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1.
Cell ; 169(2): 243-257.e25, 2017 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-28388409

RESUMO

Of all known cultured stem cell types, pluripotent stem cells (PSCs) sit atop the landscape of developmental potency and are characterized by their ability to generate all cell types of an adult organism. However, PSCs show limited contribution to the extraembryonic placental tissues in vivo. Here, we show that a chemical cocktail enables the derivation of stem cells with unique functional and molecular features from mice and humans, designated as extended pluripotent stem (EPS) cells, which are capable of chimerizing both embryonic and extraembryonic tissues. Notably, a single mouse EPS cell shows widespread chimeric contribution to both embryonic and extraembryonic lineages in vivo and permits generating single-EPS-cell-derived mice by tetraploid complementation. Furthermore, human EPS cells exhibit interspecies chimeric competency in mouse conceptuses. Our findings constitute a first step toward capturing pluripotent stem cells with extraembryonic developmental potentials in culture and open new avenues for basic and translational research. VIDEO ABSTRACT.


Assuntos
Técnicas de Cultura de Células/métodos , Células-Tronco Pluripotentes/citologia , Animais , Blastocisto/citologia , Linhagem Celular , Quimera/metabolismo , Dimetideno/farmacologia , Humanos , Indicadores e Reagentes/química , Camundongos , Minociclina/química , Minociclina/farmacologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Poli(ADP-Ribose) Polimerase-1/metabolismo
2.
Development ; 145(11)2018 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-29848638

RESUMO

The postmeiotic development of male germ cells, also known as spermiogenesis, features the coordinated expression of a large number of spermatid-specific genes. However, only a limited number of key transcription factors have been identified and the underlying regulatory mechanisms remain largely unknown. Here, we report that SOX30, the most-divergent member of the Sry-related high-motility group box (SOX) family of transcription factors, is essential for mouse spermiogenesis. The SOX30 protein was predominantly expressed in spermatids, while its transcription was regulated by retinoic acid and by MYBL1 before and during meiosis. Sox30 knockout mice arrested spermiogenesis at step 3 round spermatids, which underwent apoptosis and abnormal chromocenter formation. We also determined that SOX30 regulated the expression of hundreds of spermatid-specific protein-coding and long non-coding RNA genes. SOX30 bound to the proximal promoter of its own gene and activated its transcription. These results reveal SOX30 as a novel key regulator of spermiogenesis that regulates its own transcription to enforce and activate this meiotic regulatory pathway.


Assuntos
Regulação da Expressão Gênica/genética , Fatores de Transcrição SOX/genética , Espermátides/metabolismo , Espermatogênese/fisiologia , Animais , Apoptose/fisiologia , Masculino , Meiose/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-myb/genética , Transativadores/genética , Tretinoína/metabolismo
3.
Biol Reprod ; 102(3): 598-606, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-31621828

RESUMO

The placenta, which originates from the trophectoderm (TE), is the first organ to form during mammalian embryogenesis. Recent studies based on bioinformatics analysis have revealed that heterogeneous gene expression initiates cell-fate decisions and directs two distinct cell fates by modulating the balance of pluripotency and differentiation as early as the four-cell stage. However, direct developmental evidence to support this is still lacking. To address at which stage the cell fate of the TE and inner cell mass (ICM) is determined, in this study, we administered a microinjection of Cre mRNA into a single blastomere of the mTmG mouse at different cleavage stages before implantation to examine the distributions of the descendants of the single-labeled cell in the mouse fetus and the placenta at E12.5. We found that the descendants of the labeled cells at the two-cell stage contributed to both the placenta and the fetus. Notably, the derivatives of the labeled cells at the four-cell stage fell into three categories: (1) distributed in both embryonic and extraembryonic lineages, (2) distributed only in mouse placental trophoblast layers, or (3) distributed only in the lineage derived from the ICM. In addition, these results fell in line with single-cell studies focusing on gene expression patterns that characterize particular lineages within the blastocyst. In conclusion, this study shows that the four-cell blastomeres differ in their individual developmental properties insofar as they contribute to either or both the ICM and trophoblast fate.


Assuntos
Linhagem da Célula/fisiologia , Implantação do Embrião/fisiologia , Desenvolvimento Embrionário/fisiologia , Placenta/citologia , Trofoblastos/citologia , Animais , Diferenciação Celular/fisiologia , Feminino , Camundongos , Placenta/metabolismo , Gravidez , Trofoblastos/metabolismo
4.
Biol Reprod ; 103(1): 60-69, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32301970

RESUMO

Wt1 gene encodes a nuclear transcription factor which is specifically expressed in ovarian granulosa cells and testicular Sertoli cells. Our previous studies demonstrated that Wt1 is required for the lineage specification of supporting cells and inactivation of Wt1 results in Sertoli cells to Leydig-like cells transformation. To test whether Wt1 is also involved in lineage maintenance of granulosa cells during ovary development, Wt1 was specifically deleted in pre-granulosa cells using Foxl2-cre. We found that the female Wt1-/flox; Foxl2-cre mice were infertile with atrophic ovaries and no growing follicles with multiple layers of granulosa cells were observed. A large number of 3ß-HSD-positive steroidogenic cells were detected in ovaries of Wt1-/flox; Foxl2-cre mice during embryonic stage and these cells were derived from Foxl2-expressing pre-granulosa cells. The quantitative results showed the expression of granulosa cell marker genes (Foxl2, Follistatin) was downregulated and steroidogenic cell marker genes (3ß-HSD, Cyp11a1, Star and Sf1) was dramatically increased in Wt1-/flox; Foxl2-cre ovaries. We also found that the meiosis of germ cells in Wt1-/flox; Foxl2-cre ovaries was delayed but not arrested. This study demonstrates that Wt1 is required for lineage maintenance of granulosa cells and inactivation of Wt1 results in pre-granulosa cells to steroidogenic cells transformation which in turn causes the defect of ovary development.


Assuntos
Diferenciação Celular/fisiologia , Células da Granulosa/fisiologia , Ovário/crescimento & desenvolvimento , Esteroides/biossíntese , Proteínas WT1/deficiência , Proteínas WT1/fisiologia , 3-Hidroxiesteroide Desidrogenases/análise , Animais , Reprogramação Celular , Cruzamentos Genéticos , Feminino , Proteína Forkhead Box L2/genética , Proteína Forkhead Box L2/fisiologia , Células da Granulosa/enzimologia , Infertilidade Feminina/etiologia , Masculino , Meiose/fisiologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Folículo Ovariano/crescimento & desenvolvimento , Ovário/enzimologia , Diferenciação Sexual/fisiologia , Proteínas WT1/genética
5.
J Biol Chem ; 292(43): 17577-17586, 2017 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-28900034

RESUMO

Sertoli and granulosa cells are two major types of somatic cells in male and female gonads, respectively. Previous studies have shown that Sertoli and granulosa cells are derived from common progenitor cells and that differentiation of these two cell types is regulated by sex differentiation genes. The signaling pathway including the adhesion and transcription factor Ctnnb1 (cadherin-associated protein, ß1, also known as ß-catenin) regulates differentiation of granulosa cells in the absence of the transcription factor Sry, and overactivation of ß-catenin in the presence of Sry leads to granulosa prior to sex determination. Surprisingly, our previous study found that ß-catenin overactivation in Sertoli cells after sex determination can also cause disruption of the testicular cord and aberrant testis development. However, the underlying molecular mechanism was unclear. In this study, we found that constitutive activation of Ctnnb1 in Sertoli cells led to ectopic expression of the granulosa cell-specific marker FOXL2 in testes. Co-staining experiments revealed that FOXL2-positive cells were derived from Sertoli cells, and Sertoli cells were transformed into granulosa-like cells after Ctnnb1 overactivation. Further studies demonstrated that CTNNB1 induced Foxl2 expression by directly binding to transcription factor Tcf/Lef-binding sites in the FOXL2 promoter region. We also found that direct overexpression of Foxl2 decreased the expression of Sertoli cell-specific genes in primary Sertoli cells. Taken together, these results demonstrate that repression of ß-catenin (CTNNB1) signaling is required for lineage maintenance of Sertoli cells. Our study provides a new mechanism for Sertoli cell lineage maintenance during gonad development.


Assuntos
Transdiferenciação Celular , Fatores de Transcrição Forkhead/biossíntese , Regulação da Expressão Gênica , Células da Granulosa/metabolismo , Células de Sertoli/metabolismo , Transdução de Sinais , beta Catenina/biossíntese , Animais , Feminino , Proteína Forkhead Box L2 , Fatores de Transcrição Forkhead/genética , Células da Granulosa/citologia , Masculino , Camundongos , Camundongos Transgênicos , Células de Sertoli/citologia , Proteína da Região Y Determinante do Sexo/genética , Proteína da Região Y Determinante do Sexo/metabolismo , beta Catenina/genética
6.
Cell Prolif ; 57(5): e13580, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38230761

RESUMO

The mammalian Pre-B cell leukaemia transcription factors 1-4 (PBX1-4) constitutes the PBC class of the homeodomain (HD)-containing proteins, which play important roles in diverse developmental processes. The functions and the underlying molecular mechanisms of PBX1-3 but not PBX4 have been extensively studied, and they have been reported to direct essential morphogenetic processes and organogenesis. In the present study, we generated knockin mice of FLAG-tagged PBX4 and the Pbx4 knockout (KO) mice and carried out in-depth characterisation of PBX4 expression and function. PBX4 was initially detected only in the testis among several organs of the adult mice and was expressed in spermatocytes and spermatids. However, no abnormality in spermatogenesis, but growth retardation and premature death after birth were observed in most adult Pbx4 KO mice. These animals were inactive and had shorter hindlimbs and lower numbers of reticulocytes and lymphocytes, probably caused by abnormalities at earlier developmental stages. Pbx4 mRNAs were indeed detected in several embryonic cell types related to limb development by in situ hybridisation and single-cell RNA-sequencing analysis. Pbx4 protein was also detected in the bone marrow of adult mice with a lower level compared with that in the testis. PBX4 preferentially binds to the promoters of a large number of genes including those for other HD-containing proteins and ribosomal proteins whose mutations are related to anaemia. PBX4-binding sites are enriched in motifs similar to those of other HD-containing proteins such as PKNOX1 indicating that PBX4 may also act as a co-transcription factor like other PBC proteins. Together, these results show that PBX4 participates in limb development and haematopoiesis while its function in spermatogenesis has not been revealed by gene KO probably due to the complementary effects of other genes.


Assuntos
Proteínas de Ligação a DNA , Extremidades , Regulação da Expressão Gênica no Desenvolvimento , Hematopoese , Proteínas de Homeodomínio , Animais , Masculino , Camundongos , Hematopoese/genética , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Regiões Promotoras Genéticas/genética , Testículo/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo
7.
J Genet Genomics ; 50(2): 99-107, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36494057

RESUMO

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has affected more than 600 million people worldwide. Several organs including lung, intestine, and brain are infected by SARS-CoV-2. It has been reported that SARS-CoV-2 receptor angiotensin-converting enzyme-2 (ACE2) is expressed in human testis. However, whether testis is also affected by SARS-CoV-2 is still unclear. In this study, we generate a human ACE2 (hACE2) transgenic mouse model in which the expression of hACE2 gene is regulated by hACE2 promoter. Sertoli and Leydig cells from hACE2 transgenic mice can be infected by SARS-CoV-2 pseudovirus in vitro, and severe pathological changes are observed after injecting the SARS-CoV-2 pseudovirus into the seminiferous tubules. Further studies reveal that Sertoli and Leydig cells from hACE2 transgenic mice are also infected by authentic SARS-CoV-2 virus in vitro. After testis interstitium injection, authentic SARS-CoV-2 viruses are first disseminated to the interstitial cells, and then detected inside the seminiferous tubules which in turn cause germ cell loss and disruption of seminiferous tubules. Our study demonstrates that testis is most likely a target of SARS-CoV-2 virus. Attention should be paid to the reproductive function in SARS-CoV-2 patients.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Masculino , Camundongos , Animais , Testículo/metabolismo , Enzima de Conversão de Angiotensina 2/metabolismo , Camundongos Transgênicos , Modelos Animais de Doenças
8.
Sci Adv ; 8(21): eabn1606, 2022 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-35613276

RESUMO

The chromatin state, which undergoes global changes during spermatogenesis, is critical to meiotic initiation and progression. However, the key regulators involved and the underlying molecular mechanisms remain to be uncovered. Here, we report that mouse BEND2 is specifically expressed in spermatogenic cells around meiotic initiation and that it plays an essential role in meiotic progression. Bend2 gene knockout in male mice arrested meiosis at the transition from zygonema to pachynema, disrupted synapsis and DNA double-strand break repair, and induced nonhomologous chromosomal pairing. BEND2 interacted with chromatin-associated proteins that are components of certain transcription-repressor complexes. BEND2-binding sites were identified in diverse chromatin states and enriched in simple sequence repeats. BEND2 inhibited the expression of genes involved in meiotic initiation and regulated chromatin accessibility and the modification of H3K4me3. Therefore, our study identified BEND2 as a previously unknown key regulator of meiosis, gene expression, and chromatin state during mouse spermatogenesis.

9.
Cells ; 11(23)2022 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-36497134

RESUMO

A developmental niche vacancy in host embryos is necessary for stem cell complementation-based organ regeneration (SCOG). Thyroid transcription factor 1 (TTF-1) is a tissue-specific transcription factor that regulates the embryonic development and differentiation of the thyroid and, more importantly, lungs; thus, it has been considered as a master gene to knockout in order to develop a lung vacancy host. TTF-1 knockout mice were originally produced by inserting a stop codon in Exon 3 of the gene (E3stop) through embryonic stem cell-based homologous recombination. The main problems of utilizing E3stop host embryos for lung SCOG are that these animals all have a tracheoesophageal fistula (TEF), which cannot be corrected by donor stem cells, and most of them have monolateral sac-like lungs. To improve the mouse model towards achieving SCOG-based lung generation, in this project, we used the CRISPR/Cas9 tool to remove Exon 2 of the gene by zygote microinjection and successfully produced TTF-1 knockout (E2del) mice. Similar to E3stop, E2del mice are birth-lethal due to retarded lung development with sac-like lungs and only a rudimentary bronchial tree, increased basal cells but without alveolar type II cells and blood vessels, and abnormal thyroid development. Unlike E3stop, 57% of the E2del embryos presented type I tracheal agenesis (TA, a kind of human congenital malformation) with a shortened trachea and clear separations of the trachea and esophagus, while the remaining 43% had TEF. Furthermore, all the E2del mice had bilateral sac-like lungs. Both TA and bilateral sac-like lungs are preferred in SCOG. Our work presents a new strategy for producing SCOG host embryos that may be useful for lung regeneration.


Assuntos
Sistemas CRISPR-Cas , Fator Nuclear 1 de Tireoide , Fístula Traqueoesofágica , Animais , Feminino , Humanos , Camundongos , Gravidez , Sistemas CRISPR-Cas/genética , Modelos Animais de Doenças , Pulmão , Camundongos Knockout , Fator Nuclear 1 de Tireoide/genética
10.
J Cell Biol ; 221(7)2022 07 04.
Artigo em Inglês | MEDLINE | ID: mdl-35674692

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causal pathogen of the ongoing global pandemic of coronavirus disease 2019 (COVID-19). Loss of smell and taste are symptoms of COVID-19, and may be related to cilia dysfunction. Here, we found that the SARS-CoV-2 ORF10 increases the overall E3 ligase activity of the CUL2ZYG11B complex by interacting with ZYG11B. Enhanced CUL2ZYG11B activity by ORF10 causes increased ubiquitination and subsequent proteasome-mediated degradation of an intraflagellar transport (IFT) complex B protein, IFT46, thereby impairing both cilia biogenesis and maintenance. Further, we show that exposure of the respiratory tract of hACE2 mice to SARS-CoV-2 or SARS-CoV-2 ORF10 alone results in cilia-dysfunction-related phenotypes, and the ORF10 expression in primary human nasal epithelial cells (HNECs) also caused a rapid loss of the ciliary layer. Our study demonstrates how SARS-CoV-2 ORF10 hijacks CUL2ZYG11B to eliminate IFT46 and leads to cilia dysfunction, thereby offering a powerful etiopathological explanation for how SARS-CoV-2 causes multiple cilia-dysfunction-related symptoms specific to COVID-19.


Assuntos
Cílios , SARS-CoV-2 , Ubiquitina-Proteína Ligases , Animais , Células Cultivadas , Cílios/metabolismo , Cílios/patologia , Proteínas do Citoesqueleto , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Humanos , Camundongos , SARS-CoV-2/patogenicidade , Olfato , Ubiquitina-Proteína Ligases/metabolismo
11.
Front Cell Dev Biol ; 9: 655145, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33898455

RESUMO

3-hydroxybutyrate dehydrogenase-2 (Bdh2), a short-chain dehydrogenase, catalyzes a rate-limiting step in the biogenesis of the mammalian siderophore, playing a key role in iron homeostasis, energy metabolism and apoptosis. However, the function of Bdh2 in embryonic stem cells (ESCs) remains unknown. To gain insights into the role of Bdh2 on pluripotency and cell fate decisions of mouse ESCs, we generated Bdh2 homozygous knockout lines for both mouse advanced embryonic stem cell (ASC) and ESC using CRISPR/Cas9 genome editing technology. Bdh2 deficiency in both ASCs and ESCs had no effect on expression of core pluripotent transcription factors and alkaline phosphatase activity, suggesting dispensability of Bdh2 for self-renewal and pluripotency of ESCs. Interestingly, cells with Bdh2 deficiency exhibited potency of endoderm differentiation in vitro; with upregulated endoderm associated genes revealed by RNA-seq and RT-qPCR. We further demonstrate that Bdh2 loss inhibited expression of multiple methyltransferases (DNMTs) at both RNA and protein level, suggesting that Bdh2 may be essentially required to maintain DNA methylation in ASCs and ESCs. Overall, this study provides valuable data and resources for understanding how Bdh2 regulate earliest cell fate decision and DNA methylation in ASCs/ESCs.

12.
Front Med (Lausanne) ; 8: 793437, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-35071273

RESUMO

SARS-CoV-2 is an emerging coronavirus threatening human health and the economy worldwide. As an RNA virus, variants emerge during the pandemic and potentially influence the efficacy of the anti-viral drugs and vaccines. Eight spike variants harboring highly recurrent mutations were selected and introduced into a replication-competent recombinant VSV in place of the original G protein (rVSV-SARS-CoV-2). The resulting mutant viruses displayed similar growth curves in vitro as the wild-type virus and could be neutralized by sera from convalescent COVID-19 patients. Several variants, especially Beta strain, showed resistance to human neutralizing monoclonal antibodies targeting the receptor-binding domain (RBD). A single dose of rVSV-SARS-CoV-2 Beta variant could elicit enhanced and broad-spectrum neutralizing antibody responses in human ACE2 knock-in mice and golden Syrian hamsters, while other mutants generated antibody levels comparable to the wild-type. Therefore, our results will be of value to the development of next-generation vaccines and therapeutic antibodies.

13.
Signal Transduct Target Ther ; 6(1): 389, 2021 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-34759261

RESUMO

SARS-CoV-2 and SARS-CoV are genetically related coronavirus and share the same cellular receptor ACE2. By replacing the VSV glycoprotein with the spikes (S) of SARS-CoV-2 and SARS-CoV, we generated two replication-competent recombinant viruses, rVSV-SARS-CoV-2 and rVSV-SARS-CoV. Using wild-type and human ACE2 (hACE2) knock-in mouse models, we found a single dose of rVSV-SARS-CoV could elicit strong humoral immune response via both intranasal (i.n.) and intramuscular (i.m.) routes. Despite the high genetic similarity between SARS-CoV-2 and SARS-CoV, no obvious cross-neutralizing activity was observed in the immunized mice sera. In macaques, neutralizing antibody (NAb) titers induced by one i.n. dose of rVSV-SARS-CoV-2 were eight-fold higher than those by a single i.m. dose. Thus, our data indicates that rVSV-SARS-CoV-2 might be suitable for i.n. administration instead of the traditional i.m. immunization in human. Because rVSV-SARS-CoV elicited significantly stronger NAb responses than rVSV-SARS-CoV-2 in a route-independent manner, we generated a chimeric antigen by replacing the receptor binding domain (RBD) of SARS-CoV S with that from the SARS-CoV-2. rVSV expressing the chimera (rVSV-SARS-CoV/2-RBD) induced significantly increased NAbs against SARS-CoV-2 in mice and macaques than rVSV-SARS-CoV-2, with a safe Th1-biased response. Serum immunized with rVSV-SARS-CoV/2-RBD showed no cross-reactivity with SARS-CoV. hACE2 mice receiving a single i.m. dose of either rVSV-SARS-CoV-2 or rVSV-SARS-CoV/2-RBD were fully protected against SARS-CoV-2 challenge without obvious lesions in the lungs. Our results suggest that transplantation of SARS-CoV-2 RBD into the S protein of SARS-CoV might be a promising antigen design for COVID-19 vaccines.


Assuntos
Vacinas contra COVID-19/imunologia , COVID-19/prevenção & controle , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Técnicas de Introdução de Genes , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Testes de Neutralização , Proteínas Recombinantes de Fusão/imunologia , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/genética
14.
Cell Res ; 31(4): 415-432, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-32913304

RESUMO

Aging is a major risk factor for many diseases, especially in highly prevalent cardiopulmonary comorbidities and infectious diseases including Coronavirus Disease 2019 (COVID-19). Resolving cellular and molecular mechanisms associated with aging in higher mammals is therefore urgently needed. Here, we created young and old non-human primate single-nucleus/cell transcriptomic atlases of lung, heart and artery, the top tissues targeted by SARS-CoV-2. Analysis of cell type-specific aging-associated transcriptional changes revealed increased systemic inflammation and compromised virus defense as a hallmark of cardiopulmonary aging. With age, expression of the SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2) was increased in the pulmonary alveolar epithelial barrier, cardiomyocytes, and vascular endothelial cells. We found that interleukin 7 (IL7) accumulated in aged cardiopulmonary tissues and induced ACE2 expression in human vascular endothelial cells in an NF-κB-dependent manner. Furthermore, treatment with vitamin C blocked IL7-induced ACE2 expression. Altogether, our findings depict the first transcriptomic atlas of the aged primate cardiopulmonary system and provide vital insights into age-linked susceptibility to SARS-CoV-2, suggesting that geroprotective strategies may reduce COVID-19 severity in the elderly.


Assuntos
Envelhecimento , SARS-CoV-2/fisiologia , Transcriptoma , Células Epiteliais Alveolares/citologia , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/virologia , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Ácido Ascórbico/farmacologia , COVID-19/patologia , COVID-19/virologia , Linhagem Celular , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Células Endoteliais/virologia , Humanos , Interleucina-7/metabolismo , Interleucina-7/farmacologia , Macaca fascicularis , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/virologia , RNA-Seq , SARS-CoV-2/isolamento & purificação , Análise de Célula Única , Transcriptoma/efeitos dos fármacos
15.
Am J Pathol ; 175(5): 1975-83, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19808649

RESUMO

Mouse models of liver injury provide useful tools for studying hepatocyte engraftment and proliferation. A representative model of liver injury is the albumin-urokinase (Alb-uPA) transgenic model, but neonatal lethality hampers its widespread application. To overcome this problem, we generated a transgenic mouse in which transcription of the reverse tetracycline transactivator was (rtTA) driven by the mouse albumin promoter, and backcrossed the rtTA mice onto severe combined immunodeficient (SCID)/bg mice to generate immunodeficient rtTA/SCID mice. We then produced recombinant adenoviruses Ad.TRE-uPA, in which the urokinase was located downstream of the tetracycline response element (TRE). The rtTA/SCID mouse hepatocytes were then infected with Ad.TRE-uPA to establish an inducible liver injury mouse model. In the presence of doxycycline, uPA was exclusively expressed in endogenous hepatocytes and caused extensive liver injury. Enhanced green fluorescent protein-labeled mouse hepatocytes selectively repopulated the rtTA/SCID mouse liver and replaced over 80% of the recipient liver mass after repeated administration of Ad.TRE-uPA. Compared with the original uPA mice, rtTA/SCID mice did not exhibit problems regarding breeding efficiency, and the time window for transplantation was flexible. In addition, we could control the extent of liver injury to facilitate transplantation surgery by regulating the dose of Ad.TRE-uPA. Our inducible mouse model will be convenient for studies of hepatocyte transplantation and hepatic regeneration, and this system will facilitate screening for potential genetic factors critical for engraftment and proliferation of hepatocytes in vivo.


Assuntos
Hepatócitos/transplante , Fígado , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Albuminas/genética , Albuminas/metabolismo , Animais , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hepatócitos/citologia , Hepatócitos/fisiologia , Fígado/citologia , Fígado/enzimologia , Fígado/lesões , Transplante de Fígado , Camundongos , Camundongos SCID , Camundongos Transgênicos , Modelos Animais , Tetraciclina/metabolismo , Transaminases/sangue , Ativador de Plasminogênio Tipo Uroquinase/genética
16.
Stem Cells ; 27(2): 383-9, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19056907

RESUMO

Most mouse embryonic stem (ES) cells are derived from a 129 or C57BL/6 background, whereas the derivation efficiency of ES cells is extremely low on certain refractory types of background for which ES cells are highly desired. Here we report an optimized, highly efficient protocol by combining pluripotin, a small molecule, and leukemia inhibitory factor (LIF) for the derivation of mouse ES cells. With this method, we successfully isolated ES cell lines from five strains of mice, with an efficiency of 57% for NOD-scid, 63% for SCID beige, 80% for CD-1, and 100% for two F1 strains from C57BL/6xCD-1. By tracking the Oct4-positive cells in the Oct4-green fluorescent protein embryos in the process of ES cell isolation, we found that pluripotin combined with LIF improved the efficiency of ES cell isolation by selectively maintaining the Oct4-positive cells in the outgrowth. To our knowledge, this is the first report of ES cells being efficiently derived from immunodeficient mice on refractory backgrounds (NOD-scid on a NOD background and SCID beige on a BALB/c background).


Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Fator Inibidor de Leucemia/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Imuno-Histoquímica , Cariotipagem , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Camundongos Transgênicos , Reação em Cadeia da Polimerase Via Transcriptase Reversa
17.
Cell Death Dis ; 10(6): 461, 2019 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-31189875

RESUMO

miR-21 is aberrantly expressed, and plays a role in various types of tumors and many other diseases. However, the mechanism of miR-21 in LPS-induced septic shock is still unclear. In this study, we investigated the mechanism of miR-21 in LPS-induced pyroptosis and septic shock. Here, we show that miR-21 deficiency inhibited NLRP3, ASC, and caspase-1 expression, as well as inflammasome activation in myeloid cells from both mice and humans. We found that the NF-κB pathway was regulated by miR-21, and that A20 was a direct target of miR-21. Furthermore, miR-21 deficiency inhibited the ASC pyroptosome, which restrained caspase-1 activation and GSDMD cleavage, thereby preventing LPS-induced pyroptosis and septic shock. miR-21 deficiency resulted in an increase in A20, which led to decreased IL-1ß production and caspase-1 activation. Caspase-1-mediated GSDMD cleavage was consequently decreased, which prevented pyroptosis in LPS-induced sepsis in mice. Our results demonstrate that miR-21 is a critical positive regulator of the NF-κB pathway and NLRP3 inflammasomes in pyroptosis and septic shock via A20. In addition, by analyzing published miRNA expression profiles in the Gene Expression Omnibus database, we found that the miR-21 levels in peripheral blood from patients with septic shock were elevated. Thus, miR-21 may serve as a potential treatment target in patients with septic shock.


Assuntos
Inflamassomos/metabolismo , MicroRNAs/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Piroptose/genética , Choque Séptico/metabolismo , Animais , Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Caspase 1/metabolismo , Citocinas/metabolismo , Feminino , Humanos , Inflamassomos/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lipopolissacarídeos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , NF-kappa B/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteínas de Ligação a Fosfato/genética , Proteínas de Ligação a Fosfato/metabolismo , Piroptose/efeitos dos fármacos , Choque Séptico/induzido quimicamente , Choque Séptico/genética , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/genética , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/metabolismo
18.
Cell Rep ; 22(9): 2279-2293, 2018 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-29490266

RESUMO

The precise function and role of nucleosome assembly protein 1-like 1 (Nap1l1) in brain development are unclear. Here, we find that Nap1l1 knockdown decreases neural progenitor cell (NPC) proliferation and induces premature neuronal differentiation during cortical development. A similar deficiency in embryonic neurogenesis was observed in Nap1l1 knockout (KO) mice, which were generated using the CRISPR-Cas9 system. RNA sequencing (RNA-seq) analysis indicates that Ras-associated domain family member 10 (RassF10) may be the downstream target of Nap1l1. Furthermore, we found that Nap1l1 regulates RassF10 expression by promoting SETD1A-mediated H3K4 trimethylation at the RassF10 promoter. Nap1l1 KO defects may be rescued by RassF10 overexpression, suggesting that Nap1l1 controls NPC differentiation through RassF10. Our findings reveal an essential role for the Nap1l1 histone chaperone in cortical neurogenesis during early embryonic brain development.


Assuntos
Encéfalo/citologia , Encéfalo/embriologia , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Proteína 1 de Modelagem do Nucleossomo/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Córtex Cerebral/embriologia , Córtex Cerebral/metabolismo , Feminino , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Inativação Gênica , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Metilação , Camundongos Endogâmicos ICR , Camundongos Knockout , Mitose/genética , Neurogênese/genética , Proteína 1 de Modelagem do Nucleossomo/genética , Regiões Promotoras Genéticas
19.
Cell Stem Cell ; 23(1): 31-45.e7, 2018 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-29937202

RESUMO

Chemical reprogramming provides a powerful platform for exploring the molecular dynamics that lead to pluripotency. Although previous studies have uncovered an intermediate extraembryonic endoderm (XEN)-like state during this process, the molecular underpinnings of pluripotency acquisition remain largely undefined. Here, we profile 36,199 single-cell transcriptomes at multiple time points throughout a highly efficient chemical reprogramming system using RNA-sequencing and reconstruct their progression trajectories. Through identifying sequential molecular events, we reveal that the dynamic early embryonic-like programs are key aspects of successful reprogramming from XEN-like state to pluripotency, including the concomitant transcriptomic signatures of two-cell (2C) embryonic-like and early pluripotency programs and the epigenetic signature of notable genome-wide DNA demethylation. Moreover, via enhancing the 2C-like program by fine-tuning chemical treatment, the reprogramming process is remarkably accelerated. Collectively, our findings offer a high-resolution dissection of cell fate dynamics during chemical reprogramming and shed light on mechanistic insights into the nature of induced pluripotency.


Assuntos
Reprogramação Celular/efeitos dos fármacos , Desenvolvimento Embrionário/genética , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Análise de Sequência de RNA , Análise de Célula Única , Animais , Células Cultivadas , Desenvolvimento Embrionário/efeitos dos fármacos , Ensaios de Triagem em Larga Escala , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transcriptoma
20.
Cell Death Dis ; 8(1): e2532, 2017 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-28055014

RESUMO

Globozoospermia is a common reproductive disorder that causes male infertility in humans, and the malformation or loss of acrosomes is the prominent feature of this disease. Although the acrosome is thought to be derived from the Golgi apparatus, the detailed molecular mechanisms remain unclear. GM130 is a cis-side localized Golgi matrix protein,whereas the physiological functions of this protein remain elusive. Here we showed that inactivation of GM130-caused male infertility in mouse model. The primary defects were the absence of acrosomes, round sperm heads, and aberrant assembly of the mitochondrial sheath, which comprise the characteristic features of human globozoospermia. Further investigation indicated that loss of GM130 did not affect the secretion of pro-acrosomic vesicles, whereas the vesicles failed to fuse into a single large acrosome vesicle. Co-localization of the adaptor protein complex AP1 and trans-Golgi network (TGN) protein TGN46 was disrupted, suggesting that the malformation of acrosomes is most likely due to the defect in the sorting and coating of Golgi-derived pro-acrosomic vesicles. Thus, the GM130-deficient mouse provides a valuable model for investigating the etiology of human globozoospermia.


Assuntos
Acrossomo/metabolismo , Autoantígenos/genética , Infertilidade Masculina/genética , Proteínas de Membrana/genética , Teratozoospermia/genética , Acrossomo/patologia , Animais , Modelos Animais de Doenças , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Humanos , Infertilidade Masculina/patologia , Masculino , Proteínas de Membrana/antagonistas & inibidores , Camundongos , Espermatogênese/genética , Teratozoospermia/patologia
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