Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 18 de 18
Filtrar
1.
BMC Biol ; 20(1): 14, 2022 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-35027054

RESUMO

BACKGROUND: Infectious diseases of farmed and wild animals pose a recurrent threat to food security and human health. The macrophage, a key component of the innate immune system, is the first line of defence against many infectious agents and plays a major role in shaping the adaptive immune response. However, this phagocyte is a target and host for many pathogens. Understanding the molecular basis of interactions between macrophages and pathogens is therefore crucial for the development of effective strategies to combat important infectious diseases. RESULTS: We explored how porcine pluripotent stem cells (PSCs) can provide a limitless in vitro supply of genetically and experimentally tractable macrophages. Porcine PSC-derived macrophages (PSCdMs) exhibited molecular and functional characteristics of ex vivo primary macrophages and were productively infected by pig pathogens, including porcine reproductive and respiratory syndrome virus (PRRSV) and African swine fever virus (ASFV), two of the most economically important and devastating viruses in pig farming. Moreover, porcine PSCdMs were readily amenable to genetic modification by CRISPR/Cas9 gene editing applied either in parental stem cells or directly in the macrophages by lentiviral vector transduction. CONCLUSIONS: We show that porcine PSCdMs exhibit key macrophage characteristics, including infection by a range of commercially relevant pig pathogens. In addition, genetic engineering of PSCs and PSCdMs affords new opportunities for functional analysis of macrophage biology in an important livestock species. PSCs and differentiated derivatives should therefore represent a useful and ethical experimental platform to investigate the genetic and molecular basis of host-pathogen interactions in pigs, and also have wider applications in livestock.


Assuntos
Vírus da Febre Suína Africana , Doenças Transmissíveis , Vírus da Febre Suína Africana/genética , Animais , Interações Hospedeiro-Patógeno/genética , Macrófagos , Células-Tronco , Suínos
2.
Am J Transplant ; 20(4): 988-998, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31733031

RESUMO

Porcine xenografts lacking swine leukocyte antigen (SLA) class I are thought to be protected from human T cell responses. We have previously shown that SLA class I deficiency can be achieved in pigs by CRISPR/Cas9-mediated deletion of ß2 -microglobulin (B2M). Here, we characterized another line of genetically modified pigs in which targeting of the B2M locus did not result in complete absence of B2M and SLA class I but rather in significantly reduced expression levels of both molecules. Residual SLA class I was functionally inert, because no proper differentiation of the CD8+ T cell subset was observed in B2Mlow pigs. Cells from B2Mlow pigs were less capable in triggering proliferation of human peripheral blood mononuclear cells in vitro, which was mainly due to the nonresponsiveness of CD8+ T cells. Nevertheless, cytotoxic effector cells developing from unaffected cell populations (eg, CD4+ T cells, natural killer cells) lysed targets from both SLA class I+ wildtype and SLA class Ilow pigs with similar efficiency. These data indicate that the absence of SLA class I is an effective approach to prevent the activation of human CD8+ T cells during the induction phase of an anti-xenograft response. However, cytotoxic activity of cells during the effector phase cannot be controlled by this approach.


Assuntos
Linfócitos T CD8-Positivos , Leucócitos Mononucleares , Animais , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe II , Humanos , Imunidade , Fenótipo , Suínos
3.
Xenotransplantation ; 26(6): e12525, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31119817

RESUMO

BACKGROUND: Despite major improvements in pig-to-primate xenotransplantation, long-term survival of xenografts is still challenging. The major histocompatibility complex (MHC) class I, which is crucial in cellular immune response, is an important xenoantigen. Abrogating MHC class I expression on xenografts might be beneficial for extending graft survival beyond current limits. METHODS: In this study, we employed the CRISPR/Cas9 system to target exon 2 of the porcine beta-2-microglobulin (B2M) gene to abrogate SLA class I expression on porcine cells. B2M-KO cells served as donor cells for somatic cell nuclear transfer, and cloned embryos were transferred to three recipient sows. The offspring were genotyped for mutations at the B2M locus, and blood samples were analyzed via flow cytometry for the absence of SLA class I molecules. RESULTS: Pregnancies were successfully established and led to the birth of seven viable piglets. Genomic sequencing proved that all piglets carried biallelic modifications at the B2M locus leading to a frameshift, a premature stop codon, and ultimately a functional knockout. However, survival times of these animals did not exceed 4 weeks due to unexpected disease processes. CONCLUSION: Here, we demonstrate the feasibility of generating SLA class I knockout pigs by targeting the porcine beta-2-microglobulin gene using the CRISPR/Cas9 system. Additionally, our findings indicate for the first time that this genetic modification might have a negative impact on the viability of the animals. These issues need to be solved to unveil the real value for xenotransplantation in the future.


Assuntos
Galactosiltransferases/genética , Antígenos de Histocompatibilidade Classe I/genética , Transplante Heterólogo , Microglobulina beta-2/genética , Animais , Animais Geneticamente Modificados , Sistemas CRISPR-Cas , Feminino , Técnicas de Inativação de Genes/métodos , Técnicas de Transferência Nuclear , Gravidez , Suínos , Transplante Heterólogo/métodos
4.
Nat Protoc ; 19(6): 1710-1749, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38509352

RESUMO

Pigs share anatomical and physiological traits with humans and can serve as a large-animal model for translational medicine. Bona fide porcine pluripotent stem cells (PSCs) could facilitate testing cell and drug therapies. Agriculture and biotechnology may benefit from the ability to produce immune cells for studying animal infectious diseases and to readily edit the porcine genome in stem cells. Isolating porcine PSCs from preimplantation embryos has been intensively attempted over the past decades. We previously reported the derivation of expanded potential stem cells (EPSCs) from preimplantation embryos and by reprogramming somatic cells of multiple mammalian species, including pigs. Porcine EPSCs (pEPSCs) self-renew indefinitely, differentiate into embryonic and extra-embryonic lineages, and permit precision genome editing. Here we present a highly reproducible experimental procedure and data of an optimized and robust porcine EPSC culture system and its use in deriving new pEPSC lines from preimplantation embryos and reprogrammed somatic cells. No particular expertise is required for the protocols, which take ~4-6 weeks to complete. Importantly, we successfully established pEPSC lines from both in vitro fertilized and somatic cell nuclear transfer-derived embryos. These new pEPSC lines proliferated robustly over long-term passaging and were amenable to both simple indels and precision genome editing, with up to 100% targeting efficiency. The pEPSCs differentiated into embryonic cell lineages in vitro and teratomas in vivo, and into porcine trophoblast stem cells in human trophoblast stem cell medium. We show here that pEPSCs have unique epigenetic features, particularly H3K27me3 levels substantially lower than fibroblasts.


Assuntos
Blastocisto , Reprogramação Celular , Animais , Blastocisto/citologia , Suínos , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Células-Tronco Pluripotentes/citologia , Feminino
5.
Front Cell Dev Biol ; 11: 1111684, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37261075

RESUMO

Domestic pigs (Sus scrofa) share many genetic, anatomical, and physiological traits with humans and therefore constitute an excellent preclinical animal model. Fundamental understanding of the cellular and molecular processes governing early porcine cardiogenesis is critical for developing advanced porcine models used for the study of heart diseases and new regenerative therapies. Here, we provide a detailed characterization of porcine cardiogenesis based on fetal porcine hearts at various developmental stages and cardiac cells derived from porcine expanded pluripotent stem cells (pEPSCs), i.e., stem cells having the potential to give rise to both embryonic and extraembryonic tissue. We notably demonstrate for the first time that pEPSCs can differentiate into cardiovascular progenitor cells (CPCs), functional cardiomyocytes (CMs), epicardial cells and epicardial-derived cells (EPDCs) in vitro. Furthermore, we present an enhanced system for whole-embryo culture which allows continuous ex utero development of porcine post-implantation embryos from the cardiac crescent stage (ED14) up to the cardiac looping (ED17) stage. These new techniques provide a versatile platform for studying porcine cardiac development and disease modeling.

6.
Nat Biotechnol ; 41(12): 1787-1800, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37012447

RESUMO

The epicardium, the mesothelial envelope of the vertebrate heart, is the source of multiple cardiac cell lineages during embryonic development and provides signals that are essential to myocardial growth and repair. Here we generate self-organizing human pluripotent stem cell-derived epicardioids that display retinoic acid-dependent morphological, molecular and functional patterning of the epicardium and myocardium typical of the left ventricular wall. By combining lineage tracing, single-cell transcriptomics and chromatin accessibility profiling, we describe the specification and differentiation process of different cell lineages in epicardioids and draw comparisons to human fetal development at the transcriptional and morphological levels. We then use epicardioids to investigate the functional cross-talk between cardiac cell types, gaining new insights into the role of IGF2/IGF1R and NRP2 signaling in human cardiogenesis. Finally, we show that epicardioids mimic the multicellular pathogenesis of congenital or stress-induced hypertrophy and fibrotic remodeling. As such, epicardioids offer a unique testing ground of epicardial activity in heart development, disease and regeneration.


Assuntos
Coração , Pericárdio , Humanos , Pericárdio/metabolismo , Miocárdio , Diferenciação Celular/genética , Linhagem da Célula/genética , Biologia
7.
Nat Commun ; 14(1): 1722, 2023 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-37012244

RESUMO

Cardiogenesis relies on the precise spatiotemporal coordination of multiple progenitor populations. Understanding the specification and differentiation of these distinct progenitor pools during human embryonic development is crucial for advancing our knowledge of congenital cardiac malformations and designing new regenerative therapies. By combining genetic labelling, single-cell transcriptomics, and ex vivo human-mouse embryonic chimeras we uncovered that modulation of retinoic acid signaling instructs human pluripotent stem cells to form heart field-specific progenitors with distinct fate potentials. In addition to the classical first and second heart fields, we observed the appearance of juxta-cardiac field progenitors giving rise to both myocardial and epicardial cells. Applying these findings to stem-cell based disease modelling we identified specific transcriptional dysregulation in first and second heart field progenitors derived from stem cells of patients with hypoplastic left heart syndrome. This highlights the suitability of our in vitro differentiation platform for studying human cardiac development and disease.


Assuntos
Células-Tronco Pluripotentes , Tretinoína , Humanos , Animais , Camundongos , Tretinoína/farmacologia , Coração , Miocárdio , Diferenciação Celular , Miócitos Cardíacos
8.
Reprod Fertil Dev ; 25(1): 103-28, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23244833

RESUMO

Pluripotent cells, such as embryonic stem (ES) cells, embryonic germ cells and embryonic carcinoma cells are a unique type of cell because they remain undifferentiated indefinitely in in vitro culture, show self-renewal and possess the ability to differentiate into derivatives of the three germ layers. These capabilities make them a unique in vitro model for studying development, differentiation and for targeted modification of the genome. True pluripotent ESCs have only been described in the laboratory mouse and rat. However, rodent physiology and anatomy differ substantially from that of humans, detracting from the value of the rodent model for studies of human diseases and the development of cellular therapies in regenerative medicine. Recently, progress in the isolation of pluripotent cells in farm animals has been made and new technologies for reprogramming of somatic cells into a pluripotent state have been developed. Prior to clinical application of therapeutic cells differentiated from pluripotent stem cells in human patients, their survival and the absence of tumourigenic potential must be assessed in suitable preclinical large animal models. The establishment of pluripotent cell lines in farm animals may provide new opportunities for the production of transgenic animals, would facilitate development and validation of large animal models for evaluating ESC-based therapies and would thus contribute to the improvement of human and animal health. This review summarises the recent progress in the derivation of pluripotent and reprogrammed cells from farm animals. We refer to our recent review on this area, to which this article is complementary.


Assuntos
Animais Domésticos/fisiologia , Reprogramação Celular , Células-Tronco Pluripotentes Induzidas/citologia , Animais , Animais Domésticos/embriologia , Animais Domésticos/genética , Animais Geneticamente Modificados , Fusão Celular/veterinária , Embrião de Mamíferos/citologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genitália/citologia , Genitália/embriologia , Humanos , Masculino , Técnicas de Transferência Nuclear/veterinária , Especificidade da Espécie
9.
Microsc Microanal ; 17(4): 474-97, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21682936

RESUMO

Pluripotent cells are unique because of their ability to differentiate into the cell lineages forming the entire organism. True pluripotent stem cells with germ line contribution have been reported for mice and rats. Human pluripotent cells share numerous features of pluripotentiality, but confirmation of their in vivo capacity for germ line contribution is impossible due to ethical and legal restrictions. Progress toward derivation of embryonic stem cells from domestic species has been made, but the derived cells were not able to produce germ line chimeras and thus are termed embryonic stem-like cells. However, domestic animals, in particular the domestic pig (Sus scrofa), are excellent large animals models, in which the clinical potential of stem cell therapies can be studied. Reprogramming technologies for somatic cells, including somatic cell nuclear transfer, cell fusion, in vitro culture in the presence of cell extracts, in vitro conversion of adult unipotent spermatogonial stem cells into germ line derived pluripotent stem cells, and transduction with reprogramming factors have been developed with the goal of obtaining pluripotent, germ line competent stem cells from domestic animals. This review summarizes the present state of the art in the derivation and maintenance of pluripotent stem cells in domestic animals.


Assuntos
Animais Domésticos , Células-Tronco Pluripotentes/fisiologia , Transplante de Células-Tronco/métodos , Animais , Pesquisa Biomédica/métodos , Pesquisa Biomédica/tendências , Camundongos , Ratos
10.
Virus Res ; 294: 198295, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33422555

RESUMO

Expanded potential stem cells (EPSCs) have been recently derived from porcine preimplantation embryos (Gao et al., 2019). These cells were shown to express key pluripotency genes, to be genetically stable and differentiate to derivatives of the three germ layers and additionally to trophoblast. Their molecular features and expanded potency to contribute to all embryonic and extra-embryonic cell lineages are generally not seen in the embryo-derived or induced pluripotent stem cells (iPSCs). Therefore porcine EPSCs represent a unique state of cellular potency. In the past it had been shown that human and murine embryonic stem cells (ESCs) show an increased expression of murine and human endogenous retroviruses, respectively, and retroviral expression patterns were used as markers of ESC pluripotency. An increased expression of porcine endogenous retroviruses (PERVs) was also detected in porcine iPSCs. Here we investigated 24 passages of five different clones of porcine EPSCs derived from German landrace pigs and show that they harbour PERV-A, PERV-B and PERV-C, but their expression was very low and did not change during cultivation. No recombinant PERV-A/Cs were found in these cells. The low expression despite the presence of spliced mRNA, and negative infection assay and electron microscopy results indicate that no PERV particles were released. Therefore, the absence of PERV expression seems to be a unique feature of porcine EPSCs. Most importantly, the copy number of PERV proviruses was much lower in EPSCs than in young and older pigs (29.1 copies compared with 35.8), indicating an increase in copy number during life time.


Assuntos
Retrovirus Endógenos , Doenças dos Suínos , Animais , Retrovirus Endógenos/genética , Camundongos , Provírus/genética , RNA Mensageiro , Células-Tronco , Suínos
11.
Cell Reprogram ; 22(3): 118-133, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32429746

RESUMO

Chimeric pigs harboring organs derived from human stem cells are promising for patient-specific regenerative therapies. Induced pluripotent stem cells (iPSCs) can contribute to all cell types of the fetus, including germline after injection into embryos. However, ethical concerns prohibit testing human iPSCs in chimera assays. Here, we evaluated porcine embryos as hosts for an interspecies chimera assay using iPSCs from either cynomolgus monkeys (cyiPSCs) or mouse (miPSCs). To establish an in vitro culture system compatible for cyiPSCs and porcine embryos, we determined blastocyst development in eight different stem cell media. The highest developmental rates of blastocysts were achieved in Knockout Dulbecco's modified Eagle's medium with 20% knockout serum replacement. We found that cyiPSCs injected into porcine embryos survived in vitro and were mostly located in the trophectoderm (TE). Instead, when miPSCs were injected into porcine embryos, the cells rapidly proliferated. The behavior of chimeras developed in vitro was recapitulated in vivo; cyiPSCs were observed in the TE, but not in the porcine epiblast. However, when miPSCs were injected into in vivo derived porcine embryos, mouse cells were found in both, the epiblast and TE. These results demonstrate that porcine embryos could be useful for evaluating the interspecies chimera-forming ability of iPSCs from different species.


Assuntos
Quimera/embriologia , Técnicas de Cultura Embrionária/veterinária , Desenvolvimento Embrionário/fisiologia , Células-Tronco Pluripotentes Induzidas/citologia , Animais , Blastocisto , Meios de Cultura , Embrião de Mamíferos , Macaca fascicularis , Camundongos , Especificidade da Espécie , Suínos
12.
Nat Cell Biol ; 21(6): 687-699, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31160711

RESUMO

We recently derived mouse expanded potential stem cells (EPSCs) from individual blastomeres by inhibiting the critical molecular pathways that predispose their differentiation. EPSCs had enriched molecular signatures of blastomeres and possessed developmental potency for all embryonic and extra-embryonic cell lineages. Here, we report the derivation of porcine EPSCs, which express key pluripotency genes, are genetically stable, permit genome editing, differentiate to derivatives of the three germ layers in chimeras and produce primordial germ cell-like cells in vitro. Under similar conditions, human embryonic stem cells and induced pluripotent stem cells can be converted, or somatic cells directly reprogrammed, to EPSCs that display the molecular and functional attributes reminiscent of porcine EPSCs. Importantly, trophoblast stem-cell-like cells can be generated from both human and porcine EPSCs. Our pathway-inhibition paradigm thus opens an avenue for generating mammalian pluripotent stem cells, and EPSCs present a unique cellular platform for translational research in biotechnology and regenerative medicine.


Assuntos
Diferenciação Celular/genética , Reprogramação Celular/genética , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes/citologia , Animais , Blastômeros/citologia , Blastômeros/metabolismo , Linhagem da Célula/genética , Células-Tronco Embrionárias/citologia , Camadas Germinativas/crescimento & desenvolvimento , Camadas Germinativas/metabolismo , Humanos , Camundongos , Medicina Regenerativa , Transdução de Sinais/genética , Suínos , Trofoblastos/citologia , Trofoblastos/metabolismo
13.
Mol Reprod Dev ; 75(5): 731-43, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18058811

RESUMO

Histone modification genes in bovine embryos: The mRNA expression pattern of histone-related genes was determined in bovine oocytes and embryos. We compared immature and in vitro-matured oocytes, either before or after enucleation and activation, in vitro produced embryos (zygotes, 8-16 cell stages, blastocysts), embryos cloned with female or male donor cells; parthenogenetic embryos, and in vivo-derived blastocysts to detect deviations from the normal expression pattern. A sensitive semi-quantitative endpoint RT-PCR assay was used to reveal differences in histone deacetylation [histone deacetylase 2 (HDAC2)]; histone acetylation [histone acetyltransferase 1 (HAT1)]; histone methylation [histone methyltransferases (SUV39H1, G9A)]; heterochromatin formation [heterochromatin protein 1 (HP1)]; and chromatin-mediated transcription regulation [zygote arrest 1 (ZAR1)]. With the exception of ZAR1, these mRNAs were present throughout preimplantation development. The relative abundance of mRNAs for histone methyltransferases (SUV39H1 and G9A) and for heterochromatin-associated protein (HP1) differed significantly before and after activation of the bovine embryonic genome. The similarity of HAT1 gene expression in 8-16 cell embryos and blastocysts suggests that histone acetylation is primarily affected by in vitro culture only prior to embryonic genome activation. HDAC2 gene mRNA expression was not affected by in vitro culture and/or cloning before and after activation of the embryonic genome. The donor cell line affected mRNA expression patterns of genes involved in reprogramming cloned embryos suggesting epigenetic dysregulation. Results show that both in vitro production and somatic cloning alter the mRNA expression of histone modifying genes in bovine embryos.


Assuntos
Blastocisto/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Histonas/metabolismo , Proteínas Nucleares/biossíntese , Processamento de Proteína Pós-Traducional/fisiologia , RNA Mensageiro/biossíntese , Acetilação , Animais , Blastocisto/citologia , Bovinos , Clonagem de Organismos/métodos , Técnicas de Cultura Embrionária/métodos , Epigênese Genética/fisiologia , Feminino , Histonas/genética , Masculino , Proteínas Nucleares/genética , Partenogênese/fisiologia , RNA Mensageiro/genética , Especificidade da Espécie
14.
Stem Cells Dev ; 25(5): 386-94, 2016 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-26691930

RESUMO

The reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) is a complex process that involves significant epigenetic alterations in the reprogrammed cells. Epigenetic modifiers such as histone deacetylase (HDAC) inhibitors have been shown to increase the efficiency of derivation of iPSCs in humans and mice. In this study, we used three HDAC inhibitors, valproic acid, sodium butyrate, and suberoylanilide hydroxamic acid, together with ascorbic acid, for derivation and long-term feeder-free culture of porcine iPS-like cells. In the absence of exogenous growth factors and/or small molecules, these inhibitors were able to maintain the expression of key pluripotency markers, including genes known to be specific for naive pluripotent state in mouse stem cells, for over 60 passages under feeder-free conditions. Surprisingly, the cells became dependent on HDAC inhibitors for the maintenance of proliferation. Moreover, despite showing successful integration into blastocysts upon injection, the cells were unable to undergo normal differentiation in vitro and in vivo in the form of teratomas. Our results suggest that HDAC inhibitors maintain pluripotency gene expression of porcine iPSC-like cells in long-term culture, but prevent lineage specification, requiring further optimization of culture conditions for porcine iPSC derivation.


Assuntos
Técnicas de Cultura de Células/métodos , Células Alimentadoras/citologia , Inibidores de Histona Desacetilases/farmacologia , Células-Tronco Pluripotentes Induzidas/citologia , Animais , Diferenciação Celular/efeitos dos fármacos , Forma Celular , Células Cultivadas , Desenvolvimento Embrionário/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Camundongos Nus , Sus scrofa , Teratoma/patologia
15.
Stem Cells Dev ; 22(1): 124-35, 2013 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-22989381

RESUMO

The domestic pig is an important large animal model for preclinical testing of novel cell therapies. Recently, we produced pluripotency reporter pigs in which the Oct4 promoter drives expression of the enhanced green fluorescent protein (EGFP). Here, we reprogrammed Oct4-EGFP fibroblasts employing the nonviral Sleeping Beauty transposon system to deliver the reprogramming factors Oct4, Sox2, Klf4, and cMyc. Successful reprogramming to a pluripotent state was indicated by changes in cell morphology and reactivation of the Oct4-EGFP reporter. The transposon-reprogrammed induced pluripotent stem (iPS) cells showed long-term proliferation in vitro over >40 passages, expressed transcription factors typical of embryonic stem cells, including OCT4, NANOG, SOX2, REX1, ESRRB, DPPA5, and UTF1 and surface markers of pluripotency, including SSEA-1 and TRA-1-60. In vitro differentiation resulted in derivatives of the 3 germ layers. Upon injection of putative iPS cells under the skin of immunodeficient mice, we observed teratomas in 3 of 6 cases. These results form the basis for in-depth studies toward the derivation of porcine iPS cells, which hold great promise for preclinical testing of novel cell therapies in the pig model.


Assuntos
Elementos de DNA Transponíveis/genética , Células-Tronco Pluripotentes Induzidas/fisiologia , Animais , Antígenos de Diferenciação/metabolismo , Transformação Celular Neoplásica , Células Cultivadas , Técnicas de Cocultura , Fibroblastos/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células-Tronco Pluripotentes Induzidas/transplante , Células-Tronco Pluripotentes Induzidas/ultraestrutura , Fator 4 Semelhante a Kruppel , Camundongos , Camundongos Nus , Microscopia de Fluorescência , Neurogênese , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Regiões Promotoras Genéticas , Fatores de Transcrição SOXB1/metabolismo , Sus scrofa , Teratoma/patologia , Transcriptoma , Transgenes
16.
Stem Cells Dev ; 20(9): 1563-75, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21126163

RESUMO

The domesticated pig has emerged as an important tool for development of surgical techniques, advancement of xenotransplantation, creation of important disease models, and preclinical testing of novel cell therapies. However, germ line-competent pluripotent porcine stem cells have not yet been derived. This has been a major obstacle to genetic modification of pigs. The transcription factor Oct4 is essential for the maintenance of pluripotency and for reprogramming somatic cells to a pluripotent state. Here, we report the production of transgenic pigs carrying an 18 kb genomic sequence of the murine Oct4 gene fused to the enhanced green fluorescent protein (EGFP) cDNA (OG2 construct) to allow identification of pluripotent cells by monitoring Oct4 expression by EGFP fluorescence. Eleven viable transgenic piglets were produced by somatic cell nuclear transfer. Expression of the EGFP reporter construct was confined to germ line cells, the inner cell mass and trophectoderm of blastocysts, and testicular germ cells. Reprogramming of fibroblasts from these animals by fusion with pluripotent murine embryonic stem cells or viral transduction with human OCT4, SOX2, KLF4, and c-MYC cDNAs resulted in Oct4-EGFP reactivation. The OG2 pigs have thus proved useful for monitoring reprogramming and the induction and maintenance of pluripotency in porcine cells. In conclusion, the OG2 transgenic pigs are a new large animal model for studying the derivation and maintenance of pluripotent cells, and will be valuable for the development of cell therapy.


Assuntos
Animais Geneticamente Modificados , Proteínas de Fluorescência Verde/genética , Fator 3 de Transcrição de Octâmero/genética , Proteínas Recombinantes de Fusão/genética , Sus scrofa/genética , Animais , Blastocisto/metabolismo , Fusão Celular , Células Cultivadas , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Engenharia Genética , Células Germinativas/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Humanos , Células Híbridas/metabolismo , Células-Tronco Pluripotentes Induzidas , Rim/metabolismo , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Masculino , Camundongos , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Técnicas de Transferência Nuclear , Fator 3 de Transcrição de Octâmero/metabolismo , Gravidez , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Testículo/citologia , Testículo/metabolismo
17.
PLoS One ; 6(11): e27563, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22110672

RESUMO

Recently, we generated transposon-transgenic boars (Sus scrofa), which carry three monomeric copies of a fluorophore marker gene. Amazingly, a ubiquitous fluorophore expression in somatic, as well as in germ cells was found. Here, we characterized the prominent fluorophore load in mature spermatozoa of these animals. Sperm samples were analyzed for general fertility parameters, sorted according to X and Y chromosome-bearing sperm fractions, assessed for potential detrimental effects of the reporter, and used for inseminations into estrous sows. Independent of their genotype, all spermatozoa were uniformly fluorescent with a subcellular compartmentalization of the fluorophore protein in postacrosomal sheath, mid piece and tail. Transmission of the fluorophore protein to fertilized oocytes was shown by confocal microscopic analysis of zygotes. The monomeric copies of the transgene segregated during meiosis, rendering a certain fraction of the spermatozoa non-transgenic (about 10% based on analysis of 74 F1 offspring). The genotype-independent transmission of the fluorophore protein by spermatozoa to oocytes represents a non-genetic contribution to the mammalian embryo.


Assuntos
Proteínas de Bactérias/genética , Genótipo , Proteínas Luminescentes/genética , Espermatozoides/metabolismo , Sus scrofa/genética , Transgenes/genética , Animais , Animais Geneticamente Modificados , Elementos de DNA Transponíveis/genética , Embrião de Mamíferos , Fertilidade/genética , Fertilidade/efeitos da radiação , Luz , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espermatozoides/efeitos da radiação , Sus scrofa/embriologia , Sus scrofa/fisiologia , Transcrição Gênica/efeitos da radiação , Cromossomo X/genética , Cromossomo X/efeitos da radiação , Cromossomo Y/genética , Cromossomo Y/efeitos da radiação
18.
Cell Reprogram ; 12(1): 55-65, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20132013

RESUMO

Fusion of terminally differentiated somatic cells with pluripotent embryonic stem cells has been proposed as model for reprogramming the somatic cell genome, and may contribute to our understanding of the underlying mechanisms of this epigenetic process. We established an interspecies cell fusion model using murine embryonic stem cells (ESCs) and porcine fibroblasts. These inter-species fusion experiments yielded much lower conversion efficiency rates than murine intraspecies fusion. Nevertheless, two double-resistant mouse-pig hybrid clones could be generated. Reactivation of the porcine OCT4 gene, an essential pluripotent stem cell marker, and demethylation of the porcine OCT4 promoter in hybrid clone 1, suggested successful reprogramming of porcine chromosomes. A rapid loss of porcine chromosomes was observed during the selection phase. Spectral karyotyping (SKY) analysis showed that fusion-hybrid clone 1 carried a tetraploid mouse chromosome complement with only few pig chromosomes and/or chromosomal fragments. Hybrid clone 2 had a diploid set of murine chromosomes complements and also contained an interspecies chromosome fusion product. Interspecies cell fusion results in hybrid cells that retained the complement of mouse chromosomes and preferentially lose porcine chromosomes during colony expansion. Neither species-specific chromosomal segregation nor reprogrammed diploid porcine cells were observed. These findings indicate that generation of reprogrammed pluripotent diploid cells by cell fusion may require additional supporting provisions.


Assuntos
Reprogramação Celular , Cromossomos de Mamíferos , Células Híbridas , Animais , Animais Geneticamente Modificados , Fusão Celular/métodos , Linhagem Celular , Células Cultivadas , Embrião de Mamíferos/citologia , Células-Tronco Embrionárias/fisiologia , Estudos de Viabilidade , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Células-Tronco Pluripotentes/fisiologia , Sus scrofa/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA