RESUMO
During early development, gene expression is tightly regulated. However, how genome organization controls gene expression during the transition from naïve embryonic stem cells to epiblast stem cells is still poorly understood. Using single-molecule microscopy approaches to reach nanoscale resolution, we show that genome remodeling affects gene transcription during pluripotency transition. Specifically, after exit from the naïve pluripotency state, chromatin becomes less compacted, and the OCT4 transcription factor has lower mobility and is more bound to its cognate sites. In epiblast cells, the active transcription hallmark, H3K9ac, decreases within the Oct4 locus, correlating with reduced accessibility of OCT4 and, in turn, with reduced expression of Oct4 nascent RNAs. Despite the high variability in the distances between active pluripotency genes, distances between Nodal and Oct4 decrease during epiblast specification. In particular, highly expressed Oct4 alleles are closer to nuclear speckles during all stages of the pluripotency transition, while only a distinct group of highly expressed Nodal alleles are in close proximity to Oct4 when associated with a nuclear speckle in epiblast cells. Overall, our results provide new insights into the role of the spatiotemporal genome remodeling during mouse pluripotency transition and its correlation with the expression of key pluripotency genes.
Assuntos
Genoma , Camadas Germinativas , Células-Tronco Embrionárias Murinas , Fator 3 de Transcrição de Octâmero , Animais , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Camadas Germinativas/citologia , Camadas Germinativas/metabolismo , Genoma/genética , Regulação da Expressão Gênica no Desenvolvimento , Cromatina/metabolismo , Cromatina/genética , Diferenciação Celular/genética , Imagem Individual de Molécula/métodos , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/citologia , Histonas/metabolismo , Histonas/genética , Montagem e Desmontagem da CromatinaRESUMO
The linear sequence of DNA provides invaluable information about genes and their regulatory elements along chromosomes. However, to fully understand gene function and regulation, we need to dissect how genes physically fold in the three-dimensional nuclear space. Here we describe immuno-OligoSTORM, an imaging strategy that reveals the distribution of nucleosomes within specific genes in super-resolution, through the simultaneous visualization of DNA and histones. We combine immuno-OligoSTORM with restraint-based and coarse-grained modeling approaches to integrate super-resolution imaging data with Hi-C contact frequencies and deconvoluted micrococcal nuclease-sequencing information. The resulting method, called Modeling immuno-OligoSTORM, allows quantitative modeling of genes with nucleosome resolution and provides information about chromatin accessibility for regulatory factors, such as RNA polymerase II. With Modeling immuno-OligoSTORM, we explore intercellular variability, transcriptional-dependent gene conformation, and folding of housekeeping and pluripotency-related genes in human pluripotent and differentiated cells, thereby obtaining the highest degree of data integration achieved so far to our knowledge.
Assuntos
Nuclease do Micrococo , Nucleossomos , Cromatina/genética , DNA/genética , Histonas/genética , Humanos , Nuclease do Micrococo/metabolismo , Nucleossomos/genética , RNA Polimerase II/genéticaRESUMO
PIWI-interacting RNAs (piRNAs) are a class of non-coding RNAs initially thought to be restricted exclusively to germline cells. In recent years, accumulating evidence has demonstrated that piRNAs are actually expressed in pluripotent, neural, cardiac and even cancer cells. However, controversy remains around the existence and function of somatic piRNAs. Using small RNA-seq samples from H9 pluripotent cells differentiated to mesoderm progenitors and cardiomyocytes we identified the expression of 447 piRNA transcripts, of which 241 were detected in pluripotency, 218 in mesoderm and 171 in cardiac cells. The majority of them originated from the sense strand of protein coding and lncRNAs genes in all stages of differentiation, though no evidences of amplification loop (ping-pong) were found. Genes hosting piRNA transcripts in cardiac samples were related to critical biological processes in the heart, like contraction and cardiac muscle development. Our results indicate that these piRNAs might have a role in fine-tuning the expression of genes involved in differentiation of pluripotent cells to cardiomyocytes.
Assuntos
Células-Tronco Embrionárias Humanas/citologia , Miócitos Cardíacos/citologia , RNA Interferente Pequeno/genética , Adulto , Diferenciação Celular , Linhagem Celular , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Miócitos Cardíacos/metabolismoRESUMO
The stem cell niche has a strong influence in the differentiation potential of human pluripotent stem cells with integrins playing a major role in communicating cells with the extracellular environment. However, it is not well understood how interactions between integrins and the extracellular matrix are involved in cardiac stem cell differentiation. To evaluate this, we performed a profile of integrins expression in two stages of cardiac differentiation: mesodermal progenitors and cardiomyocytes. We found an active regulation of the expression of different integrins during cardiac differentiation. In particular, integrin α5 subunit showed an increased expression in mesodermal progenitors, and a significant downregulation in cardiomyocytes. To analyze the effect of α5 subunit, we modified its expression by using a CRISPRi technique. After its downregulation, a significant impairment in the process of epithelial-to-mesenchymal transition was seen. Early mesoderm development was significantly affected due to a downregulation of key genes such as T Brachyury and TBX6. Furthermore, we observed that repression of integrin α5 during early stages led to a reduction in cardiomyocyte differentiation and impaired contractility. In summary, our results showed the link between changes in cell identity with the regulation of integrin α5 expression through the alteration of early stages of mesoderm commitment.
Assuntos
Células-Tronco Embrionárias Humanas/citologia , Integrina alfa5/genética , Miócitos Cardíacos/citologia , Sistemas CRISPR-Cas , Diferenciação Celular , Linhagem Celular , Regulação para Baixo , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Miócitos Cardíacos/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Nicho de Células-TroncoRESUMO
Leukemia inhibitory factor (LIF) is a growth factor with pleiotropic biological functions. It has been reported that LIF acts at different stages during mesoderm development. Also, it has been shown that LIF has a cytoprotective effect on neonatal murine cardiomyocytes (CMs) in culture, but little is known about the role of LIF during human cardiogenesis. Thus, we analyzed the effects of LIF on human pluripotent stem cells (PSC) undergoing cardiac differentiation. We first showed that LIF is expressed in the human heart during early development. We found that the addition of LIF within a precise time window during the in vitro differentiation process significantly increased CMs viability. This finding was associated to a decrease in the expression of pro-apoptotic protein Bax, which coincides with a reduction of the apoptotic rate. Therefore, the addition of LIF may represent a promising strategy for increasing CMs survival derived from PSCs.
Assuntos
Diferenciação Celular , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Fator Inibidor de Leucemia/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Células-Tronco Pluripotentes/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Embrionárias Humanas/patologia , Humanos , Fator Inibidor de Leucemia/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Fenótipo , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/patologia , Fatores de Tempo , Proteína X Associada a bcl-2/metabolismoRESUMO
Please note that Carolina Blüguermann's surname was misspelled (as Blugüermann) in this article as originally published.
RESUMO
MicroRNAs are small non-coding RNAs involved in post-transcriptional regulation of gene expression related to many cellular functions. We performed a small-RNAseq analysis of cardiac differentiation from pluripotent stem cells. Our analyses identified some new aspects about microRNA expression in this differentiation process. First, we described a dynamic expression profile of microRNAs where some of them are clustered according to their expression level. Second, we described the extensive network of isomiRs and ADAR modifications. Third, we identified the microRNAs families and clusters involved in the establishment of cardiac lineage and define the mirRNAome based on these groups. Finally, we were able to determine a more accurate miRNAome associated with cardiomyocytes by comparing the expressed microRNAs with other mature cells. MicroRNAs exert their effect in a complex and interconnected way, making necessary a global analysis to better understand their role. Our data expands the knowledge of microRNAs and their implications in cardiomyogenesis.
Assuntos
Biomarcadores/metabolismo , Linhagem da Célula/genética , Regulação da Expressão Gênica , Mesoderma/metabolismo , MicroRNAs/genética , Miócitos Cardíacos/metabolismo , Células-Tronco Pluripotentes/metabolismo , Diferenciação Celular , Células Cultivadas , Genoma Humano , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mesoderma/citologia , Miócitos Cardíacos/citologia , Células-Tronco Pluripotentes/citologiaRESUMO
Cell reprogramming has been well described in mouse and human cells. The expression of specific microRNAs has demonstrated to be essential for pluripotent maintenance and cell differentiation, but not much information is available in domestic species. We aim to generate horse iPSCs, characterize them and evaluate the expression of different microRNAs (miR-302a,b,c,d, miR-205, miR-145, miR-9, miR-96, miR-125b and miR-296). Two equine iPSC lines (L2 and L3) were characterized after the reprogramming of equine fibroblasts with the four human Yamanaka's factors (OCT-4/SOX-2/c-MYC/KLF4). The pluripotency of both lines was assessed by phosphatase alkaline activity, expression of OCT-4, NANOG and REX1 by RT-PCR, and by immunofluorescence of OCT-4, SOX-2 and c-MYC. In vitro differentiation to embryo bodies (EBs) showed the capacity of the iPSCs to differentiate into ectodermal, endodermal and mesodermal phenotypes. MicroRNA analyses resulted in higher expression of the miR-302 family, miR-9 and miR-96 in L2 and L3 vs. fibroblasts (p<0.05), as previously shown in human pluripotent cells. Moreover, downregulation of miR-145 and miR-205 was observed. After differentiation to EBs, higher expression of miR-96 was observed in the EBs respect to the iPSCs, and also the expression of miR-205 was induced but only in the EB-L2. In addition, in silico alignments of the equine microRNAs with mRNA targets suggested the ability of miR-302 family to regulate cell cycle and epithelial mesenchymal transition genes, miR-9 and miR-96 to regulate neural determinant genes and miR-145 to regulate pluripotent genes, similarly as in humans. In conclusion, we could obtain equine iPSCs, characterize them and determine for the first time the expression level of microRNAs in equine pluripotent cells.
Assuntos
Cavalos , Células-Tronco Pluripotentes Induzidas/metabolismo , MicroRNAs/genética , Animais , Diferenciação Celular/genética , Fibroblastos/citologia , Perfilação da Expressão Gênica , Células-Tronco Pluripotentes Induzidas/citologia , Fator 4 Semelhante a Kruppel , Técnicas de Transferência NuclearRESUMO
Human embryonic and induced pluripotent stem cells are self-renewing pluripotent stem cells (PSC) that can differentiate into a wide range of specialized cells. Basic fibroblast growth factor is essential for PSC survival, stemness and self-renewal. PI3K/AKT pathway regulates cell viability and apoptosis in many cell types. Although it has been demonstrated that PI3K/AKT activation by bFGF is relevant for PSC stemness maintenance its role on PSC survival remains elusive. In this study we explored the molecular mechanisms involved in the regulation of PSC survival by AKT. We found that inhibition of AKT with three non-structurally related inhibitors (GSK690693, AKT inhibitor VIII and AKT inhibitor IV) decreased cell viability and induced apoptosis. We observed a rapid increase in phosphatidylserine translocation and in the extent of DNA fragmentation after inhibitors addition. Moreover, abrogation of AKT activity led to Caspase-9, Caspase-3, and PARP cleavage. Importantly, we demonstrated by pharmacological inhibition and siRNA knockdown that GSK3ß signaling is responsible, at least in part, of the apoptosis triggered by AKT inhibition. Moreover, GSK3ß inhibition decreases basal apoptosis rate and promotes PSC proliferation. In conclusion, we demonstrated that AKT activation prevents apoptosis, partly through inhibition of GSK3ß, and thus results relevant for PSC survival.
Assuntos
Glicogênio Sintase Quinase 3 beta/metabolismo , Proteína Oncogênica v-akt/metabolismo , Células-Tronco Pluripotentes/fisiologia , Transdução de Sinais , Sobrevivência Celular , Células Cultivadas , Regulação da Expressão Gênica , HumanosRESUMO
INTRODUCTION: Mesenchymal stem cells (MSCs) are a promising source of cells for regenerative therapies. Although they can be isolated easily from several tissues, cell expansion is limited since their properties are lost with successive passages. Hence, pluripotent derived MSCs (PD-MSCs) arise as a suitable alternative for MSC production. Nevertheless, at present, PD-MSC derivation protocols are either expensive or not suitable for clinical purposes. METHODS: In this work we present a therapy-grade, inexpensive and simple protocol to derive MSCs from pluripotent stem cells (PSCs) based on the use of platelet lysate (PL) as medium supplement. RESULTS: We showed that the PD-MSCPL expressed multiple MSC markers, including CD90, CD73, CD105, CD166, and CD271, among others. These cells also show multilineage differentiation ability and immunomodulatory effects on pre-stimulated lymphocytes. Thorough characterization of these cells showed that a PD-MSCPL resembles an umbilical cord (UC) MSC and differs from a PSC in surface marker and extracellular matrix proteins and integrin expression. Moreover, the OCT-4 promoter is re-methylated with mesenchymal differentiation comparable with the methylation levels of UC-MSCs and fibroblasts. Lastly, the use of PL-supplemented medium generates significantly more MSCs than the use of fetal bovine serum. CONCLUSIONS: This protocol can be used to generate a large amount of PD-MSCs with low cost and is compatible with clinical therapies.