for the outer membrane of the two layers. The EVAL membrane is a semipermeable membrane with good insulin permeability, which functions as an immunoisolation membrane. The inner membrane consists of PAU-coated HD-PE (nonwoven material processed with polyaminourethan) and it is designed to function as a scaffold for cells. We used Lewis rats to determine whether the effectiveness of fibroblast growth factor 2 (bFGF) can be improved by concomitantly using bFGF with a capacity for blood vessel regeneration as well as bFGF immersed in a sheet of gelatin. We placed the BAP in the abdominal cavity and covered it with the greater omentum. We were able to significantly increase the blood flow and the number of new blood vessels in the tissue surrounding the BAP device by using gelatinized bFGF. There were only a few instances of fibrosis as a biological reaction to the EVAL membrane, and the infiltration of inflammatory cells was mild. There were no adverse effects related to implantation of the device. We confirmed in this study that the use of an implantable BAP device and bFGF allowed for a better blood flow around the BAP device. There were only minor instances of fibrosis and inflammation reaction around the BAP, thus indicating the BAP that we are currently developing to have an excellent histocompatibility.
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Abdomen/irrigación sanguínea , Factor 2 de Crecimiento de Fibroblastos/farmacología , Gelatina/química , Neovascularización Fisiológica/efectos de los fármacos , Páncreas Artificial , Abdomen/patología , Animales , Vasos Sanguíneos/patología , Gelatina/farmacología , Ensayo de Materiales , Ratas , Ratas Endogámicas LewRESUMEN
BACKGROUND: Human mesenchymal stem cells (hMSCs) are multipotent stem cells found in the adult bone marrow that have the capacity to differentiate into various mesenchymal cell types. The hMSCs may provide a potential therapy to restore damaged tissues or organs of mesenchymal origin; however, a drawback is their limited life span in vitro. METHODS: We immortalized normal hMSCs with retrovirally transmitted human telomerase reverse transcriptase cDNA. One of the immortalized clones (YKNK-12) was established, and the biological characteristics were investigated in vitro and in vivo. RESULTS: YKNK-12 cells were capable of differentiating adipocytes, osetoblasts, and chondrocytes. Osteogenically differentiated YKNK-12 cells produced significant levels of growth factors BMP4, BMP6, FGF6, FGF7, transforming growth factor-beta1, and transforming growth factor-beta3.. Microcomputer tomography T and soft X-ray assays showed an excellent calvarial bone healing in mice after transplantation of osteogenically differentiated YKNK-12 cells. These cells expressed human-specific osteocalcin and increased the gene expression of runt-related transcription factor 2, alkaline phosphatase, osteocalcin, and osterix in the bone regenerating area. YKNK-12 cell transplant corrected the bone defect without inducing any adverse effects. CONCLUSIONS: We conclude that hMSCs immortalized by transduction with human telomerase reverse transcriptase may provide an unlimited source of cells for therapeutic use in bone regeneration.
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Enfermedades Óseas/cirugía , Regeneración Ósea , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/enzimología , Osteogénesis , Cráneo/cirugía , Telomerasa/metabolismo , Adipocitos/enzimología , Animales , Biomarcadores/metabolismo , Enfermedades Óseas/diagnóstico por imagen , Enfermedades Óseas/enzimología , Enfermedades Óseas/fisiopatología , Diferenciación Celular , Línea Celular Transformada , Linaje de la Célula , Proliferación Celular , Supervivencia Celular , Condrocitos/enzimología , Modelos Animales de Enfermedad , Femenino , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Ratones , Ratones Endogámicos BALB C , Osteoblastos/enzimología , Radiografía , Cráneo/diagnóstico por imagen , Cráneo/enzimología , Cráneo/fisiopatología , Telomerasa/genética , Factores de Tiempo , Transducción GenéticaRESUMEN
Construction of a safe and functional bioartificial pancreas (BAP) that provides an adequate environment for islet cells may be an important approach to treating diabetic patients. Various types of BAP devices have been developed, but most of them involve extravascular implantation of islets in microcapsules or diffusion chambers. These devices have poor diffusive exchange between the islets and blood, and often rupture. To overcome these problems, we developed a new type of BAP composed of polyethylene-vinyl alcohol (EVAL) hollow fibers that are permeable to glucose and insulin and a poly-amino-urethane-coated, non-woven polytetrafluoroethylene (PTFE) fabric that allows cell adhesion. Porcine islets attached to the surface of the PTFE fabric, but not to the surface of the EVAL hollow fibers, allowing nutrient and oxygen exchange between blood flowing inside the fibers and cells outside. We inoculated this BAP with porcine islets and connected it to the circulation of totally pancreatectomized diabetic pigs. We found that blood glucose levels were reduced to a normal range and general health was improved, resulting in longer survival times. In addition, regulation of insulin secretion from the BAP was properly controlled in response to glucose both in vitro and in vivo. These results indicate that our newly developed BAP may be a potential therapy for the treatment of diabetes in humans.
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Órganos Bioartificiales , Diabetes Mellitus Experimental/terapia , Membranas Artificiales , Páncreas Artificial , Animales , Glucemia/análisis , Diabetes Mellitus Experimental/sangre , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/metabolismo , Ratones , PorcinosRESUMEN
A human pancreatic beta-cell line that is functionally equivalent to primary beta-cells has not been available. We established a reversibly immortalized human beta-cell clone (NAKT-15) by transfection of primary human beta-cells with a retroviral vector containing simian virus 40 large T-antigen (SV40T) and human telomerase reverse transcriptase (hTERT) cDNAs flanked by paired loxP recombination targets, which allow deletion of SV40T and TERT by Cre recombinase. Reverted NAKT-15 cells expressed beta-cell transcription factors (Isl-1, Pax 6, Nkx 6.1, Pdx-1), prohormone convertases 1/3 and 2, and secretory granule proteins, and secreted insulin in response to glucose, similar to normal human islets. Transplantation of NAKT-15 cells into streptozotocin-induced diabetic severe combined immunodeficiency mice resulted in perfect control of blood glucose within 2 weeks; mice remained normoglycemic for longer than 30 weeks. The establishment of this cell line is one step toward a potential cure of diabetes by transplantation.
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Técnicas de Cultivo de Célula/métodos , Diabetes Mellitus Tipo 1/cirugía , Trasplante de Islotes Pancreáticos/métodos , Islotes Pancreáticos/fisiología , Ingeniería de Tejidos/métodos , Animales , Línea Celular , Proliferación Celular , Mejoramiento Genético/métodos , Humanos , Ratones , Ratones SCID , Resultado del TratamientoRESUMEN
Recently, much attention has been attracted by a novel therapy for liver failure using a hybrid bioartificial liver (BAL) support device that incorporates living liver cells. Researchers in various fields have considered the following cells for potential use in BALs: human embryonic stem (ES) cells; somatic stem cells; differentiated tissue cells; and cells derived from tissues of different animal species, particularly from the pig. With their pluripotency, human ES cells are extremely useful, and many research groups are joining the race to develop BALs. One such effort involves the breeding of transgenic pigs to overcome interspecies barriers. Recent reports suggest, however, that porcine endogenous retrovirus may infect human tissues, and clinical application of transgenic pigs has become a controversial issue. To avoid such risks, we recommend that researchers adopt a reversible immortalization system that uses the Cre-loxP site-specific recombination reaction targeting human hepatocytes in their final differentiated state. This system has allowed us to establish a safe human hepatocyte line that is capable of differentiation at low cost and on a large scale. We are also designing and developing an artificial liver module made of a combination of hollow fibers and nonwoven fabrics. The objective of this review article is to report our therapeutic strategy, which aims at the earliest possible introduction of the treatment of liver failure using BALs.
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Ingeniería Biomédica/normas , Hepatocitos/trasplante , Fallo Hepático/terapia , Hígado Artificial , Animales , Ingeniería Biomédica/tendencias , Línea Celular , Trasplante de Células/métodos , Enfermedad Crónica , Modelos Animales de Enfermedad , Predicción , Rechazo de Injerto , Supervivencia de Injerto , Haplorrinos , Humanos , Fallo Hepático/diagnóstico , Pronóstico , Sensibilidad y Especificidad , PorcinosRESUMEN
Hepatocyte transplantation (HTX) has received great expectation for the treatment of a wide spectrum of liver diseases. Considering the severe shortage of human livers for hepatocyte isolation, porcine hepatocytes are an attractive alternative to normal human hepatocytes. To develop such therapy, establishment of an efficient hepatocyte isolation and transplantation model that enables accurate assessment of safety and efficacy of HTX is extremely important. Porcine hepatocytes were isolated from a surgically removed liver segment with a four-step retrograde perfusion using dispase and collagenase. The resultant hepatocytes of > 84% viability were used for transplantation experiment in a pig model of acute liver failure induced by intravenous administration of D-galactosamine (D-gal) (0.5 mg/kg). Twenty-four hours after D-gal injection, transplantation of freshly isolated porcine hepatocytes (1 x 10(9)) was safely conducted and prolonged the survival of D-gal-treated pigs. We describe an efficient porcine hepatocyte isolation and subsequent cell transplantation in pigs with D-gal-induced liver failure.
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Trasplante de Células/métodos , Hepatocitos/trasplante , Hepatopatías/terapia , Trasplante de Hígado/métodos , Trasplante Heterólogo/métodos , Animales , Separación Celular/métodos , Supervivencia Celular/fisiología , Trasplante de Células/tendencias , Colagenasas , Modelos Animales de Enfermedad , Endopeptidasas , Galactosamina , Hepatocitos/citología , Hepatocitos/fisiología , Fallo Hepático Agudo/inducido químicamente , Fallo Hepático Agudo/terapia , Trasplante de Hígado/tendencias , Masculino , Perfusión/métodos , Tasa de Supervivencia , Sus scrofa , Trasplante Heterólogo/tendencias , Resultado del TratamientoRESUMEN
Development of liver-targeted cell therapies, such as hepatocyte transplantation and bioartificial livers, requires a large amount of functional hepatocytes as needed. To achieve this development, establishing an excellent cryopreservation method of hepatocytes is an extremely important issue. Therefore, we performed a comparative review of cryoprotective effects of various cryopreservation solutions using primarily isolated porcine hepatocytes. Porcine hepatocytes were isolated with a four-step dispase and collagenase perfusion method. The obtained hepatocytes with the initial viabilities of 76%, 84%, and 96% were assigned to the following four groups for cryopreservation at -80 degrees C: Dulbecco's modified Eagle's medium (DMEM) + 10% fetal bovine serum (FBS) + 12% dimethyl sulfoxide (DMSO) (group A), University of Wisconsin (UW) solution + 12% DMSO (group B), Cell Banker 1 (group C), and Cell Banker 2 (group D). The hepatocytes in each group were thawed at 3 days, 10 days, and 5 months of cryopreservation and subjected to comparative analyses, including viability, plating efficiency, LDH release, ammonia removal test, and lentiviral gene transfer. These parameters were the most favorable in the hepatocytes cryopreserved with UW solution. Approximately 5% of thawed cryopreserved porcine hepatocytes expressed LacZ activity after lentiviral transduction. Intrasplenic transplantation of UW solution-cryopreserved hepatocytes improved the survival of rats treated with D-galactosamine. UW solution maintained the functions of cryopreserved porcine hepatocytes.
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Trasplante de Células/métodos , Criopreservación/métodos , Crioprotectores/farmacología , Hepatocitos/efectos de los fármacos , Hepatocitos/trasplante , Trasplante de Hígado/métodos , Soluciones Preservantes de Órganos , Adenosina/farmacología , Alopurinol/farmacología , Amoníaco/metabolismo , Animales , Técnicas de Cultivo de Célula/métodos , Separación Celular/métodos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Vectores Genéticos/genética , Glutatión/farmacología , Hepatocitos/fisiología , Insulina/farmacología , L-Lactato Deshidrogenasa/metabolismo , Operón Lac/genética , Hepatopatías/terapia , Masculino , Rafinosa/farmacología , Ratas , Ratas Wistar , Sus scrofa , Transducción Genética , Trasplante Heterólogo/métodosRESUMEN
Adsorption column Medisorba MG-50 (Kuraray Medical Inc.) for the treatment of myasthenia gravis (MG) is introduced. The adsorbent in this column is composed of cellulose beads as carrier material and covalent-bound synthetic peptide as a ligand that has a specific affinity to the pathogenic anti-acetylcholine receptor antibody of MG. The amino acid sequence of the peptide is modified from the segment of alpha 183-200 of the torpedo acetylcholine receptor (AChR) protein, and the segment is the acetylcholine binding site on AChR and the target site of anti-AChR antibody. The adsorbent showed specific adsorption characteristics to the anti-ACHR antibody (blocking antibody) in vitro. Clinically, MG-50 is used in plasma-perfusion therapy, and it is recognized that MG-50 specifically reduces blocking antibody titer and improves MG symptoms. MG-50 is approved in Japan.
Asunto(s)
Autoanticuerpos/sangre , Autoanticuerpos/inmunología , Técnicas de Inmunoadsorción/instrumentación , Miastenia Gravis/terapia , Receptores Colinérgicos/inmunología , Ensayos Clínicos como Asunto , Humanos , Inmunoadsorbentes/inmunología , Miastenia Gravis/inmunología , Fragmentos de Péptidos/inmunología , Sensibilidad y EspecificidadRESUMEN
Bilirubin adsorption column Medisorba BL-300 (Kuraray Medical Inc.) for the treatment of hyperbilirubinemia is introduced. The adsorbent packed in this column is the porous anion exchange resin coated with hydrophilic 2-hydroxyethyl methacrylate copolymer in order to improve blood compatibility. The adsorbent showed a high adsorption rate for direct bilirubin and indirect bilirubin in patient plasma in vitro. This column is used in plasmaperfusion therapy in clinical use. BL-300 showed high removal rate for direct bilirubin, indirect bilirubin and bile acid in clinical evaluation. BL-300 is approved in Japan and it has been used for the treatment of hyperbilirubinemia caused by many diseases such as liver failure and hepatitis.
Asunto(s)
Resinas de Intercambio Aniónico/química , Hemoperfusión , Hiperbilirrubinemia/terapia , Adsorción , Bilirrubina/sangre , Ensayos Clínicos como Asunto , Humanos , Intercambio Plasmático/métodos , Sensibilidad y Especificidad , Resultado del TratamientoRESUMEN
Kuraray has developed many kinds of apheresis devices, such as plasma separators, plasma fractionators, and apheresis monitors. In this article, apheresis membranes, especially double filtration plasmapheresis (DFPP) and plasma fractionators used in DFPP are introduced. DFPP is both clinically and cost effective apheresis therapy, and it has been used widely for the treatment of many kinds of diseases. Several types of plasma separators with various pore sizes are available. It is important to select the proper plasma separator with suitable pore size, determined by the size of the pathogenic substances to be removed. The Evaflux 5A ethylene-vinyl alcohol copolymer plasma fractionator efficiently separates low-density lipoprotein from high-density lipoprotein. DFPP with the Evaflux 5A is effective for the treatment of familiar hyperlipidemia.