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1.
Int J Mol Sci ; 22(19)2021 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-34639216

RESUMO

In the placenta the proliferative cytotrophoblast cells fuse into the terminally differentiated syncytiotrophoblast layer which undertakes several energy-intensive functions including nutrient uptake and transfer and hormone synthesis. We used Seahorse glycolytic and mitochondrial stress tests on trophoblast cells isolated at term from women of healthy weight to evaluate if cytotrophoblast (CT) and syncytiotrophoblast (ST) have different bioenergetic strategies, given their different functions. Whereas there are no differences in basal glycolysis, CT have significantly greater glycolytic capacity and reserve than ST. In contrast, ST have significantly higher basal, ATP-coupled and maximal mitochondrial respiration and spare capacity than CT. Consequently, under stress conditions CT can increase energy generation via its higher glycolytic capacity whereas ST can use its higher and more efficient mitochondrial respiration capacity. We have previously shown that with adverse in utero conditions of diabetes and obesity trophoblast respiration is sexually dimorphic. We found no differences in glycolytic parameters between sexes and no difference in mitochondrial respiration parameters other than increases seen upon syncytialization appear to be greater in females. There were differences in metabolic flexibility, i.e., the ability to use glucose, glutamine, or fatty acids, seen upon syncytialization between the sexes with increased flexibility in female trophoblast suggesting a better ability to adapt to changes in nutrient supply.


Assuntos
Feto/fisiologia , Glicólise , Mitocôndrias/fisiologia , Placenta/fisiologia , Caracteres Sexuais , Trofoblastos/fisiologia , Adulto , Respiração Celular , Feminino , Feto/citologia , Humanos , Técnicas In Vitro , Masculino , Placenta/citologia , Gravidez , Trofoblastos/citologia
2.
Nature ; 597(7875): 196-205, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34497388

RESUMO

The Human Developmental Cell Atlas (HDCA) initiative, which is part of the Human Cell Atlas, aims to create a comprehensive reference map of cells during development. This will be critical to understanding normal organogenesis, the effect of mutations, environmental factors and infectious agents on human development, congenital and childhood disorders, and the cellular basis of ageing, cancer and regenerative medicine. Here we outline the HDCA initiative and the challenges of mapping and modelling human development using state-of-the-art technologies to create a reference atlas across gestation. Similar to the Human Genome Project, the HDCA will integrate the output from a growing community of scientists who are mapping human development into a unified atlas. We describe the early milestones that have been achieved and the use of human stem-cell-derived cultures, organoids and animal models to inform the HDCA, especially for prenatal tissues that are hard to acquire. Finally, we provide a roadmap towards a complete atlas of human development.


Assuntos
Movimento Celular , Rastreamento de Células , Células/citologia , Biologia do Desenvolvimento/métodos , Embrião de Mamíferos/citologia , Feto/citologia , Disseminação de Informação , Organogênese , Adulto , Animais , Atlas como Assunto , Técnicas de Cultura de Células , Sobrevivência Celular , Visualização de Dados , Feminino , Humanos , Imageamento Tridimensional , Masculino , Modelos Animais , Organogênese/genética , Organoides/citologia , Células-Tronco/citologia
3.
Nature ; 597(7875): 250-255, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34497389

RESUMO

The cellular landscape of the human intestinal tract is dynamic throughout life, developing in utero and changing in response to functional requirements and environmental exposures. Here, to comprehensively map cell lineages, we use single-cell RNA sequencing and antigen receptor analysis of almost half a million cells from up to 5 anatomical regions in the developing and up to 11 distinct anatomical regions in the healthy paediatric and adult human gut. This reveals the existence of transcriptionally distinct BEST4 epithelial cells throughout the human intestinal tract. Furthermore, we implicate IgG sensing as a function of intestinal tuft cells. We describe neural cell populations in the developing enteric nervous system, and predict cell-type-specific expression of genes associated with Hirschsprung's disease. Finally, using a systems approach, we identify key cell players that drive the formation of secondary lymphoid tissue in early human development. We show that these programs are adopted in inflammatory bowel disease to recruit and retain immune cells at the site of inflammation. This catalogue of intestinal cells will provide new insights into cellular programs in development, homeostasis and disease.


Assuntos
Envelhecimento , Sistema Nervoso Entérico/citologia , Feto/citologia , Saúde , Intestinos/citologia , Intestinos/crescimento & desenvolvimento , Linfonodos/citologia , Linfonodos/crescimento & desenvolvimento , Adulto , Animais , Criança , Doença de Crohn/patologia , Conjuntos de Dados como Assunto , Sistema Nervoso Entérico/anatomia & histologia , Sistema Nervoso Entérico/embriologia , Sistema Nervoso Entérico/crescimento & desenvolvimento , Células Epiteliais/citologia , Feminino , Feto/anatomia & histologia , Feto/embriologia , Humanos , Intestinos/embriologia , Intestinos/inervação , Linfonodos/embriologia , Linfonodos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Organogênese , Receptores de IgG/metabolismo , Transdução de Sinais , Análise Espaço-Temporal , Fatores de Tempo
4.
Nature ; 598(7880): 327-331, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34588693

RESUMO

Haematopoiesis in the bone marrow (BM) maintains blood and immune cell production throughout postnatal life. Haematopoiesis first emerges in human BM at 11-12 weeks after conception1,2, yet almost nothing is known about how fetal BM (FBM) evolves to meet the highly specialized needs of the fetus and newborn. Here we detail the development of FBM, including stroma, using multi-omic assessment of mRNA and multiplexed protein epitope expression. We find that the full blood and immune cell repertoire is established in FBM in a short time window of 6-7 weeks early in the second trimester. FBM promotes rapid and extensive diversification of myeloid cells, with granulocytes, eosinophils and dendritic cell subsets emerging for the first time. The substantial expansion of B lymphocytes in FBM contrasts with fetal liver at the same gestational age. Haematopoietic progenitors from fetal liver, FBM and cord blood exhibit transcriptional and functional differences that contribute to tissue-specific identity and cellular diversification. Endothelial cell types form distinct vascular structures that we show are regionally compartmentalized within FBM. Finally, we reveal selective disruption of B lymphocyte, erythroid and myeloid development owing to a cell-intrinsic differentiation bias as well as extrinsic regulation through an altered microenvironment in Down syndrome (trisomy 21).


Assuntos
Células da Medula Óssea/citologia , Medula Óssea , Síndrome de Down/sangue , Síndrome de Down/imunologia , Feto/citologia , Hematopoese , Sistema Imunitário/citologia , Linfócitos B/citologia , Células Dendríticas/citologia , Síndrome de Down/metabolismo , Síndrome de Down/patologia , Células Endoteliais/patologia , Eosinófilos/citologia , Células Eritroides/citologia , Granulócitos/citologia , Humanos , Imunidade , Células Mieloides/citologia , Células Estromais/citologia
5.
Cells ; 10(9)2021 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-34572090

RESUMO

In this study, transcriptomic changes of the developing brain of pig fetuses of both sexes were investigated on gestation days (GD) 45, 60 and 90. Pig fetal brain grows rapidly around GD60. Consequently, gene expression of the fetal brain was distinctly different on GD90 compared to that of GD45 and GD60. In addition, varying numbers of differentially expressed genes (DEGs) were identified in the male brain compared to the female brain during development. The sex of adjacent fetuses also influenced gene expression of the fetal brain. Extensive changes in gene expression at the exon-level were observed during brain development. Pathway enrichment analysis showed that the ionotropic glutamate receptor pathway and p53 pathway were enriched in the female brain, whereas specific receptor-mediated signaling pathways were enriched in the male brain. Marker genes of neurons and astrocytes were significantly differentially expressed between male and female brains during development. Furthermore, comparative analysis of gene expression patterns between fetal brain and placenta suggested that genes related to ion transportation may play a key role in the regulation of the brain-placental axis in pig. Collectively, the study suggests potential application of pig models to better understand influence of fetal sex on brain development.


Assuntos
Encéfalo/metabolismo , Feto/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Placenta/metabolismo , Caracteres Sexuais , Transcriptoma , Animais , Encéfalo/citologia , Feminino , Feto/citologia , Masculino , Placenta/citologia , Gravidez , Transdução de Sinais , Sus scrofa
6.
Cells ; 10(8)2021 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-34440750

RESUMO

Canines are useful in mammalian preclinical studies because they are larger than rodents and share many diseases with humans. Canine fetal fibroblast cells (CFFs) are an easily accessible source of somatic cells. However, they are easily driven to senescence and become unusable with continuous in vitro culture. Therefore, to overcome these deficiencies, we investigated whether tetracycline-inducible L-myc gene expression promotes self-renewal activity and tumorigenicity in the production of induced conditional self-renewing fibroblast cells (iCSFCs). Here, we describe the characterization of a new iCSFC line immortalized by transduction with L-myc that displays in vitro self-renewal ability without tumorigenic capacity. We established conditionally inducible self-renewing fibroblast cells by transducing CFF-3 cells with L-myc under the tetracycline-inducible gene expression system. In the absence of doxycycline, the cells did not express L-myc or undergo self-renewal. The iCSFCs had a fibroblast-like morphology, normal chromosome pattern, and expressed fibroblast-specific genes and markers. However, the iCSFCs did not form tumors in a soft agar colony-forming assay. We observed higher expression of three ES modules (core pluripotency genes, polycomb repressive complex genes (PRC), and MYC-related genes) in the iCSFCs than in the CFF-3 cells; in particular, the core pluripotency genes (OCT4, SOX2, and NANOG) were markedly up-regulated compared with the PRC and MYC module genes. These results demonstrated that, in canine fetal fibroblasts, L-myc tetracycline-inducible promoter-driven gene expression induces self-renewal capacity but not tumor formation. This study suggests that L-myc gene-induced conditional self-renewing fibroblast cells can be used as an in vitro tool in a variety of biomedical studies related to drug screening.


Assuntos
Autorrenovação Celular/fisiologia , Proteínas Proto-Oncogênicas c-myc/genética , Animais , Proliferação de Células , Reprogramação Celular , Cães , Feminino , Feto/citologia , Feto/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Fase G1 , Proteínas do Grupo Polycomb/metabolismo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-myc/metabolismo
7.
Sci Rep ; 11(1): 12796, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-34140608

RESUMO

In mammals, neural crest cells populate the gut and form the enteric nervous system (ENS) early in embryogenesis. Although the basic ENS structure is highly conserved across species, we show important differences between mice and humans relating to the prenatal and postnatal development of mucosal enteric glial cells (mEGC), which are essential ENS components. We confirm previous work showing that in the mouse mEGCs are absent at birth, and that their appearance and homeostasis depends on postnatal colonization by microbiota. In humans, by contrast, a network of glial cells is already present in the fetal gut. Moreover, in xenografts of human fetal gut maintained for months in immuno-compromised mice, mEGCs persist following treatment with antibiotics that lead to the disappearance of mEGCs from the gut of the murine host. Single cell RNAseq indicates that human and mouse mEGCs differ not only in their developmental dynamics, but also in their patterns of gene expression.


Assuntos
Microbioma Gastrointestinal , Homeostase , Mucosa Intestinal/citologia , Neuroglia/metabolismo , Animais , Feminino , Feto/citologia , Regulação da Expressão Gênica , Xenoenxertos , Humanos , Camundongos , Neuroglia/citologia
8.
Reprod Domest Anim ; 56(9): 1243-1253, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34174122

RESUMO

Retention of foetal membranes (RFM) is a major reproductive disorder in dairy cows. An appropriate immune response is important for a physiological expulsion of the foetal membranes at parturition. Our study aims to provide a deeper insight into characteristics of foetal and maternal macrophages in bovine term placenta. We used transmission electron microscopy (TEM), immunohistochemistry and semi-quantitative RT-PCR to provide a deeper insight into characteristics of foetal and maternal macrophages in bovine term placenta. Semi-quantitative RT-PCR was used to define macrophage polarization in foetal and maternal compartments of normal term placenta. Gene expression of factors involved in M1 polarization [interferon regulatory factor-5 (IRF5), interleukin (IL)-12A, IL12B] and in M2 polarization (IL10) were studied. Ultrastructurally, foetal macrophages showed an irregular shape and large vacuoles, whereas the maternal macrophages were spindle shaped. By immunohistochemistry, macrophages were identified by a strong staining with the lysosomal marker Lysosome-associated membrane glycoprotein 1 (LAMP-1), while myofibroblast in the maternal stroma was positive for alpha-smooth muscle actin. We used the LAMP-1 marker to compare the density of foetal stromal macrophages in placentas of cows with RFM and in controls, but no statistically significant difference was observed. RT-PCR showed a higher expression of all studied genes in the maternal compartment of the placenta and generally a higher expression of M1-, compared to M2-associated genes. Our results indicated that at parturition placental macrophages predominantly show the pro-inflammatory M1 polarization. The higher expression of all the target genes in the maternal compartment may denote that maternal macrophages in bovine term placenta are more frequent than foetal macrophages.


Assuntos
Feto/citologia , Macrófagos/fisiologia , Placenta/citologia , Animais , Bovinos , Feminino , Feto/imunologia , Macrófagos/ultraestrutura , Parto , Placenta/imunologia , Gravidez , Transcriptoma
9.
Gene ; 790: 145706, 2021 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-33979681

RESUMO

Previous studies have shown that extracellular vesicles (EVs) containing proteins, lipids, nucleic acids and other biological components exist in all kinds of body fluids. EVs, as an intercellular communication carrier, regulate the functions of its target cells by transporting biomacromolecules between cells. In this study, a total of six female Dazu black goats were divided into NP group (NP, non-pregnant group) and P30 (P30, 30-day pregnant group). The goats in NP group (n = 3) were in estrus, but failed to fertilize; the other goats in P30 group (n = 3) were fertilized by natural mating. Firstly, goats plasma-derived EVs were isolated using ultracentrifugation. Secondly, EVs were identified by transmission electron microscope (TEM), dynamic light scattering (DLS), and by testing its markers (CD9 and CD63) using west blotting in NP and P30 groups, respectively. Thirdly, EVs related miRNAs were sequenced and analyzed by bioinformatics method. Data shows that miR-31-5p, miR-137-3p, novel_miR_1355, novel_miR_734 and novel_miR_736 exclusively were expressed in P30 group. Their target genes were significantly enriched in the axon guidance, the Notch signaling pathway, the Wnt signaling pathway, tight junction and the Hippo signaling pathway. And miRNA-mRNA interactive network analysis reveals potential regulatory functions of miRNAs for goat during early pregnancy. These findings provided theretical references for studying the regulation of plasma-derived EVs between the fetal and placental development, and these candidate miRNAs identified might be as markers for diagnosis of goat early pregnancy.


Assuntos
MicroRNA Circulante/sangue , MicroRNA Circulante/genética , Vesículas Extracelulares/metabolismo , Feto/metabolismo , Placentação , RNA Mensageiro/metabolismo , Animais , Biologia Computacional , Vesículas Extracelulares/genética , Feminino , Feto/citologia , Cabras , Gravidez , RNA Mensageiro/genética
10.
Nature ; 595(7865): 85-90, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33981037

RESUMO

The ontogeny of the human haematopoietic system during fetal development has previously been characterized mainly through careful microscopic observations1. Here we reconstruct a phylogenetic tree of blood development using whole-genome sequencing of 511 single-cell-derived haematopoietic colonies from healthy human fetuses at 8 and 18 weeks after conception, coupled with deep targeted sequencing of tissues of known embryonic origin. We found that, in healthy fetuses, individual haematopoietic progenitors acquire tens of somatic mutations by 18 weeks after conception. We used these mutations as barcodes and timed the divergence of embryonic and extra-embryonic tissues during development, and estimated the number of blood antecedents at different stages of embryonic development. Our data support a hypoblast origin of the extra-embryonic mesoderm and primitive blood in humans.


Assuntos
Linhagem da Célula/genética , Desenvolvimento Embrionário/genética , Sistema Hematopoético/embriologia , Sistema Hematopoético/metabolismo , Mutação , Células Sanguíneas/citologia , Células Sanguíneas/metabolismo , Células Clonais/citologia , Células Clonais/metabolismo , Análise Mutacional de DNA , Feto/citologia , Feto/embriologia , Feto/metabolismo , Camadas Germinativas/citologia , Camadas Germinativas/metabolismo , Saúde , Sistema Hematopoético/citologia , Humanos , Cariotipagem , Masculino , Mesoderma/citologia , Mesoderma/embriologia , Mesoderma/metabolismo , Taxa de Mutação , Especificidade de Órgãos/genética , Fatores de Tempo , Sequenciamento Completo do Genoma , Fluxo de Trabalho
11.
Sci Rep ; 11(1): 9356, 2021 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-33931678

RESUMO

The endocannabinoid system (ECS) plays a complex role in the development of neural circuitry during fetal brain development. The cannabinoid receptor type 1 (CB1) controls synaptic strength at both excitatory and inhibitory synapses and thus contributes to the balance of excitatory and inhibitory signaling. Imbalances in the ratio of excitatory to inhibitory synapses have been implicated in various neuropsychiatric disorders associated with dysregulated central nervous system development including autism spectrum disorder, epilepsy, and schizophrenia. The role of CB1 in human brain development has been difficult to study but advances in induced pluripotent stem cell technology have allowed us to model the fetal brain environment. Cortical spheroids resemble the cortex of the dorsal telencephalon during mid-fetal gestation and possess functional synapses, spontaneous activity, an astrocyte population, and pseudo-laminar organization. We first characterized the ECS using STORM microscopy and observed synaptic localization of components similar to that which is observed in the fetal brain. Next, using the CB1-selective antagonist SR141716A, we observed an increase in excitatory, and to a lesser extent, inhibitory synaptogenesis as measured by confocal image analysis. Further, CB1 antagonism increased the variability of spontaneous activity within developing neural networks, as measured by microelectrode array. Overall, we have established that cortical spheroids express ECS components and are thus a useful model for exploring endocannabinoid mediation of childhood neuropsychiatric disease.


Assuntos
Encéfalo/fisiologia , Córtex Cerebral/fisiologia , Feto/fisiologia , Receptor CB1 de Canabinoide/antagonistas & inibidores , Rimonabanto/farmacologia , Esferoides Celulares/fisiologia , Sinapses/fisiologia , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Astrócitos/fisiologia , Encéfalo/citologia , Encéfalo/efeitos dos fármacos , Antagonistas de Receptores de Canabinoides/farmacologia , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , Feto/citologia , Feto/efeitos dos fármacos , Humanos , Transdução de Sinais , Esferoides Celulares/citologia , Esferoides Celulares/efeitos dos fármacos , Sinapses/efeitos dos fármacos
12.
PLoS One ; 16(4): e0249695, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33857205

RESUMO

A major challenge for cell-based non-invasive prenatal testing (NIPT) is to distinguish individual presumptive fetal cells from maternal cells in female pregnancies. We have sought a rapid, robust, versatile, and low-cost next-generation sequencing method to facilitate this process. Toward this goal, single isolated cells underwent whole genome amplification prior to genotyping. Multiple highly polymorphic genomic regions (including HLA-A and HLA-B) with 10-20 very informative single nucleotide polymorphisms (SNPs) within a 200 bp interval were amplified with a modified method based on other publications. To enhance the power of cell identification, approximately 40 Human Identification SNP (Applied Biosystems) test amplicons were also utilized. Using SNP results to compare to sex chromosome data from NGS as a reliable standard, the true positive rate for genotyping was 83.4%, true negative 6.6%, false positive 3.3%, and false negative 6.6%. These results would not be sufficient for clinical diagnosis, but they demonstrate the general validity of the approach and suggest that deeper genotyping of single cells could be completely reliable. A paternal DNA sample is not required using this method. The assay also successfully detected pathogenic variants causing Tay Sachs disease, cystic fibrosis, and hemoglobinopathies in single lymphoblastoid cells, and disease-causing variants in three cell-based NIPT cases. This method could be applicable for any monogenic diagnosis.


Assuntos
DNA/genética , Feto/citologia , Doenças Genéticas Inatas/diagnóstico , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Teste Pré-Natal não Invasivo/métodos , Análise de Célula Única/métodos , Trofoblastos/citologia , Feminino , Feto/metabolismo , Doenças Genéticas Inatas/genética , Testes Genéticos/métodos , Humanos , Gravidez , Trofoblastos/metabolismo
13.
Neurochem Res ; 46(8): 1933-1940, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33914233

RESUMO

Agmatine, an endogenous derivative of arginine, has been found to be effective in treating idiopathic pain, convulsion, stress-mediated behavior, and attenuate the withdrawal symptoms of drugs like morphine. In the early stages of ischemic brain injury in animals, exogenous agmatine treatment was found to be neuroprotective. Agmatine is also considered as a putative neurotransmitter and is still an experimental drug. Chemically, agmatine is called agmatine 1-(4-aminobutyl guanidine). Crystallographic study data show that positively-charged guanidine can bind to the protein containing Gly and Asp residues, and the amino group can interact with the complimentary sites of Glu and Ser. In this study, we blocked the amino end of the agmatine by conjugating it with FITC, but the guanidine end was unchanged. We compared the neuroprotective function of the agmatine and agmatine-FITC by treating them in neurons after excitotoxic stimulation. We found that even the amino end blocked neuronal viability in the excitotoxic condition, by NMDA treatment for 1 h, was increased by agmatine-FITC, which was similar to that of agmatine. We also found that the agmatine-FITC treatment reduced the expression of nitric oxide production in NMDA-treated cells. This study suggests that even if the amino end of agmatine is blocked, it can perform its neuroprotective function.


Assuntos
Agmatina/farmacologia , Neurônios/efeitos dos fármacos , Neuroproteção/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Agmatina/química , Animais , Células Cultivadas , Córtex Cerebral/citologia , Feminino , Feto/citologia , Fluoresceína-5-Isotiocianato/química , Fluoresceína-5-Isotiocianato/farmacologia , Camundongos Endogâmicos ICR , N-Metilaspartato/toxicidade , Fármacos Neuroprotetores/química , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo
14.
Science ; 371(6535): 1245-1248, 2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33737484

RESUMO

Mosaic mutations can be used to track cell lineages in humans. We used cell cloning to analyze embryonic cell lineages in two living individuals and a postmortem human specimen. Of 10 reconstructed postzygotic divisions, none resulted in balanced contributions of daughter lineages to tissues. In both living individuals, one of two lineages from the first cleavage was dominant across tissues, with 90% frequency in blood. We propose that the efficiency of DNA repair contributes to lineage imbalance. Allocation of lineages in postmortem brain correlated with anterior-posterior axis, associating lineage history with cell fate choices in embryos. We establish a minimally invasive framework for defining cell lineages in any living individual, which paves the way for studying their relevance in health and disease.


Assuntos
Blastômeros/citologia , Divisão Celular , Linhagem da Célula , Desenvolvimento Embrionário , Adulto , Idoso , Blastocisto/citologia , Células Sanguíneas , Diferenciação Celular , Linhagem Celular , Reparo do DNA , Feminino , Feto/citologia , Variação Genética , Genoma Humano , Humanos , Mutação INDEL , Células-Tronco Pluripotentes Induzidas/citologia , Masculino , Células-Tronco Neurais/citologia , Polimorfismo de Nucleotídeo Único
15.
Int J Mol Sci ; 22(4)2021 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-33669517

RESUMO

Despite low levels of vascular endothelial growth factor (VEGF)-A, the secretome of human Wharton's jelly (WJ) mesenchymal stromal cells (MSCs) effectively promoted proangiogenic responses in vitro, which were impaired upon the depletion of small (~140 nm) extracellular vesicles (EVs). The isolated EVs shared the low VEGF-A profile of the secretome and expressed five microRNAs, which were upregulated compared to fetal dermal MSC-derived EVs. These upregulated microRNAs exclusively targeted the VEGF-A gene within 54 Gene Ontology (GO) biological processes, 18 of which are associated with angiogenesis. Moreover, 15 microRNAs of WJ-MSC-derived EVs were highly expressed (Ct value ≤ 26) and exclusively targeted the thrombospondin 1 (THBS1) gene within 75 GO biological processes, 30 of which are associated with the regulation of tissue repair. The relationship between predicted microRNA target genes and WJ-MSC-derived EVs was shown by treating human umbilical-vein endothelial cells (HUVECs) with appropriate doses of EVs. The exposure of HUVECs to EVs for 72 h significantly enhanced the release of VEGF-A and THBS1 protein expression compared to untreated control cells. Finally, WJ-MSC-derived EVs stimulated in vitro tube formation along with the migration and proliferation of HUVECs. Our findings can contribute to a better understanding of the molecular mechanisms underlying the proangiogenic responses induced by human umbilical cord-derived MSCs, suggesting a key regulatory role for microRNAs delivered by EVs.


Assuntos
Vesículas Extracelulares/metabolismo , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/metabolismo , Neovascularização Fisiológica , Fator A de Crescimento do Endotélio Vascular/metabolismo , Geleia de Wharton/citologia , Movimento Celular , Proliferação de Células , Separação Celular , Feto/citologia , Fluoresceínas/metabolismo , Ontologia Genética , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Imunofenotipagem , MicroRNAs/genética , Nanopartículas/química , Reprodutibilidade dos Testes , Pele/citologia , Succinimidas/metabolismo , Trombospondina 1/metabolismo , Cordão Umbilical/citologia
16.
Sci Rep ; 11(1): 6867, 2021 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-33767268

RESUMO

Significant shape changes in the human facial skeleton occur in the early prenatal period, and understanding this process is critical for studying a myriad of congenital facial anomalies. However, quantifying and visualizing human fetal facial growth has been challenging. Here, we applied quantitative geometric morphometrics (GM) to high-resolution magnetic resonance images of human embryo and fetuses, to comprehensively analyze facial growth. We utilized non-linear growth estimation and GM methods to assess integrated epigenetic growth between masticatory muscles and associated bones. Our results show that the growth trajectory of the human face in the early prenatal period follows a curved line with three flexion points. Significant antero-posterior development occurs early, resulting in a shift from a mandibular prognathic to relatively orthognathic appearance, followed by expansion in the lateral direction. Furthermore, during this time, the development of the zygoma and the mandibular ramus is closely integrated with the masseter muscle.


Assuntos
Face/fisiologia , Feto/citologia , Mandíbula/crescimento & desenvolvimento , Músculo Masseter/crescimento & desenvolvimento , Músculos da Mastigação/crescimento & desenvolvimento , Crânio/crescimento & desenvolvimento , Face/anatomia & histologia , Feminino , Feto/fisiologia , Humanos , Masculino , Gravidez
17.
Cells ; 10(3)2021 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-33668852

RESUMO

The phenomenon of the reprogramming of terminally differentiated cells can be achieved by various means, like somatic cell nuclear transfer, cell fusion with a pluripotent cell, or the introduction of pluripotency genes. Here, we present the evidence that somatic cells can attain the expression of pluripotency markers after their introduction into early embryos. Mouse embryonic fibroblasts introduced between blastomeres of cleaving embryos, within two days of in vitro culture, express transcription factors specific to blastocyst lineages, including pluripotency factors. Analysis of donor tissue marker DNA has revealed that the progeny of introduced cells are found in somatic tissues of foetuses and adult chimaeras, providing evidence for cell reprogramming. Analysis of ploidy has shown that in the chimaeras, the progeny of introduced cells are either diploid or tetraploid, the latter indicating cell fusion. The presence of donor DNA in diploid cells from chimaeric embryos proved that the non-fused progeny of introduced fibroblasts persisted in chimaeras, which is evidence of reprogramming by embryonic niche. When adult somatic (cumulus) cells were introduced into early cleavage embryos, the extent of integration was limited and only cell fusion-mediated reprogramming was observed. These results show that both cell fusion and cell interactions with the embryonic niche reprogrammed somatic cells towards pluripotency.


Assuntos
Envelhecimento/fisiologia , Biomarcadores/metabolismo , Reprogramação Celular , Quimera/fisiologia , Embrião de Mamíferos/citologia , Células-Tronco Pluripotentes/metabolismo , Animais , Blastocisto/citologia , Blastômeros/citologia , Fusão Celular , Linhagem Celular , Células do Cúmulo/citologia , Diploide , Técnicas de Cultura Embrionária , Desenvolvimento Embrionário , Feminino , Feto/citologia , Corantes Fluorescentes/metabolismo , Camundongos , Mórula/citologia , Células-Tronco Pluripotentes/citologia , Gravidez , Tetraploidia
18.
Biomolecules ; 11(2)2021 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-33572428

RESUMO

Diverse cell therapy approaches constitute prime developmental prospects for managing acute or degenerative cartilaginous tissue affections, synergistically complementing specific surgical solutions. Bone marrow stimulation (i.e., microfracture) remains a standard technique for cartilage repair promotion, despite incurring the adverse generation of fibrocartilagenous scar tissue, while matrix-induced autologous chondrocyte implantation (MACI) and alternative autologous cell-based approaches may partly circumvent this effect. Autologous chondrocytes remain standard cell sources, yet arrays of alternative therapeutic biologicals present great potential for regenerative medicine. Cultured human epiphyseal chondro-progenitors (hECP) were proposed as sustainable, safe, and stable candidates for chaperoning cartilage repair or regeneration. This study describes the development and industrial transposition of hECP multi-tiered cell banking following a single organ donation, as well as preliminary preclinical hECP safety. Optimized cell banking workflows were proposed, potentially generating millions of safe and sustainable therapeutic products. Furthermore, clinical hECP doses were characterized as non-toxic in a standardized chorioallantoic membrane model. Lastly, a MACI-like protocol, including hECPs, was applied in a three-month GLP pilot safety evaluation in a caprine model of full-thickness articular cartilage defect. The safety of hECP transplantation was highlighted in xenogeneic settings, along with confirmed needs for optimal cell delivery vehicles and implantation techniques favoring effective cartilage repair or regeneration.


Assuntos
Cartilagem Articular/fisiologia , Transplante de Células , Terapia Baseada em Transplante de Células e Tecidos , Feto/citologia , Xenoenxertos , Medicina Regenerativa , Células-Tronco/citologia , Animais , Cabras/embriologia , Humanos , Modelos Animais
19.
FEBS J ; 288(14): 4394-4411, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33524211

RESUMO

Cattle have emerged as one of the most important domestic animals widely used for meat, milk, and fur. Derivation of bovine pluripotent stem cells (PSCs) can be applied in drug selecting and human disease modeling and facilitated agriculture-related applications such as production of genetically excellent cattle by gene editing. Extended PSCs (EPSCs), capable of differentiating into embryonic and extraembryonic parts, have been generated in mouse, human, and pig. Whether bovine EPSCs could be generated, and their chimeric competency remains unclear. This study focused on derivation of bovine EPSCs using LCDM medium and exploring the characteristics of EPSCs among different species, including bovine, mouse, and human EPSCs. Here, using LCDM medium (consisting of hLIF, CHIR99021, (S)-(+)-dimethindene maleate, and minocycline hydrochloride) enables the derivation of bovine EPSCs from induced PSCs (iPSCs) and bovine fetal fibroblasts (BFF) with stable morphology, pluripotent marker expression, and in vitro differentiation ability. Notably, bovine EPSCs exhibited interspecies chimeric contribution to embryonic and extraembryonic tissues in pre-implantation blastocysts and postimplantation bovine-mouse chimeras. Transcriptome analysis revealed the unique molecular characteristics of bovine EPSCs compared with iPSCs. The similarities and differences in molecular features across bovine, human, and mouse EPSCs were also described by transcriptome analysis. Taken together, the LCDM culture system containing chemical cocktails can be used for the establishment and long-term passaging of bovine EPSCs with embryonic and extraembryonic potency in bovine-mouse chimeras. Our findings lay the foundation of generating PSCs in domestic animals and open avenues for basic and applied research in biology, medicine, and agriculture. DATABASE: Gene expression data of bovine EPSCs and bovine iPSCs are available in the GEO databases under the accession number PRJNA693452.


Assuntos
Técnicas de Cultura de Células/métodos , Diferenciação Celular , Meios de Cultura/farmacologia , Embrião de Mamíferos/citologia , Feto/citologia , Fibroblastos/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Animais , Bovinos , Quimera , Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/metabolismo , Feminino , Feto/efeitos dos fármacos , Feto/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Camundongos , RNA-Seq
20.
Development ; 148(5)2021 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-33574039

RESUMO

In mice, the entry of germ cells into meiosis crucially depends on the expression of stimulated by retinoic acid gene 8 (Stra8). Stra8 is expressed specifically in pre-meiotic germ cells of females and males, at fetal and postnatal stages, respectively, but the mechanistic details of its spatiotemporal regulation are yet to be defined. In particular, there has been considerable debate regarding whether retinoic acid is required, in vivo, to initiate Stra8 expression in the mouse fetal ovary. We show that the distinctive anterior-to-posterior pattern of Stra8 initiation, characteristic of germ cells in the fetal ovary, is faithfully recapitulated when 2.9 kb of the Stra8 promoter is used to drive eGFP expression. Using in vitro transfection assays of cutdown and mutant constructs, we identified two functional retinoic acid responsive elements (RAREs) within this 2.9 kb regulatory element. We also show that the transcription factor DMRT1 enhances Stra8 expression, but only in the presence of RA and the most proximal RARE. Finally, we used CRISPR/Cas9-mediated targeted mutation studies to demonstrate that both RAREs are required for optimal Stra8 expression levels in vivo.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Células Germinativas/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Sítios de Ligação , Sistemas CRISPR-Cas/genética , Feminino , Desenvolvimento Fetal/genética , Feto/citologia , Feto/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células Germinativas/citologia , Meiose , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutagênese , Ovário/citologia , Ovário/metabolismo , Regiões Promotoras Genéticas , Receptores X de Retinoides/genética , Receptores X de Retinoides/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/farmacologia , Tretinoína/farmacologia
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