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Background and Objectives@#Embryologically, mesodermal development is closely related to the development of various organs such as muscles, blood vessels, and hearts, which are the main organs that make up the body. However, treatment for mesoderm developmental disorders caused by congenital or acquired factors has so far relied on surgery and drug treatment for symptom relief, and more fundamentally, treatment for mesoderm developmental disorders is needed. @*Methods@#and Results: In our study, microRNA (miRNA), which plays an important role in the mesoderm development process, was identified and the developmental function was evaluated. miRNAs consist of small nucleotides, which act as transcription factors that bind to the 3’ untranslated region and suppressed target gene expression. We constructed the human embryonic stem cell (hESC) knockout cell line and analyzed the function and characteristics of miR-5739, which plays an important role in mesoderm lineage. miR-5739 acts as a transcription factor targeting SMA, Brachyury T, Hand1, which controls muscle proliferation and differentiation, and KDR gene, which regulates vessel formation in vitro. In vivo results suggest a role in regulating muscle proliferation and differentiation. Gene ontology analysis confirmed that the miR-5739 is closely related to genes that regulate muscle and vessel proliferation and differentiation. Importantly, abnormal expression of miR-5739 was detected in somatic cells derived from patients with congenital muscle disease. @*Conclusions@#Our study demonstrate that miR-5739 gene function significantly affects transcriptional circuits that regu-late muscle and vascular differentiation during embryonic development.
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Background and Objectives@#Currently, safety pharmacological tests for the central nervous system depend on animal behavioral analysis. However, due to the subjectivity of behavioral analysis and differences between species, there is a limit to appropriate nervous system toxicity assessment, therefore a new neurotoxicity assessment that can simulate the human central nervous system is required. @*Methods@#and Results: In our study, we developed an in vitro neurotoxicity assessment focusing on neuronal function. To minimize the differences between species and fast screening, hiPSC-derived neurons and a microelectrode array (MEA) that could simultaneously measure the action potentials of the neuronal networks were used. After analyzing the molecular and electrophysiological characters of our neuronal network, we conducted a neurotoxicity assessment on neurotransmitters, neurotoxicants, illicit drugs, and new psychoactive substances (NPS). We found that most substances used in our experiments responded more sensitively to our MEA-based neurotoxicity assessment than to the conventional neurotoxicity assessment. Also, this is the first paper that evaluates various illicit drugs and NPS using MEA-based neurotoxicity assessment using hiPSC-derived neurons. @*Conclusions@#Our study expanded the scope of application of neurotoxicity assessment using hiPSC-derived neurons to NPS, and accumulated evaluation data of various toxic substances for hiPSC-derived neurons.
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Cell labeling technologies are required to monitor the fate of transplanted cells in vivo and to select target cells for the observation of certain changes in vitro. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been transplanted for the treatment of heart injuries or used in vitro for preclinical cardiac safety assessments. Cardiomyocyte (CM) labeling has been used in these processes to facilitate target cell monitoring. However, the functional effect of the labeling agent on hiPSC-CMs has not been studied. Therefore, we investigated the effects of labeling agents on CM cellular functions. 3’-Dioctadecyloxacarbocyanine perchlorate (DiO), quantum dots (QDs), and a DNA plasmid expressing EGFP using Lipo2K were used to label hiPSC-CMs. We conclude that the hiPSC-CM labeling with DiO and QDs does not induce arrhythmogenic effects but rather improves the mRNA expression of cardiac ion channels and Ca2+ influx by L-type Ca2+ channels. Thus, DiO and QD labeling agents may be useful tools to monitor transplanted CMs, and further in vivo influences of the labeling agents should be investigated in the future.
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All-trans retinoic acid (ATRA) is a highly effective treatment for acute promyelocytic leukemia (APL), a cytogenetically distinct subtype of acute myeloid leukemia (AML). However, ATRA-based treatment is not effective in other subtypes of AML. In non-APL AML, ATRA signaling pathway is impaired or downmodulated, and consequently fails to respond to pharmacological doses of ATRA. Therefore, complementary treatment strategies are needed to improve ATRA responsiveness in non-APL AML. In this study, we investigated the combined effect of ATRA and bromodomain inhibitor JQ1, proven to have potent anti-cancer activity mainly through inhibition of c-Myc. We showed that the combination of ATRA with JQ1 synergistically inhibited proliferation of AML cells. The synergistic growth inhibition was resulted from differentiation or apoptosis depending on the kind of AML cells. Concomitantly, the combined treatment of ATRA and JQ1 caused greater depletion of c-Myc and hTERT expression than each agent alone in AML cells. Taken together, these findings support the rationale for the use of the combination of ATRA and JQ1 as a therapeutic strategy for the treatment of AML.
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Apoptose , Leucemia , Leucemia Mieloide Aguda , Leucemia Promielocítica Aguda , TretinoínaRESUMO
Acute myeloid leukemia (AML) is a fatal hematological malignancy which is resistant to a variety of chemotherapy drugs. Extracellular signal-regulated kinase 5 (ERK5) plays a novel role in chemoresistance in some cancer cells and this pathway is a central mediator of cell survival and apoptotic regulation. The aim of this study was to investigate the effect of ERK5 inhibitor, XMD8-92, on proliferation and apoptosis in AML cell lines. Findings showed that XMD8-92 inhibited the activation of ERK5 by G-CSF and decreased the expression of c-Myc and Cyclin D1. The treatment of XMD8-92 reduced the phosphorylation of ERK5 leading to a distinct inhibition of cell proliferation and increased apoptosis in Kasumi-1 and HL-60 cells. Taken together, our study suggests that the inhibition of ERK5 by XMD8-92 can trigger apoptosis and inhibit proliferation in AMLs. Therefore, the inhibition of ERK5 may be an effective adjuvant in AML chemotherapy.
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Humanos , Apoptose , Ciclo Celular , Linhagem Celular , Proliferação de Células , Sobrevivência Celular , Ciclina D1 , Tratamento Farmacológico , Fator Estimulador de Colônias de Granulócitos , Neoplasias Hematológicas , Células HL-60 , Leucemia Mieloide Aguda , Proteína Quinase 7 Ativada por Mitógeno , FosforilaçãoRESUMO
PURPOSE: To evaluate the therapeutic effect of human embryonic stem cell (hESC)-derived multipotent mesenchymal stem cells (M-MSCs) on ketamine-induced cystitis (KC) in rats. METHODS: To induce KC, 10-week-old female rats were injected with 25-mg/kg ketamine hydrochloride twice weekly for 12 weeks. In the sham group, phosphate buffered saline (PBS) was injected instead of ketamine. One week after the final injection of ketamine, the indicated doses (0.25, 0.5, and 1×106 cells) of M-MSCs (KC+M-MSC group) or PBS vehicle (KC group) were directly injected into the bladder wall. One week after M-MSC injection, the therapeutic outcomes were evaluated via cystometry, histological analyses, and measurement of gene expression. Next, we compared the efficacy of M-MSCs at a low dose (1×105 cells) to that of an identical dose of adult bone marrow (BM)-derived MSCs. RESULTS: Rats in the KC group exhibited increased voiding frequency and reduced bladder capacity compared to rats of the sham group. However, these parameters recovered after transplantation of M-MSCs at all doses tested. KC bladders exhibited markedly increased mast cell infiltration, apoptosis, and tissue fibrosis. Administration of M-MSCs significantly reversed these characteristic histological alterations. Gene expression analyses indicated that several genes associated with tissue fibrosis were markedly upregulated in KC bladders. However the expression of these genes was significantly suppressed by the administration of M-MSCs. Importantly, M-MSCs ameliorated bladder deterioration in KC rats after injection of a low dose (1×105) of cells, at which point BM-derived MSCs did not substantially improve bladder function. CONCLUSIONS: This study demonstrates for the first time the therapeutic efficacy of hESC-derived M-MSCs on KC in rats. M-MSCs restored bladder function more effectively than did BM-derived MSCs, protecting against abnormal changes including mast cell infiltration, apoptosis and fibrotic damage.
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Adulto , Animais , Feminino , Humanos , Ratos , Apoptose , Medula Óssea , Cistite , Fibrose , Expressão Gênica , Células-Tronco Embrionárias Humanas , Ketamina , Mastócitos , Células-Tronco Mesenquimais , Células-Tronco Multipotentes , Dor Pélvica , Bexiga UrináriaRESUMO
Human cardiomyocytes (CMs) cease to proliferate and remain terminally differentiated thereafter, when humans reach the mid-20s. Thus, any damages sustained by myocardium tissue are irreversible, and they require medical interventions to regain functionality. To date, new surgical procedures and drugs have been developed, albeit with limited success, to treat various heart diseases including myocardial infarction. Hence, there is a pressing need to develop more effective treatment methods to address the increasing mortality rate of the heart diseases. Functional CMs are not only an important in vitro cellular tool to model various types of heart diseases for drug development, but they are also a promising therapeutic agent for cell therapy. However, the limited proliferative capacity entails difficulties in acquiring functional CMs in the scale that is required for pathological studies and cell therapy development. Stem cells, human pluripotent stem cells (hPSCs) in particular, have been considered as an unlimited cellular source for providing functional CMs for various applications. Notable progress has already been made: the first clinical trials of hPSCs derived CMs (hPSC-CMs) for treating myocardial infarction was approved in 2015, and their potential use in disease modeling and drug discovery is being fully explored. This concise review gives an account of current development of differentiation, purification and maturation techniques for hPSC-CMs, and their application in cell therapy development and pharmaceutical industries will be discussed with the latest experimental evidence.
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Humanos , Terapia Baseada em Transplante de Células e Tecidos , Descoberta de Drogas , Indústria Farmacêutica , Cardiopatias , Técnicas In Vitro , Mortalidade , Infarto do Miocárdio , Miocárdio , Miócitos Cardíacos , Células-Tronco Pluripotentes , Células-TroncoRESUMO
Hemangioblasts or blood islands only arise in early development thereby the sources to obtain these bi-potential cells are limited. While previous studies have isolated both lineages in vitro through the hemangioblast, derivation efficiency was rather low due to cellular damage attributed by enzyme usage and fluorescent activated cell sorting (FACS). This study focused on avoiding the use of damaging factors in the derivation of endothelial cells (ECs). Single cell H9-human embryonic stem cells (hESCs) were obtained by using a mild dissociation protocol then human embryoid body (hEB) formation was performed under hemangioblast differentiation conditions. The hEBs were subjected to a two-stage cytokine treatment procedure. Subsequent culture of the adhesive cells in day 4 hEBs gave arise to a seemingly pure population of ECs. The hESC-derived ECs were characterized by identifying signature endothelial gene and protein markers as well as testing for in vitro functionality. Furthermore, in vivo functionality was also confirmed by transplanting the cells in hindlimb ischemic murine models. We demonstrate that the genetic change required for EC derivation precedes blast colony formation. Furthermore, cell damage was prevented by abating enzyme usage and FACS, resulting in a high yield of ECs upon adhesion. Under this method, confluent cultures of ECs were obtainable 4 days after hEB formation which is significantly faster than previous protocols.
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Animais , Humanos , Adesivos , Corpos Embrioides , Células-Tronco Embrionárias , Células Endoteliais , Hemangioblastos , Membro Posterior , Células-Tronco Embrionárias Humanas , Técnicas In Vitro , Ilhas , MétodosRESUMO
Many researchers have focused on the role of adipocytes in increasing efficient bone tissue engineering and osteogenic differentiation of stem cells. Previous reports have not reached a definite consensus on whether adipocytes positively influence in vitro osteogenic differentiation and in vivo bone formation. We investigated the adipocyte influence on osteogenic differentiation from adipose-derived stromal cells (ADSCs) and bone formation through histological analysis in vitro and in vivo. Using the direct co-culture system, we analyzed the influence of adipocytes to promote the differentiation fate of ADSCs. Using co-transplantation of ADSC-derived adipocytes and osteoblasts into the dorsal region of mice, the osteogenesis and bone quality were determined by histological morphology, radiography, and the measurement of the Ca²⁺ concentration. The adipocyte negatively affected the osteoblast differentiation of ADSCs in the in vitro system and induced osteogenesis of osteoblasts in the in vivo system through co-transplantation. Interestingly, in the co-transplanted adipocytes and osteoblasts, the bone formation areas decreased in the osteoblast only group compared with the mixed adipocytes and osteoblast group 6 weeks after transplantation. Conversely, co-transplantation and osteoblast transplantation had similar degrees of calcification as observed from radiography analysis and the measurement of the Ca²⁺ concentrations. Our results revealed that adipocytes inhibited osteoblast differentiation in vitro but enhanced the efficacy of osteogenesis in vivo. In addition, the adipocytes controlled the activity of osteoclasts in the newly formed bone tissue. Our approach can be used to reconstruct bone using stem cell-based tissue engineering and to enhance the understanding of the role adipocytes play.
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Animais , Camundongos , Adipócitos , Osso e Ossos , Técnicas de Cocultura , Consenso , Técnicas In Vitro , Osteoblastos , Osteoclastos , Osteogênese , Radiografia , Células-Tronco , Células Estromais , Engenharia TecidualRESUMO
BACKGROUND: The use of growth factors in skin rejuvenation is emerging as a novel anti-aging treatment. While the role of growth factors in wound healing is well established, their use in skin rejuvenation has only recently been to be studied and no controlled trials have been performed. OBJECTIVE: We evaluated the anti-aging effects of secretory factors of endothelial precursor cells differentiated from human embryonic stem cells (hESC-EPC) in Asian skin. METHODS: A total of 25 women were included in this randomized, controlled split-face study. The right and left sides of each participant's face were randomly allocated to hESC-EPC conditioned medium (CM) or saline. To enhance epidermal penetration, a 0.25-mm microneedle roller was used. Five treatment sessions were repeated at 2-week intervals. RESULTS: Physician's global assessment of pigmentation and wrinkles after treatment revealed statistically significant effects of microneedling plus hESC-EPC CM compared to microneedling alone (p<0.05). Skin measurements by Mexameter and Visiometer also revealed statistically significant effects of microneedling plus hESC-EPC CM on both pigmentation and wrinkles (p<0.05). The only minimal adverse event was mild desquamation in one participant. CONCLUSION: Secretory factors of hESC-EPC improve the signs of skin aging and could be a potential option for skin rejuvenation.
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Feminino , Humanos , Envelhecimento , Povo Asiático , Meios de Cultivo Condicionados , Células-Tronco Embrionárias , Peptídeos e Proteínas de Sinalização Intercelular , Pigmentação , Rejuvenescimento , Envelhecimento da Pele , Pele , Células-Tronco , CicatrizaçãoRESUMO
Recent advances in stem cell biology, including the development of optimized cell type-specific culture systems, and the broader understandings of biochemical and molecular signals involved in cell self-renewal and differentiation have brought cell-based therapy closer to practical application. As of now, at least 250 adult stem cell therapies are being used or tested in clinical situations. Stem cells have two important properties that distinguish them from other types of cells; they can both proliferate without changing their phenotypes indefinitely, and they also can differentiate into one or more new kinds of cells depending on their culture conditions. Thus, stem cell therapy could be most effective for treating the diseases that are marked by the loss of cells. The typical examples are Parkinson's disease, Alzheimer's disease, diabetes, heart failure, blindness, spinal cord injury, and stroke. Additionally, stem cell derivatives can be used in drug discovery as well. In the last decade, various types of stem cells have been identified from preimplantation stage embryos, fetuses, placentas, and adult tissues. Moreover, it is now almost a common practice to produce induced pluripotent stem (iPS) cells from various adult somatic cells using only a few defined factors. Thus, it is feasible that patient-specific stem cells will be generated with less controversy in the near future. However, human embryonic stem (ES) cells firmly remain "the gold standard" because of their greatest potential to become any type of cell in the body. The vast knowledge obtained from human ES cell research in the past decade has made cell-based therapy more promising than ever. Even the recent establishment of iPS cell technology is the culmination of human ES cells research. In our laboratory, interesting human cardiovascular cells including endothelial precursor cells and beating myocardiac cells, artificial blood cells, and retinal pigment epithelial cells were successfully differentiated and their therapeutic potential was confirmed after cell transplantation into animal models. Thus, here, the current research status of human embryonic stem cell-based therapy will be introduced and the future directions of stem cell applications in clinical trials will be discussed.
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Adulto , Humanos , Células-Tronco Adultas , Doença de Alzheimer , Células Artificiais , Biologia , Cegueira , Células Sanguíneas , Transplante de Células , Descoberta de Drogas , Células-Tronco Embrionárias , Estruturas Embrionárias , Células Epiteliais , Feto , Insuficiência Cardíaca , Células-Tronco Pluripotentes Induzidas , Modelos Animais , Doença de Parkinson , Fenótipo , Placenta , Retinaldeído , Traumatismos da Medula Espinal , Células-Tronco , Acidente Vascular Cerebral , TransplantesRESUMO
The authors would like to amend a reference (Lee et al., 2003) that was cited in "Cell culture" section of "Materials and Methods". Instead of "(Lee et al., 2003)", we would like to change the reference to "(Kim et al., 2003)". In "References", it also needs to include the following reference. Kim YY, Seol HW, Ahn HJ. Temporal expression of differentiation markers in embryoid bodies from various human embryonic stem cell line. International Society for Stem Cell Research 1st Annual Meeting, Washington, DC. U.S.A. June 8-11, 2003, Abstract No. 35. The authors apologize for any inconvenience.
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OBJECTIVE: To understand the crucial requirement for the normal early folliculogenesis, we evaluated molecular as well as physiological differences during in vitro ovarian culture. Among the important regulators for follicle development, anti-Mullerian hormone (AMH) and FSH Receptor (FSHR) have been known to be expressed in the cuboidal granulosa cells. Meanwhile, it is known that c-kit is germ cell-specific and GDF-9 is also oocyte-specific regulator. To evaluate the functional requirement for the competence of normal follicular development, we investigated the differential mRNA expression of several factors secreted from granulosa cells and oocytes between in vivo and in vitro developed ovaries. MATERIALS AND METHODS: Ovaries from ICR neonates (the day of birth) were cultured for 4 days (for primordial to primary transition) or 8 days (for secondary follicle formation) in alpha-MEM glutamax supplemented with 3 mg/ml BSA without serum or growth factors. The mRNA levels of the several factors were investigated by quantitative real-time PCR analysis. Freshly isolated 0-, 4-, and 8-day-old ovaries were used as control. RESULTS: The mRNA of AMH and FSHR as granulosa cell factors was highly increased according to the ovarian development in both of 4- and 8-day-old control. However, the mRNA expression was not induced in both of 4- and 8-day in vitro cultured ovaries. The mRNA expression of GDF-9 known to regulate follicle growth as an oocyte factor was different between in vivo and in vitro developed ovaries. In addition, the transcript of GDF-9 was expressed in the primordial follicles of mouse ovaries. The mRNA expression of c-kit was not significantly different during the early folliculogenesis in vitro. CONCLUSION: This is the first report regarding endogenous AMH and FSHR expression during the early folliculogenesis in vitro. In conclusion, it will be very valuable to evaluate cuboidal granulosa cell factors as functional marker(s) for normal early folliculogenesis in vitro.
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Animais , Feminino , Humanos , Recém-Nascido , Camundongos , Hormônio Antimülleriano , Células da Granulosa , Fator 9 de Diferenciação de Crescimento , Peptídeos e Proteínas de Sinalização Intercelular , Competência Mental , Oócitos , Ovário , Reação em Cadeia da Polimerase em Tempo Real , Receptores do FSH , RNA MensageiroRESUMO
Nanog is a newly identified divergent homeodomain protein that directs the infinite propagation and sustains the pluripotency of embryonic stem cells. It has been reported that murine Nanog has two potent transactivation domains in N-terminal and C-terminal regions. Human Nanog (hNanog) polypeptide shares about 58% and 87% identity to the open reading frame and homeodomain of murine Nanog, respectively. However, the functional domains and molecular mechanisms of hNanog are poorly understood. In this study, for the first time, we presented that only C-terminus of hNanog contains a potent transactivation domain. Based on the amino acid sequences of homeobox domain, we roughly divided hNanog open reading frame into the three regions such as N-terminal, homeodomain and C-terminal regions and constructed either the fusion proteins between hNanog individual and Gal4 DNA binding domain or the context of native hNanog protein. Reporter assays by using reporter plamid containing Gal4 or Nanog binding site revealed that the only C-terminal region exhibited the significant fold induction of transactivation. However, interestingly, there was no significant activation through N-terminal region unlike murine Nanog, suggesting that C-terminal region may have more critical roles in the transcriptional activation of target genes. Taken together, the finding of a putative transactivation domain in hNanog may contribute to the further understanding of molecular mechanism on the regulation of downstream genes involved in self-renewal and pluripotency of human stem cells.
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Animais , Humanos , Camundongos , Células COS , Chlorocebus aethiops , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Células HeLa , Proteínas de Homeodomínio/genética , Rim/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/genética , Proteínas de Saccharomyces cerevisiae/genética , Deleção de Sequência , Ativação Transcricional , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
Human embryonic stem (hES) cells are capable of differentiating into pluralistic cell types, however, spontaneous differentiation generally gives rise to a limited number of specific differentiated cell types and a large degree of cell heterogeneity. In an effort to increase the efficiency of specified hES cell differentiation, we performed a series of transient transfection of hES cells with EGFP expression vectors driven by different promoter systems, including human cellular polypeptide chain elongation factor 1 alpha (hEF1alpha), human cytomegalo-virus, and chicken beta-actin. All these promoters were found to lead reporter gene expression in undifferentiated hES cells, but very few drug-selectable transfectants were obtained and failed to maintain stable expression of the transgene with either chemical or electroporation methods. In an attempt to increase transfection efficiency and obtain stable transgene expression, differentiated hES cells expressing both mesodermal and ectodermal markers were derived using a defined medium. Differentiated hES cells were electroporated with a hEF1alpha promoter-driven EGFP or human noggin expression vector. Using RT-PCR, immunocytochemistry and fluorescence microscopy, the differentiated hES cells transfected with foreign genes were confirmed to retain stable gene and protein expression during prolonged culture. These results may provide a new tool for introducing exogenous genes readily into hES cells, thereby facilitating more directed differentiation into specific and homogenous cell populations.
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Animais , Humanos , Actinas/genética , Proteínas Morfogenéticas Ósseas/genética , Diferenciação Celular , Galinhas , Citomegalovirus/genética , Sistemas de Liberação de Medicamentos , Estruturas Embrionárias/citologia , Terapia Genética , Proteínas de Fluorescência Verde/genética , Técnicas Imunoenzimáticas , Microscopia de Fluorescência , Fator 1 de Elongação de Peptídeos/genética , Células-Tronco Pluripotentes/citologia , Regiões Promotoras Genéticas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica/genéticaRESUMO
Mouse embryonic stem (mES) cells are capable of undergoing chondrogenesis in vitro. To enhance this process, the human SOX9 (hSOX9) cDNA was delivered into mES cells and the clones overexpressing hSOX9 (denoted as mES-hSOX9 cells) were verified by Western blot analysis. The transcripts of collagen IIA (a juvenile form), aggrecan and Pax1 were expressed in mES-hSOX9 cells grown on feeder layers, suggesting the immediate effect of exogenous SOX9 on chondrogenesis. However, SOX9 overexpression did not affect the cell cycle distribution in undifferentiated mES cells. Upon differentiation, collagen IIB (an adult form) was detected in day 3 immature embryoid bodies. In addition, the overexpression of exogenous SOX9 significantly induced transcriptional activity driven by SOX9 binding site. Taken together, we for the first time demonstrated that constitutive overexpression of exogenous SOX9 in undifferentiated mES cells might have dual potentials to induce both chondrogenic commitment and growth capacity in the undifferentiated status.
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Animais , Humanos , Camundongos , Diferenciação Celular/genética , Linhagem Celular , Condrogênese , Colágeno Tipo II/genética , Estruturas Embrionárias/citologia , Elementos Facilitadores Genéticos/genética , Proteínas da Matriz Extracelular/genética , Marcadores Genéticos/genética , Proteínas de Grupo de Alta Mobilidade/genética , Lectinas Tipo C/genética , Fatores de Transcrição Box Pareados/genética , Proteoglicanas/genética , Células-Tronco/metabolismo , Ativação Transcricional , Fatores de Transcrição/genéticaRESUMO
OBJECTIVE: The aim of this study were to compare the effects of EG and PROH on cryopreservation of mouse and human embryos, and to find the optimal protocol for embryo freezing. METHODS: Human embryos derived from fertilized eggs showing 3 pronuclei (PN) and mouse embryos were divided into two groups respectively: dehydrated with 1.5 M EG+0.2 M sucrose or 1.5 M PROH+0.2 M sucrose using the slow freezing method. Moreover mouse embryos were controlled the exposure time of cryoprotectant during dehydration or rehydration steps. RESULTS: The survival rates of human embryos were 79.2% (84/106) in EG group and 77.9% (88/113) in PROH group. In mouse embryos, the survival and development rates up to blastocyst were 70.6% (245/347), 44.1% (123/279) in EG group and 62.1% (198/319), 45.1% (123/279) in PROH group, respectively. However, in EG group, partially damaged embryos after thawing were decreased compared to PROH group. In combination group, when the exposure time during dehydration and rehydration were reduced, the survival and embryonic developments were increased slightly, but not significant. CONCLUSION: Cryopreservation of mouse and human embryos at cleavage stage by using EG or PROH exhibited no statistical difference in the survival rate and/or developmental rate to blastocyst. However, the use of EG for cryopreservation of embryos might reduce the exposure time of the cryoprotectant because of a high permeation of EG and result in lessen its toxic effects.
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Animais , Feminino , Humanos , Camundongos , Gravidez , Blastocisto , Criopreservação , Desidratação , Desenvolvimento Embrionário , Estruturas Embrionárias , Etilenoglicol , Hidratação , Congelamento , Propilenoglicol , Sacarose , Taxa de Sobrevida , ZigotoRESUMO
OBJECTIVES: Controversial arguments exists on both the case for and against on the accumulation of mitochondrial DNA (mtDNA) deletion in association to tissue and age. The debate continues as to whether this mutation is a major contributor to the phenotypic expression of aging and common degenerative diseases or simply a clinical insignificant epiphenomenon. The objective of this study was to determine whether the accumulation of mtDNA deletion is correlated with age-related and tissue-specific variation. MATERIALS AND METHODS: One hundred and fifty-seven tissues from blood, ovary, uterine muscle, and abdominal muscle were obtained from patients ranging in age from 31~60 years. After reviewing the clinical reports, patients with mitochondrial disorder were excluded from this study. The tissues were obtained at gynecological surgeries with the consent of the patient. Total DNA isolated from blood, ovary, uterine muscle, and abdominal muscle was amplified by two rounds of PCR using two pairs of primers corresponding to positions 8225-8247 (sense), 13551-13574 (antisense) for the area around deleted mtDNA and 8421-8440 (sense), 13520-13501 (antisense) for nested PCR product. A statistical analysis was performed by c2-test. RESULTS: About 0% of blood, 94.8% of ovary, 71.4% of uterine muscle, and 86.1% abdominal muscle harbored mtDNA deletion. When we examined the proportion of deleted mtDNA according to age deletion rate was 90% of ovary, 63.6% of uterine muscle, 77.7% of abdominal muscle in thirties and 100% of all tissue in fifties. CONCLUSION: The findings of this study suggest that the mtDNA deletion is varied in tissue-specific pattern and increases with aging.
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Animais , Feminino , Humanos , Camundongos , Músculos Abdominais , Envelhecimento , DNA , DNA Mitocondrial , Procedimentos Cirúrgicos em Ginecologia , Doenças Mitocondriais , Miométrio , Ovário , Reação em Cadeia da PolimeraseRESUMO
Monozygotic twinning is a relatively rare event in vivo conception, being estimated to occur in 0.42% of all birth. The underlying mechanism for monozygotic twin formation is the division of the embryo early in its development. Separation of cells may theoretically occur before or after inner cell mass formation. The incidence of monozygotic twinning following assisted reproduction techniques is higher than the commonly accepted incidence after in vivo conception. Patients at particular risk of monozygotic twins are those aged > 35 years and those who had manipulation techniques for assisted fertilization. Hence, it seems prudent to counsel these patients about the potential obstetric complications of monozygotic multiple gestations prior to the initiation of their treatment.
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Humanos , Estruturas Embrionárias , Fertilização , Fertilização in vitro , Incidência , Parto , Técnicas Reprodutivas , Gemelaridade Monozigótica , Gêmeos MonozigóticosRESUMO
OBJECTIVE: To report the pregnancy which was made by in vitro fertilization using recombinant follicle stimulating hormone and gonadotropin releasing hormone antagonist. MATERIAL AND METHOD: Case report. RESULTS: Six oocytes were retrieved and all were fertilized by intracytoplasmic sperm injection. Six embryos were transferred and the pregnancy was confirmed. CONCLUSION: It is envisaged that the availability of recombinant gonadotropins and gonadotropin releasing hormone antagonists will ultimately lead to shorter, cheaper and safer treatments, using reduced dosages.