RESUMO
Human embryonic stem cells (hESCs) are pluripotent cell types derived from the inner cell mass of human blastocysts. Recent data indicate that the majority of established female XX hESC lines have undergone X chromosome inactivation (XCI) prior to differentiation, and XCI of hESCs can be either XIST-dependent (class II) or XIST-independent (class III). XCI of female hESCs precludes the use of XX hESCs as a cell-based model for examining mechanisms of XCI, and will be a challenge for studying X-linked diseases unless strategies are developed to reactivate the inactive X. In order to recover nuclei with two active X chromosomes (class I), we developed a reprogramming strategy by supplementing hESC media with the small molecules sodium butyrate and 3-deazaneplanocin A (DZNep). Our data demonstrate that successful reprogramming can occur from the XIST-dependent class II nuclear state but not class III nuclear state. To determine whether these small molecules prevent XCI, we derived six new hESC lines under normoxic conditions (UCLA1-UCLA6). We show that class I nuclei are present within the first 20 passages of hESC derivation prior to cryopreservation, and that supplementation with either sodium butyrate or DZNep preserve class I nuclei in the self-renewing state. Together, our data demonstrate that self-renewal and survival of class I nuclei are compatible with normoxic hESC derivation, and that chemical supplementation after derivation provides a strategy to prevent epigenetic progression and retain nuclei with two active X chromosomes in the self-renewing state.
Assuntos
Cromatina/efeitos dos fármacos , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Epigênese Genética/efeitos dos fármacos , Adenosina/análogos & derivados , Adenosina/farmacologia , Butiratos/farmacologia , Técnicas de Cultura de Células , Diferenciação Celular , Linhagem Celular , Reprogramação Celular/efeitos dos fármacos , Reprogramação Celular/genética , Cromatina/química , Cromatina/metabolismo , Cromossomos Humanos X/genética , Células-Tronco Embrionárias/efeitos dos fármacos , Feminino , Humanos , RNA Longo não Codificante , RNA não Traduzido/genéticaRESUMO
The cell intrinsic programming that regulates mammalian primordial germ cell (PGC) development in the pre-gonadal stage is challenging to investigate. To overcome this we created a transgene-free method for generating PGCs in vitro (iPGCs) from mouse embryonic stem cells (ESCs). Using labeling for SSEA1 and cKit, two cell surface molecules used previously to isolate presumptive iPGCs, we show that not all SSEA1+/cKit+ double positive cells exhibit a PGC identity. Instead, we determined that selecting for cKit(bright) cells within the SSEA1+ fraction significantly enriches for the putative iPGC population. Single cell analysis comparing SSEA1+/cKit(bright) iPGCs to ESCs and embryonic PGCs demonstrates that 97% of single iPGCs co-express PGC signature genes Blimp1, Stella, Dnd1, Prdm14 and Dazl at similar levels to e9.5-10.5 PGCs, whereas 90% of single mouse ESC do not co-express PGC signature genes. For the 10% of ESCs that co-express PGC signature genes, the levels are significantly lower than iPGCs. Microarray analysis shows that iPGCs are transcriptionally distinct from ESCs and repress gene ontology groups associated with mesoderm and heart development. At the level of chromatin, iPGCs contain 5-methyl cytosine bases in their DNA at imprinted and non-imprinted loci, and are enriched in histone H3 lysine 27 trimethylation, yet do not have detectable levels of Mvh protein, consistent with a Blimp1-positive pre-gonadal PGC identity. In order to determine whether iPGC formation is dependent upon Blimp1, we generated Blimp1 null ESCs and found that loss of Blimp1 significantly depletes SSEA1/cKit(bright) iPGCs. Taken together, the generation of Blimp1-positive iPGCs from ESCs constitutes a robust model for examining cell-intrinsic regulation of PGCs during the Blimp1-positive stage of development.
Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Células Germinativas/citologia , Células Germinativas/metabolismo , Análise de Célula Única/métodos , Fatores de Transcrição/metabolismo , Animais , Biomarcadores/metabolismo , Diferenciação Celular/genética , Separação Celular , Corpos Embrioides/citologia , Corpos Embrioides/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Gônadas/citologia , Antígenos CD15/metabolismo , Mesoderma/metabolismo , Camundongos , Modelos Biológicos , Fator 3 de Transcrição de Octâmero/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fator 1 de Ligação ao Domínio I Regulador Positivo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Transcrição Gênica , TransgenesRESUMO
Glucocorticoids are beneficial in many muscular dystrophies but they are ineffective in treating dysferlinopathy, a rare muscular dystrophy caused by loss of dysferlin. We sought to understand the molecular basis for this disparity by studying the effects of a glucocorticoid on differentiation of the myoblast cell line, C2C12, and dysferlin-deficient C2C12s. We found that pharmacologic doses of dexamethasone enhanced the myogenic fusion efficiency of C2C12s and increased the induction of dysferlin, along with specific myogenic transcription factors, sarcolemmal and structural proteins. In contrast, the dysferlin-deficient C2C12 cell line demonstrated a reduction in long myotubes and early induction of particular muscle differentiation proteins, most notably, myosin heavy chain. Dexamethasone partially reversed the defect in myogenic fusion in the dysferlin-deficient C2C12 cells. We hypothesize that a key therapeutic benefit of glucocorticoids may be the up-regulation of dysferlin as an important component of glucocorticoid-enhanced myogenic differentiation.
Assuntos
Dexametasona/farmacologia , Proteínas de Membrana/agonistas , Desenvolvimento Muscular/efeitos dos fármacos , Doenças Musculares/tratamento farmacológico , Mioblastos/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Linhagem Celular , Dexametasona/uso terapêutico , Relação Dose-Resposta a Droga , Disferlina , Glucocorticoides/farmacologia , Glucocorticoides/uso terapêutico , Proteínas de Membrana/biossíntese , Proteínas de Membrana/deficiência , Camundongos , Desenvolvimento Muscular/fisiologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Proteínas Musculares/efeitos dos fármacos , Proteínas Musculares/metabolismo , Doenças Musculares/metabolismo , Doenças Musculares/fisiopatologia , Mioblastos/metabolismo , Cadeias Pesadas de Miosina/efeitos dos fármacos , Cadeias Pesadas de Miosina/metabolismo , Fatores de Transcrição/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
The inactive X chromosome of female mammals displays several properties of heterochromatin including late replication, histone H4 hypoacetylation, histone H3 hypomethylation at lysine-4, and methylated CpG islands. We show that cre-Lox-mediated excision of 21 kb from both Xist alleles in female mouse fibroblasts led to the appearance of two histone modifications throughout the inactive X chromosome usually associated with euchromatin: histone H4 acetylation and histone H3 lysine-4 methylation. Despite these euchromatic properties, the inactive X chromosome was replicated even later in S phase than in wild-type female cells. Homozygosity for the deletion also caused regions of the active X chromosome that are associated with very high concentrations of LINE-1 elements to be replicated very late in S phase. Extreme late replication is a property of fragile sites and the 21-kb deletions destabilized the DNA of both X chromosomes, leading to deletions and translocations. This was accompanied by the phosphorylation of p53 at serine-15, an event that occurs in response to DNA damage, and the accumulation of gamma-H2AX, a histone involved in DNA repair, on the X chromosome. The Xist locus therefore maintains the DNA stability of both X chromosomes.