RESUMEN
Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs), which is a highly heterogeneous process. Here we report the cell fate continuum during somatic cell reprogramming at single-cell resolution. We first develop SOT to analyze cell fate continuum from Oct4/Sox2/Klf4- or OSK-mediated reprogramming and show that cells bifurcate into two categories, reprogramming potential (RP) or non-reprogramming (NR). We further show that Klf4 contributes to Cd34+/Fxyd5+/Psca+ keratinocyte-like NR fate and that IFN-γ impedes the final transition to chimera-competent pluripotency along the RP cells. We analyze more than 150,000 single cells from both OSK and chemical reprograming and identify additional NR/RP bifurcation points. Our work reveals a generic bifurcation model for cell fate decisions during somatic cell reprogramming that may be applicable to other systems and inspire further improvements for reprogramming.
Asunto(s)
Diferenciación Celular/genética , Linaje de la Célula/genética , Técnicas de Reprogramación Celular , Reprogramación Celular/genética , Células Madre Pluripotentes Inducidas/fisiología , Células Madre Embrionarias de Ratones/fisiología , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Animales , Femenino , Regulación del Desarrollo de la Expresión Génica , Células Madre Pluripotentes Inducidas/metabolismo , Interferón gamma/genética , Interferón gamma/metabolismo , Factor 4 Similar a Kruppel , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Transgénicos , Células Madre Embrionarias de Ratones/metabolismo , Fenotipo , Transducción de Señal , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
TGF-ß signaling family plays an essential role to regulate fate decisions in pluripotency and lineage specification. How the action of TGF-ß family signaling is intrinsically executed remains not fully elucidated. Here, we show that HBO1, a MYST histone acetyltransferase (HAT) is an essential cell intrinsic determinant for TGF-ß signaling in human embryonic stem cells (hESCs). HBO1-/- hESCs fail to response to TGF-ß signaling to maintain pluripotency and spontaneously differentiate into neuroectoderm. Moreover, HBO1 deficient hESCs show complete defect in mesendoderm specification in BMP4-triggered gastruloids or teratomas. Molecularly, HBO1 interacts with SMAD4 and co-binds the open chromatin labeled by H3K14ac and H3K4me3 in undifferentiated hESCs. Upon differentiation, HBO1/SMAD4 co-bind and maintain the mesoderm genes in BMP4-triggered mesoderm cells while lose chromatin occupancy in neural cells induced by dual-SMAD inhibition. Our data reveal an essential role of HBO1, a chromatin factor to determine the action of SMAD in both human pluripotency and mesendoderm specification.
Asunto(s)
Diferenciación Celular , Histona Acetiltransferasas , Mesodermo , Transducción de Señal , Proteína Smad4 , Humanos , Proteína Morfogenética Ósea 4/metabolismo , Proteína Morfogenética Ósea 4/genética , Línea Celular , Cromatina/metabolismo , Endodermo/citología , Endodermo/metabolismo , Histona Acetiltransferasas/metabolismo , Histona Acetiltransferasas/genética , Histonas/metabolismo , Células Madre Embrionarias Humanas/metabolismo , Células Madre Embrionarias Humanas/citología , Mesodermo/metabolismo , Mesodermo/citología , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/citología , Proteína Smad4/metabolismo , Proteína Smad4/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Cellular totipotency is critical for whole-organism generation, yet how totipotency is established remains poorly illustrated. Abundant transposable elements (TEs) are activated in totipotent cells, which is critical for embryonic totipotency. Here, we show that the histone chaperone RBBP4, but not its homolog RBBP7, is indispensable for maintaining the identity of mouse embryonic stem cells (mESCs). Auxin-induced degradation of RBBP4, but not RBBP7, reprograms mESCs to the totipotent 2C-like cells. Also, loss of RBBP4 enhances transition from mESCs to trophoblast cells. Mechanistically, RBBP4 binds to the endogenous retroviruses (ERVs) and functions as an upstream regulator by recruiting G9a to deposit H3K9me2 on ERVL elements, and recruiting KAP1 to deposit H3K9me3 on ERV1/ERVK elements, respectively. Moreover, RBBP4 facilitates the maintenance of nucleosome occupancy at the ERVK and ERVL sites within heterochromatin regions through the chromatin remodeler CHD4. RBBP4 depletion leads to the loss of the heterochromatin marks and activation of TEs and 2C genes. Together, our findings illustrate that RBBP4 is required for heterochromatin assembly and is a critical barrier for inducing cell fate transition from pluripotency to totipotency.
Asunto(s)
Heterocromatina , Células Madre Pluripotentes , Animales , Ratones , Heterocromatina/genética , Heterocromatina/metabolismo , Factores de Transcripción/metabolismo , Cromatina/metabolismo , Células Madre Pluripotentes/metabolismo , Células Madre Embrionarias de Ratones/metabolismo , Epigénesis GenéticaRESUMEN
Polycomb repressive complexes (PRCs) play critical roles in cell fate decisions during normal development as well as disease progression through mediating histone modifications such as H3K27me3 and H2AK119ub. How exactly PRCs recruited to chromatin remains to be fully illuminated. Here, we report that YTHDF1, the N6-methyladenine (m6 A) RNA reader that was previously known to be mainly cytoplasmic, associates with RNF2, a PRC1 protein that mediates H2AK119ub in human embryonic stem cells (hESCs). A portion of YTHDF1 localizes in the nuclei and associates with RNF2/H2AK119ub on a subset of gene loci related to neural development functions. Knock-down YTHDF1 attenuates H2AK119ub modification on these genes and promotes neural differentiation in hESCs. Our findings provide a noncanonical mechanism that YTHDF1 participates in PRC1 functions in hESCs.
Asunto(s)
Proteínas de Ciclo Celular , Células Madre Embrionarias Humanas , Proteínas de Unión al ARN , Humanos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromatina , Células Madre Embrionarias Humanas/metabolismo , Complejo Represivo Polycomb 1/genética , Complejo Represivo Polycomb 1/metabolismo , Proteínas del Grupo Polycomb/genética , Proteínas del Grupo Polycomb/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Histonas/genética , Histonas/metabolismoRESUMEN
MicroRNAs (miRNAs) are posttranscriptional modulators of gene expression and play an important role in many developmental processes. We report here that expression of microRNA-145 (miR-145) is low in self-renewing human embryonic stem cells (hESCs) but highly upregulated during differentiation. We identify the pluripotency factors OCT4, SOX2, and KLF4 as direct targets of miR-145 and show that endogenous miR-145 represses the 3' untranslated regions of OCT4, SOX2, and KLF4. Increased miR-145 expression inhibits hESC self-renewal, represses expression of pluripotency genes, and induces lineage-restricted differentiation. Loss of miR-145 impairs differentiation and elevates OCT4, SOX2, and KLF4. Furthermore, we find that the miR-145 promoter is bound and repressed by OCT4 in hESCs. This work reveals a direct link between the core reprogramming factors and miR-145 and uncovers a double-negative feedback loop involving OCT4, SOX2, KLF4, and miR-145.
Asunto(s)
Diferenciación Celular , Células Madre Embrionarias/citología , Regulación del Desarrollo de la Expresión Génica , Factores de Transcripción de Tipo Kruppel/genética , MicroARNs/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , Células Madre Pluripotentes/citología , Factores de Transcripción SOXB1/genética , Regiones no Traducidas 3'/metabolismo , Línea Celular , Células Madre Embrionarias/metabolismo , Humanos , Factor 4 Similar a Kruppel , Células Madre Pluripotentes/metabolismoRESUMEN
CLCN2 encodes a two-pore homodimeric chloride channel protein (CLC-2) that is widely expressed in human tissues. The association between Clcn2 and the retina is well-established in mice, as loss-of-function of CLC-2 can cause retinopathy in mice; however, the ocular phenotypes caused by CLCN2 mutations in humans and the underlying mechanisms remain unclear. The present study aimed to define the ocular features and reveal the pathogenic mechanisms of CLCN2 variants associated with retinal degeneration in humans using an in vitro overexpression system, as well as patient-induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium (RPE) cells and retinal organoids (ROs). A patient carrying the homozygous c.2257C > T (p.R753X) nonsense CLCN2 mutation was followed up for > 6 years. Ocular features were comprehensively characterized with multimodality imaging and functional examination. The patient presented with severe bilateral retinal degeneration with loss of photoreceptor and RPE. In vitro, mutant CLC-2 maintained the correct subcellular localization, but with reduced channel function compared to wild-type CLC-2 in HEK293T cells. Additionally, patient iPSC-derived RPE cells carrying the CLCN2 mutation exhibited dysfunctional ClC-2 chloride channels and outer segment phagocytosis. Notably, these functions were rescued following the repair of the CLCN2 mutation using the CRISPR-Cas9 system. However, this variant did not cause significant photoreceptor degeneration in patient-derived ROs, indicating that dysfunctional RPE is likely the primary cause of biallelic CLCN2 variant-mediated retinopathy. This study is the first to establish the confirmatory ocular features of human CLCN2-related retinal degeneration, and reveal a pathogenic mechanism associated with biallelic CLCN2 variants, providing new insights into the cause of inherited retinal dystrophies.
Asunto(s)
Células Madre Pluripotentes Inducidas , Distrofias Retinianas , Animales , Humanos , Ratones , Canales de Cloruro/genética , Codón sin Sentido , Células HEK293 , Mutación , Fagocitosis/genética , Especies Reactivas de Oxígeno/metabolismo , Distrofias Retinianas/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patologíaRESUMEN
Tick-borne encephalitis virus (TBEV) is the causative agent of a potentially fatal neurological infection in humans. Investigating virus-host interaction is important for understanding the pathogenesis of TBEV and developing effective antiviral drugs against this virus. Here, we report that mammalian ste20-like kinase 3 (MST3) is involved in the regulation of TBEV infection. The knockdown or knockout of MST3, but not other mammalian ste20-like kinase family members, inhibited TBEV replication. The knockdown of MST3 also significantly reduced TBEV replication in mouse primary astrocytes. Life cycle analysis indicated that MST3 remarkably impaired virion assembly efficiency and specific infectivity by respectively 59% and 95% in MST3-knockout cells. We further found that MST3 interacts with the viral proteins NS2A and prM; and MST3 enhances the interaction of NS2A-NS4A. Thus, MST3-NS2A complex plays a major role in recruiting prM-E heterodimers and NS4A and mediates the virion assembly. Additionally, we found that MST3 was biotinylated and combined with other proteins (e.g., ATG5, Sec24A, and SNX4) that are associated with the cellular membrane required for TBEV infection. Overall, our study revealed a novel function for MST3 in TBEV infection and identified as a novel host factor supporting TBEV assembly.
Asunto(s)
Virus de la Encefalitis Transmitidos por Garrapatas , Encefalitis Transmitida por Garrapatas , Animales , Ratones , Humanos , Virus de la Encefalitis Transmitidos por Garrapatas/genética , Proteínas Virales/metabolismo , Mamíferos/metabolismo , Proteínas de Transporte VesicularRESUMEN
Hematopoietic stem cells (HSCs)/progenitor cells (HPCs) are generated from hemogenic endothelial cells (HECs) during the endothelial-to-hematopoietic transition (EHT); however, the underlying mechanism remains poorly understood. Here, using an array of approaches, including CRSPR/Cas9 gene knockouts, RNA-Seq, ChIP-Seq, ATAC-Seq etc., we report that vitamin C (Vc) is essential in HPC generation during human pluripotent stem cell (hPSC) differentiation in defined culture conditions. Mechanistically, we found that the endothelial cells generated in the absence of Vc fail to undergo the EHT because of an apparent failure in opening up genomic loci essential for hematopoiesis. Under Vc deficiency, these loci exhibited abnormal accumulation of histone H3 trimethylation at Lys-27 (H3K27me3), a repressive histone modification that arose because of lower activities of demethylases that target H3K27me3. Consistently, deletion of the two H3K27me3 demethylases, Jumonji domain-containing 3 (JMJD3 or KDM6B) and histone demethylase UTX (UTX or KDM6A), impaired HPC generation even in the presence of Vc. Furthermore, we noted that Vc and jmjd3 are also important for HSC generation during zebrafish development. Together, our findings reveal an essential role for Vc in the EHT for hematopoiesis, and identify KDM6-mediated chromatin demethylation as an important regulatory mechanism in hematopoietic cell differentiation.
Asunto(s)
Ácido Ascórbico/metabolismo , Células Madre Hematopoyéticas/metabolismo , Histona Demetilasas/metabolismo , Animales , Animales Modificados Genéticamente , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Cromatina/metabolismo , Cromatina/fisiología , Desmetilación , Células Endoteliales/metabolismo , Histona Demetilasas/genética , Histonas/metabolismo , Humanos , Histona Demetilasas con Dominio de Jumonji/metabolismo , Lisina/metabolismo , Metilación , Células Madre Pluripotentes/metabolismo , Pez Cebra/genéticaRESUMEN
BACKGROUND & AIMS: It has been a challenge to develop fully functioning cells from human pluripotent stem cells (hPSCs). We investigated how activation of hedgehog signaling regulates derivation of enteric neural crest (NC) cells from hPSCs. METHODS: We analyzed transcriptomes of mouse and hPSC-derived enteric NCs using single-cell RNA sequencing (scRNA-seq) to identify the changes in expression associated with lineage differentiation. Intestine tissues were collected from Tg(GBS-GFP), Sufuf/f; Wnt1-cre, Ptch1+/-, and Gli3Δ699/Δ699 mice and analyzed by flow cytometry and immunofluorescence for levels of messenger RNAs encoding factors in the hedgehog signaling pathway during differentiation of enteric NCs. Human NC cells (HNK-1+p75NTR+) were derived from IMR90 and UE02302 hPSC lines. hPSCs were incubated with a hedgehog agonist (smoothened agonist [SAG]) and antagonists (cyclopamine) and analyzed for differentiation. hPSC-based innervated colonic organoids were derived from these hPSC lines and analyzed by immunofluorescence and neuromuscular coupling assay for expression of neuronal subtype markers and assessment of the functional maturity of the hPSC-derived neurons, respectively. RESULTS: Single-cell RNA sequencing analysis showed that neural fate acquisition by human and mouse enteric NC cells requires reduced expression of NC- and cell cycle-specific genes and up-regulation of neuronal or glial lineage-specific genes. Activation of the hedgehog pathway was associated with progression of mouse enteric NCs to the more mature state along the neuronal and glial lineage differentiation trajectories. Activation of the hedgehog pathway promoted development of cultured hPSCs into NCs of greater neurogenic potential by activating expression of genes in the neurogenic lineage. The hedgehog agonist increased differentiation of hPSCs into cells of the neuronal lineage by up-regulating expression of GLI2 target genes, including INSM1, NHLH1, and various bHLH family members. The hedgehog agonist increased expression of late neuronal markers and neuronal activities in hPSC-derived neurons. CONCLUSIONS: In enteric NCs from humans and mice, activation of hedgehog signaling promotes differentiation into neurons by promoting cell-state transition, expression of genes in the neurogenic lineage, and functional maturity of enteric neurons.
Asunto(s)
Diferenciación Celular , Proteínas Hedgehog/metabolismo , Células Madre Pluripotentes Inducidas/fisiología , Neuronas/fisiología , Transducción de Señal/fisiología , Animales , Línea Celular , Sistema Nervioso Entérico/citología , Perfilación de la Expresión Génica/métodos , Proteínas Hedgehog/genética , Humanos , Mucosa Intestinal/citología , Mucosa Intestinal/inervación , Masculino , Ratones , Ratones Transgénicos , Cresta Neural/citología , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodosRESUMEN
During neurogenesis, neural patterning is a critical step during which neural progenitor cells differentiate into neurons with distinct functions. However, the molecular determinants that regulate neural patterning remain poorly understood. Here we optimized the "dual SMAD inhibition" method to specifically promote differentiation of human pluripotent stem cells (hPSCs) into forebrain and hindbrain neural progenitor cells along the rostral-caudal axis. We report that neural patterning determination occurs at the very early stage in this differentiation. Undifferentiated hPSCs expressed basal levels of the transcription factor orthodenticle homeobox 2 (OTX2) that dominantly drove hPSCs into the "default" rostral fate at the beginning of differentiation. Inhibition of glycogen synthase kinase 3ß (GSK3ß) through CHIR99021 application sustained transient expression of the transcription factor NANOG at early differentiation stages through Wnt signaling. Wnt signaling and NANOG antagonized OTX2 and, in the later stages of differentiation, switched the default rostral cell fate to the caudal one. Our findings have uncovered a mutual antagonism between NANOG and OTX2 underlying cell fate decisions during neural patterning, critical for the regulation of early neural development in humans.
Asunto(s)
Diferenciación Celular , Linaje de la Célula , Proteína Homeótica Nanog/metabolismo , Células-Madre Neurales/citología , Neuronas/citología , Factores de Transcripción Otx/metabolismo , Células Madre Pluripotentes/citología , Tipificación del Cuerpo , Células Cultivadas , Regulación del Desarrollo de la Expresión Génica , Humanos , Colículos Inferiores/citología , Colículos Inferiores/metabolismo , Núcleos del Rafe Mesencefálico/citología , Núcleos del Rafe Mesencefálico/metabolismo , Células-Madre Neurales/metabolismo , Neurogénesis , Neuronas/metabolismo , Células Madre Pluripotentes/metabolismo , Prosencéfalo/citología , Prosencéfalo/metabolismo , Rombencéfalo/citología , Rombencéfalo/metabolismoRESUMEN
BACKGROUND/AIMS: Among different molecular candidates, there is growing data to support that long noncoding RNAs (lncRNAs) play a significant role in acute myeloid leukemia (AML). HOXA-AS2 is significantly overexpressed in a variety of tumors and associated with anti-cancer drug resistance, however, little is known regarding the expression and function of HOXA-AS2 in the chemoresistance of AML. In this study, we aimed to determine the role and molecular mechanism of HOXA-AS2 in adriamycin-based chemotherapy resistance in AML cells. METHODS: Quantitative real-time PCR was used to detect HOXA-AS2 expression in the BM samples and ADR cell lines, U/A and T/A cells. Furthermore, the effects of HOXA-AS2 silencing on cell proliferation and apoptosis were assessed in vitro by CCK8 and flow cytometry, and on tumor growth in vivo. Furthermore, bioinformatics online programs predicted and luciferase reporter assay were used to validate the association of HOXA-AS2 and miR-520c-3p in AML. RESULTS: In this study, we showed that HOXA-AS2 is significantly upregulated in BM samples from AML patients after treatment with adriamycin-based chemotherapy and in U/A and T/A cells. Knockdown of HOXA-AS2 inhibited ADR cell proliferation in vitro and in vivo and promoted apoptosis. Bioinformatics online programs predicted that HOXA-AS2 sponge miR-520c-3p at 3'-UTR with complementary binding sites, which was validated using luciferase reporter assay and anti-Ago2 RIP assay. HOXA-AS2 could negatively regulate the expression of miR-520c-3p in ADR cells. S100A4 was predicted as a downstream target of miR-520c-3p, which was confirmed by luciferase reporter assay. CONCLUSION: Our results suggest that HOXA-AS2 plays an important role in the resistance of AML cells to adriamycin. Thus, HOXA-AS2 may represent a therapeutic target for overcoming resistance to adriamycin-based chemotherapy in AML.
Asunto(s)
Leucemia Mieloide Aguda/patología , MicroARNs/metabolismo , ARN Largo no Codificante/metabolismo , Proteína de Unión al Calcio S100A4/metabolismo , Regiones no Traducidas 3' , Animales , Antagomirs/metabolismo , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Doxorrubicina/uso terapéutico , Resistencia a Antineoplásicos , Regulación Neoplásica de la Expresión Génica , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Masculino , Ratones , Ratones Desnudos , MicroARNs/antagonistas & inhibidores , MicroARNs/genética , Interferencia de ARN , ARN Largo no Codificante/antagonistas & inhibidores , ARN Largo no Codificante/genética , ARN Interferente Pequeño/metabolismo , ARN Interferente Pequeño/uso terapéutico , Proteína de Unión al Calcio S100A4/química , Proteína de Unión al Calcio S100A4/genéticaRESUMEN
Dissociation-induced apoptosis is a striking phenomenon in human embryonic stem cells (hESCs), but not in naive mouse ESCs. Rho-associated kinase-dependent actin-myosin hyperactivation is an underlying mechanism that triggers apoptosis in dissociated hESCs; however, in this study, we show that the Ink4A-ARF-mediated senescence pathway is another mechanism to cause apoptosis in individualized hESCs. We show that P16INK4A and P14ARF are immediately induced in hESCs upon dissociation, but not in mouse ESCs. Overexpression of BMI1, a suppressor for Ink4A-ARF, greatly promotes survival and cloning efficiency of individualized hESCs mechanistically via direct binding the H3K27me3-marked Ink4A-ARF locus. Forced expression of BMI1 in hESCs does not reduce the actin-myosin activation that is triggered by dissociation, which indicates it is an independent pathway for hESC survival. Furthermore, dual inhibition of both Ink4A-ARF and actin-myosin hyperactivation enables successful passaging of hESCs via gelatin, a nonbioactive matrix. In sum, we provide an additional mechanism that underlies cell death in individualized hESCs that might help to fully understand the differential cell characteristics between naive and primed ESCs.-Wang, W., Zhu, Y., Huang, K., Shan, Y., Du, J., Dong, X., Ma, P., Wu, P., Zhang, J., Huang, W., Zhang, T., Liao, B., Yao, D., Pan, G., Liu, J. Suppressing P16Ink4a and P14ARF pathways overcomes apoptosis in individualized human embryonic stem cells.
Asunto(s)
Apoptosis , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Células Madre Embrionarias/metabolismo , Proteína p14ARF Supresora de Tumor/metabolismo , Actinas/metabolismo , Animales , Línea Celular , Senescencia Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Células Madre Embrionarias/fisiología , Humanos , Ratones , Miosinas/metabolismo , Complejo Represivo Polycomb 1/genética , Complejo Represivo Polycomb 1/metabolismo , Proteína p14ARF Supresora de Tumor/genéticaRESUMEN
Reprogramming of somatic cells to induced pluripotent stem cells rewrites the code of cell fate at the chromatin level. Yet, little is known about this process physically. Here, we describe a fluorescence recovery after photobleaching method to assess the dynamics of heterochromatin/euchromatin and show significant heterochromatin loosening at the initial stage of reprogramming. We identify growth arrest and DNA damage-inducible protein a (Gadd45a) as a chromatin relaxer in mouse embryonic fibroblasts, which also enhances somatic cell reprogramming efficiency. We show that residue glycine 39 (G39) in Gadd45a is essential for interacting with core histones, opening chromatin and enhancing reprogramming. We further demonstrate that Gadd45a destabilizes histone-DNA interactions and facilitates the binding of Yamanaka factors to their targets for activation. Our study provides a method to screen factors that impact on chromatin structure in live cells, and identifies Gadd45a as a chromatin relaxer.
Asunto(s)
Proteínas de Ciclo Celular/genética , Reprogramación Celular , Heterocromatina/metabolismo , Células Madre Pluripotentes Inducidas/fisiología , Proteínas Nucleares/genética , Animales , Proteínas de Ciclo Celular/metabolismo , Diferenciación Celular/genética , Reprogramación Celular/genética , ADN/genética , ADN/metabolismo , Células Madre Embrionarias/metabolismo , Fibroblastos/metabolismo , Glicina/metabolismo , Heterocromatina/genética , Histonas/genética , Histonas/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Proteínas Nucleares/metabolismo , FotoblanqueoRESUMEN
The generation of personalized induced pluripotent stem cells (iPSCs) followed by targeted genome editing provides an opportunity for developing customized effective cellular therapies for genetic disorders. However, it is critical to ascertain whether edited iPSCs harbor unfavorable genomic variations before their clinical application. To examine the mutation status of the edited iPSC genome and trace the origin of possible mutations at different steps, we have generated virus-free iPSCs from amniotic cells carrying homozygous point mutations in ß-hemoglobin gene (HBB) that cause severe ß-thalassemia (ß-Thal), corrected the mutations in both HBB alleles by zinc finger nuclease-aided gene targeting, and obtained the final HBB gene-corrected iPSCs by excising the exogenous drug resistance gene with Cre recombinase. Through comparative genomic hybridization and whole-exome sequencing, we uncovered seven copy number variations, five small insertions/deletions, and 64 single nucleotide variations (SNVs) in ß-Thal iPSCs before the gene targeting step and found a single small copy number variation, 19 insertions/deletions, and 340 single nucleotide variations in the final gene-corrected ß-Thal iPSCs. Our data revealed that substantial but different genomic variations occurred at factor-induced somatic cell reprogramming and zinc finger nuclease-aided gene targeting steps, suggesting that stringent genomic monitoring and selection are needed both at the time of iPSC derivation and after gene targeting.
Asunto(s)
Reprogramación Celular , Endonucleasas/metabolismo , Endorribonucleasas/metabolismo , Marcación de Gen , Inestabilidad Genómica , Células Madre Pluripotentes Inducidas/citología , Alelos , Animales , Diferenciación Celular , Cromosomas/ultraestructura , Hibridación Genómica Comparativa , Variaciones en el Número de Copia de ADN , Eritroblastos/citología , Exoma , Eliminación de Gen , Variación Genética , Humanos , Ratones , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Dedos de Zinc/genética , Globinas beta/genética , Talasemia beta/genéticaRESUMEN
BACKGROUND & AIMS: Levels of atonal homolog 8 (ATOH8) are reduced in 48% of hepatitis B virus-associated hepatocellular carcinoma cells (HCCs). ATOH8 downregulation is associated with loss of tumor differentiation, indicating an effect mediated by cancer stem cells. We investigated the effects of loss of ATOH8 in human hepatocellular carcinoma (HCC) cells and cell lines. METHODS: HCC and adjacent nontumor tissues were collected, from 2001 through 2012, from 242 patients undergoing hepatectomy at Sun Yat-Sen University Cancer Center in China; 83% of HCCs were associated with hepatitis B virus (HBV) infection. CD133(+) cells were isolated from tumor tissues by flow cytometry. Experiments were performed in HBV-positive and HBV-negative HCC cell lines, the immortalized liver cell line LO2, and 8 other HCC cell lines. ATOH8 was expressed from lentiviral vectors in PLC8024 and Huh7 cells; levels were knocked down with small interfering RNAs in QSG7701 cells. Cells carrying empty vectors were used as controls. Gene regulation by ATOH8 was assessed in mobility shift and luciferase reporter assays. Cells were analyzed in proliferation, foci formation, and colony formation assays. The tumorigenic and chemo-resistant potential of cells were investigated by assessing growth of xenograft tumors in immunocompromised mice. Metastatic features of cells were assessed in Matrigel invasion assays and wound healing analyses. RESULTS: Levels of ATOH8 mRNA were reduced by more than 4-fold, compared to nontumor tissues, in 118 of 242 HCC samples (48.8%). Patients with tumor reductions in ATOH8 had significantly shorter times of disease-free survival (mean, 41.4 months) than patients with normal tissue levels (mean, 52.6 months). ATOH8 expression was reduced in HepG2, Huh7, PLC8024 and CRL8064 HCC cells, as well as CD133(+) cells isolated from human HCC samples. Transgenic expression of ATOH8 in HCC cell lines significantly reduced proliferation and foci colony formation, as well as their invasive and migratory abilities. Transgenic expression of ATOH8 reduced the ability of HBV-positive PLC8024 cells to form tumors in mice, compared to control cells. Cells with ATOH8 knockdown formed xenograft tumors more rapidly, in more mice, than control cells. ATOH8 repressed transcription of stem-cell associated genes including OCT4, NANOG, and CD133. Knockdown of ATOH8 in CD133-negative QSG7701 cells caused them to express CD133; acquire self-renewal, differentiation, chemo-resistance properties; form more xenograft tumors in mice; and generate induced pluripotent stem cells (based on staining for alkaline phosphatase and their ability to form embryoid bodies and teratomas). Alternatively, expression of ATOH8 in PLC8024 and Huh7 cells significantly reduced the numbers of cells expressing CD133, and increased the chemo-sensitivity of Huh7 cells to 5-fluorouracil (5-FU) and cisplatin, in vitro and in mice. CONCLUSIONS: ATOH8 appears to be a tumor suppressor that induces stem-cell features and chemoresistance in HCC cells. Strategies to restore its levels and activities might be developed to treat patients with liver cancer.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Carcinoma Hepatocelular/metabolismo , Desdiferenciación Celular , Neoplasias Hepáticas/metabolismo , Células Madre Neoplásicas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Antígeno AC133 , Animales , Antígenos CD/metabolismo , Antineoplásicos/farmacología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Movimiento Celular , Proliferación Celular , Supervivencia Celular , Supervivencia sin Enfermedad , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Resistencia a Antineoplásicos , Regulación Neoplásica de la Expresión Génica , Glicoproteínas/metabolismo , Células Hep G2 , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Estimación de Kaplan-Meier , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Masculino , Ratones SCID , Invasividad Neoplásica , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Péptidos/metabolismo , Interferencia de ARN , ARN Mensajero/metabolismo , Tratamiento con ARN de Interferencia , Transducción de Señal , Factores de Tiempo , Transcripción Genética , Transfección , Proteínas Supresoras de Tumor/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Human neural stem cells hold great promise for research and therapy in neural disease. We describe the generation of integration-free and expandable human neural progenitor cells (NPCs). We combined an episomal system to deliver reprogramming factors with a chemically defined culture medium to reprogram epithelial-like cells from human urine into NPCs (hUiNPCs). These transgene-free hUiNPCs can self-renew and can differentiate into multiple functional neuronal subtypes and glial cells in vitro. Although functional in vivo analysis is still needed, we report that the cells survive and differentiate upon transplant into newborn rat brain.
Asunto(s)
Encéfalo/citología , Diferenciación Celular , Reprogramación Celular , Células Epiteliales/citología , Células-Madre Neurales/citología , Ingeniería de Tejidos/métodos , Orina/citología , Animales , Animales Recién Nacidos , Biomarcadores/metabolismo , Western Blotting , Perfilación de la Expresión Génica , Humanos , Técnicas para Inmunoenzimas , Células-Madre Neurales/trasplante , Neuroglía/citología , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genética , Ratas , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trasplante de Células Madre , Orina/químicaRESUMEN
Differentiation of neural lineages from human pluripotent stem cells (hPSCs) raises the hope of generating functional cells for the treatment of neural diseases. However, current protocols for differentiating hPSCs into neural lineages remain inefficient and largely variable between different hPSC lines. We report that microRNA 376c (miR-376c) significantly enhanced neural differentiation of hPSCs in a defined condition by suppressing SMAD4, the co-SMAD for TGF-ß signaling. Downstream, SMAD4 directly bound and suppressed PAX6, the critical neural lineage specification factor. Interestingly, we also found that SMAD4 binds and suppresses miR-376c clusters in undifferentiated hESCs. In summary, our findings revealed a reciprocal antagonism between miR-376c and SMAD signaling that regulates cell fate during human neural differentiation.
Asunto(s)
Diferenciación Celular , MicroARNs/metabolismo , Células Madre Pluripotentes/citología , Transducción de Señal , Proteína Smad4/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Diferenciación Celular/fisiología , Células Cultivadas , Técnicas de Silenciamiento del Gen/métodos , Humanos , Células Madre Pluripotentes/metabolismo , Transducción de Señal/fisiologíaRESUMEN
CCCTC-binding factor (CTCF) is a ubiquitously expressed "master weaver" and plays multiple functions in the genome, including transcriptional activation/repression, chromatin insulation, imprinting, X chromosome inactivation, and high-order chromatin organization. It has been shown that CTCF facilitates the recruitment of the upstream binding factor onto ribosomal DNA (rDNA) and regulates the local epigenetic state of rDNA repeats. However, the mechanism by which CTCF modulates rRNA gene transcription has not been well understood. Here we found that wild-type CTCF augments the pre-rRNA level, cell size, and cell growth in cervical cancer cells. In contrast, RNA interference-mediated knockdown of CTCF reduced pre-rRNA transcription. CTCF positively regulates rRNA gene transcription in a RNA polymerase I-dependent manner. We identified an RRGR motif as a putative nucleolar localization sequence in the C-terminal region of CTCF that is required for activating rRNA gene transcription. Using mass spectrometry, we identified SMC2 and SMC4, two subunits of condensin complexes that interact with CTCF. Condensin negatively regulates CTCF-mediated rRNA gene transcription. Knockdown of SMC2 expression significantly facilitates the loading of CTCF and the upstream binding factor onto the rDNA locus and increases histone acetylation across the rDNA locus. Taken together, our study suggests that condensin competes with CTCF in binding to a specific rDNA locus and negatively regulates CTCF-mediated rRNA gene transcription.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Genes de ARNr/fisiología , Complejos Multiproteicos/metabolismo , ARN Polimerasa I/metabolismo , Precursores del ARN/biosíntesis , Proteínas Represoras/metabolismo , Iniciación de la Transcripción Genética/fisiología , Acetilación , Adenosina Trifosfatasas/genética , Factor de Unión a CCCTC , ADN Ribosómico/genética , ADN Ribosómico/metabolismo , Proteínas de Unión al ADN/genética , Femenino , Sitios Genéticos/fisiología , Células HeLa , Humanos , Complejos Multiproteicos/genética , Estructura Terciaria de Proteína , ARN Polimerasa I/genética , Precursores del ARN/genética , Proteínas Represoras/genéticaRESUMEN
ß-Thalassemia (ß-Thal) is a group of life-threatening blood disorders caused by either point mutations or deletions of nucleotides in ß-globin gene (HBB). It is estimated that 4.5% of the population in the world carry ß-Thal mutants (1), posing a persistent threat to public health. The generation of patient-specific induced pluripotent stem cells (iPSCs) and subsequent correction of the disease-causing mutations offer an ideal therapeutic solution to this problem. However, homologous recombination-based gene correction in human iPSCs remains largely inefficient. Here, we describe a robust process combining efficient generation of integration-free ß-Thal iPSCs from the cells of patients and transcription activator-like effector nuclease (TALEN)-based universal correction of HBB mutations in situ. We generated integration-free and gene-corrected iPSC lines from two patients carrying different types of homozygous mutations and showed that these iPSCs are pluripotent and have normal karyotype. We showed that the correction process did not generate TALEN-induced off targeting mutations by sequencing. More importantly, the gene-corrected ß-Thal iPS cell lines from each patient can be induced to differentiate into hematopoietic progenitor cells and then further to erythroblasts expressing normal ß-globin. Our studies provide an efficient and universal strategy to correct different types of ß-globin mutations in ß-Thal iPSCs for disease modeling and applications.
Asunto(s)
Desoxirribonucleasas/genética , Desoxirribonucleasas/uso terapéutico , Terapia Genética , Globinas beta/genética , Talasemia beta/genética , Diferenciación Celular , Células Cultivadas , Desoxirribonucleasas/metabolismo , Endonucleasas/genética , Recombinación Homóloga , Humanos , Células Madre Pluripotentes Inducidas/citología , Mutación , Globinas beta/metabolismo , Talasemia beta/patología , Talasemia beta/terapiaRESUMEN
Reprogramming of somatic cells to a pluripotent state holds huge potentials for regenerative medicine. However, a debate over which method is better, somatic cell nuclear transfer (SCNT) or induced pluripotent stem (iPS) cells, still persists. Both approaches have the potential to generate patient-specific pluripotent stem cells for replacement therapy. Yet, although SCNT has been successfully applied in various vertebrates, no human pluripotent stem cells have been generated by SCNT due to technical, legal and ethical difficulties. On the other hand, human iPS cell lines have been reported from both healthy and diseased individuals. A recent study reported the generation of triploid human pluripotent stem cells by transferring somatic nuclei into oocytes, a variant form of SCNT. In this essay, we discuss this progress and the potentials of these two reprogramming approaches for regenerative medicine.