RESUMEN
We investigated the effects of coculture with hepatic stellate cells (HSCs) on the differentiation of embryonic stem (ES) cells and embryoid bodies (EBs). Rat HSCs were incubated until becoming semi-confluent and adherent to the dish. Undifferentiated mouse ES cells and 4-day EBs were cultured in gelatin-coated or HSC-feeder dishes, then induced hepatocyte-like cells and the remaining undifferentiated ES cells were examined using immunocytochemical and RT-PCR methods. HSCs promoted the differentiation of EBs into hepatocyte-like cells, whereas they inhibited the differentiation of undifferentiated ES cells. Among EB outgrowths cocultured with HSCs, albumin-immunopositive cells were clearly and abundantly observed, while they were faintly and scarcely seen in EB outgrowths without HSCs. mRNA expressions of the hepatocyte-related markers such as albumin, transthyretin, alpha-1-antitrypsin, tryptophan-2,3-dioxygenase, phosphoenolpyruvate carboxykinase, hepatocyte nuclear factor 4α and cytochrome P4507a1 were clearly detected in EB outgrowths cocultured with HSCs, while they were only weakly detected or undetected in spontaneous EB outgrowths without HSCs. In contrast to the promoted hepatic differentiation of EBs by HSCs, undifferentiated ES cells formed cellular colonies in HSC-feeder dishes that were similar to the colonies of undifferentiated ES cells kept in maintenance medium containing leukemia inhibitory factor. In addition, ES cell colonies were immunopositive for Oct-3/4, markers of an undifferentiated state, and there were few ALB-immunopositive cells in the colonies. Thus, HSCs have contrasting effects on EBs undergoing differentiation and undifferentiated ES cells, i.e., positive and negative modulation, respectively.
Asunto(s)
Diferenciación Celular , Técnicas de Cocultivo , Cuerpos Embrioides/citología , Células Madre Embrionarias/citología , Células Estrelladas Hepáticas/citología , Animales , Línea Celular , Embrión de Mamíferos/citología , Células Madre Embrionarias/metabolismo , Ratones , Ratas , Células Madre/química , Células Madre/citologíaRESUMEN
Although mouse Wnt-10b has been shown to play various roles in a wide range of biological actions, the effects on epithelial stem/progenitor cells in the skin have not been reported. In the present study, we investigated the effects of Wnt-10b on proliferation and differentiation of murine skin-derived CD34 and CD49f double-positive (CD34(+)CD49f(+)) cells, a supposed fraction as enriched epithelial stem/progenitor cells. The cells were prepared from dorsal skin samples obtained from young adult mice as alpha6 integrin (CD49f) and CD34 double-positive cells by fluorescent activated cell sorting (FACS), and they were cultured with or without Wnt-10b to investigate its effects on proliferation and differentiation. Involvement of canonical Wnt signaling pathway was confirmed by TOPFLASH assay, and differentiation of the CD34(+)CD49f(+) cells was assessed by RT-PCR analysis and immunocytochemical examinations. The skin-derived CD34(+)CD49f(+) cells were immunopositive for Lhx2 and expressed mRNA of classical markers for bulge stem cells, including Lhx2, keratin15, Sox9, S100a6, and NFATc1. Their proliferation was suppressed by Wnt-10b, and the markers for differentiated epithelial cells became to be expressed in the culture with Wnt-10b. These results suggest that Wnt-10b promotes differentiation of epithelial stem/progenitor cells in the skin.
Asunto(s)
Antígenos CD34/metabolismo , Integrina alfa6/metabolismo , Piel/citología , Células Madre/citología , Células Madre/fisiología , Proteínas Wnt/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Aumento de la Célula/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Ratones , Piel/efectos de los fármacos , Fenómenos Fisiológicos de la Piel/efectos de los fármacos , Células Madre/efectos de los fármacos , Proteínas Wnt/administración & dosificaciónRESUMEN
A method for obtaining mouse hepatocytes by infusing collagenase solution into the left ventricle was established. This technique was shown to be equivalent to the intra-portal infusion method and more practical, especially in postnatal mice with a small body size.
Asunto(s)
Colagenasas/administración & dosificación , Ventrículos Cardíacos/metabolismo , Hepatocitos/citología , Animales , Células Cultivadas , RatonesRESUMEN
Embryonic stem (ES) cells are a potential source for treatment of spinal cord injury (SCI). Although one of the main problems of ES cell-based cell therapy is tumor formation, there is no ideal method to suppress tumor development. In this study, we examined whether transplantation with bone marrow stromal cells (BMSCs) prevented tumor formation in SCI model mice that received ES cell-derived grafts containing both undifferentiated ES cells and neural stem cells. Embryoid bodies (EBs) formed in 4-day hanging drop cultures were treated with retinoic acid (RA) at a low concentration of 5 x 10(-9) M for 4 days, in order to allow some of the ES cells to remain in an undifferentiated state. RA-treated EBs were enzymatically digested into single cells and used as ES cell-derived graft cells. Mice transplanted with ES cell-derived graft cells alone developed tumors at the grafted site and behavioral improvement ceased after day 21. In contrast, no tumor development was observed in mice cotransplanted with BMSCs, which also showed sustained behavioral improvement. In vitro results demonstrated the disappearance of SSEA-1 expression in cytochemical examinations, as well as attenuated mRNA expressions of the undifferentiated markers Oct3/4, Utf1, Nanog, Sox2, and ERas by RT-PCR in RA-treated EBs cocultured with BMSCs. In addition, MAP2-immunopositive cells appeared in the EBs cocultured with BMSCs. Furthermore, the synthesis of NGF, GDNF, and BDNF was confirmed in cultured BMSCs, while immunohistochemical examinations demonstrated the survival of BMSCs and their maintained ability of neurotrophic factor production at the grafted site for up to 5 weeks after transplantation. These results suggest that BMSCs induce undifferentiated ES cells to differentiate into a neuronal lineage by neurotrophic factor production, resulting in suppression of tumor formation. Cotransplantation of BMSCs with ES cell-derived graft cells may be useful for preventing the development of ES cell-derived tumors.
Asunto(s)
Células de la Médula Ósea/citología , Trasplante de Médula Ósea/métodos , Neoplasias/patología , Traumatismos de la Médula Espinal/terapia , Trasplante de Células Madre/métodos , Células Madre/citología , Animales , Células de la Médula Ósea/metabolismo , Línea Celular Tumoral , Células Cultivadas , Células Madre Embrionarias , Inmunohistoquímica , Ratones , Factores de Crecimiento Nervioso/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Traumatismos de la Médula Espinal/patología , Células Madre/patología , Células del Estroma/citología , Células del Estroma/metabolismo , Células del Estroma/trasplanteRESUMEN
Embryonic stem (ES) cells are a potential source for treatment of spinal cord injury (SCI). Although one of the main problems of ES cell-based cell therapy is tumor formation, there is no ideal method to suppress tumor development. In this study, we examined whether transplantation with bone marrow stromal cells (BMSCs) prevented tumor formation in SCI model mice that received ES cell-derived grafts containing both undifferentiated ES cells and neural stem cells. Embryoid bodies (EBs) formed in 4-day hanging drop cultures were treated with retinoic acid (RA) at a low concentration of 5 × 10-9 M for 4 days, in order to allow some of the ES cells to remain in an undifferentiated state. RA-treated EBs were enzymatically digested into single cells and used as ES cell-derived graft cells. Mice transplanted with ES cell-derived graft cells alone developed tumors at the grafted site and behavioral improvement ceased after day 21. In contrast, no tumor development was observed in mice cotransplanted with BMSCs, which also showed sustained behavioral improvement. In vitro results demonstrated the disappearance of SSEA-1 expression in cytochemical examinations, as well as attenuated mRNA expressions of the undifferentiated markers Oct3/4, Utf1, Nanog, Sox2, and ERas by RT-PCR in RA-treated EBs cocultured with BMSCs. In addition, MAP2-immunopositive cells appeared in the EBs cocultured with BMSCs. Furthermore, the synthesis of NGF, GDNF, and BDNF was confirmed in cultured BMSCs, while immunohistochemical examinations demonstrated the survival of BMSCs and their maintained ability of neurotrophic factor production at the grafted site for up to 5 weeks after transplantation. These results suggest that BMSCs induce undifferentiated ES cells to differentiate into a neuronal lineage by neurotrophic factor production, resulting in suppression of tumor formation. Cotransplantation of BMSCs with ES cell-derived graft cells may be useful for preventing the development of ES cell-derived tumors.
RESUMEN
We present 3 adult cases of visceral toxocariasis from the same family, who each consumed thin slices of raw bovine liver weekly, and developed eosinophilia and multiple small lesions in their livers and lungs. Serological examinations using the larval excretory-secretory product of Toxocara canis strongly indicated infection with Toxocara species larvae. The patients responded well to treatment with albendazole. Ingestion of raw liver from paratenic animals is considered to be a common transmission route of human toxocariasis, especially in adults.
Asunto(s)
Parasitología de Alimentos , Larva Migrans Visceral/diagnóstico , Hígado/parasitología , Toxocara canis , Adulto , Albendazol/uso terapéutico , Animales , Antihelmínticos/uso terapéutico , Anticuerpos Antihelmínticos/sangre , Bovinos , Eosinofilia , Femenino , Humanos , Larva Migrans Visceral/tratamiento farmacológico , Larva Migrans Visceral/parasitología , Larva Migrans Visceral/fisiopatología , Hígado/patología , Pulmón/parasitología , Pulmón/patología , Masculino , Persona de Mediana Edad , Tomografía Computarizada por Rayos X , Toxocara canis/inmunologíaRESUMEN
The effects of CoCl(2) on retinoic acid (RA)-treated embryoid bodies (EBs) were investigated. Four-day EBs were treated with 5x10(-6) M of RA for 4 d, then subjected to attached culturing for 7 d in the presence of CoCl(2) at 0, 20, and 100 microM. Differentiation into MAP2- and GFAP-immunopositive cells was inhibited by CoCl(2) in a dose-dependent manner. Next, RA-treated EBs were dissociated into single cells and cultured for 7 d at an initial cell density of 1x10(3)/cm(2). The number of cells increased in a CoCl(2)-dose dependent fashion. In cultures with 100 microM of CoCl(2), more than 90% of the cells were immunopositive for nestin and nestin-immunopositive cells formed clusters, while there were few cells immunopositive for MAP2 or GFAP. These results suggest that CoCl(2) inhibits neural differentiation of RA-treated EB cells and promotes the proliferation of nestin-immunopositive cells, i.e., embryonic stem (ES)-derived neural stem-like cells.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Cobalto/farmacología , Células Madre Embrionarias/efectos de los fármacos , Neuronas/efectos de los fármacos , Tretinoina/farmacología , Animales , Secuencia de Bases , Proliferación Celular , Cartilla de ADN , Relación Dosis-Respuesta a Droga , Células Madre Embrionarias/citología , Inmunohistoquímica , Neuronas/citología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
We report a Japanese patient with loiasis who became infected in Cameroon. Despite the clinical history and laboratory data providing adequate evidence for suspecting loiasis, microfilariae were not detected in the blood. It is important to note that most infected travelers whose home countries are in nonendemic regions are amicrofilaremic.
Asunto(s)
Loiasis/diagnóstico , Microfilarias/aislamiento & purificación , Viaje , Adulto , Animales , Antiparasitarios/uso terapéutico , Camerún , Eosinofilia/parasitología , Femenino , Humanos , Ivermectina/uso terapéutico , Loiasis/sangre , Loiasis/tratamiento farmacológico , Reacción en Cadena de la PolimerasaRESUMEN
Although Wnts are expressed in hair follicles throughout life from embryo to adult, and considered to be critical for their development and maturation, their roles remain largely unknown. In the present study, we investigated the effects of Wnts (Wnt-3a, Wnt-5a, Wnt-10b, and Wnt-11) on epithelial cell differentiation using adult mouse-derived primary skin epithelial cell (MPSEC) cultures and hair growth using hair follicle organ cultures. Only Wnt-10b showed evident promotion of epithelial cell differentiation and hair shaft growth, in contrast to Wnt-3a, 5a, and 11. Our results suggest that Wnt-10b is unique and plays an important role in differentiation of epithelial cells in the hair follicle.
Asunto(s)
Células Epiteliales/citología , Células Epiteliales/metabolismo , Folículo Piloso/citología , Folículo Piloso/metabolismo , Proteínas Wnt/metabolismo , Animales , Diferenciación Celular/fisiología , Proliferación Celular , Células Cultivadas , Ratones , Ratones Endogámicos C3HRESUMEN
We transplanted undifferentiated embryonic stem (ES) cells into the spleens of carbon tetrachloride (CCl(4))-treated mice to determine their effects on liver fibrosis. Carbon tetrachloride at 0.5 ml/kg of body weight was injected intraperitoneally into C57BL/6 mice twice weekly for up to 20 weeks. Four weeks after the first injection, the mice were divided into two groups and those in group 1 received 1 x 10(5) ES cells genetically labelled with enhanced green fluorescent protein (GFP) in the spleens, while group 2 mice received 0.1 ml of phosphate-buffered saline. In group 1, GFP-immunopositive cells were retained and found in areas of fibrosis in the liver, and reduced liver fibrosis was observed as compared with group 2. Secondary transplantation of ES cells at 12 weeks after the initial transplantation enhanced the reduction in liver fibrosis. No teratoma formation or uncontrolled growth of ES cells in organs, including the liver and spleen, was observed in any of the mice. In the livers of group 1 mice, metalloproteinase 9-immunopositive cells derived from ES cells as well as those from the recipient were observed. These cells were also found to be immunopositive for the hepatoblast marker Delta-like (DlK-1), a member of the DlK-1 family of transmembrane proteins. These results suggest that ES-based cell therapy is potentially useful for liver fibrosis treatment and that reduction in CCl(4)-induced liver fibrosis by transplantation of ES cells may be related closely to the emergence of metalloproteinase-producing hepatoblast-like cells.
Asunto(s)
Células Madre Embrionarias/trasplante , Cirrosis Hepática/cirugía , Hígado/patología , Animales , Tetracloruro de Carbono , Diferenciación Celular , Colágeno/análisis , Femenino , Proteínas Fluorescentes Verdes/administración & dosificación , Proteínas Fluorescentes Verdes/análisis , Hepatocitos/enzimología , Inmunohistoquímica , Inyecciones , Cirrosis Hepática/patología , Cirrosis Hepática Experimental , Metaloproteinasa 9 de la Matriz/análisis , Ratones , Ratones Endogámicos C57BL , BazoRESUMEN
AIM: To transplant undifferentiated embryonic stem (ES) cells into the spleens of carbon tetrachloride (CCl4)-treated mice to determine their ability to differentiate into hepatocytes in the liver. METHODS: CCl4, 0.5 mL/kg body weight, was injected into the peritoneum of C57BL/6 mice twice a week for 5 wk. In group 1 (n=12), 1 x 10(5) undifferentiated ES cells (0.1 mL of 1 x 10(6)/mL solution), genetically labeled with GFP, were transplanted into the spleens 1 d after the second injection. Group 2 mice (n=12) were injected with 0.2 mL of saline twice a week, instead of CCl4, and the same amount of ES cells was transplanted into the spleens. Group 3 mice (n=6) were treated with CCl4 and injected with 0.1 mL of saline into the spleen, instead of ES cells. Histochemical analyses of the livers were performed on post-transplantation d (PD) 10, 20, and 30. RESULTS: Considerable numbers of GFP-immunopositive cells were found in the periportal regions in group 1 mice (CCl4-treated) on PD 10, however, not in those untreated with CCl4 (group 2). The GFP-positive cells were also immunopositive for albumin (ALB), alpha-1 antitrypsin, cytokeratin 18, and hepatocyte nuclear factor 4 alpha on PD 20. Interestingly, most of the GFP-positive cells were immunopositive for DLK, a hepatoblast marker, on PD 10. Although very few ES-derived cells were demonstrated immunohistologically in the livers of group 1 mice on PD 30, improvements in liver fibrosis were observed. Unexpectedly, liver tumor formation was not observed in any of the mice that received ES cell transplantation during the experimental period. CONCLUSION: Undifferentiated ES cells developed into hepatocyte-like cells with appropriate integration into tissue, without uncontrolled cell growth.
Asunto(s)
Diferenciación Celular/fisiología , Células Madre Embrionarias/trasplante , Hepatocitos/citología , Cirrosis Hepática/patología , Bazo/cirugía , Albúminas/metabolismo , Animales , Tetracloruro de Carbono , Movimiento Celular/fisiología , Trasplante de Células/métodos , Células Madre Embrionarias/citología , Femenino , Factor Nuclear 4 del Hepatocito/metabolismo , Hepatocitos/metabolismo , Queratina-18/metabolismo , Cirrosis Hepática/inducido químicamente , Ratones , Ratones Endogámicos C57BL , Bazo/citología , Neoplasias del Bazo/inducido químicamente , Neoplasias del Bazo/prevención & controlRESUMEN
We report a case of cystic echinococcosis (CE) caused by Echinococcusgranulosus, for which a modified percutaneous evacuation (PEVAC) treatment was applied. The patient had immigrated from Peru to Japan and had 2 hydatid cystic masses, 1 located in segment (S)5 of the liver and the other in S3 (5.3 and 3.5 cm in diameter, respectively), both of which were visualized as pseudotumors by ultrasound (US) examinations. Albendazole treatment showed no effects and surgical treatment was refused. After punctuation of the S5 cyst under US guidance and S3 with CT guidance, 10- and 12-French gauge catheters, respectively, with multiple side holes were inserted. About 60 ml of the cyst contents was drawn out from the S5 lesion and 2 ml from the S3 lesion. Using repetitive manual injections and aspiration of small amounts of hypertonic saline, the remaining cyst content was removed as much as possible, after which 20 and 10 ml of 98% ethanol was injected into the S5 and S3 lesions, respectively. A short-term evaluation during the 4 month-period following the procedure using US revealed nearly complete evacuation of the S5 lesion, whereas that at S3 remained as a pseudo-solid mass. We consider that percutaneous treatment is a safe therapeutic modality for hydatid cysts. This is the first case report of CE treated percutaneously in Japan.
Asunto(s)
Equinococosis Hepática/diagnóstico por imagen , Equinococosis Hepática/terapia , Echinococcus granulosus , Emigración e Inmigración , Animales , Biopsia con Aguja Fina , Equinococosis Hepática/parasitología , Equinococosis Hepática/patología , Etanol/administración & dosificación , Humanos , Soluciones Hipertónicas/administración & dosificación , Japón , Perú , Punciones/métodos , Resultado del Tratamiento , UltrasonografíaRESUMEN
AIM: To explore whether a co-culture of cynomolgus monkey embryonic stem (cES) cells with embryonic liver cells could promote their differentiation into hepatocytes. METHODS: Mouse fetal liver-derived cells (MFLCs) were prepared as adherent cells from mouse embryos on embryonic d (ED) 14, after which undifferentiated cES cells were co-cultured with MFLCs. The induction of cES cells along a hepatic lineage was examined in MFLC-assisted differentiation, spontaneous differentiation, and growth factors (GF) and chemicals-induced differentiations (GF-induced differentiation) using retinoic acid, leukemia inhibitory factor (LIF), FGF2, FGF4, hepatocyte growth factor (HGF), oncostatin M (OSM), and dexamethasone. RESULTS: The mRNA expression of alpha-fetoprotein, albumin (ALB), alpha-1-antitrypsin, and hepatocyte nuclear factor 4alpha was observed earlier in the differentiating cES cells co-cultured with MFLCs, as compared to cES cells undergoing spontaneous differentiation and those subjected to GF-induced differentiation. The expression of cytochrome P450 7a1, a possible marker for embryonic endoderm-derived mature hepatocytes, was only observed in cES cells that had differentiated in a co-culture with MFLCs. Further, the disappearance of Oct3/4, a representative marker of an undifferentiated state, was noted in cells co-cultured with MFLCs, but not in those undergoing spontaneous or GF-induced differentiation. Immunocytochemical analysis revealed an increased ratio of ALB-immunopositive cells among cES cells co-cultured with MFLCs, while glycogen storage and urea synthesis were also demonstrated. CONCLUSION: MFLCs showed an ability to induce cES cells to differentiate toward hepatocytes. The co-culture system with MFLCs is a useful method for induction of hepatocyte-like cells from undifferentiated cES cells.
Asunto(s)
Células Madre Embrionarias/citología , Hepatocitos/citología , Hígado/citología , Hígado/embriología , Animales , Diferenciación Celular/efectos de los fármacos , Colesterol 7-alfa-Hidroxilasa/genética , Colesterol 7-alfa-Hidroxilasa/metabolismo , Técnicas de Cocultivo/métodos , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/metabolismo , Regulación de la Expresión Génica , Glucógeno/metabolismo , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Péptidos y Proteínas de Señalización Intercelular/farmacología , Hígado/efectos de los fármacos , Hígado/metabolismo , Macaca fascicularis , Ratones , Ratones Endogámicos C57BL , Proteínas de Transporte de Catión Orgánico/genética , Proteínas de Transporte de Catión Orgánico/metabolismo , Urea/metabolismoRESUMEN
We successfully established cynomolgus monkey embryonic stem (cES) cell lines expressing enhanced green fluorescent protein (GFP) by introducing a GFP-encoding gene under cytomegalovirus immediate early enhancer (CMVIE) promoter regulation into cES cells. The cells maintained the ability of in vitro differentiation toward ectodermal, mesodermal, and endodermal lineages, and produced teratomas composed of tissues derived from the three embryonic germ layers when transplanted into severe combined immunodeficient disease mice. GFP expression was also observed in the differentiated cells. These GFP-expressing cES cell lines are considered useful for basic research, including cell transplantation.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Clonación Molecular/métodos , Proteínas Fluorescentes Verdes/metabolismo , Ingeniería de Proteínas/métodos , Células Madre/citología , Células Madre/metabolismo , Ingeniería de Tejidos/métodos , Animales , Diferenciación Celular , Supervivencia Celular , Células Cultivadas , Proteínas Fluorescentes Verdes/genética , Macaca fascicularis , Ratones , Ratones SCID , Proteínas Recombinantes/metabolismoRESUMEN
We present a case of rapid onset of glycogen storage hepatomegaly, caused by a massive dose of long-acting insulin and large doses of glucose, in a type-2 diabetic patient. A 41-year-old man was admitted to our hospital because of hypoglycemia and unconsciousness following subcutaneous administration of 180 units of insulin glargine in a suicide attempt. Despite continuous hypercaloric infusion with additional intravenous glucose injections, hypoglycemia persisted for 36 hours. Although the hepatic function was normal and no hepatomegaly was detected on admission, the liver function tests became abnormal and hepatomegaly was detected on hospitalization day 3. Plain abdominal computed tomography (CT) scanning confirmed liver enlargement, with hepatic CT attenuation markedly elevated at 83.7 HU. Liver biopsy revealed hepatocytic glycogen deposition with edematous degeneration. Based on these findings, the diagnosis was made as rapid onset glycogen storage hepatomegaly caused by administration of a massive dose of long-acting insulin and supplementation with large doses of glucose. With improved glycemic control, the liver function improved, the CT findings of hepatomegaly improved, and the hepatic CT attenuation decreased. Repeat liver biopsy also confirmed almost complete disappearance of glycogen deposits. When hepatic dysfunction or hepatomegaly is detected during treatment with insulin, the possibility of hepatic glycogen deposition should be considered. CT scanning and liver biopsy were useful in diagnosing this case.