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1.
Journal of Zhejiang University. Science. B ; (12): 293-304, 2018.
Article in English | WPRIM | ID: wpr-1010389

ABSTRACT

Axon regeneration is crucial for recovery from neurological diseases. Numerous studies have identified several genes, microRNAs (miRNAs), and transcription factors (TFs) that influence axon regeneration. However, the regulatory networks involved have not been fully elucidated. In the present study, we analyzed a regulatory network of 51 miRNAs, 27 TFs, and 59 target genes, which is involved in axon regeneration. We identified 359 pairs of feed-forward loops (FFLs), seven important genes (Nap1l1, Arhgef12, Sema6d, Akt3, Trim2, Rab11fip2, and Rps6ka3), six important miRNAs (hsa-miR-204-5p, hsa-miR-124-3p, hsa-miR-26a-5p, hsa-miR-16-5p, hsa-miR-17-5p, and hsa-miR-15b-5p), and eight important TFs (Smada2, Fli1, Wt1, Sp6, Sp3, Smad4, Smad5, and Creb1), which appear to play an important role in axon regeneration. Functional enrichment analysis revealed that axon-associated genes are involved mainly in the regulation of cellular component organization, axonogenesis, and cell morphogenesis during neuronal differentiation. However, these findings need to be validated by further studies.


Subject(s)
Humans , Axons/physiology , Cell Differentiation , Cluster Analysis , Embryonic Stem Cells/cytology , Gene Expression Profiling , Gene Expression Regulation , Gene Regulatory Networks , MicroRNAs/metabolism , Nerve Regeneration , Neurons/metabolism , Software , Transcription Factors/metabolism
2.
Article in English | IMSEAR | ID: sea-157121

ABSTRACT

The ability to successfully derive human embryonic stem cells (hESC) lines from human embryos following in vitro fertilization (IVF) opened up a plethora of potential applications of this technique. These cell lines could have been successfully used to increase our understanding of human developmental biology, transplantation medicine and the emerging science of regenerative medicine. The main source for human embryos has been ‘discarded’ or ‘spare’ fresh or frozen human embryos following IVF. It is a common practice to stimulate the ovaries of women undergoing any of the assisted reproductive technologies (ART) and retrieve multiple oocytes which subsequently lead to multiple embryos. Of these, only two or maximum of three embryos are transferred while the rest are cryopreserved as per the decision of the couple. in case a couple does not desire to ‘cryopreserve’ their embryos then all the embryos remaining following embryo transfer can be considered ‘spare’ or if a couple is no longer in need of the ‘cryopreserved’ embryos then these also can be considered as ‘spare’. But, the question raised by the ethicists is, “what about ‘slightly’ over-stimulating a woman to get a few extra eggs and embryos? The decision becomes more difficult when it comes to ‘discarded’ embryos. As of today, the quality of the embryos is primarily assessed based on morphology and the rate of development mainly judged by single point assessment. Despite many criteria described in the literature, the quality assessment is purely subjective. The question that arises is on the decision of ‘discarding’ embryos. What would be the criteria for discarding embryos and the potential ‘use’ of ESC derived from the ‘abnormal appearing’ embryos? This paper discusses some of the newer methods to procure embryos for the derivation of embryonic stem cell lines which will respect the ethical concerns but still provide the source material.


Subject(s)
Cryopreservation/methods , Embryo, Mammalian/cytology , Embryonic Stem Cells/cytology , Female , Humans , Nuclear Transfer Techniques
3.
São Paulo; s.n; s.n; 2014. 181 p. tab, graf, ilus.
Thesis in Portuguese | LILACS | ID: biblio-847078

ABSTRACT

As proteína quinases C (PKC) pertencem à família das serina/treonina quinases, que vem sendo apontadas como importantes enzimas para os processos de proliferação e diferenciação das células tronco embrionárias (CTE), todavia, a função exata de cada isoforma dessa família ainda não está clara. Dados anteriores do nosso laboratório indicam que dentre as PKCs expressas em CTE, formas cataliticamente ativas da PKCßI são altamente expressas no núcleo das CTE murinas. Estas ao se diferenciarem expressam essa quinase no seu citoplasma ou deixam de expressar a mesma, e que a maioria dos alvos da PKCßI em CTE indiferenciada estão envolvidos em processos de regulação da transcrição de proteínas envolvidas em processos de proliferação/ diferenciação. Dando continuidade aos resultados anteriores do laboratório, no presente trabalho, com técnicas de proteômica e fosfoproteômica identificamos outros alvos nucleares da PKCßI em CTE indiferenciadas. Vimos que de fato inibindo-se a PKCßI diminuiu-se a fostorilação de fatores envolvidos com a indiferenciação das CTE. Dentre os alvos da PKCßI encontramos a proteína adaptadora, TIF1 que recruta proteínas remodeladoras de cromatina. Essa proteína é essencial para a manutenção do estado indiferenciado das CTE. In vitro a PKCßI foi capaz de fosforilar a TIF1ß e inibindo-se a PKCßI por RNAi vimos uma diminuição na expressão da TIF1ß e no fator de indiferenciação Nanog cuja expressão já foi demonstrada ser regulada pela TIF1ß. Além disso vimos que inibindo-se a PKCßI com o peptídeo inibidor da PKCßI aumentou a expressão de proteínas reguladas pelo c-Myc. E que o RNAi para a PKCßI aumentou a expressão de proteínas que regulam a expressão do c-Myc. Não vimos nenhum efeito na fosforilação ou expressão do c-Myc após a inibição da PKCßI o que sugere que a PKCßI ative proteínas repressoras do c-Myc. Nossos estudos sugerem que a PKCßI regula a manutenção do estado indiferenciado das CTE regulando a expressão e atividade da Tif1ß um possível alvo direto da PKCßI. Levando a modificações da cromatina e regulação da expressão de genes que mantém as CTE indiferenciadas. Outro ponto de regulação da PKCßI parece ser a nibição da atividade de c-Myc o que seria importante para a manutenção do estado indiferenciado visto que o c-Myc é um amplificador das vias de sinalização que mantém as células proliferando. Desta forma a PKCßI parece ter um papel central na regulação da expressão gênica de CTE à nível de modificações epigenéticas e a nível transcricional mantendo as CTE indiferenciadas


The Protein kinase C (PKC) family of serine/treonine kinases, are being described as important enzymes for proliferation and diferentiation of embryonic stem cells (ESC), however, the exact function of the different isoenzymes of this family still is unclear. Previous data from our laboratory indicates that amongst the PKCs expressed in ESC, catalytically active forms of PKCßI are highly expressed in nucleus of murine ESC. When these cells differentiate this kinase can be found in the cytoplasm or not expressed at all, and that the majority of PKCßI targets in undifferentiated ESC are involved in the regulation of proteins involved in transcription of proteins involved in proliferation/ diferentiation. Continuing our previous work herewith using proteomics and phosphoproteomics techniques we identified other nuclear PKCßI targets in undifferentiated ESC. We indeed saw that inhibiting PKCßI decreased the phosphorylation of factors involved with maintainance of the undifferentiated state of ESC. Amongst the targets of PKCßI we found the adaptor protein, TIF1ßI, that recruits cromatin remodeling proteins. This protein is essential for the maintenance of the undifferentiated state of ESC. In vitro PKCßI phosphorylated TIF1ß and inhibiting PKCßI with RNAi decreased the expression of TIF1ß and of the undifferentiation factor Nanog whose expression has been shown to be regulated by TIF1ß. We also saw that inhibiting PKCßI with a peptide inhibitor increased the expression of proteins regulated by c-Myc, and that RNAi for PKCßI increased the expression of proteins that regulate the expression of c-Myc. We did not see any effect on the phosphorylation or expression of c-Myc after inhibition of PKCßI suggesting that PKCßI activates c-Myc repressor proteins. Our studies sugest that PKCßI regulates the maintenance of the undiferentiated state of ESC regulating the expression and activity of Tif1ß a possibly a direct target of PKCßI, leading to chromatin modifications and regulation of genes that maintain ESC undiferentiated. Another form of regulation of PKCßI seems to be by inhibiting the activity of c-Myc which is importante to maintain ESC undifferentiated since c-Myc is na an amplifyer of signaling patheways that maintain ESC proliferating. Together PKCßI has a central role in the regulation of the gene expression of ESC at the level of epigenetic modifications and transcriptional regulation


Subject(s)
Embryonic Stem Cells/cytology , Protein Kinase C/metabolism , Cell Differentiation , Chromatin/genetics , Mass Spectrometry/methods , Phosphorylation , Protein Kinase C beta/analysis , Proteomics/instrumentation , Repressor Proteins/genetics , Substrates for Biological Treatment/classification
4.
Experimental & Molecular Medicine ; : e108-2014.
Article in English | WPRIM | ID: wpr-103502

ABSTRACT

The epithelial-mesenchymal transition (EMT) is important for embryonic development and the formation of various tissues or organs. However, EMT dysfunction in normal cells leads to diseases, such as cancer or fibrosis. During the EMT, epithelial cells are converted into more invasive and active mesenchymal cells. E-box-binding proteins, including Snail, ZEB and helix-loop-helix family members, serve as EMT-activating transcription factors. These transcription factors repress the expression of epithelial markers, for example, E-cadherin, rearrange the cytoskeleton and promote the expression of mesenchymal markers, such as vimentin, fibronectin and other EMT-activating transcription factors. Signaling pathways that induce EMT, including transforming growth factor-beta, Wnt/glycogen synthase kinase-3beta, Notch and receptor tyrosine kinase signaling pathways, interact with each other for the regulation of this process. Although the mechanism(s) underlying EMT in cancer or embryonic development have been identified, the mechanism(s) in embryonic stem cells (ESCs) remain unclear. In this review, we describe the underlying mechanisms of important EMT factors, indicating a precise role for EMT in ESCs, and characterize the relationship between EMT and ESCs.


Subject(s)
Animals , Humans , Cadherins/metabolism , Embryonic Stem Cells/cytology , Epithelial-Mesenchymal Transition , Signal Transduction , Transcription Factors/metabolism
5.
Journal of Veterinary Science ; : 61-71, 2014.
Article in English | WPRIM | ID: wpr-56432

ABSTRACT

This study was performed to assess the neurotoxic effects of methylmercury, arsanilic acid and danofloxacin by quantification of neural-specific proteins in vitro. Quantitation of the protein markers during 14 days of differentiation indicated that the mouse ESCs were completely differentiated into neural cells by Day 8. The cells were treated with non-cytotoxic concentrations of three chemicals during differentiation. Low levels of exposure to methylmercury decreased the expression of GABAA-R and Nestin during the differentiating stage, and Nestin during the differentiated stage. In contrast, GFAP, Tuj1, and MAP2 expression was affected only by relatively high doses during both stages. Arsanilic acid affected the levels of GABA(A)-R and GFAP during the differentiated stage while the changes of Nestin and Tuj1 were greater during the differentiating stage. For the neural markers (except Nestin) expressed during both stages, danofloxacin affected protein levels at lower concentrations in the differentiated stage than the differentiating stage. Acetylcholinesterase activity was inhibited by relatively low concentrations of methylmercury and arsanilic acid during the differentiating stage while this activity was inhibited only by more than 40 microM of danofloxacin in the differentiated stage. Our results provide useful information about the different toxicities of chemicals and the impact on neural development.


Subject(s)
Animals , Mice , Acetylcholinesterase/metabolism , Arsanilic Acid/toxicity , Cell Differentiation/drug effects , Embryonic Stem Cells/cytology , Environmental Pollutants/toxicity , Fluorescent Antibody Technique , Fluoroquinolones/toxicity , Gene Expression Regulation/drug effects , Methylmercury Compounds/toxicity , Nerve Tissue Proteins/metabolism , Neurons/cytology , Tetrazolium Salts/metabolism , Thiazoles/metabolism
6.
Journal of Veterinary Science ; : 519-528, 2014.
Article in English | WPRIM | ID: wpr-24548

ABSTRACT

The present study was conducted to develop an effective method for establishment of porcine parthenogenetic embryonic stem cells (ppESCs) from parthenogenetically activated oocyte-derived blastocysts. The addition of 10% fetal bovine serum (FBS) to the medium on the 3rd day of oocyte culturing improved the development of blastocysts, attachment of inner cell masses (ICMs) onto feeder cells, and formation of primitive ppESC colonies. ICM attachment was further enhanced by basic fibroblast growth factor, stem cell factor, and leukemia inhibitory factor. From these attached ICMs, seven ppESC lines were established. ppESC pluripotency was verified by strong enzymatic alkaline phosphatase activity and the expression of pluripotent markers OCT3/4, Nanog, and SSEA4. Moreover, the ppESCs were induced to form an embryoid body and teratoma. Differentiation into three germ layers (ectoderm, mesoderm, and endoderm) was confirmed by the expression of specific markers for the layers and histological analysis. In conclusion, data from the present study suggested that our modified culture conditions using FBS and cytokines are highly useful for improving the generation of pluripotent ppESCs.


Subject(s)
Animals , Blastocyst/cytology , Cell Culture Techniques/veterinary , Cell Differentiation , Cytokines/metabolism , Embryonic Stem Cells/cytology , Parthenogenesis , Pluripotent Stem Cells/cytology , Swine/physiology
7.
Indian J Exp Biol ; 2013 Mar; 51(3): 201-207
Article in English | IMSEAR | ID: sea-147583

ABSTRACT

In the experimental group (shh inhibited group), there were significant decreases in the expression of Oct4, Nanog, Shh, GATA4, Brachyury and Goosecoid, while increases were observed for TAT and Pdx1. The expression of Sox17 did not differ between two control and experimental groups. In experimental group, the amount of GSC positive cells was somehow lower but it seems that there was no difference for Sox17. Shh inhibition induces ESCs to differentiate toward definitive endoderm by committing mesendodermal lineages.


Subject(s)
Animals , Cell Differentiation , Cell Line , Cell Lineage , DNA Primers , Dithizone/pharmacology , Embryonic Stem Cells/cytology , Endoderm/metabolism , Gene Expression Regulation, Developmental , Hedgehog Proteins/metabolism , Homeodomain Proteins/metabolism , Immunohistochemistry , Mesoderm/metabolism , Mice , Microscopy, Fluorescence , Octamer Transcription Factor-3/metabolism , Reverse Transcriptase Polymerase Chain Reaction
8.
Experimental & Molecular Medicine ; : e55-2013.
Article in English | WPRIM | ID: wpr-209543

ABSTRACT

Hematopoietic stem cells (HSCs) comprise a rare population of cells that can regenerate and maintain lifelong blood cell production. This functionality is achieved through their ability to undergo many divisions without activating a poised, but latent, capacity for differentiation into multiple blood cell types. Throughout life, HSCs undergo sequential changes in several key properties. These affect mechanisms that regulate the self-renewal, turnover and differentiation of HSCs as well as the properties of the committed progenitors and terminally differentiated cells derived from them. Recent findings point to the Lin28b-let-7 pathway as a master regulator of many of these changes with important implications for the clinical use of HSCs for marrow rescue and gene therapy, as well as furthering our understanding of the different pathogenesis of childhood and adult-onset leukemia.


Subject(s)
Animals , Humans , Cell Differentiation , Cell Lineage , Embryonic Stem Cells/cytology , HMGA2 Protein/genetics , Hematopoietic Stem Cells/cytology , Leukemia/etiology , MicroRNAs/genetics , RNA-Binding Proteins/genetics
9.
Experimental & Molecular Medicine ; : e53-2013.
Article in English | WPRIM | ID: wpr-209542

ABSTRACT

The transplantation of neural precursor cells (NPCs) is known to be a promising approach to ameliorating behavioral deficits after stroke in a rodent model of middle cerebral artery occlusion (MCAo). Previous studies have shown that transplanted NPCs migrate toward the infarct region, survive and differentiate into mature neurons to some extent. However, the spatiotemporal dynamics of NPC migration following transplantation into stroke animals have yet to be elucidated. In this study, we investigated the fates of human embryonic stem cell (hESC)-derived NPCs (ENStem-A) for 8 weeks following transplantation into the side contralateral to the infarct region using 7.0T animal magnetic resonance imaging (MRI). T2- and T2*-weighted MRI analyses indicated that the migrating cells were clearly detectable at the infarct boundary zone by 1 week, and the intensity of the MRI signals robustly increased within 4 weeks after transplantation. Afterwards, the signals were slightly increased or unchanged. At 8 weeks, we performed Prussian blue staining and immunohistochemical staining using human-specific markers, and found that high percentages of transplanted cells migrated to the infarct boundary. Most of these cells were CXCR4-positive. We also observed that the migrating cells expressed markers for various stages of neural differentiation, including Nestin, Tuj1, NeuN, TH, DARPP-32 and SV38, indicating that the transplanted cells may partially contribute to the reconstruction of the damaged neural tissues after stroke. Interestingly, we found that the extent of gliosis (glial fibrillary acidic protein-positive cells) and apoptosis (TUNEL-positive cells) were significantly decreased in the cell-transplanted group, suggesting that hESC-NPCs have a positive role in reducing glia scar formation and cell death after stroke. No tumors formed in our study. We also performed various behavioral tests, including rotarod, stepping and modified neurological severity score tests, and found that the transplanted animals exhibited significant improvements in sensorimotor functions during the 8 weeks after transplantation. Taken together, these results strongly suggest that hESC-NPCs have the capacity to migrate to the infarct region, form neural tissues efficiently and contribute to behavioral recovery in a rodent model of ischemic stroke.


Subject(s)
Animals , Humans , Male , Rats , Apoptosis , Cell Differentiation , Cell Movement , Embryonic Stem Cells/cytology , Glial Fibrillary Acidic Protein/genetics , Infarction, Middle Cerebral Artery/metabolism , Neural Stem Cells/cytology , Psychomotor Performance , Rats, Sprague-Dawley , Receptors, CXCR4/genetics
10.
Experimental & Molecular Medicine ; : e56-2013.
Article in English | WPRIM | ID: wpr-152453

ABSTRACT

As the theory of stem cell plasticity was first proposed, we have explored an alternative hypothesis for this phenomenon: namely that adult bone marrow (BM) and umbilical cord blood (UCB) contain more developmentally primitive cells than hematopoietic stem cells (HSCs). In support of this notion, using multiparameter sorting we were able to isolate small Sca1+Lin-CD45- cells and CD133+Lin-CD45- cells from murine BM and human UCB, respectively, which were further enriched for the detection of various early developmental markers such as the SSEA antigen on the surface and the Oct4 and Nanog transcription factors in the nucleus. Similar populations of cells have been found in various organs by our team and others, including the heart, brain and gonads. Owing to their primitive cellular features, such as the high nuclear/cytoplasm ratio and the presence of euchromatin, they are called very small embryonic-like stem cells (VSELs). In the appropriate in vivo models, VSELs differentiate into long-term repopulating HSCs, mesenchymal stem cells (MSCs), lung epithelial cells, cardiomyocytes and gametes. In this review, we discuss the most recent data from our laboratory and other groups regarding the optimal isolation procedures and describe the updated molecular characteristics of VSELs.


Subject(s)
Animals , Humans , Cell Lineage , Cell Separation/methods , Embryonic Stem Cells/cytology , Hematopoietic Stem Cells/cytology , Mesenchymal Stem Cells/cytology , Pluripotent Stem Cells/cytology
11.
Arq. neuropsiquiatr ; 70(7): 540-546, July 2012. ilus
Article in English | LILACS | ID: lil-642981

ABSTRACT

Cell therapies, based on transplantation of immature cells, are being considered as a promising tool in the treatment of neurological disorders. Many efforts are being concentrated on the development of safe and effective stem cell lines. Nevertheless, the neurogenic potential of some cell lines, i.e., the ability to generate mature neurons either in vitro or in vivo, is largely unknown. Recent evidence indicate that this potential might be distinct among different cell lines, therefore limiting their broad use as replacement cells in the central nervous system. Here, we have reviewed the latest advancements regarding the electrophysiological maturation of stem cells, focusing our attention on fetal-derived-, embryonic-, and induced pluripotent stem cells. In summary, a large body of evidence supports the biological safety, high neurogenic potential, and in some diseases probable clinical efficiency related to fetal-derived cells. By contrast, reliable data regarding embryonic and induced pluripotent stem cells are still missing.


Terapias celulares, baseadas no transplante de células imaturas, têm sido consideradas ferramentas promissoras no tratamento de doenças neurológicas. Muitos esforços têm sido concentrados no desenvolvimento de linhas de células-tronco seguras e eficazes. No entanto, o potencial neurogênico de algumas linhagens celulares, ou seja, a habilidade de gerar neurônios maduros, in vitro ou in vivo, ainda é altamente desconhecida. Dados recentes sugerem que esse potencial é distinto entre diversos tipos celulares, o que limitaria o largo emprego como células restauradoras no sistema nervoso central. Neste relato, revisaram-se os avanços recentes relacionados à maturação eletrofisiológica de células-tronco, com foco em células derivadas de tecido fetal, células embrionárias e células pluripotentes induzidas. Em resumo, há evidências que apontam para segurança biológica de células fetais, com alto potencial neurogênico e, em se tratando de algumas doenças, provável eficiência clínica. Ao contrário, ainda não há dados confiáveis acerca de células embrionárias e pluripotentes induzidas.


Subject(s)
Humans , Embryonic Stem Cells/cytology , Nervous System Diseases/surgery , Neural Stem Cells/cytology , Pluripotent Stem Cells/cytology , Embryonic Stem Cells/transplantation , Neural Stem Cells/transplantation , Pluripotent Stem Cells/transplantation
12.
Biol. Res ; 45(3): 215-222, 2012. ilus
Article in English | LILACS | ID: lil-659279

ABSTRACT

The skin is a complex stratified organ which acts not only as a permeability barrier and defense against external agents, but also has essential thermoregulatory, sensory and metabolic functions. Due to its high versatility and activity, the skin undergoes continuous self-renewal to repair damaged tissue and replace old cells. Consequently, the skin is a reservoir for adult stem cells of different embryonic origins. Skin stem cell populations reside in the adult hair follicle, sebaceous gland, dermis and epidermis. However, the origin of most of the stem cell populations found in the adult epidermis is still unknown. Far more unknown is the embryonic origin of other stem cells that populate the other layers of this tissue. In this review we attempt to clarify the emergence, structure, markers and embryonic development of diverse populations of stem cells from the epidermis, dermis and related appendages such as the sebaceous gland and hair follicle.


Subject(s)
Humans , Embryonic Development , Embryonic Stem Cells/cytology , Skin/cytology , Skin/embryology , Cell Differentiation , Epidermis/cytology , Epidermis/embryology , Hair Follicle/embryology , Sebaceous Glands/anatomy & histology , Sebaceous Glands/cytology , Skin/growth & development
13.
Experimental & Molecular Medicine ; : 457-463, 2012.
Article in English | WPRIM | ID: wpr-167888

ABSTRACT

Embryonic stem cells (ESCs) can be propagated in vitro on feeder layers of mouse STO fibroblast cells. The STO cells secrete several cytokines that are essential for ESCs to maintain their undifferentiated state. In this study, we found significant growth inhibition of mouse ESCs (mESCs) cultured on STO cells infected with adenovirus containing a dominant-negative mutant form of IkappaB (rAd-dnIkappaB). This blockage of the NF-kappaB signal pathway in STO cells led to a significant decrease in [3H]thymidine incorporation and colony formation of mESCs. Expression profile of cytokines secreted from the STO cells revealed an increase in the bone morphogenetic protein4 (BMP4) transcript level in the STO cells infected with adenoviral vector encoding dominant negative IkappaB (rAd-dnIkappaB). These results suggested that the NF-kappaB signaling pathway represses expression of BMP4 in STO feeder cells. Conditioned medium from the rAd-dnIkappaB-infected STO cells also significantly reduced the colony size of mESCs. Addition of BMP4 prevented colony formation of mESCs cultured in the conditioned medium. Our finding suggested that an excess of BMP4 in the conditioned medium also inhibits proliferation of mESCs.


Subject(s)
Animals , Mice , Bone Morphogenetic Protein 4/genetics , Cell Differentiation/genetics , Cell Proliferation , Culture Media, Conditioned , Embryonic Stem Cells/cytology , Feeder Cells/cytology , Fibroblasts/cytology , Gene Expression Regulation/genetics , I-kappa B Proteins/genetics , Mutation , NF-kappa B/genetics , Signal Transduction
14.
Experimental & Molecular Medicine ; : 303-309, 2012.
Article in English | WPRIM | ID: wpr-186639

ABSTRACT

We previously reported the successful establishment of embryonic stem cell (ESC)-like multipotent spermatogonial stem cells (mSSCs) from neonatal mouse testis. Here, we examined the ability of mSSCs to differentiate into vascular endothelial cells and smooth muscle cells, and compared to that of mouse ESCs. We used real-time reverse transcriptase polymerase chain reaction and immunohistochemistry to examine gene expression profiles of mSSCs and ESCs during in vitro vascular differentiation. Both mSSCs and ESCs exhibited substantial increase in the expression of mesodermal markers, such as Brachyury, Flk1, Mesp1, Nkx2.5, and Islet1, and a decrease in the expression of pluripotency markers, such as Oct3/4 and Nanog during the early stage of differentiation. The mRNA levels of vascular endothelial (VE)-cadherin and CD31 gradually increased in both differentiated mSSCs and ESCs. VE-cadherin- or CD31-positive cells formed sprouting branch-like structures, as observed during embryonic vascular development. At the same time, vascular smooth muscle cell-specific markers, such as myocardin and alpha-smooth muscle actin (SMA), were also highly expressed in differentiated mSSCs and ESCs. Immunocytochemical analysis revealed that the differentiated cells expressed both alpha-SMA and SM22-alpha proteins, and exhibited the intracellular fibril structure typical of smooth muscle cells. Overall, our findings showed that mSSCs have similar vascular differentiation abilities to those of ESCs, suggesting that mSSCs may be an alternative source of autologous pluripotent stem cells for vascular regeneration.


Subject(s)
Animals , Humans , Male , Mice , Animals, Newborn , Biomarkers/metabolism , Cell Differentiation/physiology , Embryonic Stem Cells/cytology , Endothelial Cells/cytology , Gene Expression , Gene Expression Profiling , Immunohistochemistry , Muscle, Smooth, Vascular/cytology , Myocytes, Smooth Muscle/cytology , Pluripotent Stem Cells/cytology , Real-Time Polymerase Chain Reaction , Spermatogonia/cytology , Testis/cytology
15.
Experimental & Molecular Medicine ; : 379-388, 2011.
Article in English | WPRIM | ID: wpr-102686

ABSTRACT

Gangliosides have been suggested to play important roles in various functions such as adhesion, cell differentiation, growth control, and signaling. Mouse follicular development, ovulation, and luteinization during the estrous cycle are regulated by several hormones and cell-cell interactions. In addition, spermatogenesis in seminiferous tubules of adult testes is also regulated by several hormones, including follicle-stimulating hormone (FSH) and luteinizing hormone (LH) and cell-cell interactions. The regulation of these processes by hormones and cell-cell interactions provides evidence for the importance of surface membrane components, including gangliosides. During preimplantation embryo development, a mammalian embryo undergoes a series of cleavage divisions whereby a zygote is converted into a blastocyst that is sufficiently competent to be implanted in the maternal uterus and continue its development. Mouse embryonic stem (mES) cells are pluripotent cells derived from mouse embryo, specifically, from the inner cell mass of blastocysts. Differentiated neuronal cells are derived from mES cells through the formation of embryonic bodies (EBs). EBs recapitulate many aspects of lineage-specific differentiation and temporal and spatial gene expression patterns during early embryogenesis. Previous studies on ganglioside expression during mouse embryonic development (including during in vitro fertilization, ovulation, spermatogenesis, and embryogenesis) reported that gangliosides were expressed in both undifferentiated and differentiated (or differentiating) mES cells. In this review, we summarize some of the advances in our understanding of the functional roles of gangliosides during the stages of mouse embryonic development, including ovulation, spermatogenesis, and embryogenesis, focusing on undifferentiated and differentiated mES cells (neuronal cells).


Subject(s)
Animals , Mice , Cell Differentiation , Embryonic Development , Embryonic Stem Cells/cytology , Gametogenesis , Gangliosides/metabolism , Urogenital System/cytology
16.
The Korean Journal of Gastroenterology ; : 125-132, 2011.
Article in Korean | WPRIM | ID: wpr-84306

ABSTRACT

Stem cell research is a innovative technology that focuses on using undifferentiated cells able to self-renew through the asymmetrical or symmetrical divisions. Three types of stem cells have been studied in laboratory including embryonic stem cell, adult stem cells and induced pluripotent stem cells. Embryonic stem cells are pluripotent stem cells derived from the inner cell mass and it can give rise to any fetal or adult cell type. Adult stem cells are multipotent, have the ability to differentiate into a limited number of specialized cell types, and have been obtained from the bone marrow, umbilical cord blood, placenta and adipose tissue. Stem cell therapy is the most promising therapy for several degenerative and devastating diseases including digestive tract disease such as liver failure, inflammatory bowel disease, Celiac sprue, and pancreatitis. Further understanding of biological properties of stem cells will lead to safe and successful stem cell therapies.


Subject(s)
Humans , Adult Stem Cells/cytology , Embryonic Stem Cells/cytology , Induced Pluripotent Stem Cells/cytology , Stem Cells/cytology
17.
Article in English | IMSEAR | ID: sea-135478

ABSTRACT

Background & objectives: Stem cell therapy has been considered as an ideal option for the treatment of Parkinson’s disease. Murine embryonic stem cells (mESCs)-derived dopaminergic (DA) neurons may substitute the degenerated neurons in the brain. In this study we generated highly enriched cultures of neural progenitors from mESCs and grafted them into the striatum of Parkinsonian rats to evaluate their ability to improve impaired function. Methods: An animal model was developed for Parkinson’s disease in rats, using 6- hydroxy dopamine. The animals were divided into two groups: (i) the control group treated with culture medium only, and (ii) the experimental group, which was treated with a murine ESC cell-line (CCE). Transplanted cells were labelled with bromodeoxyuridine (BrdU), exposed to retinoic acid and then engrafted within the striatum of the rat model. Results: Treated ES cells by retinoic acid were found to relieve apomorphine-induced asymmetric motor behaviour. Immunohistochemistry results revealed tyrosine hydroxlase immunoreactivity in engrafted cells 15 days after transplantation. Further, the ultrastructural examination along with cresyl violet staining confirmed that the cells gained neuronal and glial appearance. Interpretation & conclusions: Our data demonstrate that retinoic acid treatment and transplanting ESC cells to the lessioned brain can lead to the generation of putative dopaminergic neurons and functional recovery in parkinsonian rat model with.


Subject(s)
Animals , Behavior, Animal , Bromodeoxyuridine , Embryonic Stem Cells/cytology , Embryonic Stem Cells/drug effects , Female , Mice , Parkinsonian Disorders/physiopathology , Parkinsonian Disorders/therapy , Rats , Rats, Sprague-Dawley , Stem Cell Transplantation
18.
Experimental & Molecular Medicine ; : 155-165, 2010.
Article in English | WPRIM | ID: wpr-203597

ABSTRACT

In developed countries, in which people have nutrient-rich diets, convenient environments, and access to numerous medications, the disease paradigm has changed. Nowadays, heart failure is one of the major causes of death. In spite of this, the therapeutic efficacies of medications are generally unsatisfactory. Although whole heart transplantation is ideal for younger patients with heart failure, many patients are deemed to be unsuitable for this type of surgery due to complications and/or age. The need for therapeutic alternatives to heart transplantation is great. Regenerative therapy is a strong option. For this purpose, several cell sources have been investigated, including intrinsic adult stem or progenitor cells and extrinsic pluripotent stem cells. Most intrinsic stem cells seem to contribute to a regenerative environment via paracrine factors and/or angiogenesis, whereas extrinsic pluripotent stem cells are unlimited sources of cardiomyocytes. In this review, we summarize the various strategies for using regenerative cardiomyocytes including our recent progressions: non-genetic approaches for the purification of cardiomyocytes and efficient transplantation. We expect that use of intrinsic and extrinsic stem cells in combination will enhance therapeutic effectiveness.


Subject(s)
Animals , Humans , Embryonic Stem Cells/cytology , Myocardium/cytology , Myocytes, Cardiac/cytology , Regeneration , Stem Cell Transplantation , Tissue Engineering
19.
Experimental & Molecular Medicine ; : 175-186, 2010.
Article in English | WPRIM | ID: wpr-203595

ABSTRACT

Recently, reactive oxygen species (ROS) have been studied as a regulator of differentiation into specific cell types in embryonic stem cells (ESCs). However, ROS role in human ESCs (hESCs) is unknown because mouse ESCs have been used mainly for most studies. Herein we suggest that ROS generation may play a critical role in differentiation of hESCs; ROS enhances differentiation of hESCs into bi-potent mesendodermal cell lineage via ROS-involved signaling pathways. In ROS-inducing conditions, expression of pluripotency markers (Oct4, Tra 1-60, Nanog, and Sox2) of hESCs was decreased, while expression of mesodermal and endodermal markers was increased. Moreover, these differentiation events of hESCs in ROS-inducing conditions were decreased by free radical scavenger treatment. hESC-derived embryoid bodies (EBs) also showed similar differentiation patterns by ROS induction. In ROS-related signaling pathway, some of the MAPKs family members in hESCs were also affected by ROS induction. p38 MAPK and AKT (protein kinases B, PKB) were inactivated significantly by buthionine sulfoximine (BSO) treatment. JNK and ERK phosphorylation levels were increased at early time of BSO treatment but not at late time point. Moreover, MAPKs family-specific inhibitors could prevent the mesendodermal differentiation of hESCs by ROS induction. Our results demonstrate that stemness and differentiation of hESCs can be regulated by environmental factors such as ROS.


Subject(s)
Humans , Biomarkers/metabolism , Cell Differentiation/drug effects , Cell Line , Cell Lineage/drug effects , Cells, Cultured , Down-Regulation/drug effects , Embryo, Mammalian/cytology , Embryonic Stem Cells/cytology , Endoderm/cytology , Enzyme Activation/drug effects , Free Radical Scavengers/pharmacology , Mesoderm/cytology , Mitogen-Activated Protein Kinases/metabolism , Pluripotent Stem Cells/cytology , Reactive Oxygen Species/metabolism , Up-Regulation/drug effects
20.
Yonsei Medical Journal ; : 622-632, 2010.
Article in English | WPRIM | ID: wpr-46869

ABSTRACT

MicroRNAs (miRNAs) are small non-coding RNAs that regulate messenger RNAs at the post-transcriptional level. They play an important role in the control of cell physiological functions, and their alterations have been related to cancer, where they can function as oncogenes or tumor suppressor genes. Recently, they have emerged as key regulators of "stemness", collaborating in the maintenance of pluripotency, control of self-renewal, and differen-tiation of stem cells. The miRNA pathway has been shown to be crucial in embryonic development and in embryonic stem (ES) cells, as shown by Dicer knockout analysis. Specific patterns of miRNAs have been reported to be expressed only in ES cells and in early phases of embryonic development. Moreover, many cancers present small populations of cells with stem cell characteristics, called cancer stem cells (CSCs). CSCs are responsible for relapse and treatment failure in many cancer patients, and the comparative analysis of expression patterns between ES cells and tumors can lead to the identification of a miRNA signature to define CSCs. Most of the key miRNAs identified to date in ES cells have been shown to play a role in tumor diagnosis or prognosis, and may well prove to be essential in cancer therapy in the foreseeable future.


Subject(s)
Humans , Embryonic Stem Cells/cytology , MicroRNAs/genetics , Models, Biological , Neoplastic Stem Cells/cytology , Signal Transduction/genetics
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