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1.
Cell Tissue Res ; 375(3): 697-707, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30284085

RESUMO

Cancer stem cells (CSCs) are specific targets for therapeutic applications, but the rarity of CSCs within tumors makes the isolation of CSCs difficult. To overcome these problems, we generated CSCs in vitro using established reprogramming techniques. We transduced four previously established reprogramming factors, Oct3/4, Sox2, Klf4, and L-myc, into the colon cancer cell lines LoVo and OUMS-23, and investigated the biological characteristics of these lines. Tra-1-60+ cells were obtained from reprogrammed induced pluripotent stem (iPS) cell-like colonies and showed CSC properties, including colony formation, maintenance of colonies by repeated passages, and feeder cell dependency, as well as increased expressions of CSC markers such as CD133 and ALDH1. The CSC-like cells showed increased chemoresistance to 5-fluorouracil and elevated tumorigenicity upon transplantation into kidneys of immune-deficient mice. These tumors shifted to a poorly differentiated stage with many atypical cells, cytoplasmic mucin, and focal papillary components, with demonstrated dedifferentiation. The principal component analysis from DNA microarrays showed that though both cell lines moved to iPS cells after reprogramming, they were not completely identical to iPS cells. Significantly elevated gene expression of Decorin and CD90 was observed in CSC-like cells. Together, these results show that reprogramming of cancer cells produced not pluripotent stem cells but CSC-like cells, and these findings will provide biological information about genuine CSCs and help establish new CSC-targeted therapies.


Assuntos
Reprogramação Celular , Neoplasias do Colo/patologia , Células-Tronco Neoplásicas/metabolismo , Animais , Biomarcadores Tumorais/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Fator 4 Semelhante a Kruppel , Camundongos SCID , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
2.
Biochem Biophys Res Commun ; 482(4): 549-555, 2017 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-27856246

RESUMO

Retinoblastoma is the most common intraocular malignancy in pediatric patients. It develops rapidly in the retina and can be fatal if not treated promptly. It has been proposed that a small population of cancer cells, termed cancer stem cells (CSCs), initiate tumorigenesis from immature tissue stem cells or progenitor cells. Reprogramming technology, which can convert mature cells into pluripotent stem cells (iPS), provides the possibility of transducing malignant cancer cells back to CSCs, a type of early stage of cancer. We herein took advantage of reprogramming technology to induce CSCs from retinoblastoma cancer cells. In the present study, the 4 Yamanaka transcription factors, Oct4, Sox2, Klf4 and c-myc, were transduced into retinoblastoma cells (Rbc51). iPS-like colonies were observed 15 days after transduction and showed significantly enhanced CSC properties. The gene and protein expression levels of pluripotent stem cell markers (Tra-1-60, Oct4, Nanog) and cancer stem cell markers (CD133, CD44) were up-regulated in transduced Rbc51 cells compared to control cells. Moreover, iPS-like CSCs could be sorted using the Magnetic-activated cell sorting (MACS) method. A sphere formation assay demonstrated spheroid formation in transduced Rbc51 cells cultured in serum free media, and these spheroids could be differentiated into Pax6-, Nestin-positive neural progenitors and rhodopsin- and recoverin-positive mature retinal cells. The cell viability after 5-Fu exposure was higher in transduced Rbc51 cells. In conclusion, CSCs were generated from retinoblastoma cancer cells using reprogramming technology. Our novel method can generate CSCs, the study of which can lead to better understanding of cancer-specific initiation, cancer epigenetics, and the overlapping mechanisms of cancer development and pluripotent stem cell behavior.


Assuntos
Técnicas de Reprogramação Celular/métodos , Reprogramação Celular , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Neoplásicas/citologia , Neoplasias da Retina/genética , Retinoblastoma/genética , Linhagem Celular Tumoral , Regulação da Expressão Gênica no Desenvolvimento , Regulação Neoplásica da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Fator 4 Semelhante a Kruppel , Células-Tronco Neoplásicas/metabolismo , Retina/citologia , Retina/metabolismo , Fatores de Transcrição/genética , Transdução Genética
3.
Cell Tissue Res ; 354(3): 751-9, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23996199

RESUMO

The major molecular signals of pancreatic exocrine development are largely unknown. We examine the role of fibroblast growth factor 7 (FGF7) in the final induction of pancreatic amylase-containing exocrine cells from induced-pancreatic progenitor cells derived from human embryonic stem (hES) cells. Our protocol consisted in three steps: Step I, differentiation of definitive endoderm (DE) by activin A treatment of hES cell colonies; Step II, differentiation of pancreatic progenitor cells by re-plating of the cells of Step I onto 24-well plates at high density and stimulation with all-trans retinoic acid; Step III, differentiation of pancreatic exocrine cells with a combination of FGF7, glucagon-like peptide 1 and nicotinamide. The expression levels of pancreatic endodermal markers such as Foxa2, Sox17 and gut tube endoderm marker HNF1ß were up-regulated in both Step I and II. Moreover, in Step III, the induced cells expressed pancreatic markers such as amylase, carboxypeptidase A and chymotrypsinogen B, which were similar to those in normal human pancreas. From day 8 in Step III, cells immunohistochemically positive for amylase and for carboxypeptidase A, a pancreatic exocrine cell product, were induced by FGF7. Pancreatic progenitor Pdx1-positive cells were localized in proximity to the amylase-positive cells. In the absence of FGF7, few amylase-positive cells were identified. Thus, our three-step culture protocol for human ES cells effectively induces the differentiation of amylase- and carboxypeptidase-A-containing pancreatic exocrine cells.


Assuntos
Amilases/biossíntese , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Fator 7 de Crescimento de Fibroblastos/metabolismo , Pâncreas Exócrino/citologia , Pâncreas Exócrino/efeitos dos fármacos , Ativinas/farmacologia , Comunicação Celular/efeitos dos fármacos , Comunicação Celular/fisiologia , Contagem de Células , Técnicas de Cultura de Células/métodos , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Células Cultivadas , Técnicas Citológicas , Células-Tronco Embrionárias/metabolismo , Fator 7 de Crescimento de Fibroblastos/genética , Fator 7 de Crescimento de Fibroblastos/farmacologia , Peptídeo 1 Semelhante ao Glucagon/farmacologia , Humanos , Niacinamida/farmacologia , Pâncreas Exócrino/enzimologia , Pâncreas Exócrino/metabolismo , Tretinoína/farmacologia
4.
Int J Food Sci Nutr ; 64(8): 988-92, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23848379

RESUMO

There is an increasing interest in dietary polyphenols for risk reduction in cardiovascular diseases. The aim of this study was to evaluate acute and chronic flow-mediated dilation (FMD) and blood pressure responses to daily intake of boysenberry juice. FMD of the brachial artery was measured in six subjects in the initial, intermediate and follow-up stages of a 4-week open-label intervention study. The intake of boysenberry juice (180 ml/d) increased FMD with progression of intervention stage, and FMD differed in the follow-up stage compared with pre-intake baseline (p = 0.0163 < 0.0167 = 0.1/6) using Bonferroni correction. Changes in systolic blood pressure (SBP) correlated negatively with SBP before intake only in the follow-up stage (r = -0.961 and p = 0.0007 at 3.5 h), indicating a greater SBP reduction in subjects with higher SBP. These results suggest that daily intake of boysenberry juice is beneficial for reducing cardiovascular risk.


Assuntos
Pressão Sanguínea/efeitos dos fármacos , Doenças Cardiovasculares/prevenção & controle , Frutas/química , Hipertensão/dietoterapia , Polifenóis/farmacologia , Rosaceae/química , Vasodilatação/efeitos dos fármacos , Adulto , Idoso , Bebidas , Pressão Sanguínea/fisiologia , Artéria Braquial , Doenças Cardiovasculares/etiologia , Doenças Cardiovasculares/fisiopatologia , Dilatação Patológica , Feminino , Humanos , Hipertensão/fisiopatologia , Masculino , Pessoa de Meia-Idade , Fitoterapia , Preparações de Plantas/farmacologia , Fatores de Risco
5.
Biosci Biotechnol Biochem ; 76(9): 1694-701, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22972352

RESUMO

The antihypertensive effect of a single oral administration of a boysenberry seed polyphenol extract to spontaneously hypertensive rats was evaluated at different doses (100 and 200 mg/kg), and a significant decrease in systolic blood pressure (SBP) was observed up to 6 h post administration. The extract was separated into proanthocyanidin-rich and ellagitannin fractions by solvent partition. A significant decrease in SBP was observed only after administering the proanthocyanidin-rich fraction, and this decrease was abolished by an N(G)-nitro-L-arginine methyl ester (L-NAME) injection. An analysis of the orally absorbable components showed that intact dimeric and trimeric procyanidins and propelargonidins were detectable in the plasma with a maximal concentration 2 h post administration. The vasorelaxant activity of the extract was also confirmed by in vitro assay using rat aorta rings. These results suggest that proanthocyanidins (PAs) in boysenberry seeds may have played an important role in the observed antihypertensive effect.


Assuntos
Anti-Hipertensivos/isolamento & purificação , Hipertensão/tratamento farmacológico , Proantocianidinas/isolamento & purificação , Rosaceae/química , Sementes/química , Vasodilatadores/isolamento & purificação , Absorção , Administração Oral , Animais , Anti-Hipertensivos/administração & dosagem , Aorta/efeitos dos fármacos , Pressão Sanguínea/efeitos dos fármacos , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Masculino , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Extratos Vegetais/química , Proantocianidinas/administração & dosagem , Ratos , Ratos Endogâmicos SHR , Técnicas de Cultura de Tecidos , Vasodilatadores/administração & dosagem
6.
Cryobiology ; 64(1): 12-22, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22133891

RESUMO

Cryopreservation is an essential technique in basic research and clinical applications of human embryonic stem (hES) cells. Cryopreserved hES cells are fragile and undergo post-thaw apoptosis. We performed gene pathway analysis on cryopreserved and thawed hES cells to examine the effect of Y-27632, a Rho-associated kinase (ROCK) inhibitor, on apoptosis and associated molecular events. Y-27632 was added to the cryopreservation solution and/or the post-thaw medium of two hES cell lines (KhES-1, KhES-3). Post-thaw apoptosis was recorded as a function of time using Giemsa staining and the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Apoptosis plateaued 12h after the untreated hES cells were thawed. Gene pathway analysis showed the activation of IL-1ß, TGF-ß, and their respective receptors (IL-1R, ACVR1C) in the mitogen-activated protein kinase (MAPK) pathway, which resulted in the upregulation of caspase-8 and -10. Quantitative RT-PCR confirmed the upregulation of IL-1ß, TGF-ß, their respective receptors, and caspase-10 and -3. As these molecules were suppressed by Y-27632, gene pathways involving these molecules probably depend on ROCK activation. The TGF-ß receptor antagonist, SB-431542, and an inhibitor of p38MAPK, SB-203580, did not affect apoptosis. Combining Y-27632 with SB-203580, however, resulted in an increase in the survival rate compared with the control. This suggests that the initiation of apoptosis depends on cytokine interactions and multiple ways exist to reduce post-thaw apoptosis in hES cells. Y-27632 can suppress cytokine interactions and the MAPK pathway, thereby reducing the occurrence of apoptosis, and is an effective cryoprotectant for hES cells.


Assuntos
Amidas/farmacologia , Apoptose/efeitos dos fármacos , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/fisiologia , Piridinas/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Benzamidas/farmacologia , Caspase 10/biossíntese , Caspase 3/biossíntese , Caspase 8/biossíntese , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Criopreservação , Crioprotetores/metabolismo , Dioxóis/farmacologia , Células-Tronco Embrionárias/citologia , Humanos , Imidazóis/farmacologia , Interleucina-1beta/biossíntese , Fator de Crescimento Transformador beta/biossíntese , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
7.
Biochem Biophys Res Commun ; 410(3): 608-13, 2011 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-21684256

RESUMO

Mouse embryonic stem cells (ESCs) can be induced to form pancreatic exocrine enzyme-producing cells in vitro in a stepwise fashion that recapitulates the development in vivo. However, there is no protocol for the differentiation of pancreatic-like cells from human ESCs (hESCs). Based upon the mouse ESC model, we have induced the in vitro formation of pancreatic exocrine enzyme-producing cells from hESCs. The protocol took place in four stages. In Stage 1, embryoid bodies (EBs) were formed from dissociated hESCs and then treated with the growth factor activin A, which promoted the expression of Foxa2 and Sox17 mRNAs, markers of definitive endoderm. In Stage 2, the cells were treated with all-trans retinoic acid which promoted the transition to cells that expressed gut tube endoderm mRNA marker HNF1b. In Stage 3, the cells were treated with fibroblast growth factor 7 (FGF7), which induced expression of Pdx1 typical of pancreatic progenitor cells. In Stage 4, treatment with FGF7, glucagon-like peptide 1, and nicotinamide induced the expression amylase (AMY) mRNA, a marker for mature pancreatic exocrine cells. Immunohistochemical staining showed the expression of AMY protein at the edges of cell clusters. These cells also expressed other exocrine secretory proteins including elastase, carboxypeptidase A, chymotrypsin, and pancreatic lipase in culture. Production of these hESC-derived pancreatic enzyme-producing cells represents a critical step in the study of pancreatic organogenesis and in the development of a renewable source of human pancreatic-like exocrine cells.


Assuntos
Técnicas de Cultura de Células , Diferenciação Celular , Corpos Embrioides/citologia , Pâncreas Exócrino/citologia , Ativinas/farmacologia , Amilases/biossíntese , Carboxipeptidases A/biossíntese , Quimotripsina/biossíntese , Corpos Embrioides/efeitos dos fármacos , Corpos Embrioides/enzimologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/enzimologia , Fator 7 de Crescimento de Fibroblastos/farmacologia , Peptídeo 1 Semelhante ao Glucagon/farmacologia , Fator 3-beta Nuclear de Hepatócito/biossíntese , Humanos , Lipase/biossíntese , Niacinamida/farmacologia , Pâncreas Exócrino/enzimologia , Elastase Pancreática/biossíntese , Fatores de Transcrição SOXF/biossíntese , Tretinoína/farmacologia
8.
Cryo Letters ; 32(6): 516-24, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22227712

RESUMO

Poor adhesion of single human embryonic stem (hES) cells after freeze-thawing causes death. To investigate mechanisms responsible for this, Rho-dependent protein kinase (ROCK) inhibitor Y-27632-treated and untreated single hES cells were analyzed for E-cadherin and F-actin distribution by immunostaining and phalloidin staining respectively and for G13 signaling pathway components by DNA microarray and quantitative polymerase chain reaction (PCR). Y-27632-treated cells clustered rapidly and maintained E-cadherin and F-actin distribution without losing Oct-3/4. Immediately after thawing, E-cadherin in untreated hES cells dotted along the membrane and then displayed eccentric cytoplasmic localization. Bleb formation and early Oct-3/4 loss occurred after F-actin network condensation in the cytoplasm. Microarray analyses and quantitative PCR indicated upregulation of two actin reorganization-associated components of the G13 signaling pathway, Arhgdib and Cdc42, in untreated cells. Considering these findings and that cell death was partly interrupted by Y-27632, E-cadherin and actin cytoskeleton network disruption through the G13 signaling pathway may cause hES cell death after freeze-thawing.


Assuntos
Actinas/metabolismo , Caderinas/biossíntese , Células-Tronco Embrionárias/citologia , Congelamento , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/metabolismo , Transdução de Sinais , Sequência de Bases , Células Cultivadas , Primers do DNA , Humanos , Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase Via Transcriptase Reversa
9.
Biochem Biophys Res Commun ; 394(4): 877-83, 2010 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-20206598

RESUMO

PURPOSE: Photoreceptors cannot regenerate and recover their functions once disordered. Transplantation of retinal pigment epithelium (RPE) has recently become a possible therapeutic approach for retinal degeneration. In the present study, we investigated the induction of photoreceptors by coculturing primate embryonic stem cells (ESCs) with ESC-derived RPE cells. METHODS: RPE cells were derived by coculturing ESCs and Sertoli cells. Photoreceptors were then induced by using ESC-derived RPE cells and retinoic acid (RA) RESULTS: RPE cell generation was confirmed by morphological analysis, which revealed highly pigmented polygonal cells with a compact cell-cell arrangement. After coculturing ESCs and RPE cells, some ESC derivatives became immunopositive for rhodopsin. RT-PCR analysis demonstrated the expression of retina-related gene markers such as Pax6, CRX, IRBP, rhodopsin, rhodopsin kinase, and Muschx10A. When RA was added, a distinct increase in the expression of photoreceptor-specific proteins and genes was found. In addition, the differentiation of bipolar horizontal cells was demonstrated by protein and gene expression. The ESCs that were cocultured with RPE cells and treated with RA were transplanted into the renal capsule or intra-vitreal space of nude mice. Grafted ESC derivatives demonstrated extensive rhodopsin expression, and they survived and organized into recipient tissues, although they formed teratomas. CONCLUSION: These results indicate that coculturing ESCs with ESC-derived RPE cells is a useful and efficient method for inducing photoreceptors and providing an insight into the use of ESCs for retina regeneration.


Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/citologia , Regeneração , Epitélio Pigmentado da Retina/fisiologia , Epitélio Pigmentado da Retina/transplante , Animais , Técnicas de Cultura de Células , Técnicas de Cocultura , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Nus , Epitélio Pigmentado da Retina/metabolismo , Células de Sertoli/citologia , Células de Sertoli/fisiologia , Tretinoína/farmacologia
10.
Biochem Biophys Res Commun ; 391(3): 1477-82, 2010 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-20034473

RESUMO

To establish an effective induction method for hepatic differentiation using serum-free media, the effects of activin in serum-containing and serum-free conditions on embryoid body (EB) induction into mesendoderm were investigated by Western blot analysis and real-time reverse transcription-polymerase chain reaction (RT-PCR) as a first step. The expression of P-smad2 and mesendodermal markers was markedly enhanced by 100ng/ml activin under serum-free conditions but were inhibited or masked under serum-containing conditions. Next, serum-free Lanford medium was used to attempt the direct induction of activin-treated EBs expressing mesendodermal markers into hepatic lineage cells and this induction was compared to that induced using Iscove's Modified Dulbecco's medium containing 20% fetal bovine serum. Once immersed in the Lanford medium, EBs began to show typical hepatic features by day 17, including Alb, AFP, TTR, and AAT expression detected by RT-PCR, and ALB, AFP, and CK18 expression detected by immunostaining. On day 22, these cells were of high quality characterized by the expression of metabolizing enzymes, including Ugt1a1, Slcola4, cyp3a11, cyp2b10, and cyp7a1 detected by real-time PCR, a 50-fold greater cyp3A11 response than control to 100muM dexamethasone stimulation, specific cellular uptake of indocyanine green, and glycogen storage in the cytoplasm. These results indicate that this simple two-step induction method under serum-free conditions induces hepatic lineage cells with high quality directly from mouse embryonic stem (ES) cell-derived mesendoderm.


Assuntos
Diferenciação Celular , Linhagem da Célula , Células-Tronco Embrionárias/efeitos dos fármacos , Hepatócitos/citologia , Animais , Técnicas de Cultura de Células , Meios de Cultura Livres de Soro/farmacologia , Células-Tronco Embrionárias/enzimologia , Células-Tronco Embrionárias/fisiologia , Endoderma/citologia , Endoderma/enzimologia , Endoderma/fisiologia , Hepatócitos/enzimologia , Mesoderma/citologia , Mesoderma/enzimologia , Mesoderma/fisiologia , Camundongos
11.
Biochem Biophys Res Commun ; 393(4): 877-82, 2010 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-20171952

RESUMO

Embryonic stem cells (ESCs) are potentially powerful tools for regenerative medicine and establishment of disease models. The recent progress in ESC technologies is noteworthy, but ESC differentiation into renal lineages is relatively less established. The present study aims to differentiate mouse ESCs (mESCs) into a renal progenitor pool, the intermediate mesoderm (IM), without addition of exogenous cytokines and embryoid formation. First, we treated mESCs with a combination of small molecules (Janus-associated tyrosine kinase inhibitor 1, LY294002, and CCG1423) and differentiated them into BMP7-positive cells, BMP7 being the presumed inducing factor for IM. When these cells were cultured with adding retinoic acid, expression of odd-skipped related 1 (Osr1), which is essential to IM differentiation, was enhanced. To simplify the differentiation protocol, the abovementioned four small molecules (including retinoic acid) were combined and added to the culture. Under this condition, more than one-half of the cells were positive for Osr1, and at the same time, Pax2 (another IM marker) was detected by real-time PCR. Expressions of ectodermal marker and endodermal marker were not enhanced, while mesodermal marker changed. Moreover, expression of genes indispensable to kidney development, i.e., Lim1 and WT1, was detected by RT-PCR. These results indicate the establishment of a specific, effective method for differentiation of the ESC monolayer into IM using a combination of small molecules, resulting in an attractive cell source that could be experimentally differentiated to understand nephrogenic mechanisms and cell-to-cell interactions in embryogenesis.


Assuntos
Proteína Morfogenética Óssea 7/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células-Tronco Embrionárias/efeitos dos fármacos , Rim/fisiologia , Mesoderma/citologia , Regeneração , Anilidas/farmacologia , Animais , Benzamidas/farmacologia , Comunicação Celular , Linhagem da Célula , Cromonas/farmacologia , Técnicas de Cocultura , Células-Tronco Embrionárias/classificação , Expressão Gênica , Marcadores Genéticos , Janus Quinases/antagonistas & inibidores , Rim/citologia , Camundongos , Morfolinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia
12.
ScientificWorldJournal ; 9: 884-90, 2009 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-19734962

RESUMO

To induce hepatocytes from human embryonic stem (hES) cells easily and effectively, a simple suspension culture method that separates ES colonies with a scraper and transfers them into newly developed, nonadherent MPC (2-methacryloyloxyethyl phosphorylcholine) plates, and the staged-additional cocktail method, including growth factors, cytokines, and Lanford serum-free medium, were developed and evaluated mainly by morphological analysis. The formed embryoid bodies (EBs) showed compact cellular agglomeration until day 4 and later formed coeloms in their interior. RT-PCR (reverse transcriptase-polymerase chain reaction) analysis showed that they are gene markers of the three germ layers. Mesenchymal cells with rough endoplasmic reticulum (rER) and extracellular matrix (ECM), and without junctions, were recognized in the interior of the EBs by transmission electron microscopy (TEM) in addition to epithelial cells. When they were stimulated by the staged-additional cocktail, they expressed albumin-positive immunoreactivity, indocyanine green (ICG) uptake, and typical ultrastructures of the hepatocytes, including bile canaliculi. These results indicate that these combined methods promote EB formation and hepatocyte differentiation from hES cells.


Assuntos
Células-Tronco Embrionárias/citologia , Hepatócitos/citologia , Sequência de Bases , Linhagem Celular , Primers do DNA , Células-Tronco Embrionárias/ultraestrutura , Humanos , Microscopia Eletrônica de Transmissão , Reação em Cadeia da Polimerase Via Transcriptase Reversa
13.
PLoS One ; 11(9): e0162693, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27632182

RESUMO

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are potential resources for the regeneration of defective organs, including the liver. However, some obstacles must be overcome before this becomes reality. Undifferentiated cells that remain following differentiation have teratoma-forming potential. Additionally, practical applications require a large quantity of differentiated cells, so the differentiation process must be economical. Here we describe a DNA microarray-based global analysis of the gene expression profiles of differentiating human pluripotent stem cells. We identified differences and commonalities among six human pluripotent stem cell lines: the hESCs KhES1, KhES2, KhES3, and H1, and the iPSCs 201B7 and 243G1. Embryoid bodies (EBs) formed without requiring supplementation with inducing factors. EBs also expressed some liver-specific metabolic genes including the ammonia-metabolizing enzymes glutamine synthetase and carbamoyl-phosphate synthase 1. Real-time PCR analysis revealed hepatocyte-like differentiation of EBs treated with ammonia in Lanford medium. Analysis of DNA microarray data suggested that hepatocyte-like cells were the most abundant population in ammonia-treated cells. Furthermore, expression levels of undifferentiated pluripotent stem cell markers were drastically reduced, suggesting a reduced teratoma-forming capacity. These results indicate that treatment of EBs with ammonia in Lanford medium may be an effective inducer of hepatic differentiation in absence of expensive inducing factors.


Assuntos
Amônia/farmacologia , Hepatócitos/efeitos dos fármacos , Células-Tronco Pluripotentes/efeitos dos fármacos , Animais , Diferenciação Celular , Linhagem da Célula , Hepatócitos/citologia , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Células-Tronco Pluripotentes/citologia
14.
World J Stem Cells ; 7(5): 883-93, 2015 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-26131319

RESUMO

AIM: To facilitate close contacts between transplanted cardiomyocytes and host skeletal muscle using cell fusion mediated by hemagglutinating virus of Japan envelope (HVJ-E) and tissue maceration. METHODS: Cardiomyocytes (1.5 × 10(6)) from fetal rats were first cultured. After proliferation, some cells were used for fusion with adult muscle fibers using HVJ-E. Other cells were used to create cardiomyocyte sheets (area: about 3.5 cm(2) including 2.1 × 10(6) cells), which were then treated with Nile blue, separated, and transplanted between the latissimus dorsi and intercostal muscles of adult rats with four combinations of HVJ-E and/or NaOH maceration: G1: HVJ-E(+), NaOH(+), Cardiomyocytes(+); G2: HVJ-E(-), NaOH(+), Cardiomyocytes(+); G3: HVJ-E(+), NaOH(-), Cardiomyocytes(+); G4: HVJ-E(-), NaOH(-), Cardiomyocytes(-). At 1 and 2 wk after transplantation, the four groups were compared by detection of beating domains, motion images using moving target analysis software, action potentials, gene expression of MLC-2v and Mesp1 by reverse transcription-polymerase chain reaction, hematoxylin-eosin staining, and immunostaining for cardiac troponin and skeletal myosin. RESULTS: In vitro cardiomyocytes were fused with skeletal muscle fibers using HVJ-E. Cardiomyocyte sheets remained in the primary transplanted sites for 2 wk. Although beating domains were detected in G1, G2, and G3 rats, G1 rats prevailed in the number, size, motion image amplitudes, and action potential compared with G2 and G3 rats. Close contacts were only found in G1 rats. At 1 wk after transplantation, the cardiomyocyte sheets showed adhesion at various points to the myoblast layer in the latissimus dorsi muscle. At 2 wk after transplantation, close contacts were seen over a broad area. Part of the skeletal muscle sarcoplasma seemed to project into the myocardiocyte plasma and some nuclei appeared to share both sarcoplasmas. CONCLUSION: The present results show that close contacts were acquired and facilitated the beating function, thereby providing a new cellular transplantation method using HVJ-E and NaOH maceration.

15.
J Nutr Sci Vitaminol (Tokyo) ; 60(1): 43-51, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24759259

RESUMO

The effects of acute or chronic intake of boysenberry juice or artificial vinegar on blood pressure (BP) and endothelial function were investigated in spontaneous hypertensive rats (SHR). A single administration of boysenberry juice (BJ, equivalent to 0.5 mL/kg body weight) or artificial boysenberry juice vinegar (BJV, equivalent to 0.5 mL BJ and 0.10 g acetic acid/kg body weight) decreased both systolic blood pressure (SBP) and diastolic blood pressure (DBP) significantly. Reductions in SBP of the control group compared with the BJ and BJV groups reached maxima of -16.8±4.3 and -28.4±7.3 mmHg 8 h after administration, respectively. Chronic SBP- and DBP-lowering effects were also observed upon daily feedings of both BJ and BJV at 4 wk. No significant differences were found in SBP or DBP between respective acute and chronic intake of BJ and BJV, except for the decrease in DBP after 4 wk of BJV intake. This suggests that the polyphenol constituents in BJ and BJV likely play a major role in lowering SBP and DBP under these conditions and that acetic acid added to BJ exerts a DBP-lowering effect after 4 wk of BJV intake. The polyphenolic constituents of these beverages might elevate plasma NO concentration via aortic endothelial nitric oxide synthase activation, but the effects of chronic intake on blood pressure might also be at least partly mediated by the renin-angiotensin system. These results may help explain the beneficial effects of boysenberry intake on cardiovascular health, such as reduced blood pressure and improved endothelial function.


Assuntos
Bebidas/análise , Pressão Sanguínea/efeitos dos fármacos , Frutas/química , Animais , Masculino , Óxido Nítrico/sangue , Óxido Nítrico Sintase Tipo III/metabolismo , Extratos Vegetais/administração & dosagem , Polifenóis/administração & dosagem , Ratos , Ratos Endogâmicos SHR , Sistema Renina-Angiotensina/efeitos dos fármacos
16.
Ann Anat ; 195(1): 50-6, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22727933

RESUMO

To investigate the effects of the Rho-dependent protein kinase (ROCK) inhibitor Y-27632 on the kinetics of E-cadherin, F-actin, and Oct3/4 distributions in dissociated human embryonic stem (hES) cells and to analyze their interactions morphologically, Y-27632-treated [R(i) (+)] and untreated [R(i) (-)] cells were immunohistochemically stained for E-cadherin and Oct3/4 within 24h of dissociation and also for F-actin. Furthermore, the gene expression of E-cadherin, Oct3/4, and RhoA was confirmed by quantitative real-time RT-PCR. E-cadherin expression intensified linearly along the membranes of R(i) (+) cells or intercellular junctions in cell clusters. F-actin accumulated along the periphery of cells and expanded in a web-like manner along junctions in cell clusters, and Oct3/4 was restricted to the nucleus within few hours of dissociation. However, R(i) (-) cells exhibited deformation and blebbing and appeared to die over time. E-cadherin exhibited a punctate pattern along the periphery, after which it accumulated on one or both sides of the cytoplasm. Actin filaments were concentrated at the bleb bases. Oct3/4 was detected in the cytoplasm, not in the nucleus the recovery of integrated E-cadherin distribution. Quantitative real-time RT-PCR revealed RhoA upregulation and E-cadherin downregulation at 12h after dissociation. Oct3/4 gene expression was unaffected by ROCK inhibition. These results revealed that the cooperative nature of hES cells is maintained by the E-cadherin-actin cytoskeleton system along with the restricted distribution of Oct3/4 in the nucleus. RhoA activation followed by dissociation disorders this system and accelerates cell death, which is partially suppressed by ROCK inhibition.


Assuntos
Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/ultraestrutura , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Actinas/metabolismo , Amidas/farmacologia , Animais , Caderinas/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Citoplasma/metabolismo , Citoplasma/ultraestrutura , Inibidores Enzimáticos/farmacologia , Humanos , Imuno-Histoquímica , Cinética , Camundongos , Piridinas/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Quinases Associadas a rho/antagonistas & inibidores
17.
Tissue Eng Part C Methods ; 18(1): 12-20, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21902618

RESUMO

Hepatocytes derived from embryonic stem cells (ESCs) are expected to be useful for basic research and clinical applications. However, in several studies, genetic methods used to detect and obtain them are difficult and pose major safety problems. Therefore, in this study, we established a novel detection system for hepatocytes by using indocyanine green (ICG), which is selectively taken up by hepatocytes, based on nongenetic manipulation. ICG has maximum light absorption near 780 nm, and it fluoresces between 800 and 900 nm. Making use of these properties, we developed flow cytometry equipped with an excitation lazer of 785 nm and specific bandpass filters and successfully detected ESC-derived ICG-positive cells that were periodic acid-Schiff positive and expressed hepatocyte phenotypic mRNAs. These results demonstrate that this detection system based on nongenetic manipulation with ICG will lead to isolate hepatocytes generated from ESCs and provide the appropriate levels of stability, quality, and safety required for cell source for cell-based therapy and pharmaceutical studies such as toxicology.


Assuntos
Técnicas de Cultura de Células , Células-Tronco Embrionárias/citologia , Hepatócitos/citologia , Verde de Indocianina/farmacologia , Animais , Células Cultivadas , Corantes/farmacologia , Citometria de Fluxo/métodos , Células HEK293 , Células Hep G2 , Humanos , Camundongos , Ácido Periódico/farmacologia , Fenótipo , Ratos , Ratos Wistar
18.
J Agric Food Chem ; 59(8): 3738-46, 2011 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-21391678

RESUMO

Proanthocyanidins and other polyphenols in the seeds and juice of boysenberry were quantitatively analyzed. Polyphenolic extracts were prepared from the waste seeds and commercial juice by chromatographic fractionation. Compositional analysis revealed that both extracts contained six polyphenolic classes: flavanol monomers, proanthocyanidins, anthocyanins, ellagic acid, ellagitannins, and flavonol glycosides. Ellagitannins were the most abundant polyphenols in both extracts. Proanthocyanidins were present as short oligomers consisting of dimeric and trimeric procyanidins and propelargonidins, with the most abundant component being procyanidin B4 in both extracts. Quantification by high-performance liquid chromatography-mass spectrometry (HPLC-MS) revealed that the seeds contained a 72-fold higher amount of proanthocyanidins than the juice. These results indicate that boysenberry fruits contain short oligomeric proanthocyanidins along with flavanol monomers and the seeds represent a good source of short oligomeric proanthocyanidins.


Assuntos
Biopolímeros/química , Flavonoides/química , Frutas/química , Fenóis/química , Proantocianidinas/química , Biopolímeros/análise , Cromatografia Líquida de Alta Pressão , Flavonoides/análise , Fenóis/análise , Polifenóis , Proantocianidinas/análise , Espectrometria de Fluorescência , Espectrometria de Massas em Tandem
19.
Stem Cells Dev ; 20(6): 1071-8, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20887097

RESUMO

Cultivation of functional pancreatic cells isolated from adult mammalian pancreas remains difficult. We developed a differentiation protocol that gradually induced the formation of mouse pancreatic exocrine cells from embryonic stem cells (ESCs). This process mimicked in vivo pancreatic development by directing cells through definitive endoderm (DE), gut tube endoderm, and pancreatic progenitor cells to differentiated cells that expressed pancreatic exocrine enzymes. Mouse ESCs were cultured in hanging drops to form embryoid bodies. Treatment of embryoid bodies with activin A induced the formation of DE cells that expressed marker mRNAs Goosecoid and Mixl1 and that were double-positive with Foxa2 and Sox17 proteins. Subsequent treatment of the DE cells by retinoic acid induced the formation of gut tube endoderm cells that expressed the specific marker Hnf1b. Expression of Goosecoid and Mixl1 was downregulated during this period. Fibroblast growth factor 7 (FGF7) promoted differentiation of PDX1-expressing pancreatic progenitor cells that also expressed Foxa2 mRNA, an endodermal marker, suggesting derivation from the DE cells. Exocrine cell differentiation was induced with FGF7, glucagon-like peptide-1, and nicotinamide. The differentiated cells expressed mature pancreatic exocrine cell mRNAs, such as Amylase, Elastase, and Carboxypeptidase A. Additionally, they produced pancreatic elastase, amylase, carboxypeptidase A, and chymotrypsin proteins that were identified in cytoplasmic granules by immunocytochemistry. Active amylase was released into the medium. Moreover, FGF7 was associated with differentiation of pancreatic exocrine cells. The findings reported here offer a novel and effective process to develop pancreatic exocrine cells from ESCs.


Assuntos
Técnicas de Cultura de Células/métodos , Diferenciação Celular , Células-Tronco Embrionárias/citologia , Pâncreas Exócrino/citologia , Animais , Diferenciação Celular/efeitos dos fármacos , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Sistema Endócrino/citologia , Fator 7 de Crescimento de Fibroblastos/farmacologia , Camundongos , Pâncreas Exócrino/efeitos dos fármacos , Pâncreas Exócrino/enzimologia
20.
Pancreas ; 40(8): 1245-52, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21989025

RESUMO

OBJECTIVE: The objective of the study was to induce transdifferentiation of human hepatoma HepG2 cells into pancreatic-like cells without direct genetic intervention. METHODS: HepG2 cells were transfected with plasmids for the hepatocyte marker protein green fluorescent protein (albumin-GFP) and the pancreatic cell marker Discosoma spp red fluorescent protein (elastase-DsRed) to create FAE-HepG2 cells. Fluorescent marker expression was used to monitor in vitro transdifferentiation stimulated 100 mM CCl4, 2 mM D-galactosamine, or 200 µM ZnCl2. Concentrations were selected for optimal cell survival rate. Transdifferentiation was also characterized by immunohistochemical detection of amylase, glucagon, and insulin and by polymerase change reaction analysis of amylase and insulin mRNA production. RESULTS: Control cells expressed albumin-GFP but no elastase-DsRed. By 30 days of culture, all 3 agents induced expression of pancreatic-like cell marker elastase-DsRed. ZnCl2 was the most effective as most cells expressed elastase-DsRed in the absence of simultaneous expression of albumin-GFP. For CCl4 and D-galactosamine, elastase-DsRed was expressed in the same cells as albumin-GFP. Cells treated by each agent also expressed amylase, insulin, and glucagon proteins and mRNAs. CONCLUSIONS: Without direct genetic intervention, select low small molecules can induce in vitro transformation of hepatoma cells into pancreatic-like cells.


Assuntos
Tetracloreto de Carbono/farmacologia , Transdiferenciação Celular/efeitos dos fármacos , Cloretos/farmacologia , Galactosamina/farmacologia , Pâncreas/citologia , Compostos de Zinco/farmacologia , Albuminas/genética , Albuminas/metabolismo , Amilases/genética , Amilases/metabolismo , Expressão Gênica/efeitos dos fármacos , Glucagon/genética , Glucagon/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células Hep G2 , Humanos , Imuno-Histoquímica , Insulina/genética , Insulina/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência , Pâncreas/metabolismo , Elastase Pancreática/genética , Elastase Pancreática/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Transfecção
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