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BACKGROUND: Mesenchymal stem cells (MSCs) can be derived from several tissues such as bone marrow, placenta, adipose tissue, or endometrial tissue. MSCs gain a lot of attention for cell-based therapy due to their characteristics including differentiation ability and immunomodulatory effect. Preclinical and clinical studies demonstrated that MSCs can be applied to treat female infertility by improving of the functions of ovary and uterus. This mini- review focuses on the current study of treatment of endometrial infertility by using MSCs. METHODS: The present study performed a literature review focusing on the effect of MSCs for treatment of women infertility caused by endometrial dysfunction. RESULTS: Bone marrow-, umbilical cord-, adipose-, amniotic-, and menstruation-derived MSCs enhance endometrial cell proliferation, injury repairs as well as reducing scar formation. The beneficial mechanism probably via immunomodulatory, cell differentiation, stimulates endometrial cell proliferation and down-regulation of fibrosis genes. The major advantage of using MSCs is to improve endometrial functions resulting in increased implantation and pregnancy. CONCLUSIONS: MSCs exhibit a potential for endometrial infertility treatment. Adipose- and menstruation-derived stem cells show advantages over other sources because the cells can be derived easily and do not causes graft rejection after autologous transplantation.
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In a feeder-dependent culture system of human pluripotent stem cells (hPSCs), coculture with mouse embryonic fibroblasts may limit the clinical use of hPSCs. The aim of this study was to determine the feasibility of using human Caesarean scar fibroblasts (HSFs) as feeder cells for the culture of hPSCs. HSFs were isolated and characterised and cocultured with hPSCs, and the pluripotency, differentiation ability and karyotypic stability of hPSCs were determined. Inactivated HSFs expressed genes (including inhibin subunit beta A (INHBA), bone morphogenetic protein 4 (BMP4), fibroblast growth factor 2 (FGF2), transforming growth factor-ß1 (TGFB1), collagen alpha-1(I) (COL1A1) and fibronectin-1 (FN1) that have been implicated in the maintenance of hPSC pluripotency. When HSFs were used as feeder cells, the pluripotency and karyotypic stability of hPSC lines did not change after prolonged coculture. Interestingly, exogenous FGF2 could be omitted from the culture medium when HSFs were used as feeder cells for hESCs but not hiPSCs. hESCs cocultured with HSF feeder cells in medium without FGF2 supplementation maintained their pluripotency (as confirmed by the expression of pluripotency markers and genes), differentiated invitro into embryonic germ layers and maintained their normal karyotype. The present study demonstrates that HSFs are a novel feeder cell type for culturing hPSCs and that supplementation of exogenous FGF2 is not necessary for the Chula2.hES line.
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Cesárea/efeitos adversos , Cicatriz/metabolismo , Células Alimentadoras/metabolismo , Fibroblastos/metabolismo , Comunicação Parácrina , Células-Tronco Pluripotentes/metabolismo , Diferenciação Celular , Linhagem Celular , Cicatriz/etiologia , Cicatriz/patologia , Técnicas de Cocultura , Estudos de Viabilidade , Células Alimentadoras/patologia , Feminino , Fibroblastos/patologia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Cariótipo , Fenótipo , Gravidez , Transdução de SinaisRESUMO
PURPOSE: Increasing intracellular energy storage by chemically activating adenosine monophosphate-activated protein kinase (AMPKα) prior to sperm cryopreservation may improve post-thawed sperm function. Using the domestic cat as a biomedical model, the objectives were to (1) confirm the expression of AMPKα and its regulatory kinases in epididymal spermatozoa and (2) assess the influence of AMPK activator, 5'-aminoimidasole-4-carboxamide-1-ß-d-ribofuranoside (AICAR) on epididymal sperm function before and after cryopreservation. METHODS: In study I, sperm samples of different qualities were obtained from cauda epididymides of domestic cats and evaluated for AMPKα expression. In study II, epididymal spermatozoa were equilibrated for either 30 or 60 min in the presence of 0 (control), 0.5, 2.0, and 5.0 mM AICAR and sperm functions were assessed before and after cryopreservation. In study III, epididymal spermatozoa were treated as in study II and evaluated for AMPKα signaling protein expressions (phospho-AMPKα Thr172 and GLUT1) as well as ATP levels. RESULTS: AMPKα protein expression was higher in high-motility vs poor-motility samples. Thirty-minute equilibration with 0.5 mM AICAR improved motion characteristics and fertilizing ability of cryopreserved sperm to the control. Increased expressions of phospho-AMPKα Thr172 and GLUT1 as well as intracellular ATP level were confirmed in sperm samples equilibrated with 0.5 or 2.0 mM AICAR for 30 min. CONCLUSIONS: Presence and role of AMPKα protein in cat regulating sperm function were demonstrated before and after cryopreservation. Findings could be used to potentially enhance cryopreserved sperm function in sub-fertile men.
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Criopreservação , Metabolismo Energético/genética , Proteínas Quinases/genética , Espermatozoides/crescimento & desenvolvimento , Quinases Proteína-Quinases Ativadas por AMP , Animais , Gatos , Feminino , Fertilização/genética , Fertilização/fisiologia , Humanos , Masculino , Preservação do Sêmen/métodos , Motilidade dos Espermatozoides/genética , Motilidade dos Espermatozoides/fisiologia , Espermatozoides/metabolismoRESUMO
Because the diploid human embryonic stem cells (hESCs) can be successfully derived from tripronuclear zygotes thus, they can serve as an alternative source of derivation of normal karyotype hESC lines. The aim of the present study was to compare the pluripotency and trophoblast differentiation ability of hESCs derived from tripronuclear zygotes and diploid hESCs. In the present study, a total of 20 tripronuclear zygotes were cultured; 8 zygotes developed to the blastocyst stage and 1 hESC line was generated. Unlike the previous studies, chromosomal correction of tripronuclear zygotes during derivation of hESCs did not occur. The established line carries 3 sets of chromosomes and showed a numerical aberration. Although the cell line displayed an abnormal chromosome number, it was found the cell line has been shown to be pluripotent with the ability to differentiate into 3 embryonic germ layers both in vitro and in vivo. The expression of X inactive specific transcript (XIST) in mid-passage (passage 42) of undifferentiated triploid hESCs was detected, indicating X chromosome inactivation of the cell line. Moreover, when this cell line was induced to differentiate toward the trophoblast lineage, morphological and functional trophoblast cells were observed, similar to the diploid hESC line.
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Células-Tronco Embrionárias Humanas/citologia , Triploidia , Trofoblastos/citologia , Zigoto/citologia , Blastocisto/citologia , Diferenciação Celular , Linhagem Celular , Linhagem da Célula , Aberrações Cromossômicas , Cromossomos/ultraestrutura , Técnicas de Cocultura , Impressões Digitais de DNA , Diploide , Técnicas de Cultura Embrionária , Fibroblastos/metabolismo , Células Germinativas/citologia , Humanos , Cariotipagem , Modelos TeóricosRESUMO
Human neural progenitor cells (hNPCs) are the starting material required for neuronal subtype differentiation. Proliferation of hNPCs allows researchers to study the mechanistic complexities and microenvironments present during neural differentiation and to explore potential applications for hNPCs in cell therapies. The use of enzymatic dissociation during hNPC proliferation causes dissociation-induced apoptosis; therefore, in the present study, we examined the effect of the p-160-Rho-associated coiled-coil kinase (ROCK) inhibitor Y-26732 on dissociation-induced apoptosis of hNPCs. We generated hNPCs via embryoid body formation using serum-free culture medium supplemented with noggin. The established hNPCs were characterized and the effect of the ROCK inhibitor on hNPC dissociation was studied. We demonstrated that supplementation of the culture media with 10 µM Y-26732 efficiently reduced apoptosis of dissociated hNPCs; this supplementation was effective when the inhibitor was applied either at (i) 24 h before dissociation of the cells and at 24 h after plating the cells or (ii) at 24 h after plating of the cells only. In addition to reducing apoptosis, both supplementation conditions with Y-26732 enhanced the proliferation of dissociated hNPCs. Our findings provide the optimal time window for ROCK treatment of hNPC dissociation in respect to apoptosis and cell proliferation.
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Células-Tronco Embrionárias/citologia , Células-Tronco Neurais/citologia , Inibidores de Proteínas Quinases/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Agregação Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Corpos Embrioides/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Humanos , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Quinases Associadas a rho/metabolismoRESUMO
Human ovarian follicular fluid (HOFF) contains proteins, extracellular matrixes necessary for growth and maturation of oocytes as well as granulosa cells. Epithelial cells and stem cells can be isolated from HOFF. However, information regarding stem cells derived from HOFF is still lacking. The objectives of the present study were to isolate, characterize, and differentiate cells derived from HOFF. HOFF was collected during the routine aspiration of oocytes in an assisted fertilization program and subjected to cell isolation, characterization, and in vitro culture. After 24 h of culture, different cell morphologies including epithelial-like-, neural-like- and fibroblast-like cells were observed. Immunocytochemistry reveals the expression of pluripotent stem cell markers (OCT4, NANOG, SSEA4), epithelial marker (CK18), FSH- and LH-receptor. For in vitro culture, the isolated cells were continuously cultured in a growth medium; alpha MEM containing 10% FBS and epidermal growth factor (EGF). After 2 weeks of in vitro culture, cells with fibroblast-like morphology dominantly grow in the culture vessels and resemble mesenchymal stem cells (MSCs). HOFF-derived cells exhibited MSC expression of CD44, CD73, CD90, CD105, CD146, and STRO-1, and were capable of differentiation into osteoblasts, chondrocytes, and adipocytes. After induction of neural differentiation, HOFF-derived cells formed spheroidal structures and expressed neural stem cell markers including Nestin, ß-tubulin III, and O4. Besides, the oocyte-like structure was observed after prolonged culture of HOFF. In conclusion, cells derived from follicular fluid exhibited stem cell characteristics, which could be useful for regenerative medicine applications and cell-based therapies.
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Terapia Baseada em Transplante de Células e Tecidos/métodos , Líquido Folicular/citologia , Folículo Ovariano/citologia , Células-Tronco Pluripotentes , Antígenos CD/metabolismo , Antígenos de Superfície/metabolismo , Diferenciação Celular , Separação Celular , Células Cultivadas , Feminino , Fertilização in vitro , Líquido Folicular/química , Humanos , Nestina/metabolismo , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/fisiologia , Medicina Regenerativa/métodos , Tubulina (Proteína)/metabolismoRESUMO
OBJECTIVE: Using domestic cats as a biomedical research model for fertility preservation, the present study aimed to characterize the influences of ovarian tissue encapsulation in biodegradable hydrogel matrix (fibrinogen/thrombin) on resilience to cryopreservation, and static versus non-static culture systems following ovarian tissue encapsulation and cryopreservation on follicle quality. METHODS: In experiment I, ovarian tissues (n=21 animals; 567 ovarian fragments) were assigned to controls or hydrogel encapsulation with 5 or 10 mg/mL fibrinogen (5 or 10 FG). Following cryopreservation (slow freezing or vitrification), follicle viability, morphology, density, and key protein phosphorylation were assessed. In experiment II (based on the findings from experiment I), ovarian tissues (n=10 animals; 270 ovarian fragments) were encapsulated with 10 FG, cryopreserved, and in vitro cultured under static or non-static systems for 7 days followed by similar follicle quality assessments. RESULTS: In experiment I, the combination of 10 FG encapsulation/slow freezing led to greater post-thawed follicle quality than in the control group, as shown by follicle viability (66.9%±2.2% vs. 61.5%±3.1%), normal follicle morphology (62.2%±2.1% vs. 55.2%±3.5%), and the relative band intensity of vascular endothelial growth factor protein phosphorylation (0.58±0.06 vs. 0.42±0.09). Experiment II demonstrated that hydrogel encapsulation promoted follicle survival and maintenance of follicle development regardless of the culture system when compared to fresh controls. CONCLUSION: These results provide a better understanding of the role of hydrogel encapsulation and culture systems in ovarian tissue cryopreservation and follicle quality outcomes using an animal model, paving the way for optimized approaches to human fertility preservation.
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From 2005 to 2008, the authors assessed the medical ethics of 779 medical students in the Department of Obstetrics and Gynecology at the Faculty of Medicine, Chulalongkorn University by using the Chula Method. This was conducted through a written examination asking the students to express their opinions about ethical issues. Their answers were rated as either Satisfactory (S) or Unsatisfactory (U). It was found that 750 students (96.3%) earned S while 29 students (3.7%) earned U. The results from this study can not be compared with the results from studies published in medical journals. Thus, knowledge about medical ethics is not complete even though it is intensively taught in medical schools and has been practiced for a long time. The authors would like to propose a new assessment of the medical ethics so that it can be systematically applied.
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Educação de Graduação em Medicina/métodos , Ética Médica/educação , Avaliação Educacional , Feminino , Ginecologia/educação , Humanos , Masculino , Obstetrícia/educação , Faculdades de Medicina , Estudantes de Medicina , Adulto JovemRESUMO
OBJECTIVE: To establish human embryonic stem (hES) cells from human embryos. DESIGN: Experimental study. SETTING: Reproductive Medicine Unit, Department of Obstetrics and Gynecology, Faculty of Medicine, Chulalongkorn University. MATERIAL AND METHOD: Abnormal and normal fertilization embryos were cultured in vitro until reaching blastocyst stage. Four different methods for isolation of ICMs were used. Immunosurgery, mechanical isolation, laser assists, and whole blastocyst culture were performed. The feeder layers used in the present study were fibroblasts, isolated from either mouse or human. Mechanical splitting of ICM outgrowths or hES-like cells was performed for propagation of cells. Characterization of hES-like cells was conducted by morphology, detection of immunostaining of Oct-4, and enzymatic activity of alkaline phosphatase (AP). HES-like cells were spontaneously differentiated through suspension culture of embryoid body (EB). Subsequent differentiation was done on gelatin-coated dishes. MAIN OUTCOME MEASURE: Establishment of hES cells. RESULTS: By using abnormal fertilization embryos, 80.0% (8/10) of blastocysts were able to attach on the feeder layers, 50% (4/8) formed ICM outgrowths, but no hES-like cells were established. By using normal fertilization embryos, 84.6% (22/26) of blastocysts were able to attach on feeder layers, 18.2% (4/22) formed ICM outgrowths. One hES-like cell line was successfully established by using mechanical isolation of ICMs and human adult skin fibroblasts as feeder layers. This hES-like cells exhibited typical morphology of hES cells, positive staining for Oct-4 and AP. hES-like cells were able to form EB and differentiated into neural-like cells. CONCLUSION: This is the first report in Thailand that hES-like cells can be isolated from normal development human embryos at blastocysts-stage using mechanical isolation of ICM and culture with human adult skin fibroblast as feeder layers.
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Massa Celular Interna do Blastocisto/citologia , Blastocisto/citologia , Técnicas de Cultura Embrionária/métodos , Células-Tronco Embrionárias/citologia , Fibroblastos/citologia , Fosfatase Alcalina/metabolismo , Animais , Blastocisto/metabolismo , Massa Celular Interna do Blastocisto/fisiologia , Diferenciação Celular , Linhagem Celular , Criopreservação , Embrião de Mamíferos/citologia , Células-Tronco Embrionárias/metabolismo , Fertilização in vitro , Fibroblastos/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos , Pele/citologia , TailândiaRESUMO
Two clones of human induced pluripotent stem cells (iPSCs) were generated from dermal fibroblasts isolated from a one-year-old Thai patient with X-linked osteogenesis imperfecta. The patient harbored a mutation, p.N459S, in the MBTPS2 gene. The cells were reprogrammed using an integration-free Sendai virus containing KLF4, c-MYC, OCT4 and SOX2. Both of the established iPSC lines (MDCUi001-A and MDCUi001-B) maintained normal karyotype, expressed pluripotent markers and differentiated into all three germ layers.
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Células-Tronco Pluripotentes Induzidas/metabolismo , Osteogênese Imperfeita/genética , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Linhagem Celular , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Cariótipo , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Masculino , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Vírus Sendai/genética , TailândiaRESUMO
OBJECTIVE: To determine the effect of leukemia inhibitory factor (LIF) on the quality of in vitro produced mouse blastocyst and the efficiency of embryonic stem (ES) cell derivation. DESIGN: Experimental study SETTING: Reproductive Medicine Unit, Department of Obstetrics and Gynecology, Faculty of Medicine, Chulalongkorn University MATERIAL AND METHOD: In vivo fertilized zygotes were collected and subjected to in vitro culture in potassium simplex optimized medium (KSOM) containing 1,000 unit/ml LIF. The developmental ability of the zygote to blastocyst-stage and the cell numbers in blastocysts were evaluated Expanded blastocysts developed in different culture media were subsequently subjected to ES cell derivation. MAIN OUTCOME MEASURE (s): The influence of LIF on the quality of and the total cell numbers of blastocyst developed in vitro. RESULTS: Supplementation of LIF in KSOM increased the rate of hatching blastocysts (63.8% vs. 53.7%; p < 0.05) and total cell numbers (91.4 +/- 15.0 vs. 85.1 +/- 7.7; p < 0.05) compared to KSOM alone. ES cells were obtained 66.7% from blastocysts developed in KSOM-LIF versus 41.7% in KSOM (p > 0.05). Established ES cell lines showed typical colony and characteristics of pluripotent murine ES cells. CONCLUSION: LIF improved the quality of in vitro produced blastocysts but not enhanced ES cell derivation.
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Blastocisto , Células-Tronco Embrionárias , Fator Inibidor de Leucemia , Animais , Desenvolvimento Embrionário , Feminino , Humanos , Técnicas In Vitro , Camundongos , ZigotoRESUMO
Embryonic stem cell (ESC) has been recognized as one of the most promising therapeutic tools for the next decade. However, there are many controversial issues in bioethics for this challenging research area. Each country has its own distinct regulations and policies for ESC research due to their differences in cultural background, religious belief and political influence. These differences will eventually play an important role on international ESC research collaboration. The present article will provide a concise summary of the different policies and regulations regarding bioethical points for ESC research worldwide and show current progress towards establishing standard bioethical guidelines for ESC research on the international level.
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Bioética/tendências , Células-Tronco Embrionárias , Ética em Pesquisa , Comparação Transcultural , HumanosRESUMO
Cell therapy is a promising therapeutic tool for the next decade. It has a potential to cure a number of chronic diseases and conditions related to aging processes or degenerative changes. In addition, it could be used to replace cells and tissues in injured organs. Furthermore, it may provide a novel approach to congenital anomalies and genetic disorders where current therapeutic options are limited However, many crucial questions need answers to ensure a safe, effective and successful solution in the field of cell therapy. In Thailand, innovative knowledge and expertise in stem cell biology and technology are required as the key elements to make cell therapy a "real" hope.
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Envelhecimento , Terapia Baseada em Transplante de Células e Tecidos/tendências , Células-Tronco , Envelhecimento/patologia , Humanos , TailândiaRESUMO
Although human pluripotent stem cells (hPSCs) can proliferate robustly on the feeder-free culture system, genetic instability of hPSCs has been reported in such environment. Alternatively, feeder cells enable hPSCs to maintain their pluripotency. The feeder cells are usually grown in a culture medium containing fetal bovine serum (FBS) prior to coculture with hPSCs. The use of FBS might limit the clinical application of hPSCs. Recently, human cord blood-derived serum (hUCS) showed a positive effect on culture of mesenchymal stem cells. It is interesting to test whether hUCS can be used for culture of feeder cells of hPSCs. This study was aimed to replace FBS with hUCS for culturing the human foreskin fibroblasts (HFFs) prior to feeder cell preparation. The results showed that HFFs cultured in hUCS-containing medium (HFF-hUCS) displayed fibroblastic features, high proliferation rates, short population doubling times, and normal karyotypes after prolonged culture. Inactivated HFF-hUCS expressed important genes, including Activin A, FGF2, and TGFß1, which have been implicated in the maintenance of hPSC pluripotency. Moreover, hPSC lines maintained pluripotency, differentiation capacities, and karyotypic stability after being cocultured for extended period with inactivated HFF-hUCS. Therefore, the results demonstrated the benefit of hUCS for hPSCs culture system.
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OBJECTIVE: The long interspersed elements (LINE-1, L1s) are a group of genetic elements found in large numbers in the human genome that can translate into phenotype by controlling genes. Growing evidence supports the role of epigenetic in polycystic ovary syndrome (PCOS). The purpose of this study is to evaluate the DNA methylation levels in LINE-1 in a tissue-specific manner using cumulus cells from patients with PCOS compared with normal controls. METHODS: The study included 19 patients with PCOS and 22 control patients who were undergoing controlled ovarian hyperstimulation. After oocyte retrieval, cumulus cells were extracted. LINE-1 DNA methylation levels were analysed by bisulfite treatment, polymerase chain reaction, and restriction enzyme digestion. The Connection Up- and Down-Regulation Expression Analysis of Microarrays software package was used to compare the gene regulatory functions of intragenic LINE-1. RESULTS: The results showed higher LINE-1 DNA methylation levels in the cumulus cells of mature oocytes in PCOS patients, 79.14 (±2.66) vs. 75.40 (±4.92); p=0.004, but no difference in the methylation of cumulus cells in immature oocytes between PCOS and control patients, 70.33 (±4.79) vs. 67.79 (±5.17); p=0.155. However, LINE-1 DNA methylation levels were found to be higher in the cumulus cells of mature oocytes than in those of immature oocytes in both PCOS and control patients. CONCLUSION: These findings suggest that the epigenetic modification of LINE-1 DNA may play a role in regulating multiple gene expression that affects the pathophysiology and development of mature oocytes in PCOS.
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Transgene-free human HS5-SV.hiPS line was generated from human cesarean scar-derived fibroblasts using temperature-sensitive Sendai virus vectors carrying Oct4, Sox2, cMyc and Klf4 exogenous transcriptional factors. The viral constructs were eliminated from HS5-SV.hiPS line through heat treatment. Transgene-free HS5-SV.hiPS cells expressed pluripotent associated transcription factors Oct4, Nanog, Sox2, Rex1 and surface markers SSEA-4, TRA-1-60 and OCT4. HS5-SV.hiPS cells formed embryoid bodies and differentiated into three embryonic germ layers in vivo. HS5-SV.hiPS cells maintained their normal karyotype (46, XX) after culture for extended period. HS5-SV.hiPS displayed the similar pattern of DNA fingerprinting to the parenteral scar-derived fibroblasts.
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Técnicas de Cultura de Células/métodos , Linhagem Celular/citologia , Cesárea , Cicatriz/patologia , Fibroblastos/patologia , Células-Tronco Pluripotentes Induzidas/citologia , Transgenes , Animais , Feminino , Humanos , Cariotipagem , Fator 4 Semelhante a Kruppel , Camundongos NusRESUMO
To reduce the cost of cytokines and growth factors in stem cell research, a simple method for the production of soluble and biological active human basic fibroblast growth factor (hbFGF) fusion protein in Escherichia coli was established. Under optimal conditions, approximately 60-80 mg of >95% pure hbFGF fusion proteins (Trx-6xHis-hbFGF and 6xHis-hbFGF) were obtained from 1 liter of culture broth. The purified hbFGF proteins, both with and without the fusion tags, were biologically active, which was confirmed by their ability to stimulate proliferation of NIH3T3 cells. The fusion proteins also have the ability to support several culture passages of undifferentiated human embryonic stem cells and induce pluripotent stem cells. This paper describes a low-cost and uncomplicated method for the production and purification of biologically active hbFGF fusion proteins.
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Técnicas de Cultura de Células/métodos , Células-Tronco Embrionárias/citologia , Fator 2 de Crescimento de Fibroblastos/biossíntese , Fator 2 de Crescimento de Fibroblastos/isolamento & purificação , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Fosfatase Alcalina/metabolismo , Animais , Técnicas de Cultura de Células/economia , Células-Tronco Embrionárias/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/farmacologia , Regulação Bacteriana da Expressão Gênica , Vetores Genéticos , Humanos , Imuno-Histoquímica , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Camundongos , Células NIH 3T3 , Reação em Cadeia da Polimerase , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologiaRESUMO
Central nervous system (CNS) dysfunction caused by neurovirulent influenza viruses is a dreaded complication of infection, and may play a role in some neurodegenerative conditions, such as Parkinson-like diseases and encephalitis lethargica. Although CNS infection by highly pathogenic H5N1 virus has been demonstrated, it is unknown whether H5N1 infects neural progenitor cells, nor whether such infection plays a role in the neuroinflammation and neurodegeneration. To pursue this question, we infected human neural progenitor cells (hNPCs) differentiated from human embryonic stem cells in vitro with H5N1 virus, and studied the resulting cytopathology, cytokine expression, and genes involved in the differentiation. Human embryonic stem cells (BG01) were maintained and differentiated into the neural progenitors, and then infected by H5N1 virus (A/Chicken/Thailand/CUK2/04) at a multiplicity of infection of 1. At 6, 24, 48, and 72 hours post-infection (hpi), cytopathic effects were observed. Then cells were characterized by immunofluorescence and electron microscopy, supernatants quantified for virus titers, and sampled cells studied for candidate genes.The hNPCs were susceptible to H5N1 virus infection as determined by morphological observation and immunofluorescence. The infection was characterized by a significant up-regulation of TNF-α gene expression, while expressions of IFN-α2, IFN-ß1, IFN-γ and IL-6 remained unchanged compared to mock-infected controls. Moreover, H5N1 infection did not appear to alter expression of neuronal and astrocytic markers of hNPCs, such as ß-III tubulin and GFAP, respectively. The results indicate that hNPCs support H5N1 virus infection and may play a role in the neuroinflammation during acute viral encephalitis.
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Citocinas/biossíntese , Encefalite Viral/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Virus da Influenza A Subtipo H5N1/fisiologia , Influenza Humana/metabolismo , Células-Tronco Neurais/metabolismo , Tropismo Viral/fisiologia , Diferenciação Celular , Linhagem Celular , Encefalite Viral/patologia , Células-Tronco Embrionárias Humanas/patologia , Células-Tronco Embrionárias Humanas/virologia , Humanos , Influenza Humana/patologia , Células-Tronco Neurais/patologia , Células-Tronco Neurais/virologiaRESUMO
OBJECTIVE: To investigate the effects of initiation time of clomiphene citrate (CC) on the endometrium of women with regular menstrual cycles. DESIGN: Randomized, double-blind, cross-over study. SETTING: Department of Obstetrics and Gynecology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand. PATIENT(S): Thirty-three healthy female volunteers with regular menstrual cycles. INTERVENTION(S): The volunteers were randomized to receive either 100 mg of CC on days 1-5 and placebo on days 5-9 (study group) or placebo on days 1-5 and CC on days 5-9 (control group). After a wash-out period of 1 month of CC treatment, the medication was switched in each group. Ultrasonography was performed daily after day 10 of the cycle to detect ovulation. Ultrasonography for endometrial appearance and thickness, endometrial sampling, and blood samples obtained for determination of E(2) and P levels were performed 7 days after ovulation in both groups. MAIN OUTCOME MEASURE(S): Morphometric analysis, histologic dating, and ultrasonographic appearance and thickness of the endometrium. RESULT(S): Morphometric parameters, histologic dating, and ultrasonographic appearance and thickness of the endometrium were similar in both groups. CONCLUSION(S): Starting CC on either day 1 or day 5 of the menstrual cycle did not have any differential effects on the endometrium of women with regular menstrual cycles, particularly regarding the morphometric analysis, histologic dating, or ultrasonographic appearance.
Assuntos
Clomifeno/administração & dosagem , Endométrio/efeitos dos fármacos , Antagonistas de Estrogênios/administração & dosagem , Ciclo Menstrual/fisiologia , Adulto , Clomifeno/farmacologia , Estudos Cross-Over , Método Duplo-Cego , Esquema de Medicação , Endométrio/anatomia & histologia , Endométrio/diagnóstico por imagem , Antagonistas de Estrogênios/farmacologia , Feminino , Humanos , Valores de Referência , UltrassonografiaRESUMO
A prospective randomized, double blind, single centre study was conducted to compare the efficacy, efficiency and clinical side effects of daily fixed dose regimen of either 100 IU or 200 IU of recombinant follicle stimulating hormone(rFSH) Follitropin beta in down-regulated women undergoing controlled ovarian hyperstimulation(COH) for either conventional in vitro fertilization(IVF) or intracytoplasmic sperm injection(ICSI). A total of sixty women were randomly allocated according to the criteria for the treatment by either 100 IU(n = 30) or 200 IU (n = 30) of FSH. Although more cycle cancellations due to low response were observed in the 100 IU group (n = 9 vs n = 2), two cases of mild and moderate ovarian hyperstimulation syndrome were noted in the higher dose group. Subjects in the group treated with 200 IU appeared to yield more follicles > 17 mm (4.4 vs 3.3, p = 0.05) and more oocytes compared to the group treated with 100 IU (9.2 versus 6.0 oocytes, NS). The total dosage required to develop at least three follicles according to the protocol was significantly lower in the group treated with 100 IU (1203.33 versus 2106. 67, P < 0.0001). In conclusion, a fixed daily dose of 200 IU of rFSH Follitropin beta compared to a fixed daily dose of 100 IU is more effective in terms of follicles > 17 mm development and the number of oocytes retrieved along with a lower cancellation rate, but less efficient as indicated by a higher total rFSH dose needed