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1.
Science ; 367(6482)2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-32054698

RESUMO

Sex determination of germ cells is vital to creating the sexual dichotomy of germ cell development, thereby ensuring sexual reproduction. However, the underlying mechanisms remain unclear. Here, we show that ZGLP1, a conserved transcriptional regulator with GATA-like zinc fingers, determines the oogenic fate in mice. ZGLP1 acts downstream of bone morphogenetic protein, but not retinoic acid (RA), and is essential for the oogenic program and meiotic entry. ZGLP1 overexpression induces differentiation of in vitro primordial germ cell-like cells (PGCLCs) into fetal oocytes by activating the oogenic programs repressed by Polycomb activities, whereas RA signaling contributes to oogenic program maturation and PGC program repression. Our findings elucidate the mechanism for mammalian oogenic fate determination, providing a foundation for promoting in vitro gametogenesis and reproductive medicine.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Oócitos/fisiologia , Oogênese/genética , Diferenciação Sexual/genética , Fatores de Transcrição/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Feminino , Feto/citologia , Masculino , Meiose/genética , Camundongos , Camundongos Knockout , Oócitos/citologia , Proteínas do Grupo Polycomb/metabolismo , Processos de Determinação Sexual , Transdução de Sinais , Fatores de Transcrição/genética , Transcriptoma , Tretinoína/fisiologia
2.
Int J Nanomedicine ; 14: 7323-7338, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31686809

RESUMO

Background: Gemcitabine (GEM) is a chemotherapeutic agent, which is known to battle cancer but challenging due to its hydrophilic nature. Nanoemulsion is water-in-oil (W/O) nanoemulsion shows potential as a carrier system in delivering gemcitabine to the cancer cell. Methods: The behaviour of GEM in MCT/surfactants/NaCl systems was studied in the ternary system at different ratios of Tween 80 and Span 80. The system with surfactant ratio 3:7 of Tween 80 and Span 80 was chosen for further study on the preparation of nanoemulsion formulation due to the highest isotropic region. Based on the selected ternary phase diagram, a composition of F1 was chosen and used for optimization by using the D-optimal mixture design. The interaction variables between medium chain triglyceride (MCT), surfactant mixture Tween 80: Span 80 (ratio 3:7), 0.9 % sodium chloride solution and gemcitabine were evaluated towards particle size as a response. Results: The results showed that NaCl solution and GEM gave more effects on particle size, polydispersity index and zeta potential of 141.57±0.05 nm, 0.168 and -37.10 mV, respectively. The optimized nanoemulsion showed good stability (no phase separation) against centrifugation test and storage at three different temperatures. The in vitro release of gemcitabine at different pH buffer solution was evaluated. The results showed the release of GEM in buffer pH 6.5 (45.19%) was higher than GEM in buffer pH 7.4 (13.62%). The cytotoxicity study showed that the optimized nanoemulsion containing GEM induced cytotoxicity towards A549 cell and at the same time reduced cytotoxicity towards MRC5 when compared to the control (GEM solution).


Assuntos
Desoxicitidina/análogos & derivados , Emulsões/química , Feto/citologia , Fibroblastos/citologia , Neoplasias Pulmonares/tratamento farmacológico , Pulmão/embriologia , Nanopartículas/química , Células A549 , Análise de Variância , Morte Celular/efeitos dos fármacos , Linhagem Celular , Desoxicitidina/farmacologia , Desoxicitidina/uso terapêutico , Fibroblastos/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Cinética , Tamanho da Partícula , Transição de Fase , Polissorbatos/química , Tensoativos/química
3.
Rev Med Suisse ; 15(668): 1914-1919, 2019 Oct 23.
Artigo em Francês | MEDLINE | ID: mdl-31643151

RESUMO

Following the introduction of non-invasive tests, antenatal screening for trisomy 21 underwent important changes. Clinicians had to rapidly adapt their practice, especially in the field of antenatal counseling. On a population wide scale, new strategies and guidelines have been implemented. This article reviews the basic concepts of antenatal screening, including the use of non-invasive cell-free fetal DNA testing.


Assuntos
Diagnóstico Pré-Natal/métodos , Síndrome de Down/diagnóstico , Síndrome de Down/genética , Feminino , Feto/citologia , Feto/metabolismo , Humanos , Gravidez
4.
Vet Immunol Immunopathol ; 217: 109955, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31639586

RESUMO

The purpose of this work was to characterize the cellular phenotype in inflammatory infiltrates of fetal tissues from pregnant heifers immunized and experimentally challenged with Neospora caninum. Fetuses from 20 heifers separated into 5 groups were obtained. The experiment was designed as follow: Group A, heifers inoculated intravenously with live tachyzoites of Argentine strain NC-6 (n = 4); Group B heifers inoculated subcutaneously with soluble native antigen from the same strain formulated with immune stimulant complexes (ISCOMs) (n = 4); Group C heifers inoculated with recombinant proteins, rNcSAG1, rNcHSP20, rNcGRA7 formulated with ISCOMs (n = 4), Group D heifers inoculated subcutaneously with sterile phosphate buffered solution (n = 4) and Group E heifers inoculated subcutaneously with antigen-free ISCOMs (n = 4). Experimental challenge was performed at 70 days of gestation and all heifers were euthanized 34 days later. Fetal tissues were taken for histological studies. Inflammatory lesions were observed in brain and lung, and immunhistochemistry was used to identify CD3+, CD20+ and MHC II+ cells. The majority of the cells that infiltrate and circumscribe the lesions in the brain and lung tissue expressed MHC II antigen; varying between 70-90% of the total cellular infiltrate. CD3+ cells were also present within the lesions, contributing to up to 30% of the inflammatory cells. CD20+ cells appeared as a marginal group, in some cases, with a range between 10 and 25%. As expected, the immunolabeling of MHC II + and CD3 + cells in fetal tissues was associated with fetal infection with N. caninum. There were statistically significant differences in the distribution and population of the inflammatory infiltrate in relation to the immunogenic treatment and the type of tissue, with inflammatory cells being markedly less extensive fetuses from group A (dams previously exposed to N. caninum) and in brain tissue. This work showed that Neospora-infection induced MHC II+ and CD3+ cells in bovine fetuses from dams receiving experimental vaccines.


Assuntos
Coccidiose/imunologia , Feto/imunologia , Imunização/veterinária , Neospora/imunologia , Vacinas Protozoárias/imunologia , Animais , Encéfalo/citologia , Encéfalo/imunologia , Bovinos , Doenças dos Bovinos/imunologia , Feminino , Feto/citologia , Imuno-Histoquímica , Pulmão/citologia , Pulmão/imunologia , Gravidez
5.
Blood ; 134(13): 1059-1071, 2019 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-31383639

RESUMO

Human lymphopoiesis is a dynamic lifelong process that starts in utero 6 weeks postconception. Although fetal B-lymphopoiesis remains poorly defined, it is key to understanding leukemia initiation in early life. Here, we provide a comprehensive analysis of the human fetal B-cell developmental hierarchy. We report the presence in fetal tissues of 2 distinct CD19+ B-progenitors, an adult-type CD10+ve ProB-progenitor and a new CD10-ve PreProB-progenitor, and describe their molecular and functional characteristics. PreProB-progenitors and ProB-progenitors appear early in the first trimester in embryonic liver, followed by a sustained second wave of B-progenitor development in fetal bone marrow (BM), where together they form >40% of the total hematopoietic stem cell/progenitor pool. Almost one-third of fetal B-progenitors are CD10-ve PreProB-progenitors, whereas, by contrast, PreProB-progenitors are almost undetectable (0.53% ± 0.24%) in adult BM. Single-cell transcriptomics and functional assays place fetal PreProB-progenitors upstream of ProB-progenitors, identifying them as the first B-lymphoid-restricted progenitor in human fetal life. Although fetal BM PreProB-progenitors and ProB-progenitors both give rise solely to B-lineage cells, they are transcriptionally distinct. As with their fetal counterparts, adult BM PreProB-progenitors give rise only to B-lineage cells in vitro and express the expected B-lineage gene expression program. However, fetal PreProB-progenitors display a distinct, ontogeny-related gene expression pattern that is not seen in adult PreProB-progenitors, and they share transcriptomic signatures with CD10-ve B-progenitor infant acute lymphoblastic leukemia blast cells. These data identify PreProB-progenitors as the earliest B-lymphoid-restricted progenitor in human fetal life and suggest that this fetal-restricted committed B-progenitor might provide a permissive cellular context for prenatal B-progenitor leukemia initiation.


Assuntos
Feto/citologia , Linfopoese , Neprilisina/análise , Células Precursoras de Linfócitos B/citologia , Adulto , Medula Óssea/embriologia , Medula Óssea/metabolismo , Células Cultivadas , Feto/embriologia , Feto/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Fígado/embriologia , Fígado/metabolismo , Neprilisina/genética , Células Precursoras de Linfócitos B/metabolismo , Transcriptoma
6.
Microsc Res Tech ; 82(11): 1928-1940, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31423711

RESUMO

Tannic acid (TA) is a phenolic compound that might act directly on osteoblast metabolism. The study was performed to investigate the effects of TA on the proliferation, mineralization, and morphology of human fetal osteoblast cells (hFOB 1.19). The cells were divided into TA-treated, untreated, and pamidronate-treated (control drug) groups. Half maximal effective concentration (EC50 ) values for TA and pamidronate were measured using MTT assay. The EC50 of hFOB 1.19 cells treated with TA was 2.94 M. This concentration was more effective compared to the pamidronate (15.27 M). Cell proliferation assay was performed to compare cell viability from Day 1 until Day 14. The morphology of hFOB 1.19 was observed via inverted microscope and scanning electron microscope. Calcium (Ca) and phosphate (P) were assessed using energy-dispersive X-ray (EDX) analysis. Furthermore, the mineralization of hFOB 1.19 was determined by von Kossa staining (P depositions) and Alizarin Red S staining (Ca depositions). The number of cells treated with TA was significantly higher than the two control groups at Day 10 and Day 14. The morphology of cells treated with TA was uniformly fusiform-shaped with filopodia extensions. Besides, globular-like structures of deposited minerals were observed in the TA-treated group. In line with other findings, EDX spectrum analysis confirmed the presence of Ca and P. The cells treated with TA had significantly higher percentage of both minerals at Day 3 and Day 10 compared to the two control groups. In conclusion, TA enhances cell proliferation and causes cell morphology changes, as well as improved mineralization.


Assuntos
Calcificação Fisiológica/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feto/citologia , Osteoblastos/metabolismo , Taninos/farmacologia , Cálcio/análise , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Humanos , Microscopia Eletrônica de Varredura , Osteoblastos/citologia , Pamidronato/farmacologia , Fosfatos/análise , Espectrometria por Raios X
7.
Nat Commun ; 10(1): 3031, 2019 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-31292453

RESUMO

Maternal immune dysregulation seems to affect fetal or postnatal immune development. Preeclampsia is a pregnancy-associated disorder with an immune basis and is linked to atopic disorders in offspring. Here we show reduction of fetal thymic size, altered thymic architecture and reduced fetal thymic regulatory T (Treg) cell output in preeclamptic pregnancies, which persists up to 4 years of age in human offspring. In germ-free mice, fetal thymic CD4+ T cell and Treg cell development are compromised, but rescued by maternal supplementation with the intestinal bacterial metabolite short chain fatty acid (SCFA) acetate, which induces upregulation of the autoimmune regulator (AIRE), known to contribute to Treg cell generation. In our human cohorts, low maternal serum acetate is associated with subsequent preeclampsia, and correlates with serum acetate in the fetus. These findings suggest a potential role of acetate in the pathogenesis of preeclampsia and immune development in offspring.


Assuntos
Acetatos/sangue , Feto/imunologia , Pré-Eclâmpsia/imunologia , Efeitos Tardios da Exposição Pré-Natal/imunologia , Linfócitos T Reguladores/imunologia , Acetatos/administração & dosagem , Acetatos/imunologia , Acetatos/metabolismo , Adulto , Animais , Animais Recém-Nascidos , Estudos de Casos e Controles , Desenvolvimento Infantil , Pré-Escolar , Suplementos Nutricionais , Feminino , Feto/citologia , Feto/diagnóstico por imagem , Microbioma Gastrointestinal/imunologia , Vida Livre de Germes/imunologia , Humanos , Tolerância Imunológica/imunologia , Lactente , Recém-Nascido , Estudos Longitudinais , Troca Materno-Fetal/imunologia , Camundongos , Tamanho do Órgão/imunologia , Pré-Eclâmpsia/sangue , Pré-Eclâmpsia/diagnóstico , Gravidez , Efeitos Tardios da Exposição Pré-Natal/patologia , Efeitos Tardios da Exposição Pré-Natal/prevenção & controle , Estudos Prospectivos , Timo/citologia , Timo/diagnóstico por imagem , Timo/crescimento & desenvolvimento , Timo/imunologia , Fatores de Transcrição/imunologia , Fatores de Transcrição/metabolismo , Ultrassonografia Pré-Natal , Adulto Jovem
8.
Reprod Domest Anim ; 54(9): 1258-1264, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31283039

RESUMO

Spermatogonial stem cells (SSC) are promising resources for genetic preservation and restoration of male germ cells in humans and animals. However, no studies have used SSC as donor nuclei in pig somatic cell nuclear transfer (SCNT). This study investigated the potential for use of porcine SSC as a nuclei donor for SCNT and developmental competence of SSC-derived cloned embryos. In addition, demecolcine was investigated to determine whether it could prevent rupture of SSC during SCNT. When the potential of SSC to support embryonic development after SCNT was compared with that of foetal fibroblasts (FF), SSC-derived SCNT embryos showed a higher (p < .05) developmental competence to the blastocyst stage (47.8%) than FF-derived embryos (25.6%). However, when SSC were used as donor nuclei in the SCNT process, cell fusion rates were lower (p < .05) than when FF were used (61.9% vs. 75.8%). Treatment of SSC with demecolcine significantly (p < .05) decreased rupture of SSC during the SCNT procedure (7.5% vs. 18.8%) and increased fusion of cell-oocyte couplets compared with no treatment (74.6% vs. 61.6%). In addition, SSC-derived SCNT embryos showed higher blastocyst formation (48.4%) than FF-derived embryos without (28.4%) and with demecolcine treatment (17.4%), even after demecolcine treatment. Our results demonstrate that porcine SSC are a desirable donor cell type for production of SCNT pig embryos and that demecolcine increases production efficiency of cloned embryos by inhibiting rupture of nuclei donor SSC during SCNT.


Assuntos
Células-Tronco Germinativas Adultas , Clonagem de Organismos/veterinária , Técnicas de Transferência Nuclear/veterinária , Suínos/embriologia , Animais , Clonagem de Organismos/métodos , Demecolcina/farmacologia , Técnicas de Cultura Embrionária/veterinária , Desenvolvimento Embrionário , Feto/citologia , Fibroblastos/citologia , Moduladores de Tubulina/farmacologia
9.
PLoS One ; 14(7): e0218536, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31306414

RESUMO

BACKGROUND: Stem cell therapy is the next generation a well-established technique. Cell therapy with mesenchymal stem cells (MSC) has been demonstrated to enhance wound healing in diabetic mice, at least partly due to improved growth factor production. However, it is unclear whether MSC can biomechanically affect wound closure. Utilizing the well-established cell-populated collagen gel contraction model we investigated the interactions between MSC and the extracellular matrix. METHODS: Murine fetal liver-derived Mesenchymal Stem Cells (MSCs) or fetal Dermal Fibroblasts (DFs) were cultured in cell-populated collagen gels (CPCGs). The effect of cell density, conditioned media, growth factors (TGF-B1, FGF, PDGF-BB), cytoskeletal disruptors (colchicine, cytochalasin-D), and relative hypoxia on gel contraction were evaluated. Finally, we also measured the expression of integrin receptors and some growth factors by MSCs within the contracting gels. RESULTS: Our results show that at different densities, MSCs induced a higher gel contraction compared to DFs. Higher cell density resulted in faster and more complete contraction of CPCGs. Cytoskeletal inhibitors either inhibited or prevented MSC-mediated contraction in a dose dependent fashion. Growth factors, conditioned media from both MSC and DF, and hypoxia all influenced CPCG contraction. DISCUSSION: The results suggest that MSCs are capable of directly contributing to wound closure through matrix contraction, and they are more effective than DF. In addition, this study demonstrates the importance of how other factors such as cell concentration, cytokines, and oxygen tension can provide potential modulation of therapies to correct wound healing impairments.


Assuntos
Colágeno/metabolismo , Derme/metabolismo , Feto/metabolismo , Fibroblastos/metabolismo , Fígado/metabolismo , Células-Tronco Mesenquimais/metabolismo , Animais , Terapia Baseada em Transplante de Células e Tecidos , Células Cultivadas , Derme/citologia , Feto/citologia , Fibroblastos/citologia , Fígado/citologia , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Transgênicos , Especificidade de Órgãos/fisiologia
10.
Bull Exp Biol Med ; 167(1): 154-158, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31183654

RESUMO

We compared phagocytic activity of macrophages of monocyte origin and Kupffer cells under the influence of M1 and M2 inducers and without activation. Cultures of monocyte-derived macrophages and Kupffer cells were characterized by intensive expression of CD68 that was not affected by activation factors. At the same time, these cultures demonstrated different dynamics of phagocytic activity. Monocyte-derived macrophages initially had more pronounced absorption capacity that gradually increased during the experiment. Kupffer cells were characterized by abrupt fluctuations of phagocytic activity: sharp growth and rapid saturation. Despite these differences, the endosomes produced by monocyte-derived macrophages and Kupffer cells had similar degrees of maturity.


Assuntos
Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Fagocitose/fisiologia , Animais , Células Cultivadas , Tomografia com Microscopia Eletrônica , Feto/citologia , Imuno-Histoquímica , Macrófagos do Fígado/citologia , Macrófagos do Fígado/metabolismo , Fígado/citologia , Fígado/metabolismo , Masculino , Ratos , Ratos Wistar
11.
Nature ; 572(7767): 67-73, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31043743

RESUMO

Study of the origin and development of cerebellar tumours has been hampered by the complexity and heterogeneity of cerebellar cells that change over the course of development. Here we use single-cell transcriptomics to study more than 60,000 cells from the developing mouse cerebellum and show that different molecular subgroups of childhood cerebellar tumours mirror the transcription of cells from distinct, temporally restricted cerebellar lineages. The Sonic Hedgehog medulloblastoma subgroup transcriptionally mirrors the granule cell hierarchy as expected, while group 3 medulloblastoma resembles Nestin+ stem cells, group 4 medulloblastoma resembles unipolar brush cells, and PFA/PFB ependymoma and cerebellar pilocytic astrocytoma resemble the prenatal gliogenic progenitor cells. Furthermore, single-cell transcriptomics of human childhood cerebellar tumours demonstrates that many bulk tumours contain a mixed population of cells with divergent differentiation. Our data highlight cerebellar tumours as a disorder of early brain development and provide a proximate explanation for the peak incidence of cerebellar tumours in early childhood.


Assuntos
Neoplasias Cerebelares/genética , Neoplasias Cerebelares/patologia , Evolução Molecular , Feto/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação Neoplásica da Expressão Gênica , Transcrição Genética , Animais , Neoplasias Cerebelares/classificação , Cerebelo/citologia , Cerebelo/embriologia , Cerebelo/metabolismo , Criança , Feminino , Feto/citologia , Glioma/classificação , Glioma/genética , Glioma/patologia , Humanos , Meduloblastoma/classificação , Meduloblastoma/genética , Meduloblastoma/patologia , Camundongos , Análise de Sequência de RNA , Análise de Célula Única , Fatores de Tempo , Transcriptoma/genética
12.
PLoS One ; 14(5): e0216795, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31083674

RESUMO

The genetic programs responsible for pulmonary lymphatic maturation prior to birth are not known. To address this gap in knowledge, we developed a novel cell sorting strategy to collect fetal pulmonary lymphatic endothelial cells (PLECs) for global transcriptional profiling. We identified PLECs based on their unique cell surface immunophenotype (CD31+/Vegfr3+/Lyve1+/Pdpn+) and isolated them from murine lungs during late gestation (E16.5, E17.5, E18.5). Gene expression profiling was performed using whole-genome microarrays, and 1,281 genes were significantly differentially expressed with respect to time (FDR q < 0.05) and grouped into six clusters. Two clusters containing a total of 493 genes strongly upregulated at E18.5 were significantly enriched in genes with functional annotations corresponding to innate immune response, positive regulation of angiogenesis, complement & coagulation cascade, ECM/cell-adhesion, and lipid metabolism. Gene Set Enrichment Analysis identified several pathways coordinately upregulated during late gestation, the strongest of which was the type-I IFN-α/ß signaling pathway. Upregulation of canonical interferon target genes was confirmed by qRT-PCR and in situ hybridization in E18.5 PLECs. We also identified transcriptional events consistent with a prenatal PLEC maturation program. This PLEC-specific program included individual genes (Ch25h, Itpkc, Pcdhac2 and S1pr3) as well as a set of chemokines and genes containing an NF-κB binding site in their promoter. Overall, this work reveals transcriptional insights into the genes, signaling pathways and biological processes associated with pulmonary lymphatic maturation in the fetal lung.


Assuntos
Células Endoteliais/metabolismo , Desenvolvimento Fetal/fisiologia , Feto/embriologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Transdução de Sinais/fisiologia , Transcriptoma/fisiologia , Animais , Células Endoteliais/citologia , Feto/citologia , Perfilação da Expressão Gênica , Camundongos
13.
Nature ; 570(7759): 107-111, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31092921

RESUMO

Adult intestinal stem cells are located at the bottom of crypts of Lieberkühn, where they express markers such as LGR51,2 and fuel the constant replenishment of the intestinal epithelium1. Although fetal LGR5-expressing cells can give rise to adult intestinal stem cells3,4, it remains unclear whether this population in the patterned epithelium represents unique intestinal stem-cell precursors. Here we show, using unbiased quantitative lineage-tracing approaches, biophysical modelling and intestinal transplantation, that all cells of the mouse intestinal epithelium-irrespective of their location and pattern of LGR5 expression in the fetal gut tube-contribute actively to the adult intestinal stem cell pool. Using 3D imaging, we find that during fetal development the villus undergoes gross remodelling and fission. This brings epithelial cells from the non-proliferative villus into the proliferative intervillus region, which enables them to contribute to the adult stem-cell niche. Our results demonstrate that large-scale remodelling of the intestinal wall and cell-fate specification are closely linked. Moreover, these findings provide a direct link between the observed plasticity and cellular reprogramming of differentiating cells in adult tissues following damage5-9, revealing that stem-cell identity is an induced rather than a hardwired property.


Assuntos
Linhagem da Célula , Intestinos/citologia , Células-Tronco/citologia , Animais , Diferenciação Celular , Reprogramação Celular , Feminino , Feto/citologia , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Intestinos/crescimento & desenvolvimento , Masculino , Camundongos , Receptores Acoplados a Proteínas-G/metabolismo , Regeneração , Nicho de Células-Tronco
14.
Lipids Health Dis ; 18(1): 122, 2019 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-31138220

RESUMO

BACKGROUND: Melanocortin 3 receptor (MC3R), a rhodopsin-like G protein-coupled receptor, is an important regulator of metabolism. Although MC3R knock-out (KO) mice and rats were generated in earlier studies, the function of MC3R remains elusive. Since pig models have many advantages over rodents in metabolism research, we generated an MC3R-KO pig using a CRSPR/Cas9-based system combined with somatic cell nuclear transfer (SCNT) technology. METHOD: Four CRSPR/Cas9 target vectors were constructed and then their cleavage efficiency was tested in porcine fetal fibroblasts (PFFs). The pX330-sgRNA1 and pX330-sgRNA4 vectors were used to co-transfect PFFs to obtain positive colonies. PCR screening and sequencing were conducted to identify the genotype of the colonies. The biallelically modified colonies and wild-type control colonies were used simultaneously as donor cells for SCNT. A total of 1203 reconstructed embryos were transferred into 6 surrogates, of which one became pregnant. The genotypes of the resulting piglets were determined by PCR and sequencing, and off-target effects in the MC3R KO piglets were detected by sequencing. Then, offspring were obtained through breeding and six male KO pigs were used for the growth performance analysis. RESULTS: Four vectors were constructed successfully, and their cleavage efficiencies were 27.96, 44.89, 32.72 and 38.86%, respectively. A total of 21 mutant colonies, including 11 MC3R-/- and 10 MC3R+/- clones, were obtained, corresponding to a gene targeting efficiency of 29.17%, with 15.28% biallelic mutations. A total of 6 piglets were born, and only two MC3R KO piglets were generated, one with malformations and a healthy one. No off-target effects were detected by sequencing in the healthy mutant. Six male MC3R KO pigs were obtained in the F2 generation and their body weight and body fat were both increased compared to wild-type full siblings. CONCLUSION: A MC3R KO pig strain was generated using the CRSIPR/Cas9-based system, which makes it possible to study the biological function of MC3R in a non-rodent model.


Assuntos
Sistemas CRISPR-Cas/genética , Técnicas de Inativação de Genes , Técnicas de Transferência Nuclear , Receptor Tipo 3 de Melanocortina/deficiência , Adiposidade , Animais , Animais Geneticamente Modificados , Sequência de Bases , Peso Corporal , Feto/citologia , Fibroblastos/metabolismo , Marcação de Genes , Vetores Genéticos/metabolismo , RNA Guia/metabolismo , Receptor Tipo 3 de Melanocortina/metabolismo , Suínos
15.
J Vis Exp ; (145)2019 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-30958455

RESUMO

Current knowledge of extracellular matrix (ECM)-cell communication translates to large two-dimensional (2D) in vitro culture studies where ECM components are presented as a surface coating. These culture systems constitute a simplification of the complex nature of the tissue ECM that encompasses biochemical composition, structure, and mechanical properties. To better emulate the ECM-cell communication shaping the cardiac microenvironment, we developed a protocol that allows for the decellularization of the whole fetal heart and adult left ventricle tissue explants simultaneously for comparative studies. The protocol combines the use of a hypotonic buffer, a detergent of anionic surfactant properties, and DNase treatment without any requirement for specialized skills or equipment. The application of the same decellularization strategy across tissue samples from subjects of various age is an alternative approach to perform comparative studies. The present protocol allows the identification of unique structural differences across fetal and adult cardiac ECM mesh and biological cellular responses. Furthermore, the herein methodology demonstrates a broader application being successfully applied in other tissues and species with minor adjustments, such as in human intestine biopsies and mouse lung.


Assuntos
Técnicas de Cultura de Células/métodos , Matriz Extracelular/metabolismo , Feto/citologia , Miocárdio/citologia , Adulto , Animais , Humanos , Camundongos , Engenharia Tecidual
16.
Int J Mol Sci ; 20(7)2019 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-30934825

RESUMO

One of the main aims in regenerative medicine is to find stem cells that are easy to obtain and are safe and efficient in either an autologous or allogenic host when transplanted. This review provides an overview of the potential use of the fetal annexes in regenerative medicine: we described the formation of the annexes, their immunological features, the new advances in the phenotypical characterization of fetal annexes-derived stem cells, the progressions obtained in the analysis of both their differentiative potential and their secretoma, and finally, the potential use of decellularized fetal membranes. Normally discarded as medical waste, the umbilical cord and perinatal tissue not only represent a rich source of stem cells but can also be used as a scaffold for regenerative medicine, providing a suitable environment for the growth and differentiation of stem cells.


Assuntos
Feto/citologia , Medicina Regenerativa , Diferenciação Celular , Humanos , Comunicação Parácrina , Células-Tronco/citologia , Geleia de Wharton/citologia
17.
Int J Mol Sci ; 20(6)2019 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-30889841

RESUMO

Human cells, when exposed to both real and simulated microgravity (s-µg), form 3D tissue constructs mirroring in vivo architectures (e.g., cartilage, intima constructs, cancer spheroids and others). In this study, we exposed human foetal osteoblast (hFOB 1.19) cells to a Random Positioning Machine (RPM) for 7 days and 14 days, with the purpose of investigating the effects of s-µg on biological processes and to engineer 3D bone constructs. RPM exposure of the hFOB 1.19 cells induces alterations in the cytoskeleton, cell adhesion, extra cellular matrix (ECM) and the 3D multicellular spheroid (MCS) formation. In addition, after 7 days, it influences the morphological appearance of these cells, as it forces adherent cells to detach from the surface and assemble into 3D structures. The RPM-exposed hFOB 1.19 cells exhibited a differential gene expression of the following genes: transforming growth factor beta 1 (TGFB1, bone morphogenic protein 2 (BMP2), SRY-Box 9 (SOX9), actin beta (ACTB), beta tubulin (TUBB), vimentin (VIM), laminin subunit alpha 1 (LAMA1), collagen type 1 alpha 1 (COL1A1), phosphoprotein 1 (SPP1) and fibronectin 1 (FN1). RPM exposure also induced a significantly altered release of the cytokines and bone biomarkers sclerostin (SOST), osteocalcin (OC), osteoprotegerin (OPG), osteopontin (OPN), interleukin 1 beta (IL-1ß) and tumour necrosis factor 1 alpha (TNF-1α). After the two-week RPM exposure, the spheroids presented a bone-specific morphology. In conclusion, culturing cells in s-µg under gravitational unloading represents a novel technology for tissue-engineering of bone constructs and it can be used for investigating the mechanisms behind spaceflight-related bone loss as well as bone diseases such as osteonecrosis or bone injuries.


Assuntos
Osso e Ossos/fisiologia , Feto/citologia , Osteoblastos/citologia , Engenharia Tecidual/métodos , Proteína Morfogenética Óssea 2/metabolismo , Forma Celular , Células Cultivadas , Citoesqueleto/metabolismo , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Humanos , Organoides/citologia , Osteoblastos/metabolismo , Osteogênese , Ligação Proteica , Transdução de Sinais , Solubilidade , Frações Subcelulares/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Simulação de Ausência de Peso
18.
Differentiation ; 106: 9-14, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30826473

RESUMO

The aim of this review is to summarize and give an overview on the findings of signaling between hepatic and hematopoietic progenitors of the liver. To date, there are not many findings published in the field, and the aim of this review is to cover all current publications in this area. The liver is the main site of hematopoiesis during fetal development. However, little is known about how hepatic and other non-hematopoietic progenitors potentially influence hematopoiesis and vice versa. The concurrent peaks of hepatic and hematopoietic progenitor proliferation during development indicate interactions that could possibly be mediated through cell-cell contact, extracellular matrices, cytokines and growth factors, or other signaling molecules. For example, hepatic progenitors, such as hepatic stem cells and hepatoblasts, possess characteristic surface markers that can be cleaved, giving rise to fragments of various lengths. A surface molecule of hepatoblasts has been demonstrated to play an essential role in hematopoiesis. Particularly, these effects on hematopoiesis were distinct, depending on whether it was membrane-bound or cleaved. In this review, the various hepatic and hematopoietic progenitor cell types are concisely described, and the current findings of their potential interactions are summarized.


Assuntos
Diferenciação Celular , Feto/citologia , Células-Tronco Hematopoéticas/citologia , Fígado/citologia , Animais , Feto/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Humanos , Fígado/fisiologia
19.
Biochem Biophys Res Commun ; 512(2): 326-330, 2019 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-30890337

RESUMO

Umbilical cord blood contains mesenchymal stem/stromal cells (MSCs) in addition to hematopoietic stem cells, serving as an attractive tool for regenerative medicine. As umbilical cord blood originates from fetus, abundant MSCs are expected to circulate in fetus. However, the properties of circulating MSCs in fetus have not been fully examined. In the present study, we aimed to analyze circulating MSCs, marked by the expression of platelet-derived growth factor receptor α (PDGFRα), during fetal development. Using PDGFRα GFP knock-in mice, we quantified the number of circulating PDGFRα positive MSCs during development. We further performed whole transcriptome analysis of circulating MSCs at single cell levels. We found that abundant PDGFRα positive cells circulate in embryo and diminish immediately after birth. In addition, single cell RNA-sequencing revealed transcriptional heterogeneity of MSCs in fetal circulation. These data lay a foundation to analyze the function of circulating MSCs during development.


Assuntos
Sangue Fetal/citologia , Sangue Fetal/metabolismo , Feto/citologia , Feto/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Animais , Contagem de Células , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Gravidez , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Medicina Regenerativa , Análise de Célula Única , Transcrição Genética
20.
Stem Cell Res ; 35: 101405, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30776675

RESUMO

Human induced pluripotent stem cell line was generated from commercially available primary human prostate fibroblasts HPrF derived from a fetus, aged 18-24 weeks of gestation. The fibroblast cell line was reprogrammed with Yamanaka factors (OCT4, SOX2, c-MYC, KLF4) using CytoTune™-iPS 2.0 Sendai Reprogramming Kit. Pluripotency of the derived transgene-free iPS cell line was confirmed both in vitro by detecting the expression of factors of pluripotency on a single-cell level, and in vivo using teratoma formation assay. This iPS cell line will be a useful tool for studying both normal prostate development and prostate cancer disease.


Assuntos
Técnicas de Reprogramação Celular , Feto , Fibroblastos , Células-Tronco Pluripotentes Induzidas , Próstata , Reprogramação Celular , Feto/citologia , Feto/embriologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Próstata/citologia , Próstata/embriologia
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