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1.
Journal of Veterinary Science ; : 101-104, 2017.
Artigo em Inglês | WPRIM | ID: wpr-122304

RESUMO

The expression of stage-specific embryonic antigens (SSEAs) was determined in several types of canine cancer cells. Flow cytometry showed SSEA-1 expression in glioblastoma, melanoma, and mammary cancer cells, although none expressed SSEA-3 or SSEA-4. Expression of SSEA-1 was not detected in lymphoma, osteosarcoma, or hemangiosarcoma cell lines. Relatively stable SSEA-1 expression was observed between 24 and 72 h of culture. After 8 days in culture, sorted SSEA-1⁻ and SSEA-1⁺ cells re-established SSEA-1 expression to levels comparable to those observed in unsorted cells. Our results document, for the first time, the expression of SSEA-1 in several canine cancer cell lines.


Assuntos
Antígenos CD15 , Neoplasias da Mama , Linhagem Celular , Citometria de Fluxo , Glioblastoma , Hemangiossarcoma , Linfoma , Melanoma , Osteossarcoma , Antígenos Embrionários Estágio-Específicos
2.
Journal of Korean Medical Science ; : 171-177, 2016.
Artigo em Inglês | WPRIM | ID: wpr-133743

RESUMO

We investigated the potential of human dental pulp stem cells (hDPSCs) to differentiate into dopaminergic neurons in vitro as an autologous stem cell source for Parkinson's disease treatment. The hDPSCs were expanded in knockout-embryonic stem cell (KO-ES) medium containing leukemia inhibitory factor (LIF) on gelatin-coated plates for 3-4 days. Then, the medium was replaced with KO-ES medium without LIF to allow the formation of the neurosphere for 4 days. The neurosphere was transferred into ITS medium, containing ITS (human insulin-transferrin-sodium) and fibronectin, to select for Nestin-positive cells for 6-8 days. The cells were then cultured in N-2 medium containing basic fibroblast growth factor (FGF), FGF-8b, sonic hedgehog-N, and ascorbic acid on poly-l-ornithine/fibronectin-coated plates to expand the Nestin-positive cells for up to 2 weeks. Finally, the cells were transferred into N-2/ascorbic acid medium to allow for their differentiation into dopaminergic neurons for 10-15 days. The differentiation stages were confirmed by morphological, immunocytochemical, flow cytometric, real-time PCR, and ELISA analyses. The expressions of mesenchymal stem cell markers were observed at the early stages. The expressions of early neuronal markers were maintained throughout the differentiation stages. The mature neural markers showed increased expression from stage 3 onwards. The percentage of cells positive for tyrosine hydroxylase was 14.49%, and the amount was 0.526 ± 0.033 ng/mL at the last stage. hDPSCs can differentiate into dopaminergic neural cells under experimental cell differentiation conditions, showing potential as an autologous cell source for the treatment of Parkinson's disease.


Assuntos
Animais , Humanos , Camundongos , Encéfalo/patologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Meios de Cultura/química , Polpa Dentária/citologia , Neurônios Dopaminérgicos/citologia , Ensaio de Imunoadsorção Enzimática , Proteína Glial Fibrilar Ácida/genética , Camundongos Endogâmicos ICR , Proteína Básica da Mielina/genética , Reação em Cadeia da Polimerase em Tempo Real , Antígenos Embrionários Estágio-Específicos/genética , Células-Tronco/citologia , Tubulina (Proteína)/genética , Tirosina 3-Mono-Oxigenase/análise
3.
Journal of Korean Medical Science ; : 171-177, 2016.
Artigo em Inglês | WPRIM | ID: wpr-133742

RESUMO

We investigated the potential of human dental pulp stem cells (hDPSCs) to differentiate into dopaminergic neurons in vitro as an autologous stem cell source for Parkinson's disease treatment. The hDPSCs were expanded in knockout-embryonic stem cell (KO-ES) medium containing leukemia inhibitory factor (LIF) on gelatin-coated plates for 3-4 days. Then, the medium was replaced with KO-ES medium without LIF to allow the formation of the neurosphere for 4 days. The neurosphere was transferred into ITS medium, containing ITS (human insulin-transferrin-sodium) and fibronectin, to select for Nestin-positive cells for 6-8 days. The cells were then cultured in N-2 medium containing basic fibroblast growth factor (FGF), FGF-8b, sonic hedgehog-N, and ascorbic acid on poly-l-ornithine/fibronectin-coated plates to expand the Nestin-positive cells for up to 2 weeks. Finally, the cells were transferred into N-2/ascorbic acid medium to allow for their differentiation into dopaminergic neurons for 10-15 days. The differentiation stages were confirmed by morphological, immunocytochemical, flow cytometric, real-time PCR, and ELISA analyses. The expressions of mesenchymal stem cell markers were observed at the early stages. The expressions of early neuronal markers were maintained throughout the differentiation stages. The mature neural markers showed increased expression from stage 3 onwards. The percentage of cells positive for tyrosine hydroxylase was 14.49%, and the amount was 0.526 ± 0.033 ng/mL at the last stage. hDPSCs can differentiate into dopaminergic neural cells under experimental cell differentiation conditions, showing potential as an autologous cell source for the treatment of Parkinson's disease.


Assuntos
Animais , Humanos , Camundongos , Encéfalo/patologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Meios de Cultura/química , Polpa Dentária/citologia , Neurônios Dopaminérgicos/citologia , Ensaio de Imunoadsorção Enzimática , Proteína Glial Fibrilar Ácida/genética , Camundongos Endogâmicos ICR , Proteína Básica da Mielina/genética , Reação em Cadeia da Polimerase em Tempo Real , Antígenos Embrionários Estágio-Específicos/genética , Células-Tronco/citologia , Tubulina (Proteína)/genética , Tirosina 3-Mono-Oxigenase/análise
4.
Asian Pacific Journal of Tropical Medicine ; (12): 639-644, 2014.
Artigo em Inglês | WPRIM | ID: wpr-820640

RESUMO

OBJECTIVE@#To compare the expression levels of pluripotent genes among incomplete reprogrammed colonies and induced pluripotent stem cells (iPSCs), to explore the relationship between the expression of pluripotent genes and incomplete reprogramming.@*METHODS@#Four genes (Oct4, Sox2, Klf4, C-Myc) were introduced into human foreskin fibroblasts (HFFs) by retroviruses. The HFFs were induced to reprogramming. Different forms of colonies were picked up, analyzed, and compared with iPSCs from different aspects, including the morphology of clones, alkaline phosphatase (AP) staining, immuno-fluorescence, and Q-PCR.@*RESULTS@#In the reprogramming process, different colonies were emerged, some of them exhibited typical human embryonic stem cell morphology (eg., compact colonies, high nucleus-to-cytoplasm ratios, and prominent nucleoli). However, these colonies couldn't maintain these characters after passage. There was an intermediate state, named partially reprogramming. Through analysis and identification, AP staining results were weakly positive, compared with iPSC colonies. The immuno-fluorescence staining demonstrated these colonies just expressed pluripotent protein Oct4. Q-PCR indicated that the expression of exogenous transcription factors was inappropriate, either at a high level or at a low level. Most of the endogenous pluripotency genes were expressed at a low level.@*CONCLUSIONS@#It may be one of the causes of incomplete reprogramming that the exogenous pluripotent gene is low-expressed or over-expressed, and successful reprogramming may depend on a specific stoichiometric balance of Oct4, Sox2, Klf4 and c-Myc.


Assuntos
Animais , Criança , Humanos , Masculino , Camundongos , Linhagem Celular , Células Cultivadas , Reprogramação Celular , Genética , Fibroblastos , Células-Tronco Pluripotentes Induzidas , Biologia Celular , Fisiologia , Camundongos Endogâmicos ICR , Fator 3 de Transcrição de Octâmero , Genética , Metabolismo , Retroviridae , Genética , Antígenos Embrionários Estágio-Específicos , Genética , Metabolismo , Transfecção , Métodos
5.
International Journal of Stem Cells ; : 26-36, 2013.
Artigo em Inglês | WPRIM | ID: wpr-86614

RESUMO

BACKGROUND AND OBJECTIVES: Use of somatic cells as a feeder layer to maintain the embryonic stem cells (ESCs) in undifferentiated state limits the stem cell research design, since experimental data may result from a combined ESCs and feeder cell response to various stimuli. Therefore, present study was designed to evaluate the developmental competence of the buffalo ESCs over different homogenous feeders and compare with various extracellular matrices using different concentrations of LIF. METHODS AND RESULTS: Inner cell masses (ICMs) of in vitro hatched blastocysts were cultured onto homologous feeders viz. fetal fibroblast, granulosa and oviductal cell feeder layers and synthetic matrices viz. fibronectin, collagen type I and matrigel in culture medium. Developmental efficiency was found higher for ESCs cultured on fetal fibroblast and granulosa layers (83.33%) followed by fibronectin (77.78%) at 30 ng LIF. Oviductal feeder was found to be the least efficient feeder showing only 11.11% undifferentiated primary ESC colonies at 30 ng LIF. However, neither feeder layer nor synthetic matrix could support the development of primary colonies at 10 ng LIF. Expression of SSEA-4, TRA-1-60 and Oct-4 were found positive in ESC colonies from all the feeders and synthetic matrices with 20 ng and 30 ng LIF. CONCLUSIONS: Fetal fibroblast and granulosa cell while, amongst synthetic matrices, fibronectin were found to be equally efficient to support the growth and maintenance of ESCs pluripotency with 30 ng LIF. This well-defined culture conditions may provide an animal model for culturing human embryonic stem cells in the xeno-free or feeder-free conditions for future clinical applications.


Assuntos
Animais , Feminino , Humanos , Blastocisto , Búfalos , Colágeno , Colágeno Tipo I , Combinação de Medicamentos , Células-Tronco Embrionárias , Matriz Extracelular , Células Alimentadoras , Fibroblastos , Fibronectinas , Células da Granulosa , Laminina , Competência Mental , Modelos Animais , Oviductos , Proteoglicanas , Antígenos Embrionários Estágio-Específicos , Pesquisa com Células-Tronco
6.
Journal of Korean Medical Science ; : 1418-1426, 2010.
Artigo em Inglês | WPRIM | ID: wpr-112652

RESUMO

This study was done to evaluate the stemness of human mesenchymal stem cells (hMSCs) derived from placenta according to the development stage and to compare the results to those from adult bone marrow (BM). Based on the source of hMSCs, three groups were defined: group I included term placentas, group II included first-trimester placentas, and group III included adult BM samples. The stemness was evaluated by the proliferation capacity, immunophenotypic expression, mesoderm differentiation, expression of pluripotency markers including telomerase activity. The cumulative population doubling, indicating the proliferation capacity, was significantly higher in group II (P<0.001, 31.7+/-5.8 vs. 15.7+/-6.2 with group I, 9.2+/-4.9 with group III). The pattern of immunophenotypic expression and mesoderm differentiation into adipocytes and osteocytes were similar in all three groups. The expression of pluripotency markers including ALP, SSEA-4, TRA-1-60, TRA-1-81, Oct-4, and telomerase were strongly positive in group II, but very faint positive in the other groups. In conclusions, hMSCs from placentas have different characteristics according to their developmental stage and express mesenchymal stemness potentials similar to those from adult human BMs.


Assuntos
Feminino , Humanos , Gravidez , Antígenos de Superfície/metabolismo , Células da Medula Óssea/citologia , Proliferação de Células , Imunofenotipagem , Células-Tronco Mesenquimais/citologia , Mesoderma/citologia , Fator 3 de Transcrição de Octâmero/metabolismo , Placenta/citologia , Primeiro Trimestre da Gravidez , Proteoglicanas/metabolismo , Antígenos Embrionários Estágio-Específicos/metabolismo , Telomerase/metabolismo
7.
Acta Academiae Medicinae Sinicae ; (6): 468-472, 2009.
Artigo em Chinês | WPRIM | ID: wpr-301670

RESUMO

<p><b>OBJECTIVE</b>To compare the effects of different types of feeder cells on supporting undifferentiation and high proliferation of human embryonic stem cells (hESC).</p><p><b>METHODS</b>hESC were seeded on mouse embryonic fibroblasts (MEF), human marrow stromal cells (hMSC), and human foreskin fibroblasts (hFF), respectively. Colony number, cell quantity after digestion, and survival rate were observed by alkaline phosphatase (AP) staining and Trypan blue, and the biological properties of hESC after 5 passages were observed by immunofluorescence staining.</p><p><b>RESULTS</b>Although all the three feeder layers could support the formation of hESC colonies and maintain pluripotency, the morphology of colonies on different feeder layers remarkably varied. The stage-specific embryonic antigen-3 and AP staining were positive on three types of feeders. The number of colonies, number of cells produced, and cell survival rates were significantly higher on MEF than on human feeder cells (P < 0.01). Furthermore, the number of AP-positive colonies and cell quantity were also significantly higher on hMSC than on hFF (P < 0.01).</p><p><b>CONCLUSIONS</b>All three types of feeder cells are able to support the growth of hMSC, although MEF are more favourable for the proliferation. Two types of human feeder cells lay the foundation for the removal of animal-derived hESC culture system. hMSC is superior to hFF in supporting the proliferation of hESC.</p>


Assuntos
Animais , Humanos , Camundongos , Antígenos Glicosídicos Associados a Tumores , Metabolismo , Técnicas de Cocultura , Métodos , Células-Tronco Embrionárias , Células Alimentadoras , Fibroblastos , Antígenos Embrionários Estágio-Específicos , Metabolismo
8.
Korean Journal of Obstetrics and Gynecology ; : 700-706, 2009.
Artigo em Coreano | WPRIM | ID: wpr-193714

RESUMO

OBJECTIVE: To determine which compartments of placenta in the term pregnancy express the embryonic stem cell markers. METHODS: We have used immunohistochemical methods with antibodies to embryonic stem cell surface antigens, TRA 1-60 (Tumor rejection antigen 1-60), TRA 1-81 (Tumor rejection antigen 1-81), SSEA-3 (stage-specific embryonic antigen-3) and SSEA-4 (stage-specific embryonic antigen-4), to identify and localize stem cells in the term placenta. RESULTS: Stem cell marker-positive cells were found in all layer of placenta. Amnionic epithelial cells was immunoreactive with TRA 1-60, TRA 1-81. Amnionic mesenchymal stromal cells was immunoreactive with TRA 1-81. Chorionic mesenchymal stromal cells was immunoreactive with TRA 1-60 and TRA 1-81. SSEA-3 and SSEA-4 were not stained at any compartment of the term placenta. Compartment that was stained most strongly by TRA 1-60 was the amnionic epithelial cells layer. Compartment that was stained most strongly by TRA 1-81 was the chorionic mesenchymal stromal cells layer. CONCLUSION: The mesenchymal stroma cells of the amnion and chorion as well as amnionic epithelial cells may be useful source of pluripotent stem cells in the term placenta.


Assuntos
Humanos , Gravidez , Âmnio , Anticorpos , Antígenos de Superfície , Antígenos Glicosídicos Associados a Tumores , Córion , Células-Tronco Embrionárias , Células Epiteliais , Células-Tronco Mesenquimais , Placenta , Células-Tronco Pluripotentes , Rejeição em Psicologia , Antígenos Embrionários Estágio-Específicos , Células-Tronco
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