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1.
J Virol ; 89(21): 11159-64, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26292329

RESUMO

Congenital human cytomegalovirus (HCMV) infection is associated with neurodevelopmental disabilities. To dissect the earliest events of infection in the developing human brain, we studied HCMV infection during controlled differentiation of human embryonic stem cells (hESC) into neural precursors. We traced a transition from viral restriction in hESC, mediated by a block in viral binding, toward HCMV susceptibility in early hESC-derived neural precursors. We further revealed the role of platelet-derived growth factor receptor alpha (PDGFRα) as a determinant of the developmentally acquired HCMV susceptibility.


Assuntos
Diferenciação Celular/fisiologia , Infecções por Citomegalovirus/fisiopatologia , Citomegalovirus/fisiologia , Células-Tronco Embrionárias/citologia , Células-Tronco Neurais/virologia , Ligação Viral , Fatores Etários , Infecções por Citomegalovirus/prevenção & controle , Células-Tronco Embrionárias/fisiologia , Humanos , Células-Tronco Neurais/fisiologia
2.
Stem Cell Reports ; 17(11): 2565-2578, 2022 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-36240774

RESUMO

Pelvic organ prolapse (POP) is common among post-menopausal women and is associated with bladder, bowel, and sexual dysfunction. Surgical repair with the patients' native tissues is sub-optimal with high reoperation rates, potentially due to diminished age-related healing. We demonstrate that systemic transplantation of mesenchymal stem cells (MSCs) improves healing of full-thickness vaginal incision in the vaginal wall of old rats, as suggested by both histological and functional analysis. Transplanted MSCs homed and survived at the surgical vaginal site. Attenuation of the injury-induced inflammatory response, increased angiogenesis, and reduced matrix metalloproteinase 9 expression were observed at the surgical site of transplanted rats. Most importantly, the functional biomechanical properties of the healed vagina, at day 30 post-injury, were improved in MSC-transplanted, compared with sham-operated non-transplanted, old rats. These results may pave the way to further translational studies toward clinical transplantation of MSCs adjuvant to POP repair for the improvement of surgical outcome.


Assuntos
Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Prolapso de Órgão Pélvico , Ratos , Feminino , Animais , Vagina/cirurgia , Vagina/metabolismo , Vagina/patologia , Prolapso de Órgão Pélvico/cirurgia , Prolapso de Órgão Pélvico/complicações , Células-Tronco Mesenquimais/patologia
3.
Stem Cell Reports ; 17(12): 2732-2744, 2022 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-36427492

RESUMO

Biological sex is a fundamental trait influencing development, reproduction, pathogenesis, and medical treatment outcomes. Modeling sex differences is challenging because of the masking effect of genetic variability and the hurdle of differentiating chromosomal versus hormonal effects. In this work we developed a cellular model to study sex differences in humans. Somatic cells from a mosaic Klinefelter syndrome patient were reprogrammed to generate isogenic induced pluripotent stem cell (iPSC) lines with different sex chromosome complements: 47,XXY/46,XX/46,XY/45,X0. Transcriptional analysis of the hiPSCs revealed novel and known genes and pathways that are sexually dimorphic in the pluripotent state and during early neural development. Female hiPSCs more closely resembled the naive pluripotent state than their male counterparts. Moreover, the system enabled differentiation between the contributions of X versus Y chromosome to these differences. Taken together, isogenic hiPSCs present a novel platform for studying sex differences in humans and bear potential to promote gender-specific medicine in the future.


Assuntos
Células-Tronco Pluripotentes Induzidas , Humanos , Feminino , Masculino , Caracteres Sexuais , Células Cultivadas , Diferenciação Celular/genética
4.
F S Rep ; 3(1): 47-56, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-35386499

RESUMO

Objective: To develop an efficient, clinical-grade, freezing protocol toward experimental clinical cryopreservation of testicular tissues in prepubertal boys suffering from cancer. Design: Experimental cryopreservation of testicular tissue. Setting: University Medical Center. Patients: Adult patients undergoing orchiectomy for various tumors and prepubertal boys scheduled for gonadotoxic treatment. Interventions: None. Main Outcome Measures: Histopathological analysis of tissue architecture, structural integrity, and cellular morphology was performed for control and frozen-thawed cryopreserved tissues.The number of seminiferous tubules per testicular section was calculated. The survival of spermatogonial stem cells (SSCs) and Sertoli cells of the control and frozen-thawed cryopreserved tissues was analyzed by immunofluorescence staining. Results: Uncontrolled Slow Freezing, Controlled slow freezing, and vitrification similarly preserved the integrity of the adult testicular tissues and the survival of SSCs and Sertoli cells. Controlled slow freezing of prepubertal testicular tissues effectively preserved their architecture, the number of tubules, SSCs, and Sertoli cells. In addition, we observed SSC loss after chemotherapy in prepubertal boys, reemphasizing the importance of fertility preservation before gonadotoxic treatment. Conclusions: Future fertility restoration for male survivors of pediatric cancers depends on the development of an optimal prepubertal testicular tissue cryopreservation method. Our findings demonstrate the effectiveness of controlled slow freezing for cryopreservation of human prepubertal testicular tissues and may contribute to more effective banking of these tissues and potential fertility restoration. Clinical Trial Registration Number: NIH research clinical trials number: NCT02529826.

5.
Stem Cell Reports ; 17(12): 2643-2660, 2022 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-36368331

RESUMO

In the mammalian embryo, a formative pluripotent phase is proposed to exist at the early post-implantation period, during the transition from the pre-implantation naive-to the post-implantation primed-epiblast. By recapitulating a laminin component of the extracellular matrix niche during embryonic formative transition, and defined culture conditions, we generated cultures highly enriched for self-renewing human pluripotent stem cells (hPSCs), exhibiting properties of early post-implantation epiblast cells. These hPSCs display post-implantation-epiblast gene expression profiles. FGF and TGF-ß signaling maintain their self-renewal for multiple passages. They have inactive canonical Wnt signaling, do not express primitive streak markers, and are competent to initiate differentiation toward germline and somatic fates. hPSCs exhibiting early post-implantation epiblast properties may shed light on human embryonic PSCs development and may serve for initiating somatic and germ cell specification.


Assuntos
Camadas Germinativas , Células-Tronco Pluripotentes , Animais , Humanos , Células-Tronco Pluripotentes/metabolismo , Embrião de Mamíferos , Linha Primitiva , Diferenciação Celular , Via de Sinalização Wnt , Mamíferos
6.
PLoS One ; 14(6): e0218081, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31194823

RESUMO

The beneficial effect of mesenchymal stem cells (MSCs) on wound healing is mostly attributed to a trophic effect that promotes angiogenesis. Whether MSCs can contribute to the formation of new blood vessels by direct differentiation is still controversial. Pelvic floor dysfunction (PFD) is a group of disorders that negatively affect the quality of women's lives. Traditional vaginal surgical repair provides disappointing anatomical outcome. Stem cell transplantation may be used to supplement surgery and improve its outcome. Here we aimed to examine the engraftment, survival, differentiation and angiogenic effect of transplanted MSCs in a vaginal injury rat model. MSCs were obtained from the bone marrow of Sprague Drawley (SD) rats, expanded and characterized in vitro. The MSCs expressed CD90 and CD29, did not express CD45, CD34, CD11b and CD31 and could differentiate into osteogenic, chondrogenic and adipogenic lineages. Cells were labeled with either PKH-26 or GFP and transplanted systemically or locally to female SD rats, just after a standardized vaginal incision was made. Engraftment after local transplantation was less efficient at all-time points compared to systemic administration. In the systemically transplanted animal group, MSCs migrated to the injury site and were present in the healed vagina for at least 30 days. Both systemic and local MSCs transplantation promoted host angiogenesis. Systemically transplanted MSCs created new vascular-like structures by direct differentiation into endothelium. These findings pave the way to further studies of the potential role of MSCs transplantation in improving surgical outcome in women with PFD.


Assuntos
Transplante de Células-Tronco Mesenquimais , Vagina/lesões , Animais , Vasos Sanguíneos/crescimento & desenvolvimento , Diferenciação Celular , Modelos Animais de Doenças , Endotélio Vascular/citologia , Feminino , Células-Tronco Mesenquimais/citologia , Distúrbios do Assoalho Pélvico/terapia , Ratos , Ratos Sprague-Dawley
7.
DNA Repair (Amst) ; 6(1): 128-34, 2007 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-17178256

RESUMO

Ataxia-telangiectasia (A-T) is a multi-system genomic instability syndrome that is caused by loss or inactivation of the ATM protein kinase. ATM is largely nuclear in proliferating cells, and activates an extensive network of pathways in response to double strand breaks (DSBs) in the DNA by phosphorylating key proteins in these pathways. The prominent symptom of A-T is neuronal degeneration, making the elucidation of ATM's functions in neurons essential to understanding the disease. It has been suggested that ATM is cytoplasmic in neurons and functions in processes that are not associated with the DNA damage response. Recently we showed that in human neuron-like cells obtained by in vitro differentiation of neuroblastomas, ATM was largely nuclear and mediated the DSB response as in proliferating cells. We have now extended these studies to two additional model systems: neurons derived from human embryonic stem cells, and cortical neurons derived from neural stem cells. The results substantiate the notion that ATM is nuclear in human neurons and mediates the DSB response, the same as it does in proliferating cells. We present here unique and powerful model systems to further study the ATM-mediated network in neurons.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/fisiologia , Neurônios/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ataxia Telangiectasia , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Núcleo Celular/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Humanos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/genética
8.
FASEB J ; 21(13): 3522-33, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17575264

RESUMO

A femtosecond laser beam gene transduction (SG-LBGT) system is described as a novel and efficient method of intradermal (i.d.) nonviral gene delivery in mice by permeabilizing cells utilizing femtosecond laser pulses. Using this approach, significant gene expression and efficient dermal transduction lasting for >7 months were obtained. The ability of this new DNA gene transfer method to enhance genetic vaccination was tested in BALB/C mice. A single i.d. injection of a plasmid (10 microg) containing the hepatitis B virus (HBV) surface antigen (HBsAg), followed by pulses of laser, induced high titers of HBsAg-specific antibodies lasting for >210 days and increased levels of IgG1, IgG2a, IFNgamma, and IL-4, indicating the activation of both Th1 and Th2 cells. Moreover, mice vaccinated using the SG-LBGT followed by challenge with pHBV showed increased protection against viral challenge, as detected by decreased levels of HBV DNA, suggesting an efficient Th1 effect against HBV-infected replicating cells. Tumor growth retardation was induced in vaccinated mice challenged with an HBsAg-expressing syngeneic tumor. In most of the parameters tested, administration of plasmid followed by laser application was significantly more effective and prolonged than that of plasmid alone. Tissue damage was not detected and integration of the plasmid into the host genomic DNA probably did not occur. We suggest that the LBGT method is an efficient and safe technology for in vivo gene expression and vaccination and emphasizes its potential therapeutic applications for i.d. nonviral gene delivery.


Assuntos
DNA/administração & dosagem , Expressão Gênica , Vacinas de DNA/administração & dosagem , Animais , Células Cultivadas , Antígenos de Superfície da Hepatite B/genética , Antígenos de Superfície da Hepatite B/imunologia , Vírus da Hepatite B/imunologia , Imunoglobulina G/metabolismo , Interferon gama/metabolismo , Interleucina-4/metabolismo , Lasers , Camundongos , Camundongos Endogâmicos BALB C , Células Th1/imunologia , Células Th1/metabolismo , Células Th2/imunologia , Células Th2/metabolismo
9.
Cloning Stem Cells ; 9(3): 339-45, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17907944

RESUMO

Genetic modifications of human embryonic stem cells (hESCs) that will efficiently promote stable homogenous gene silencing, and will also allow monitoring of the silencing level, may be invaluable for the study of function of genes in early human embryogenesis, differentiation, and maintenance of pluripotency of hESCs. RNA-mediated interference (RNAi) emerges as a highly efficient tool for specific knockdown of gene expression. Lentiviruses are efficient vectors for the delivery and stable expression of transgenes in hESCs. We sought to develop a lentiviral-RNAi-based system that will efficiently induce homogenous gene silencing and will allow the monitoring of its relative level in hESCs. Dual-promoter lentiviral vectors coexpressing an RNAi cassette and a reporter gene were initially used for efficient and stable induction of heterogeneous levels of gene silencing in polyclonal hESCs. This step was further combined with the isolation of transduced clones with different homogenous levels of gene silencing. The level of silencing in each of the clones correlated and could be monitored by the level of expression of the vector's reporter transgene. Thus, our system allows easy identification of clones with relatively different homogenous levels of gene silencing. Our approach would be valuable for the study of function of genes, in particular those whose role in hESCs biology depends on their level of expression.


Assuntos
Células-Tronco Embrionárias/fisiologia , Inativação Gênica , Lentivirus/genética , Interferência de RNA , Linhagem Celular , Clonagem Molecular , Genes Reporter , Vetores Genéticos , Proteínas de Fluorescência Verde/metabolismo , Humanos
10.
Int J Dev Biol ; 61(3-4-5): 285-292, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28621425

RESUMO

Fragile X syndrome is the most frequent cause of inherited intellectual disability. The primary molecular defect in this disease is the expansion of a CGG repeat in the 5' region of the fragile X mental retardation1 (FMR1) gene, leading to de novo methylation of the promoter and inactivation of this otherwise normal gene, but little is known about how these epigenetic changes occur during development. In order to gain insight into the nature of this process, we have used cell fusion technology to recapitulate the events that occur during early embryogenesis. These experiments suggest that the naturally occurring Fragile XFMR1 5' region undergoes inactivation post implantation in a Dicer/Ago-dependent targeted process which involves local SUV39H-mediated tri-methylation of histone H3K9. It thus appears that Fragile X syndrome may come about through inadvertent siRNA-mediated heterochromatinization.


Assuntos
Metilação de DNA , Epigênese Genética , Proteína do X Frágil da Deficiência Intelectual/genética , Síndrome do Cromossomo X Frágil/genética , Regulação da Expressão Gênica no Desenvolvimento , Regiões 5' não Traduzidas , Animais , Diferenciação Celular , Desenvolvimento Embrionário , Células-Tronco Embrionárias/metabolismo , Fibroblastos/metabolismo , Heterocromatina/química , Histonas/metabolismo , Humanos , Camundongos , Proteínas do Tecido Nervoso/genética , Fenótipo , Regiões Promotoras Genéticas , RNA/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo
11.
Methods Enzymol ; 420: 64-81, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-17161694

RESUMO

Human embryonic stem cells (hESCs) are pluripotent cells derived from the inner cell mass of preimplantation embryos. These cells can be cultured for long periods as undifferentiated cells and still retain their potential to give rise to cell types representing all three germinal layers. Given their unique properties, hESCs are expected to serve as an invaluable tool for basic and applied research. However, to exploit their remarkable potentials, the development of effective strategies for genetic modification of hESCs is required. Lentiviral-based vectors offer an attractive system for efficient gene delivery into hESCs. These vectors are derived from lentiviruses, a group of complex retroviruses that cause slow chronic immunodeficiency diseases in humans and animals. Gene delivery into hESCs by vectors derived from lentiviruses has the following advantages: (1) lentiviral vectors efficiently transduce hESCs; (2) they integrate into the host-cell genome, thus promoting stable transgene expression; (3) transgene expression is not significantly silenced in hESCs; and (4) transduced hESCs retain their self-renewal and pluripotent potential. In recent years, we and others have developed protocols for efficient transduction of hESCs by advanced modified replication-defective lentiviral-based vectors. Transduction of hESCs by these vectors resulted in high and stable transgene expression that was maintained over long periods of undifferentiated cultivation and after differentiation. This chapter focuses on methods for the use of lentiviral-based vectors for gene delivery into hESCs.


Assuntos
Células-Tronco Embrionárias/citologia , Técnicas de Transferência de Genes , Vetores Genéticos , Lentivirus/genética , Humanos , Transdução Genética/métodos
12.
Oncotarget ; 6(33): 34691-703, 2015 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-26415227

RESUMO

The function of imprinted H19 long non-coding RNA is still controversial. It is highly expressed in early embryogenesis and decreases after birth and re-expressed in cancer. To study the role of H19 in oncogenesis and pluripotency, we down-regulated H19 expression in vitro and in vivo in pluripotent human embryonic carcinoma (hEC) and embryonic stem (hES) cells. H19 knockdown resulted in a decrease in the expression of the pluripotency markers Oct4, Nanog, TRA-1-60 and TRA-1-81, and in the up-regulation of SSEA1; it further attenuated cell proliferation, decreased cell-matrix attachment, and up-regulated E-Cadherin expression. SCID-Beige mice transplanted with H19 down-regulated hEC cells exhibited slower kinetics of tumor formation, resulting in an increased animal survival. Tumors derived from H19 down-regulated cells showed a decrease in the expression of pluripotency markers and up-regulation of SSEA-1 and E-cadherin. Our results suggest that H19 oncogenicity in hEC cells is mediated through the regulation of the pluripotency state.


Assuntos
Transformação Celular Neoplásica/genética , Células-Tronco Embrionárias , Células-Tronco Pluripotentes , RNA Longo não Codificante/genética , Animais , Western Blotting , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Xenoenxertos , Humanos , Imuno-Histoquímica , Camundongos , Camundongos SCID , Microscopia de Fluorescência , Reação em Cadeia da Polimerase , RNA Interferente Pequeno , Transfecção
13.
PLoS One ; 7(9): e45532, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23049812

RESUMO

Teratoma tumor formation is an essential criterion in determining the pluripotency of human pluripotent stem cells. However, currently there is no consistent protocol for assessment of teratoma forming ability. Here we present detailed characterization of a teratoma assay that is based on subcutaneous co-transplantation of defined numbers of undifferentiated human embryonic stem cells (hESCs) with mitotically inactivated feeder cells and Matrigel into immunodeficient mice. The assay was highly reproducible and 100% efficient when 100,000 hESCs were transplanted. It was sensitive, promoting teratoma formation after transplantation of 100 hESCs, though larger numbers of animals and longer follow-up were required. The assay could detect residual teratoma forming cells within differentiated hESC populations however its sensitivity was decreased in the presence of differentiated cells. Our data lay the foundation, for standardization of a teratoma assay for pluripotency analysis. The assay can also be used for bio-safety analysis of pluripotent stem cell-derived differentiated progeny.


Assuntos
Bioensaio/normas , Células-Tronco Embrionárias/patologia , Células-Tronco Pluripotentes/patologia , Teratoma/patologia , Animais , Biomarcadores/metabolismo , Contagem de Células , Diferenciação Celular , Colágeno/administração & dosagem , Combinação de Medicamentos , Células-Tronco Embrionárias/transplante , Células Alimentadoras/citologia , Células Alimentadoras/transplante , Fibroblastos/citologia , Fibroblastos/transplante , Humanos , Injeções Subcutâneas , Cariotipagem , Laminina/administração & dosagem , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Pluripotentes/transplante , Proteoglicanas/administração & dosagem , Sensibilidade e Especificidade , Taxa de Sobrevida , Teratoma/mortalidade
14.
Nat Biotechnol ; 29(12): 1132-44, 2011 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-22119741

RESUMO

The International Stem Cell Initiative analyzed 125 human embryonic stem (ES) cell lines and 11 induced pluripotent stem (iPS) cell lines, from 38 laboratories worldwide, for genetic changes occurring during culture. Most lines were analyzed at an early and late passage. Single-nucleotide polymorphism (SNP) analysis revealed that they included representatives of most major ethnic groups. Most lines remained karyotypically normal, but there was a progressive tendency to acquire changes on prolonged culture, commonly affecting chromosomes 1, 12, 17 and 20. DNA methylation patterns changed haphazardly with no link to time in culture. Structural variants, determined from the SNP arrays, also appeared sporadically. No common variants related to culture were observed on chromosomes 1, 12 and 17, but a minimal amplicon in chromosome 20q11.21, including three genes expressed in human ES cells, ID1, BCL2L1 and HM13, occurred in >20% of the lines. Of these genes, BCL2L1 is a strong candidate for driving culture adaptation of ES cells.


Assuntos
Células-Tronco Embrionárias/citologia , Crescimento/genética , Células-Tronco Pluripotentes Induzidas/citologia , Proteínas de Ligação a RNA/metabolismo , Proteína bcl-X/metabolismo , Diferenciação Celular/genética , Linhagem Celular , Cromossomos Humanos Par 20/genética , Evolução Clonal/genética , Metilação de DNA , Etnicidade/genética , Regulação da Expressão Gênica no Desenvolvimento , Variação Genética , Genótipo , Humanos , Proteína 1 Inibidora de Diferenciação/genética , Proteína 1 Inibidora de Diferenciação/metabolismo , Polimorfismo de Nucleotídeo Único , Proteínas de Ligação a RNA/genética , Seleção Genética/genética , Proteína bcl-X/genética
15.
Cell Stem Cell ; 5(4): 396-408, 2009 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-19796620

RESUMO

Dysfunction and loss of retinal pigment epithelium (RPE) leads to degeneration of photoreceptors in age-related macular degeneration and subtypes of retinitis pigmentosa. Human embryonic stem cells (hESCs) may serve as an unlimited source of RPE cells for transplantation in these blinding conditions. Here we show the directed differentiation of hESCs toward an RPE fate under defined culture conditions. We demonstrate that nicotinamide promotes the differentiation of hESCs to neural and subsequently to RPE fate. In the presence of nicotinamide, factors from the TGF-beta superfamily, which presumably pattern RPE development during embryogenesis, further direct RPE differentiation. The hESC-derived pigmented cells exhibit the morphology, marker expression, and function of authentic RPE and rescue retinal structure and function after transplantation to an animal model of retinal degeneration caused by RPE dysfunction. These results are an important step toward the future use of hESCs to replenish RPE in blinding diseases.


Assuntos
Células-Tronco Embrionárias/citologia , Células Epiteliais/citologia , Epitélio Pigmentado da Retina/citologia , Receptores de Ativinas Tipo I/farmacologia , Receptores de Activinas Tipo II/farmacologia , Ativinas/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Transplante de Células , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/ultraestrutura , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/ultraestrutura , Fator 2 de Crescimento de Fibroblastos/farmacologia , Citometria de Fluxo , Humanos , Imunofenotipagem , Microscopia Eletrônica de Transmissão , Microscopia de Contraste de Fase , Reação em Cadeia da Polimerase , Ratos , Fator de Crescimento Transformador beta/farmacologia
16.
Mol Ther ; 7(2): 281-7, 2003 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-12597917

RESUMO

Human embryonic stem (hES) cells are pluripotent cells derived from the inner cell mass of the early preimplantation embryo. An efficient strategy for stable genetic modification of hES cells may be highly valuable for manipulating the cells in vitro and may promote the study of hES cell biology, human embryogenesis, and the development of cell-based therapies. Here, we demonstrate that vectors derived from self-inactivating (SIN) human immunodeficiency virus type 1 (HIV-1) are efficient tools for stable genetic modification of hES cells. Transduction of hES cells by a modified vector derived from SIN HIV-1 and containing the woodchuck hepatitis regulatory element (WPRE) and the central polypurine tract (cPPT) sequence facilitated stable transgene expression during prolonged (38 weeks) undifferentiated proliferation in vitro. Southern blot analysis revealed that the viral vector had integrated into the host cells' DNA. Transgene expression was maintained throughout differentiation into progeny of all three germ layers both in vitro and in vivo in teratomas. Thus, the transduced hES cells retained the capability for self-renewal and their pluripotent potential. Genetic modification of hES cells by lentiviral vectors provides a powerful tool for basic and applied research in the area of human ES cells.


Assuntos
Vetores Genéticos , HIV-1/genética , Lentivirus/genética , Células-Tronco/metabolismo , Animais , Southern Blotting , Diferenciação Celular , Linhagem Celular , Separação Celular , Embrião de Mamíferos/citologia , Citometria de Fluxo , Proteínas de Fluorescência Verde , Humanos , Imuno-Histoquímica , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos SCID , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Teratoma/metabolismo , Fatores de Tempo , Transfecção , Transgenes
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