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1.
Am J Physiol Lung Cell Mol Physiol ; 306(6): L534-42, 2014 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-24441869

RESUMO

MicroRNAs are small noncoding RNAs that inhibit protein expression. We have previously shown that the inhibition of the microRNA let-7d in epithelial cells caused changes consistent with epithelial-to-mesenchymal transition (EMT) both in vitro and in vivo. The aim of this study was to determine whether the introduction of let-7d into fibroblasts alters their mesenchymal properties. Transfection of primary fibroblasts with let-7d caused a decrease in expression of the mesenchymal markers α-smooth muscle actin, N-cadherin, fibroblast-specific protein-1, and fibronectin, as well as an increase in the epithelial markers tight junction protein-1 and keratin 19. Phenotypic changes were also present, including a delay in wound healing, reduced motility, and proliferation of fibroblasts following transfection. In addition, we examined the effects of transfection on fibroblast responsiveness to TGF-ß, an important factor in many fibrotic processes such as lung fibrosis and found that let-7d transfection significantly attenuated high-mobility group-A2 protein induction by TGF-ß. Our results indicate that administration of the epithelial microRNA let-7d can significantly alter the phenotype of primary fibroblasts.


Assuntos
Transição Epitelial-Mesenquimal , Fibroblastos/citologia , Pulmão/metabolismo , MicroRNAs/genética , Miofibroblastos/metabolismo , Fibrose Pulmonar/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Actinas/metabolismo , Caderinas/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Movimento Celular/genética , Proliferação de Células , Células Cultivadas , Fibroblastos/metabolismo , Fibronectinas/metabolismo , Proteína HMGA2/metabolismo , Proteína HMGB2/metabolismo , Humanos , Fibrose Pulmonar Idiopática/metabolismo , Fibrose Pulmonar Idiopática/patologia , Queratina-19/metabolismo , Pulmão/citologia , Alvéolos Pulmonares/metabolismo , Fibrose Pulmonar/genética , Proteína A4 de Ligação a Cálcio da Família S100 , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transfecção , Cicatrização/genética , Proteína da Zônula de Oclusão-1/metabolismo
2.
Reprod Fertil Dev ; 27(1): 89-92, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25472048

RESUMO

Generating gametes from pluripotent stem cells (PSCs) has many scientific justifications and several biomedical rationales. Here, we consider several strategies for deriving gametes from PSCs from mice and primates (human and non-human) and their anticipated strengths, challenges and limitations. Although the 'Weismann barrier', which separates the mortal somatic cell lineages from the potentially immortal germline, has long existed, breakthroughs first in mice and now in humans are artificially creating germ cells from somatic cells. Spermatozoa with full reproductive viability establishing multiple generations of seemingly normal offspring have been reported in mice and, in humans, haploid spermatids with correct parent-of-origin imprints have been obtained. Similar progress with making oocytes has been published using mouse PSCs differentiated in vitro into primordial germ cells, which are then cultured after xenografting reconstructed artificial ovaries. Progress in making human oocytes artificially is proving challenging. The usefulness of these artificial gametes, from assessing environmental exposure toxicity to optimising medical treatments to prevent negative off-target effects on fertility, may prove invaluable, as may basic discoveries on the fundamental mechanisms of gametogenesis.


Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/citologia , Células Germinativas/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Técnicas de Transferência Nuclear , Animais , Células-Tronco Embrionárias/fisiologia , Células Germinativas/fisiologia , Humanos , Técnicas In Vitro , Células-Tronco Pluripotentes Induzidas/fisiologia , Camundongos , Primatas
3.
Sci Rep ; 14(1): 4808, 2024 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-38413710

RESUMO

Transforming acidic acid coiled-coil protein 3 (TACC3) and cytoskeleton associated protein 5 (cKAP5; or colonic hepatic tumor overexpressed gene, chTOG) are vital for spindle assembly and stabilization initiated through TACC3 Aurora-A kinase interaction. Here, TACC3 and cKAP5/chTOG localization with monospecific antibodies is investigated in eGFP-centrin-2- expressing mouse meiotic spermatocytes. Both proteins bind spermatocyte spindle poles but neither kinetochore nor interpolar microtubules, unlike in mitotic mouse fibroblasts or female meiotic oocyte spindles. Spermatocytes do not display a liquid-like spindle domain (LISD), although fusing them into maturing oocytes generates LISD-like TACC3 condensates around sperm chromatin but sparse microtubule assembly. Microtubule inhibitors do not reduce TACC3 and cKAP5/chTOG spindle pole binding. MLN 8237 Aurora-A kinase inhibitor removes TACC3, not cKAP5/chTOG, disrupting spindle organization, chromosome alignment, and impacting spindle pole γ-tubulin intensity. The LISD disruptor 1,6-hexanediol abolished TACC3 in spermatocytes, impacting spindle bipolarity and chromosome organization. Cold microtubule disassembly and rescue experiments in the presence of 1,6-hexanediol reinforce the concept that spermatocyte TACC3 spindle pole presence is not required for spindle pole microtubule assembly. Collectively, meiotic spermatocytes without a LISD localize TACC3 and cKAP5/chTOG exclusively at spindle poles to support meiotic spindle pole stabilization during male meiosis, different from either female meiosis or mitosis.


Assuntos
Aurora Quinase A , Glicóis , Proteínas Associadas aos Microtúbulos , Animais , Feminino , Masculino , Camundongos , Aurora Quinase A/genética , Aurora Quinase A/metabolismo , Proteínas de Ciclo Celular/metabolismo , Meiose , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Oócitos/metabolismo , Sêmen/metabolismo , Fuso Acromático/metabolismo , Polos do Fuso/metabolismo
4.
J Cell Mol Med ; 17(2): 242-51, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23301772

RESUMO

In this study, we used a large non-human primate model, the baboon, to establish a step-wise protocol to generate CD34+ endothelial progenitor cells (EPCs) from embryonic stem cells (ESCs) and to demonstrate their reparative effects. Baboon ESCs were sequentially differentiated from embryoid body cultures for 9 days and then were specified into EPCs by culturing them in monolayer for 12 days. The resulting EPCs expressed CD34, CXCR4 and UEA-1, but neither CD31 nor CD117. The EPCs were able to form intact lumen structures when seeded on Matrigel, took up Dil-LDL, and responded to TNF-α. Angioblasts specified in EGM-2 medium and ECGS medium had 6.41 ± 1.16% (n = 3) and 9.32 ± 3.73% CD34+ cells (n = 3). The efficiency of generating CD34+ EPCs did not differ significantly from ECGS to EGM-2 culture media, however, angioblasts specified in ECGS medium expressed a higher percentage of CD34+/CXCR4+ cells (3.49 ± 1.32%, n = 3) than those specified in EGM-2 medium (0.49 ± 0.52%, n = 3). To observe their reparative capacity, we purified CD34+ progenitors after specification by EGM-2 medium; inoculated fluorescently labelled CD34+ EPCs into an arterial segment denuded of endothelium in an ex vivo system. After 14 days of ex vivo culture, the grafted cells had attached and integrated to the denuded surface; in addition, they had matured further and expressed terminally differentiated endothelial markers including CD31 and CD146. In conclusion, we have proved that specified CD34+ EPCs are promising therapeutic agents for repairing damaged vasculature.


Assuntos
Antígenos CD34/metabolismo , Diferenciação Celular , Células-Tronco Embrionárias/citologia , Endotélio Vascular/citologia , Neovascularização Fisiológica , Células-Tronco/citologia , Animais , Western Blotting , Proliferação de Células , Células Cultivadas , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Citometria de Fluxo , Imunofluorescência , Humanos , Papio , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Fator de Necrose Tumoral alfa/farmacologia
5.
Reprod Biomed Online ; 27(1): 75-80, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23664220

RESUMO

Recent advances in assisted reproduction treatment have enabled some couples with severe infertility issues to conceive, but the methods are not successful in all cases. Notwithstanding the significant financial burden of assisted reproduction treatment, the emotional scars from an inability to conceive a child enacts a greater toll on affected couples. While methods have circumvented some root causes for male and female infertility, often the underlying causes cannot be treated, thus true cures for restoring a patient's fertility are limited. Furthermore, the procedures are only available if the affected patients are able to produce gametes. Patients rendered sterile by medical interventions, exposure to toxicants or genetic causes are unable to utilize assisted reproduction to conceive a child - and often resort to donors, where permitted. Stem cells represent a future potential avenue for allowing these sterile patients to produce offspring. Advances in stem cell biology indicate that stem cell replacement therapies or in-vitro differentiation may be on the horizon to treat and could cure male and female infertility, although significant challenges need to be met before this technology can reach clinical practice. This article discusses these advances and describes the impact that these advances may have on treating infertility.


Assuntos
Infertilidade Feminina/terapia , Infertilidade Masculina/terapia , Técnicas de Reprodução Assistida , Transplante de Células-Tronco , Animais , Diferenciação Celular , Criopreservação , Feminino , Preservação da Fertilidade , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Células-Tronco Pluripotentes Induzidas/transplante , Infertilidade Masculina/genética , Masculino
6.
J Neuroinflammation ; 9: 138, 2012 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-22727020

RESUMO

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) induces neuronal dysfunction through host cellular factors and viral proteins including viral protein R (Vpr) released from infected macrophages/microglia. Vpr is important for infection of terminally differentiated cells such as macrophages. The objective of this study was to assess the effect of Vpr in the context of infectious virus particles on neuronal death through proinflammatory cytokines released from macrophages. METHODS: Monocyte-derived macrophages (MDM) were infected with either HIV-1 wild type (HIV-1wt), Vpr deleted mutant (HIV-1∆Vpr) or mock. Cell lysates and culture supernatants from MDMs were analyzed for the expression and release of proinflammatory cytokines by quantitative reverse transcription-PCR and enzyme-linked immunosorbent assay respectively. Mitogen-activated protein kinases (MAPK) were analyzed in activated MDMs by western blots. Further, the effect of Vpr on neuronal apoptosis was examined using primary neurons exposed to culture supernatants from HIV-1wt, HIV-1∆Vpr or mock-infected MDMs by Annexin-V staining, MTT and Caspase - Glo® 3/7 assays. The role of interleukin (IL)-1ß, IL-8 and tumor necrosis factor (TNF)-α on neuronal apoptosis was also evaluated in the presence or absence of neutralizing antibodies against these cytokines. RESULTS: HIV-1∆Vpr-infected MDMs exhibited reduced infection over time and specifically a significant downregulation of IL-1ß, IL-8 and TNF-α at the transcriptional and/or protein levels compared to HIV-1wt-infected cultures. This downregulation was due to impaired activation of p38 and stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) in HIV-1∆Vpr-infected MDMs. The association of SAPK/JNK and p38 to IL-1ß and IL-8 production was confirmed by blocking MAPKs that prevented the elevation of IL-1ß and IL-8 in HIV-1wt more than in HIV-1∆Vpr-infected cultures. Supernatants from HIV-1∆Vpr-infected MDMs containing lower concentrations of IL-1ß, IL-8 and TNF-α as well as viral proteins showed a reduced neurotoxicity compared to HIV-1wt-infected MDM supernatants. Reduction of neuronal death in the presence of anti-IL-1ß and anti-IL-8 antibodies only in HIV-1wt-infected culture implies that the effect of Vpr on neuronal death is in part mediated through released proinflammatory factors. CONCLUSION: Collectively, these results demonstrate the ability of HIV-1∆Vpr to restrict neuronal apoptosis through dysregulation of multiple proinflammatory cytokines in the infected target cells either directly or indirectly by suppressing viral replication.


Assuntos
Apoptose/fisiologia , Redes Reguladoras de Genes/fisiologia , Infecções por HIV/metabolismo , Mediadores da Inflamação/fisiologia , Neurônios/fisiologia , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/fisiologia , Células Cultivadas , Células HEK293 , Infecções por HIV/genética , Infecções por HIV/virologia , Humanos , Mediadores da Inflamação/administração & dosagem , Interleucina-8/antagonistas & inibidores , Interleucina-8/biossíntese , Neurônios/patologia , Neurônios/virologia , Inativação de Vírus , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/administração & dosagem
8.
Biochem Biophys Res Commun ; 414(3): 631-4, 2011 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-22005468

RESUMO

Human exhibit sexual dimorphism early in development and throughout life. Here we stringently analyzed gene expression in inbred non-human primate embryonic stem cells (nhpESCs) searching for sexually dimorphisms. We utilized location-specific probes solely, thus avoiding probe cross-reactivity between members of gene families and genomic gene duplications. Seventeen sexually dimorphic transcripts (15 genes, out of which 9 autosomals) were identified, of which five were verified using real-time q-PCR. We compared these results from pedigreed nhpESCs with available human ESCs datasets. Three human X-linked genes show sexual dimorphism. Thus, these results enhance our knowledge and deepen our understanding on early development processes for sexual dimorphism.


Assuntos
Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica , Caracteres Sexuais , Animais , Linhagem Celular , Feminino , Perfilação da Expressão Gênica , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Primatas , Transcrição Gênica
9.
Nat Cell Biol ; 4 Suppl: s19-22, 2002 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-12479610

RESUMO

Assisted reproductive technologies (ART) are exceptional among clinical therapies, as unlike most medical procedures, ART have generational consequences. Further, human embryo research in the US has been sponsored solely by the private sector and, until recent biotechnology forays into human embryonic stem cell (hESC) and cloning research, exclusively by infertility clinics. Additionally, the relatively brief clinical history of ART has made it difficult for practitioners and researchers to agree on criteria for its safety and success. Against this backdrop, market pressure on biotechnology companies to create hESC lines and on clinical practices to occupy the innovative forefront has resulted in arguably risky experiments with human embryo cloning, as well as in unintentional germ-line genetic modifications during ART and perhaps during gene therapy. Reproduction, once governed largely by passions and instinct, now seems to need further governance. Some argue that it could now be time for the biomedical community, especially in the US, to take further steps to safeguard ART.


Assuntos
Técnicas de Reprodução Assistida/ética , Técnicas de Reprodução Assistida/legislação & jurisprudência , Técnicas de Reprodução Assistida/tendências , Envelhecimento , Criopreservação , Feminino , Fertilização in vitro , Humanos , Masculino , Oócitos/fisiologia , Gravidez , Espermatozoides/fisiologia
11.
Exp Cell Res ; 316(17): 2747-59, 2010 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-20599958

RESUMO

Impaired DSB repair has been implicated as a molecular mechanism contributing to the accelerating aging phenotype in Hutchinson-Gilford progeria syndrome (HGPS), but neither the extent nor the cause of the repair deficiency has been fully elucidated. Here we perform a quantitative analysis of the steady-state number of DSBs and the repair kinetics of ionizing radiation (IR)-induced DSBs in HGPS cells. We report an elevated steady-state number of DSBs and impaired repair of IR-induced DSBs, both of which correlated strongly with abnormal nuclear morphology. We recreated the HGPS cellular phenotype in human coronary artery endothelial cells for the first time by lentiviral transduction of GFP-progerin, which also resulted in impaired repair of IR-induced DSBs, and which correlated with abnormal nuclear morphology. Farnesyl transferase inhibitor (FTI) treatment improved the repair of IR-induced DSBs, but only in HGPS cells whose nuclear morphology was also normalized. Interestingly, FTI treatment did not result in a statistically significant reduction in the higher steady-state number of DSBs. We also report a delay in localization of phospho-NBS1 and MRE11, MRN complex repair factors necessary for homologous recombination (HR) repair, to DSBs in HGPS cells. Our results demonstrate a correlation between nuclear structural abnormalities and the DSB repair defect, suggesting a mechanistic link that may involve delayed repair factor localization to DNA damage. Further, our results show that similar to other HGPS phenotypes, FTI treatment has a beneficial effect on DSB repair.


Assuntos
Núcleo Celular/patologia , Quebras de DNA de Cadeia Dupla , Reparo do DNA/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Farnesiltranstransferase/antagonistas & inibidores , Fibroblastos/patologia , Progéria/patologia , Estudos de Casos e Controles , Células Cultivadas , Inibidores Enzimáticos/uso terapêutico , Fibroblastos/efeitos dos fármacos , Humanos , Progéria/tratamento farmacológico , Síndrome
12.
Nature ; 436(7051): 641, 2005 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-16079832

RESUMO

Several mammals--including sheep, mice, cows, goats, pigs, rabbits, cats, a mule, a horse and a litter of three rats--have been cloned by transfer of a nucleus from a somatic cell into an egg cell (oocyte) that has had its nucleus removed. This technology has not so far been successful in dogs because of the difficulty of maturing canine oocytes in vitro. Here we describe the cloning of two Afghan hounds by nuclear transfer from adult skin cells into oocytes that had matured in vivo. Together with detailed sequence information generated by the canine-genome project, the ability to clone dogs by somatic-cell nuclear transfer should help to determine genetic and environmental contributions to the diverse biological and behavioural traits associated with the many different canine breeds.


Assuntos
Clonagem de Organismos , Cães/crescimento & desenvolvimento , Cães/genética , Envelhecimento , Animais , Cães/classificação , Cães/embriologia , Transferência Embrionária , Feminino , Fibroblastos/citologia , Masculino , Repetições de Microssatélites/genética , Técnicas de Transferência Nuclear , Doação de Oócitos , Oócitos/citologia , Oócitos/fisiologia , Oócitos/transplante , Gravidez , Pele/citologia , Fatores de Tempo
13.
F S Sci ; 2(4): 365-375, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34970648

RESUMO

OBJECTIVE: To demonstrate that functional spermatids can be derived in vitro from nonhuman primate pluripotent stem cells. DESIGN: Green fluorescent protein-labeled, rhesus macaque nonhuman primate embryonic stem cells (nhpESCs) were differentiated into advanced male germ cell lineages using a modified serum-free spermatogonial stem cell culture medium. In vitro-derived round spermatid-like cells (rSLCs) from differentiated nhpESCs were assessed for their ability to fertilize rhesus oocytes by intracytoplasmic sperm(atid) injection. SETTING: Multiple academic laboratory settings. PATIENTS: Not applicable. INTERVENTIONS: Intracytoplasmic sperm(atid) injection of in vitro-derived spermatids from nhpESCs into rhesus macaque oocytes. MAIN OUTCOME MEASURES: Differentiation into spermatogenic cell lineages was measured through multiple assessments including ribonucleic acid sequencing and immunocytochemistry for various spermatogenic markers. In vitro spermatids were assessed for their ability to fertilize oocytes by intracytoplasmic sperm(atid) injection by assessing early fertilization events such as spermatid deoxyribonucleic acid decondensation and pronucleus formation/apposition. Preimplantation embryo development from the one-cell zygote stage to the blastocyst stage was also assessed. RESULTS: Nonhuman primate embryonic stem cells can be differentiated into advanced germ cell lineages, including haploid rSLCs. These rSLCs undergo deoxyribonucleic acid decondensation and pronucleus formation/apposition when microinjected into rhesus macaque mature oocytes, which, after artificial activation and coinjection of ten-eleven translocation 3 protein, undergo embryonic divisions with approximately 12% developing successfully into expanded blastocysts. CONCLUSIONS: This work demonstrates that rSLCs, generated in vitro from primate pluripotent stem cells, mimic many of the capabilities of in vivo round spermatids and perform events essential for preimplantation development. To our knowledge, this work represents, for the first time, that functional spermatid-like cells can be derived in vitro from primate pluripotent stem cells.


Assuntos
Injeções de Esperma Intracitoplásmicas , Espermátides , Animais , Blastocisto , DNA , Desenvolvimento Embrionário , Células-Tronco Embrionárias , Feminino , Fertilização , Humanos , Macaca mulatta , Masculino , Gravidez
14.
Stem Cells ; 27(8): 1822-35, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19544417

RESUMO

Human embryonic stem (ES) cells are highly sensitive to environmental insults including DNA damaging agents, responding with high levels of apoptosis. To understand the response of human ES cells to DNA damage, we investigated the function of the ataxia telangiectasia mutated (ATM) DNA damage signaling pathway in response to gamma-irradiation. Here, we demonstrate for the first time in human ES cells that ATM kinase is phosphorylated and properly localized to the sites of DNA double-strand breaks within 15 minutes of irradiation. Activation of ATM kinase resulted in phosphorylation of its downstream targets: Chk2, p53, and Nbs1. In contrast to murine ES cells, Chk2 and p53 were localized to the nucleus of irradiated human ES cells. We further show that irradiation resulted in a temporary arrest of the cell cycle at the G(2), but not G(1), phase. Human ES cells resumed cycling approximately 16 hours after irradiation, but had a fourfold higher incidence of aberrant mitotic figures compared to nonirradiated cells. Finally, we demonstrate an essential role of ATM in establishing G(2) arrest since inhibition with the ATM-specific inhibitor KU55933 resulted in abolishment of G(2) arrest, evidenced by an increase in the number of cycling cells 2 hours after irradiation. In summary, these results indicate that human ES cells activate the DNA damage checkpoint, resulting in an ATM-dependent G(2) arrest. However, these cells re-enter the cell cycle with prominent mitotic spindle defects.


Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/genética , Células-Tronco Embrionárias/efeitos da radiação , Fase G1/efeitos da radiação , Fase G2/efeitos da radiação , Células-Tronco Pluripotentes/efeitos da radiação , Proteínas Serina-Treonina Quinases/genética , Proteínas Supressoras de Tumor/genética , Proteínas Mutadas de Ataxia Telangiectasia , Western Blotting , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta à Radiação , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Fase G1/genética , Fase G2/genética , Raios gama , Humanos , Imuno-Histoquímica , Microscopia Confocal , Fosforilação , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Reação em Cadeia da Polimerase , Proteínas Serina-Treonina Quinases/metabolismo , Radiação Ionizante , Transdução de Sinais/efeitos da radiação , Proteínas Supressoras de Tumor/metabolismo
15.
Cells Tissues Organs ; 192(1): 39-49, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20197653

RESUMO

We describe hollow fiber-based three-dimensional (3D) dynamic perfusion bioreactor technology for embryonic stem cells (ESC) which is scalable for laboratory and potentially clinical translation applications. We added 2 more compartments to the typical 2-compartment devices, namely an additional media capillary compartment for countercurrent 'arteriovenous' flow and an oxygenation capillary compartment. Each capillary membrane compartment can be perfused independently. Interweaving the 3 capillary systems to form repetitive units allows bioreactor scalability by multiplying the capillary units and provides decentralized media perfusion while enhancing mass exchange and reducing gradient distances from decimeters to more physiologic lengths of <1 mm. The exterior of the resulting membrane network, the cell compartment, is used as a physically active scaffold for cell aggregation; adjusting intercapillary distances enables control of the size of cell aggregates. To demonstrate the technology, mouse ESC (mESC) were cultured in 8- or 800-ml cell compartment bioreactors. We were able to confirm the hypothesis that this bioreactor enables mESC expansion qualitatively comparable to that obtained with Petri dishes, but on a larger scale. To test this, we compared the growth of 129/SVEV mESC in static two-dimensional Petri dishes with that in 3D perfusion bioreactors. We then tested the feasibility of scaling up the culture. In an 800-ml prototype, we cultured approximately 5 x 10(9) cells, replacing up to 800 conventional 100-mm Petri dishes. Teratoma formation studies in mice confirmed protein expression and gene expression results with regard to maintaining 'stemness' markers during cell expansion.


Assuntos
Técnicas de Cultura de Células/métodos , Células-Tronco Embrionárias/citologia , Animais , Reatores Biológicos , Técnicas de Cultura de Células/instrumentação , Processos de Crescimento Celular/fisiologia , Células Cultivadas , Humanos , Camundongos , Perfusão
18.
Tohoku J Exp Med ; 220(4): 247-58, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20379121

RESUMO

Fertilization is a lengthy process that culminates when the male and female pronuclei fuse in the oocyte cytoplasm. The final stage of fertilization is mediated by the sperm centrosome, which induces microtubule organization into the first mitotic spindle. Despite its critical role, only few functional analyses of the sperm centrosome have been performed until now. Here, we review the recent literature with regard to sperm centrosomal functions during human fertilization, as well as the development of functional assays for the human sperm centrosome. We then address various challenges for fertilization failure resulting from centrosomal dysfunction. Cytological analyses of oocytes that fail to complete fertilization following intracytoplasmic sperm injection (ICSI) have shown that some cases are associated with sperm centrosomal dysfunction. Human sperm can organize a sperm aster even within the oocytes of other mammals. This property has been utilized as a means of assessing the centrosome function. In some patients with teratozoospermia, the sperm does show evidence of impaired centrosomal function. Some clinical and basic challenges for overcoming the fertilization failure caused by sperm centrosomal dysfunction have been reported. The sperm centrosome plays an important role in the phase of the fertilization process after ICSI, i.e. within the oocyte's cytoplasm. The next generation of assisted reproductive technologies (ART) will likely incorporate analyses of sperm centrosomal function as well as techniques designed to counter sperm centrosome dysfunction.


Assuntos
Centrossomo/metabolismo , Fertilização , Infertilidade Masculina/fisiopatologia , Espermatozoides/citologia , Animais , Bioensaio , Centrossomo/patologia , Citoesqueleto/metabolismo , Feminino , Humanos , Masculino , Microtúbulos/metabolismo , Oócitos/citologia , Oócitos/metabolismo , Partenogênese , Gravidez , Injeções de Esperma Intracitoplásmicas , Espermatozoides/metabolismo
19.
Eur J Med Genet ; 62(8): 103682, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31150829

RESUMO

Perhaps the two most significant pioneering biomedical discoveries with immediate clinical implications during the past forty years have been the advent of assisted reproductive technologies (ART) and the genetics revolution. ART, including in vitro fertilization (IVF), intracytoplasmic sperm injection and preimplantation genetic testing, has resulted in the birth of more than 8 million children, and the pioneer of IVF, Professor Bob Edwards, was awarded the 2010 Nobel Prize. The genetics revolution has resulted in our genomes being sequenced and many of the molecular mechanisms understood, and technologies for genomic editing have been developed. With the combination of nearly routine ART protocols for healthy conceptions together with almost error-free, inexpensive and simple methods for genetic modification, the question "Are we ready for genome editing in human embryos for clinical purposes?" was debated at the 5th congress on controversies in preconception, preimplantation and Prenatal Genetic Diagnosis, in collaboration with the Ovarian Club Meeting, in November 2018 in Paris. The co-authors each presented scientific, medical and bioethical backgrounds, and the debate was chaired by Professor Alan Handyside. In this paper, we consider whether genome editing is safe and ethical. We conclude that we are currently not ready for genome editing to be used in human embryos for clinical purposes, and we call for a global debate to determine if and when this technology could be used in ART. ‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬.


Assuntos
Fertilização in vitro/tendências , Edição de Genes/tendências , Diagnóstico Pré-Implantação/tendências , Técnicas de Reprodução Assistida , Feminino , Testes Genéticos , Humanos , Gravidez , Injeções de Esperma Intracitoplásmicas
20.
Sci Rep ; 9(1): 15282, 2019 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-31653971

RESUMO

With nearly ten million babies conceived globally, using assisted reproductive technologies, fundamental questions remain; e.g., How do the sperm and egg DNA unite? Does ICSI have consequences that IVF does not? Here, pronuclear and mitotic events in nonhuman primate zygotes leading to the establishment of polarity are investigated by multidimensional time-lapse video microscopy and immunocytochemistry. Multiplane videos after ICSI show atypical sperm head displacement beneath the oocyte cortex and eccentric para-tangential pronuclear alignment compared to IVF zygotes. Neither fertilization procedure generates incorporation cones. At first interphase, apposed pronuclei align obliquely to the animal-vegetal axis after ICSI, with asymmetric furrows assembling from the male pronucleus. Furrows form within 30° of the animal pole, but typically, not through the ICSI injection site. Membrane flow drives polar bodies and the ICSI site into the furrow. Mitotic spindle imaging suggests para-tangential pronuclear orientation, which initiates random spindle axes and minimal spindle:cortex interactions. Parthenogenetic pronuclei drift centripetally and assemble astral spindles lacking cortical interactions, leading to random furrows through the animal pole. Conversely, androgenotes display cortex-only pronuclear interactions mimicking ICSI. First cleavage axis determination in primates involves dynamic cortex-microtubule interactions among male pronuclei, centrosomal microtubules, and the animal pole, but not the ICSI site.


Assuntos
Fertilização in vitro/métodos , Fertilização/fisiologia , Primatas/fisiologia , Injeções de Esperma Intracitoplásmicas/métodos , Zigoto/fisiologia , Animais , Núcleo Celular/fisiologia , Feminino , Humanos , Macaca fascicularis/fisiologia , Macaca mulatta/fisiologia , Masculino , Microtúbulos/metabolismo , Microtúbulos/fisiologia , Oócitos/citologia , Oócitos/fisiologia , Partenogênese , Corpos Polares/fisiologia , Espermatozoides/citologia , Espermatozoides/fisiologia , Fuso Acromático/fisiologia , Zigoto/citologia
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