RESUMO
Derivation of midbrain dopaminergic (DA) neurons from human embryonic stem (hES) cells has been of particular interest because of the clinical potential for DA neuron transplantation in patients with Parkinson's disease (PD). Several protocols for DA neuron differentiation from mouse embryonic stem cells and hES cells have been reported: however, protocols involving hES cells have yet to be improved. Here, we used a slightly modified stromal cell-derived inducing activity method, consisting four different culture stages, to show that KhES-1 cells differentiate into tyrosine hydroxylase (TH)-positive DA neurons. Quantitative real-time PCR analysis showed a marked induction of the DA neuron marker genes NURR1, paired-like homeodomain transcription factor 3 (PITX3), LIM homeobox transcription- factor 1, beta (LMX1B), engrailed-1 (EN1), dopamine transporter (DAT), and aromatic amino acid decarboxylase (AADC) during differentiation. Treatment with fibroblast growth factor (FGF)-20 and FGF-2 at the final differentiation stage induced the increase of DA neuron development-related transcription factors such as NURR1, PITX3, LMX1B, and EN1. FGF-20 and FGF-2 enhanced DA neuron differentiation from hES cell-derived neural progenitor cells directly without any soluble factors from PA6 cells. These results provide valuable information that will assist in efficient DA neuron differentiation from hES cells and for future transplant application.
Assuntos
Diferenciação Celular/fisiologia , Dopamina/fisiologia , Células-Tronco Embrionárias/citologia , Fatores de Crescimento de Fibroblastos/farmacologia , Neurônios/citologia , Animais , Diferenciação Celular/efeitos dos fármacos , Técnicas de Cocultura , Células-Tronco Embrionárias/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fibroblastos/citologia , Fibroblastos/fisiologia , Humanos , Camundongos , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transplante de Células-Tronco/métodos , Fatores de Transcrição/genética , Tirosina 3-Mono-Oxigenase/genéticaRESUMO
We here describe a case of advanced breast cancer (Stage IV) in which an oral S-1+TAM therapy following a primary systemic chemo-radiotherapy has been effective in maintaining the patient's QOL. A 40-year-old woman visited our hospital because of her left breast tumor. On physical examination, the tumor had invaded to the skin adjacent to the nipple forming a skin ulcer and marked deformity of the entire breast. Also noted were swollen lymph nodes in the left armpit. Subsequently, radiographic imaging tests revealed that the tumor had metastasized to the liver and lungs, as well as the skull. Accordingly, a primary systemic chemotherapy (4 series of AC/T) was started and followed by local radiation therapy (60 Gys) immediately after completing the chemotherapy. The metastasizing lesions in the liver, lungs, and skull had markedly reduced in the size and number, and the skin ulceration had healed up by these treatments. Afterwards, she has been given TAM daily and S-1 for 4 weeks with a 2-week interval. She has been quite well without any adverse effects by S-1 and TAM, and the primary as well as metastasizing lesions remain stable with normalized tumor marker levels (NC) for nearly 3 years.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Ácido Oxônico/uso terapêutico , Paclitaxel/uso terapêutico , Tegafur/uso terapêutico , Adulto , Biomarcadores Tumorais/sangue , Biópsia , Neoplasias da Mama/sangue , Neoplasias da Mama/radioterapia , Terapia Combinada , Ciclofosfamida/uso terapêutico , Citarabina/uso terapêutico , Doxorrubicina/uso terapêutico , Combinação de Medicamentos , Feminino , Humanos , Mitoxantrona/uso terapêutico , Estadiamento de Neoplasias , Fatores de Tempo , Tomografia Computadorizada por Raios X , Topotecan/uso terapêuticoRESUMO
New delta-alkyllactones (DALs) with diverse side-chain lengths (184-254 Da), which are structurally different from the widespread, naturally occurring delta-lactones of higher molecular weight (348-439 Da), such as camptothecin and sultriecin, were chemically synthesized and analyzed for their carcinostatic activity. Of the DALs with 11, 12, 13, 14, or 16 carbon atoms, delta-hexadecalactone (DH16:0) was the most carcinostatic when administered to Ehrlich ascites tumor (EAT) cells at 37 degrees C for 20 h, and measured by the mitochondrial dehydrogenase-based WST-1 assay. Prolongation of the administration period to 72 h enhanced the carcinostatic activity more markedly for DH16:0 than for other DALs. The carcinostatic activity of DALs was unexpectedly augmented by increasing the number of carbon atoms, in contrast to the conventional view that carcinostatic activity is attenuated by the addition of carbon atoms to fatty acids. Intracellular accumulation of DH16:0, as analyzed by gas chromatography, was detected (1.5 Pg/cell), whereas other DALs studied were rarely found. The results indicate a close relationship between carcinostatic activity and intracellular accumulation. Invasion of human fibrosarcoma HT-1080 cells through the reconstituted basement membrane was inhibited by several DALs, even at doses as low as 5-10% of those necessary for carcinostatic activity, suggesting an invasive mechanism different from carcinostasis. The invasion-inhibitory activity was intensified by increasing the number of carbon atoms, in a manner similar to that for the carcinostatic activity. The lifespan of EAT-cell-transplanted mice was markedly prolonged with DH16:0, presumably due to excellent distribution throughout the body and tumor cells. Thus DH16:0 may be a potent anticancer agent, in term of its carcinostatic, anti-invasive, and lifespan-prolonging activities.
Assuntos
Antineoplásicos/farmacologia , Lactonas/farmacologia , Invasividade Neoplásica , Relação Estrutura-Atividade , Antineoplásicos/química , Antineoplásicos/farmacocinética , Camptotecina/química , Camptotecina/farmacocinética , Camptotecina/farmacologia , Humanos , Lactonas/química , Lactonas/farmacocinética , Pironas , Células Tumorais CultivadasRESUMO
Neural crest cells migrate throughout the embryo and differentiate into diverse derivatives: the peripheral neurons, cranial mesenchymal cells, and melanocytes. Because the neural crest cells have critical roles in organogenesis, detailed elucidation of neural crest cell differentiation is important in developmental biology. We recently reported that melanocytes could be induced from mouse ESCs. Here, we improved the culture system and showed the existence of neural crest-like precursors. The addition of retinoic acid to the culture medium reduced the hematopoiesis and promoted the expression of the neural crest marker genes. The colonies formed contained neural crest cell derivatives: neurons and glial cells, together with melanocytes. This suggested that neural crest-like cells assuming multiple cell fates had been generated in these present cultures. To isolate the neural crest-like cells, we analyzed the expression of c-Kit, a cell-surface protein expressed in the early stage of neural crest cells in vivo. The c-Kit-positive (c-Kit(+)) cells appeared as early as day 9 of the culture period and expressed the transcriptional factors Sox10 and Snail, which are expressed in neural crest cells. When the c-Kit(+) cells were separated from the cultures and recultured, they frequently formed colonies containing neurons, glial cells, and melanocytes. Even a single c-Kit(+) cell formed colonies that contained these three cell types, confirming their multipotential cell fate. The c-Kit(+) cells were also capable of migrating along neural crest migratory pathways in vivo. These results indicate that the c-Kit(+) cells isolated from melanocyte-differentiating cultures of ESCs are closely related to neural crest cells.
Assuntos
Linhagem da Célula , Células-Tronco Embrionárias/citologia , Células-Tronco Multipotentes/citologia , Crista Neural/citologia , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Separação Celular , Transplante de Células , Células Cultivadas , Embrião de Galinha , Células-Tronco Embrionárias/efeitos dos fármacos , Citometria de Fluxo , Antígenos Comuns de Leucócito/imunologia , Melanócitos/citologia , Melanócitos/efeitos dos fármacos , Camundongos , Células-Tronco Multipotentes/efeitos dos fármacos , Crista Neural/efeitos dos fármacos , Crista Neural/transplante , Neuroglia/citologia , Neuroglia/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-kit/metabolismo , Tretinoína/farmacologiaRESUMO
Embryonic stem (ES) cells from many organisms have the capacity to generate in vitro a wide variety of cell types depending on their environment. Understanding precisely how such toti- or pluripotent cells may be driven towards a specific lineage represents a major challenge if our ambition of using ES cells to generate a ready supply of healthy cells for cell-based therapies for a range of diseases is to be realized. Recent advances have demonstrated that melanocytes and retinal pigmented epithelial (RPE) cells exhibiting the characteristics of their natural counterparts can be induced from undifferentiated ES cells grown on monolayers of specific stromal cell lines or by using a combination of Wnt3a, Endothelin-3 and SCF. The ability to induce pigment cells from ES cells promises to facilitate our understanding of the precise molecular mechanisms underlying this process and moreover enable us to distinguish the program of gene expression that underpins the choice made between generating a nerual crest-type melanocyte versus an RPE cell. Moreover, once the combination of signals required to induce a particular type of pigment cell are characterized, the way may be open for future cell-based therapy for various diseases caused by defective pigment cells.
Assuntos
Diferenciação Celular , Embrião de Mamíferos/citologia , Melanócitos/fisiologia , Células-Tronco/fisiologia , Animais , Técnicas de Cultura de Células , Linhagem da Célula , Humanos , Melanócitos/metabolismo , Crista Neural/citologia , Epitélio Pigmentado Ocular/metabolismo , Epitélio Pigmentado Ocular/transplante , Transplante de Células-Tronco , Células-Tronco/metabolismoRESUMO
The development of melanocytes from neural crest-derived precursor cells depends on signaling by the receptor tyrosine kinase KIT and the G protein-coupled endothelin receptor B (EDNRB) pathways. Loss-of-function mutations in either of these two signaling receptor molecules cause a loss or a marked reduction in the number of melanocyte precursors in the embryo and finally lead to loss of the coat color. Using cultures of embryonic stem (ES) cells to induce melanocyte differentiation in vitro, we investigated the requirement for EDNRB signaling during the entire developmental process of the melanocyte, in association with that for KIT signaling. During the 21-day period necessary for the induction of mature melanocytes from undifferentiated ES cells, endothelin 3 (EDN3), a ligand for EDNRB, increased the number of melanocytes in proportion to the period during which it was present. We tested the compensatory effect of EDNRB signaling on KIT signaling in vivo by using Kit(W-LacZ)/Kit(W-LacZ) ES cells and confirmed that the ectopic expression of EDN3 in the skin reduced the white spotting of Kit(W57)/Kit(W57)mice. KIT ligand (KITL) and EDN3 worked synergistically to induce melanocyte differentiation in vitro; however, the complete lack of EDNRB signaling attained by the use of EDN3-/- ES cells and an EDNRB antagonist, BQ788, revealed that the resulting failure of melanocyte development was not compensated by the further activation of KIT signaling by adding KITL. Simultaneous blockade of EDNRB and KIT signalings eliminated melanocyte precursors completely, suggesting that the maintenance or survival of early melanocyte precursors at least required the existence of either EDNRB or KIT signalings.
Assuntos
Diferenciação Celular , Endotelina-3/metabolismo , Melanócitos/citologia , Melanócitos/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Transdução de Sinais , Animais , Células Cultivadas , Endotelina-3/deficiência , Endotelina-3/genética , Deleção de Genes , Fator de Crescimento de Hepatócito/genética , Fator de Crescimento de Hepatócito/metabolismo , Camundongos , Camundongos Knockout , Mutação/genética , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Proto-Oncogênicas c-kit/genética , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Fator de Células-Tronco/antagonistas & inibidores , Fator de Células-Tronco/genética , Fator de Células-Tronco/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Fatores de TempoRESUMO
The integration of embryonic stem (ES) cells as well-differentiated neuronal cells into the retinas of adult mice was investigated. ES cells were transplanted in adult mouse retinas by intravitreal injections. Neuronal differentiation of the ES cells was investigated by morphological and immunohistochemical examinations on post-operative days 5 and 30. ES cell apoptosis was examined by in situ terminal dUTP-biotin nick end labeling of DNA fragments. Differentiated ES cells growing along the retinal surface developed fine neuronal cell processes around cell nuclei and generated neuronal networks into the retinal inner plexiform layer (IPL) 30 days after transplantation. The differentiated ES cells expressed retinal and neuronal markers. Many apoptotic cells were recognized in transplanted ES cells at day 5 but not at day 30 after transplantation. ES cells may be useful for neural tissue regeneration in the adult mammalian retina.
Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Multipotentes/fisiologia , Rede Nervosa/crescimento & desenvolvimento , Neurônios/fisiologia , Retina/cirurgia , Transplante de Células-Tronco/métodos , Animais , Apoptose/fisiologia , Biomarcadores , Tamanho Celular/fisiologia , Células Cultivadas , Sobrevivência de Enxerto/fisiologia , Masculino , Camundongos , Células-Tronco Multipotentes/citologia , Rede Nervosa/citologia , Regeneração Nervosa/fisiologia , Neuritos/fisiologia , Neuritos/ultraestrutura , Neurônios/citologia , Retina/citologia , Retina/fisiologiaRESUMO
BACKGROUND: We previously reported the usefulness of the alphaGal epitope as a target molecule for gene therapy against cancer. To induce cancer cell specific transcription of the alphaGal epitope, an expression vector which synthesizes the alphaGal epitope under the control of a promoter region of the human telomerase reverse transcriptase (hTERT), NK7, was constructed. METHODS: NK7 was transfected into a human pancreatic carcinoma cell line, MIA cells, and telomerase-negative SUSM-1 cells served controls. Expression of the alphaGal epitope was confirmed by flow cytometry using IB4 lectin. The susceptibility of transfected MIA cells to human natural antibodies, was examined using a complement-dependent cytotoxic cross-match test (CDC) and a flow cytometry using annexin V. RESULTS: The alphaGal epitope expression was detected only on the cell surfaces of NK7-transfected MIA cells, i.e., not on naive MIA cells or telomerase negative SUSM-1 cells. The CDC results indicated that MIA cells transfected with NK7 are susceptible to human natural antibody-mediated cell killing, and the differences, as compared to NK-7 transfected telomerase negative SUSM-1 cells or telomerase positive naïve MIA cells, were statistically significant. The flow cytometry using annexin V showed a higher number of the apoptotic cells in NK-7 transfected MIA cells than in naïve MIA cells. CONCLUSIONS: The results suggest that alphaGal epitope-expression, under the control of the hTERT-promoter, may be useful in cancer specific gene therapy.
RESUMO
To investigate the biological activity of epithelial cells in view of host defense, we analyzed the mRNA expression of inducible NOS (iNOS) as well as NO production by human gingival epithelial cells (HGEC) stimulated with IL-15. RT-PCR analysis revealed that HGEC expressed IL-15 receptor alpha-chain mRNA. In addition, stimulation with IL-15 enhanced iNOS expression by HGEC through an increase of both mRNA and protein levels. Moreover, IL-15 up-regulated the production of NO(2)(-)/NO(3)(-), a NO-derived stable end product, from HGEC. The enhanced NO production by IL-15 was inhibited by AMT, an iNOS-specific inhibitor. These results suggest that IL-15 is a potent regulator of iNOS expression by HGEC and involved in innate immunity in the mucosal epithelium.