RESUMO
Human induced pluripotent stem cells (hiPSCs) are a potential source of somatic cells for cell therapies due to their ability to self-renew and differentiate into various cells of the body. To date, the clinical application of hiPSCs has been limited due to safety issues. The present study aims to standardize the safety procedure of the derivation of GMP-compliant induced pluripotent stem cell (iPSC) lines from human fibroblasts. The hiPSC lines were generated using the nonintegrative Sendai virus method to incorporate Yamanaka reprogramming factors (OCT3/4, SOX2, KLF4 and c-MYC) into cells. A constant temperature was maintained during the cell culture, including all stages of the culture after transduction with Sendai virus. Pluripotency was proved in six independently generated hiPSC lines from adult female (47 years old) and male (57 years old) donors' derived fibroblasts via alkaline phosphatase live (ALP) staining, qPCR, and immunocytochemistry. The hiPSC lines showed a gradual decrease in the presence of the virus with each subsequent passage, and this reduction was specific to the hiPSC line. The frequency and probability of chromosomal aberrations in hiPSCs were dependent on both the iPSC clone identity and sex of the donor. In summary, the generation of hiPSC for clinical applications requires safety standards application (biosafety protocol, quality control of hiPSC lines, viral and genetic integrity screening) from the first stages of the clonal selection of hiPSC from the same donor.
Assuntos
Células-Tronco Pluripotentes Induzidas , Fator 4 Semelhante a Kruppel , Vírus Sendai , Humanos , Feminino , Masculino , Pessoa de Meia-Idade , Linhagem Celular , Fibroblastos , Diferenciação Celular/fisiologia , Transdução Genética/métodos , Fatores SexuaisRESUMO
Human trophoblast stem cells (hTSCs) can be derived from embryonic stem cells (hESCs) or be induced from somatic cells by OCT4, SOX2, KLF4 and MYC (OSKM). Here we explore whether the hTSC state can be induced independently of pluripotency, and what are the mechanisms underlying its acquisition. We identify GATA3, OCT4, KLF4 and MYC (GOKM) as a combination of factors that can generate functional hiTSCs from fibroblasts. Transcriptomic analysis of stable GOKM- and OSKM-hiTSCs reveals 94 hTSC-specific genes that are aberrant specifically in OSKM-derived hiTSCs. Through time-course-RNA-seq analysis, H3K4me2 deposition and chromatin accessibility, we demonstrate that GOKM exert greater chromatin opening activity than OSKM. While GOKM primarily target hTSC-specific loci, OSKM mainly induce the hTSC state via targeting hESC and hTSC shared loci. Finally, we show that GOKM efficiently generate hiTSCs from fibroblasts that harbor knockout for pluripotency genes, further emphasizing that pluripotency is dispensable for hTSC state acquisition.
Assuntos
Reprogramação Celular , Células-Tronco Pluripotentes Induzidas , Humanos , Reprogramação Celular/genética , Trofoblastos , Fibroblastos , Células-Tronco Embrionárias , Cromatina/genética , Fator 3 de Transcrição de Octâmero/genéticaRESUMO
Dravet syndrome (DRVT) is a rare form of neurodevelopmental disorder with a high risk of sudden unexpected death in epilepsy (SUDEP), caused mainly (>80% cases) by mutations in the SCN1A gene, coding the Nav1.1 protein (alfa-subunit of voltage-sensitive sodium channel). Mutations in SCN1A are linked to heterogenous epileptic phenotypes of various types, severity, and patient prognosis. Here we generated iPSC lines from fibroblasts obtained from three individuals affected with DRVT carrying distinct mutations in the SCN1A gene (nonsense mutation p.Ser1516*, missense mutation p.Arg1596His, and splicing mutation c.2589+2dupT). The iPSC lines, generated with the non-integrative approach, retained the distinct SCN1A gene mutation of the donor fibroblasts and were characterized by confirming the expression of the pluripotency markers, the three-germ layer differentiation potential, the absence of exogenous vector expression, and a normal karyotype. The generated iPSC lines were used to establish ventral forebrain organoids, the most affected type of neurons in the pathology of DRVT. The DRVT organoid model will provide an additional resource for deciphering the pathology behind Nav1.1 haploinsufficiency and drug screening to remediate the functional deficits associated with the disease.
Assuntos
Epilepsias Mioclônicas , Células-Tronco Pluripotentes Induzidas , Humanos , Canal de Sódio Disparado por Voltagem NAV1.1/genética , Canal de Sódio Disparado por Voltagem NAV1.1/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Epilepsias Mioclônicas/genética , Neurônios/metabolismo , Prosencéfalo/metabolismoRESUMO
Mutations in the SCN1A gene can cause a variety of phenotypes, ranging from mild forms, such as febrile seizures and generalized epilepsy with febrile seizures plus, to severe, such as Dravet and non-Dravet developmental epileptic encephalopathies. Until now, more than two thousand pathogenic variants of the SCN1A gene have been identified and different pathogenic mechanisms (loss vs. gain of function) described, but the precise molecular mechanisms responsible for the deficits exhibited by patients are not fully elucidated. Additionally, the phenotypic variability proves the involvement of other genetic factors in its final expression. This is the reason why animal models and cell line models used to explore the molecular pathology of SCN1A-related disorders are only of limited use. The results of studies based on such models cannot be directly translated to affected individuals because they do not address each patient's unique genetic background. The generation of functional neurons and glia for patient-derived iPSCs, together with the generation of isogenic controls using CRISPR/Cas technology, and finally, the 3D brain organoid models, seem to be a good way to solve this problem. Here, we review SCN1A-related encephalopathies, as well as the stem cell models used to explore their molecular basis.
Assuntos
Encefalopatias , Epilepsias Mioclônicas , Células-Tronco Pluripotentes Induzidas , Convulsões Febris , Animais , Encefalopatias/genética , Encefalopatias/terapia , Epilepsias Mioclônicas/genética , Epilepsias Mioclônicas/terapia , Canal de Sódio Disparado por Voltagem NAV1.1/genética , Convulsões Febris/genéticaRESUMO
Mitochondria are cellular organelles involved in generating energy to power various processes in the cell. Although the pivotal role of mitochondria in neurogenesis was demonstrated (first in animal models), very little is known about their role in human embryonic neurodevelopment and its pathology. In this respect human-induced pluripotent stem cells (hiPSC)-derived cerebral organoids provide a tractable, alternative model system of the early neural development and disease that is responsive to pharmacological and genetic manipulations, not possible to apply in humans. Although the involvement of mitochondria in the pathogenesis and progression of neurodegenerative diseases and brain dysfunction has been demonstrated, the precise role they play in cell life and death remains unknown, compromising the development of new mitochondria-targeted approaches to treat human diseases. The cerebral organoid model of neurogenesis and disease in vitro provides an unprecedented opportunity to answer some of the most fundamental questions about mitochondrial function in early human neurodevelopment and neural pathology. Largely an unexplored territory due to the lack of tools and approaches, this review focuses on recent technological advancements in fluorescent and molecular tools, imaging systems, and computational approaches for quantitative and qualitative analyses of mitochondrial structure and function in three-dimensional cellular assemblies-cerebral organoids. Future developments in this direction will further facilitate our understanding of the important role or mitochondrial dynamics and energy requirements during early embryonic development. This in turn will provide a further understanding of how dysfunctional mitochondria contribute to disease processes.
Assuntos
Células-Tronco Pluripotentes Induzidas , Doenças Neurodegenerativas , Animais , Feminino , Humanos , Mitocôndrias , Doenças Neurodegenerativas/metabolismo , Neurogênese , Organoides/metabolismo , GravidezRESUMO
Following fertilization, totipotent cells undergo asymmetric cell divisions, resulting in three distinct cell types in the late pre-implantation blastocyst: epiblast (Epi), primitive endoderm (PrE), and trophectoderm (TE). Here, we aim to understand whether these three cell types can be induced from fibroblasts by one combination of transcription factors. By utilizing a sophisticated fluorescent knockin reporter system, we identified a combination of five transcription factors, Gata3, Eomes, Tfap2c, Myc, and Esrrb, that can reprogram fibroblasts into induced pluripotent stem cells (iPSCs), induced trophoblast stem cells (iTSCs), and induced extraembryonic endoderm stem cells (iXENs), concomitantly. In-depth transcriptomic, chromatin, and epigenetic analyses provide insights into the molecular mechanisms that underlie the reprogramming process toward the three cell types. Mechanistically, we show that the interplay between Esrrb and Eomes during the reprogramming process determines cell fate, where high levels of Esrrb induce a XEN-like state that drives pluripotency and high levels of Eomes drive trophectodermal fate.
Assuntos
Blastocisto/fisiologia , Endoderma/fisiologia , Fibroblastos/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Trofoblastos/fisiologia , Animais , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Reprogramação Celular , Implantação do Embrião , Camundongos , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Induced pluripotent stem cells (iPSCs) undergo extensive nuclear reprogramming and are generally indistinguishable from embryonic stem cells (ESCs) in their functional capacity and transcriptome and DNA methylation profiles. However, direct conversion of cells from one lineage to another often yields incompletely reprogrammed, functionally compromised cells, raising the question of whether pluripotency is required to achieve a high degree of nuclear reprogramming. Here, we show that transient expression of Gata3, Eomes, and Tfap2c in mouse fibroblasts induces stable, transgene-independent trophoblast stem-like cells (iTSCs). iTSCs possess transcriptional profiles highly similar to blastocyst-derived TSCs, with comparable methylation and H3K27ac patterns and genome-wide H2A.X deposition. iTSCs generate trophoectodermal lineages upon differentiation, form hemorrhagic lesions, and contribute to developing placentas in chimera assays, indicating a high degree of nuclear reprogramming, with no evidence of passage through a transient pluripotent state. Together, these data demonstrate that extensive nuclear reprogramming can be achieved independently of pluripotency.
Assuntos
Linhagem da Célula , Núcleo Celular/metabolismo , Reprogramação Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Trofoblastos/citologia , Animais , Células Cultivadas , Camundongos , Camundongos Transgênicos , Trofoblastos/metabolismoRESUMO
HAX-1, a multifunctional protein involved in the regulation of apoptosis, cell migration, and calcium homeostasis, binds the 3' untranslated region motifs of specific transcripts. This suggests that HAX-1 plays a role in post-transcriptional regulation, at the level of mRNA stability/transport or translation. In this study, we analyze in detail HAX-1 colocalization with processing bodies (P-bodies) and its dependence on mRNA availability. Endogenous P-body markers DCP1 and Rck/p54 were shown to colocalize with endogenous HAX-1, but in case of the overexpressed proteins, only DCP1 displayed unperturbed colocalization with HAX-1. HAX-1 colocalization with DCP1 was observed in most of the cell lines studied, but its presence was not required for P-body formation, and its silencing caused an increase in P-body number. Preliminary mapping suggested that HAX-1 has more than one short P-body-targeting sequence. The pools of P-body-localized HAX-1 and cytosolic HAX-1 were demonstrated to dynamically exchange, suggesting steady flow of the protein. Active transcription was shown to be a factor in the localization of HAX-1 to P-bodies. Also, it was observed that HAX-1 localizes to some unidentified foci, which do not contain DCP1. In addition, it was demonstrated that HAX-1 status influences vimentin expression levels. Overall, HAX-1 was shown to colocalize with P-body markers and influence P-body number per cell in a manner dependent on mRNA availability. Presented data support the hypothesis that HAX-1 is involved in mRNA processing as an element of P-body interaction network.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Interferência de RNA , Endopeptidases/metabolismo , Células HeLa , Humanos , Células Jurkat , Células MCF-7 , Biossíntese de Proteínas , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Gênica , Vimentina/genética , Vimentina/metabolismoRESUMO
In the context of cell therapy, the epigenetic status of core stemness transcription factor (STF) genes regulating the cell proliferation/differentiation program is of primary interest. Our results confirmed that in vitro differentiation of the umbilicalcord-blood-derived-neural-stem-cells (HUCB-NSC) coincides with the progressive down-regulation of Oct3/4 and Nanog gene expression. Consistently and in parallel with the repression of gene transcription, a substantial increase in the mosaic cytosine methylation CpG dinucleotide was observed in the promoter regions of these STF genes. However none of the histone-H3 post-translational-modifications (PTM) known to be associated with transcriptionally active genes (H3Ac and H3K4me3) or repressed genes (H3K9me3 and H3K27me3) seemed to vary in relation to the progression of cell differentiation and down-regulation of STF genes. This indicates an uncoupling between STF gene expression and above mentioned histone PTMs. In contrast, the overall methylation of nuclear chromatin at repressive histone H3K9me3 was significantly higher than H3K4 trimetylation in expanding HUCB-NSC cultures and then increases through the progression of cell differentiation. These observations suggest different epigenetic programs of gene repression realized in the cell nuclei of differentiating HUCB-NSC cultures with uneven involvement of the repressive histone PTMs.
Assuntos
Diferenciação Celular/fisiologia , Proliferação de Células , Epigenômica , Sangue Fetal/citologia , Regulação da Expressão Gênica/fisiologia , Células-Tronco Neurais/fisiologia , Bucladesina/farmacologia , Imunoprecipitação da Cromatina , Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Histonas/genética , Histonas/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Proteína Homeobox Nanog , Células-Tronco Neurais/efeitos dos fármacos , Fator 3 de Transcrição de Octâmero/metabolismo , RNA Mensageiro , TransativadoresRESUMO
HAX-1 is a multi-functional protein that is involved in the regulation of apoptosis, cell motility and calcium homeostasis. It is also reported to bind RNA: it associates with structural motifs present in the 3' untranslated regions of at least two transcripts, but the functional significance of this binding remains unknown. Although HAX-1 has been detected in various cellular compartments, it is predominantly cytoplasmic. Our detailed localization studies of HAX-1 isoforms revealed partial nuclear localization, the extent of which depends on the protein isoform. Further studies demonstrated that HAX-1 is in fact a nucleocytoplasmic shuttling protein, dependent on the exportin 1 nuclear export receptor. Systematic mutagenesis allowed identification of the two nuclear export signals in the HAX-1 sequence. HAX-1 nuclear accumulation was observed after inhibition of nuclear export by leptomycin B, but also after specific cellular stress. The biological role of HAX-1 nuclear localization and shuttling remains to be established, but the HAX-1 transcript-binding properties suggest that it may be connected to mRNA processing and surveillance. In this study, HAX-1 status was shown to influence mRNA levels of DNA polymerase ß, one of the HAX-1 mRNA targets, although this effect becomes pronounced only after specific stress is applied. Moreover, HAX-1 tethering to the reporter transcript caused a significant decrease in its expression. Additionally, the HAX-1 co-localization with P-body markers, reported here, implies a role in mRNA processing. These results suggest that HAX-1 may be involved in the regulation of expression of bound transcripts, possibly as part of the stress response.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Núcleo Celular/metabolismo , Transporte Ativo do Núcleo Celular , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Animais , Arsenitos/farmacologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , DNA Polimerase beta/genética , DNA Polimerase beta/metabolismo , Regulação da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Carioferinas/metabolismo , Camundongos , Dados de Sequência Molecular , Sinais de Exportação Nuclear , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiologia , Transporte Proteico , Processamento Pós-Transcricional do RNA , Ratos , Receptores Citoplasmáticos e Nucleares/metabolismo , Estresse Fisiológico , Teratogênicos/farmacologia , Proteína Exportina 1RESUMO
Standardization of methods for obtaining iPS cells from the human somatic cells and then their successful differentiation are important in the context of their possible application in personalized cell therapy and the development of toxicological and pharmacological tests. In the present study, the influence of the small molecules representing epigenetic modulators (histone deacetylase inhibitor Trichostatin A and DNA methyltransferase inhibitor RG-108) on the process of reverting neural progenitors from HUCB-NSC (Human Umbilical Cord Blood Neural Stem Cell) line to the pluripotent state was tested. The experiments were conducted in low oxygen tension, in three different experimental layouts: (1) in the presence of reprogramming/recombinant polyarginine-tailed proteins; (2) with recombinant proteins and small molecules; (3) only in the presence of small molecules. We wanted to find out, whether it will be possible to induce pluripotent state of neural stem cells only by epigenetic modulators. Our results revealed that the inhibitors of DNA methylation and histone deacetylation used along with 5 percent oxygen tension can only transiently induce or elevate some pluripotency genes in neural progenitors with different pattern, but were not sufficient for stable reprogramming. The iPS cells from neural progenitor cells of HUCBNSC were obtained only when TSA, RG-108 and reprogramming proteins have been applied simultaneously. These cells were tested for the expression of the selected pluripotency genes and in functional assays to prove their pluripotency stage. The obtained data show that the small molecules in conjunction with reprogramming factors are the potent tools in cell reprogramming.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Reprogramação Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Sangue Fetal/citologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Células Cultivadas , Reprogramação Celular/genética , Metilação de DNA , Sangue Fetal/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Camadas Germinativas/citologia , Camadas Germinativas/efeitos dos fármacos , Humanos , Ácidos Hidroxâmicos/farmacologia , Indóis/farmacologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/fisiologia , Cariotipagem , Oxigênio/metabolismo , Ftalimidas , Células-Tronco Pluripotentes/fisiologia , Propionatos/farmacologia , Fatores de Tempo , Fatores de Transcrição/genética , Transfecção , Triptofano/análogos & derivadosRESUMO
The stem cells are characterized by self-renewal ability and potential to differentiate into other cell types of the body. They are residuing in defined microenvironments - "stem cell niches". The embryonic stem cells (ESC) are derived from embryos which exist in 3-5 percent oxygen condition. This environment is physiologically normal not only for ES cells but also for many other types of stem cells including neural stem cells (NSC). These observations suggest that low oxygen condition plays a very important role in the maintenance of cell stemness. Pluripotency is regulated by the family of hypoxia inducible factors (HIFs), which are dependent on oxygen tensions. HIF-2α is an upstream regulator of Oct4, which is one of the main transcription factors used to generate the first induced pluripotent stem cells (iPSCs). It has been shown that knock-down of HIF-2α but not HIF-1α, leads to a decrease in the expression of Oct4, Nanog and Sox2, which are important stem cells markers. The structure of hypoxia inducible factors as well as their behavior in hypoxia and normoxia was described. Therefore optimization of oxygen concentration seems to be crucial from the stem cell transplantation as well as iPS transplantation standpoint. Although many experiments with cell culture under low oxygen condition were performed, there is still much that is unknown. This short review presents some aspects on important issue of hypoxia induced regulation of stemness.