RESUMEN
Induced pluripotent stem cells (iPSCs) from patients are an attractive disease model to study tissues with poor accessibility such as the brain. Using this approach, we and others have shown that trisomy 21 results in genome-wide transcriptional dysregulations. The effects of loss of genes on chromosome 21 is much less characterized. Here, we use patient-derived neural cells from an individual with neurodevelopmental delay and a ring chromosome 21 with two deletions spanning 3.8 Mb at the terminal end of 21q22.3, containing 60 protein-coding genes. To investigate the molecular perturbations of the partial monosomy on neural cells, we established patient-derived iPSCs from fibroblasts retaining the ring chromosome 21, and we then induced iPSCs into neuroepithelial stem cells. RNA-Seq analysis of NESCs with the ring chromosome revealed downregulation of 18 genes within the deleted region together with global transcriptomic dysregulations when compared to euploid NESCs. Since the deletions on chromosome 21 represent a genetic "contrary" to trisomy of the corresponding region, we further compared the dysregulated transcriptomic profile in with that of two NESC lines with trisomy 21. The analysis revealed opposed expression changes for 23 genes on chromosome 21 as well as 149 non-chromosome 21 genes. Taken together, our results bring insights into the effects on the global and chromosome 21 specific gene expression from a partial monosomy of chromosome 21qter during early neuronal differentiation.
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In order to investigate the effects of bone marrow-derived MSCs (mesenchymal stem cells) in reversing liver fibrosis and to determine their possible mechanism of action, mouse MSCs were infused into the tail vein of a CCl(4) injection mouse chronic model. MSCs caused a decrease in liver fibrosis histopathologically, 4 weeks after transplantation. The reduction in liver collagen was confirmed by quantitative analysis. Moreover, lipid peroxidation in the CCl(4)/MSC group decreased significantly. Quantitative RT (reverse transcription)-PCR analysis showed administration of MSCs has a significant antifibrotic effect as evidenced by the decrease in expression of liver collagen and increase in MMP13 (matrix metalloproteinase 13) in the CCl(4)/MSC group when compared with the CCl(4) group, 4 weeks after transplantation. The expression of alphaSMA (smooth muscle actin) and TIMP1 was also down-regulated in the CCl(4)/MSC group. Additionally, the expression of MMP9 was significantly up-regulated in the CCl(4)-treated group; however, there was no significant change after MSC injection. Few engrafted cells in the recipient liver and were able to differentiate into albumin-positive cells. In conclusion, MSCs can enhance recovery of a CCl(4)-injured mouse liver through their influence in reducing collagen deposition by possibly affecting expression of MMPs.
Asunto(s)
Cirrosis Hepática/terapia , Metaloproteinasa 13 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Trasplante de Células Madre Mesenquimatosas , Actinas/genética , Actinas/metabolismo , Animales , Células de la Médula Ósea/citología , Tetracloruro de Carbono , Colágeno/genética , Colágeno/metabolismo , Modelos Animales de Enfermedad , Regulación hacia Abajo , Femenino , Cirrosis Hepática/inducido químicamente , Cirrosis Hepática/enzimología , Metaloproteinasa 13 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/genética , Células Madre Mesenquimatosas/citología , Ratones , Inhibidor Tisular de Metaloproteinasa-1/genética , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Regulación hacia ArribaRESUMEN
BACKGROUND: Down syndrome (DS) is characterized by neurodevelopmental abnormalities caused by partial or complete trisomy of human chromosome 21 (T21). Analysis of Down syndrome brain specimens has shown global epigenetic and transcriptional changes but their interplay during early neurogenesis remains largely unknown. We differentiated induced pluripotent stem cells (iPSCs) established from two DS patients with complete T21 and matched euploid donors into two distinct neural stages corresponding to early- and mid-gestational ages. RESULTS: Using the Illumina Infinium 450K array, we assessed the DNA methylation pattern of known CpG regions and promoters across the genome in trisomic neural iPSC derivatives, and we identified a total of 500 stably and differentially methylated CpGs that were annotated to CpG islands of 151 genes. The genes were enriched within the DNA binding category, uncovering 37 factors of importance for transcriptional regulation and chromatin structure. In particular, we observed regional epigenetic changes of the transcription factor genes ZNF69, ZNF700 and ZNF763 as well as the HOXA3, HOXB3 and HOXD3 genes. A similar clustering of differential methylation was found in the CpG islands of the HIST1 genes suggesting effects on chromatin remodeling. CONCLUSIONS: The study shows that early established differential methylation in neural iPSC derivatives with T21 are associated with a set of genes relevant for DS brain development, providing a novel framework for further studies on epigenetic changes and transcriptional dysregulation during T21 neurogenesis.
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Encéfalo/metabolismo , Metilación de ADN/genética , Síndrome de Down/genética , Epigenómica/métodos , Células Madre Pluripotentes Inducidas/metabolismo , Adulto , Encéfalo/patología , Ensamble y Desensamble de Cromatina/genética , Islas de CpG/genética , Síndrome de Down/complicaciones , Femenino , Feto/metabolismo , Feto/patología , Regulación de la Expresión Génica/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Masculino , Trastornos del Neurodesarrollo/etiología , Trastornos del Neurodesarrollo/genética , Neurogénesis/genética , Embarazo , Regiones Promotoras Genéticas , Factores de Transcripción/genética , Trisomía/genéticaRESUMEN
Embryonic stem cells (ESCs) are considered one promising new approach to generate a transplantable cell source for the treatment of liver diseases. Because traditional methods, such as the initial formation of embryoid body in the presence of serum result in all three germ layer derivatives, strategies have been utilized that favor cell-specific differentiation to generate more uniformity. Here, we have presented the use of a multistep protocol with growth factors in a serum-free adherent culture configuration to mediate the hepatocyte differentiation of human ESCs (hESCs). The differentiated cells exhibited characteristic hepatocyte morphology, ultrastructure, and expressed hepatic-related genes as shown by reverse transcription-polymerase chain reaction and displayed antibody detectable expression of markers specific for hepatic maturation. These hepatocyte-like cells also demonstrated evidence of albumin and alpha-fetoprotein secretion, glycogen storage, urea production, uptake of low-density lipoprotein, and indocyanine green. Therefore, we propose that the hepatocyte-like cells derived from hESCs by the present method may provide a useful model for the studies of key events during early liver development and a potential source of drug screening and transplantable cells for cell-replacement therapies.
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Técnicas de Cultivo de Célula/métodos , Medio de Cultivo Libre de Suero/farmacología , Células Madre Embrionarias/efectos de los fármacos , Hepatocitos/citología , Antígenos de Diferenciación/biosíntesis , Antígenos de Diferenciación/genética , Diferenciación Celular/efectos de los fármacos , Dexametasona/farmacología , Células Madre Embrionarias/citología , Perfilación de la Expresión Génica , Hepatocitos/metabolismo , Humanos , Insulina/farmacología , Péptidos y Proteínas de Señalización Intercelular/farmacología , Selenio/farmacología , Coloración y EtiquetadoRESUMEN
Down syndrome (DS) or trisomy 21 (T21) is a leading genetic cause of intellectual disability. To gain insights into dynamics of molecular perturbations during neurogenesis in DS, we established a model using induced pluripotent stem cells (iPSC) with transcriptome profiles comparable to that of normal fetal brain development. When applied on iPSCs with T21, transcriptome and proteome signatures at two stages of differentiation revealed strong temporal dynamics of dysregulated genes, proteins and pathways belonging to 11 major functional clusters. DNA replication, synaptic maturation and neuroactive clusters were disturbed at the early differentiation time point accompanied by a skewed transition from the neural progenitor cell stage and reduced cellular growth. With differentiation, growth factor and extracellular matrix, oxidative phosphorylation and glycolysis emerged as major perturbed clusters. Furthermore, we identified a marked dysregulation of a set of genes encoded by chromosome 21 including an early upregulation of the hub gene APP, supporting its role for disturbed neurogenesis, and the transcription factors OLIG1, OLIG2 and RUNX1, consistent with deficient myelination and neuronal differentiation. Taken together, our findings highlight novel sequential and differentiation-dependent dynamics of disturbed functions, pathways and elements in T21 neurogenesis, providing further insights into developmental abnormalities of the DS brain.
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Síndrome de Down/genética , Síndrome de Down/patología , Células Madre Pluripotentes Inducidas/patología , Neuronas/metabolismo , Neuronas/patología , Proteoma/metabolismo , Transcriptoma/genética , Diferenciación Celular/genética , Proliferación Celular/genética , Femenino , Humanos , Masculino , Mitocondrias/genética , Modelos Biológicos , Neuritas/metabolismo , Neurogénesis/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Factores de Tiempo , Transcripción GenéticaRESUMEN
The etiology of hereditary ataxia syndromes is heterogeneous, and the mechanisms underlying these disorders are often unknown. Here, we utilized exome sequencing in two siblings with progressive ataxia and muscular weakness and identified a novel homozygous splice mutation (c.3020-1G > A) in neurofascin (NFASC). In RNA extracted from fibroblasts, we showed that the mutation resulted in inframe skipping of exon 26, with a deprived expression of the full-length transcript that corresponds to NFASC isoform NF186. To further investigate the disease mechanisms, we reprogrammed fibroblasts from one affected sibling to induced pluripotent stem cells, directed them to neuroepithelial stem cells and finally differentiated to neurons. In early neurogenesis, differentiating cells with selective depletion of the NF186 isoform showed significantly reduced neurite outgrowth as well as fewer emerging neurites. Furthermore, whole-cell patch-clamp recordings of patient-derived neuronal cells revealed a lower threshold for openings, indicating altered Na+ channel kinetics, suggesting a lower threshold for openings as compared to neuronal cells without the NFASC mutation. Taken together, our results suggest that loss of the full-length NFASC isoform NF186 causes perturbed neurogenesis and impaired neuronal biophysical properties resulting in a novel early-onset autosomal recessive ataxia syndrome.
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Endogenous retroviruses (ERVs), which make up 8% of the human genome, have been proposed to participate in the control of gene regulatory networks. In this study, we find a region- and developmental stage-specific expression pattern of ERVs in the developing human brain, which is linked to a transcriptional network based on ERVs. We demonstrate that almost 10,000, primarily primate-specific, ERVs act as docking platforms for the co-repressor protein TRIM28 in human neural progenitor cells, which results in the establishment of local heterochromatin. Thereby, TRIM28 represses ERVs and consequently regulates the expression of neighboring genes. These results uncover a gene regulatory network based on ERVs that participates in control of gene expression of protein-coding transcripts important for brain development.
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Retrovirus Endógenos/genética , Redes Reguladoras de Genes , Células-Madre Neurales/metabolismo , Células-Madre Neurales/virología , Proteína 28 que Contiene Motivos Tripartito/metabolismo , Encéfalo/embriología , Encéfalo/virología , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Sistemas de Lectura Abierta/genética , Unión Proteica , Activación Transcripcional/genética , Regulación hacia Arriba/genéticaAsunto(s)
Colesterol/biosíntesis , Hepatocitos/fisiología , Hepatocitos/trasplante , Hiperlipoproteinemia Tipo II/terapia , Trasplante Homólogo , Adulto , Biomarcadores/metabolismo , Células de la Médula Ósea/citología , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Niño , Colesterol/genética , Femenino , Factor 4 de Crecimiento de Fibroblastos/farmacología , Mutación del Sistema de Lectura/genética , Factor de Crecimiento de Hepatocito/farmacología , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Homocigoto , Humanos , Hiperlipoproteinemia Tipo II/genética , Hiperlipoproteinemia Tipo II/inmunología , Hiperlipoproteinemia Tipo II/patología , Hiperlipoproteinemia Tipo II/fisiopatología , Hibridación Fluorescente in Situ , Masculino , Células Madre Mesenquimatosas/citología , Receptores de LDL/genéticaRESUMEN
The recent progress in stem cell techniques has broadened the horizon for in vitro disease modeling. For desired in vivo like phenotypes, not only correct cell type specification will be critical, the microenvironmental context will be essential to achieve relevant responses. We demonstrate how a three dimensional (3D) culture of stem cell derived neurons can induce in vivo like responses related to Alzheimer's disease, not recapitulated with conventional 2D cultures. To acquire a neural population of cells we differentiated neurons from neuroepithelial stem cells, derived from induced pluripotent stem cells. p21-activated kinase mediated sensing of Aß oligomers was only possible in the 3D environment. Further, the 3D phenotype showed clear effects on F-actin associated proteins, connected to the disease processes. We propose that the 3D in vitro model has higher resemblance to the AD pathology than conventional 2D cultures and could be used in further studies of the disease.
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Enfermedad de Alzheimer/patología , Células Madre Pluripotentes Inducidas/citología , Modelos Biológicos , Neuronas/citología , Quinasas p21 Activadas/metabolismo , Enfermedad de Alzheimer/enzimología , Péptidos beta-Amiloides/metabolismo , Diferenciación Celular , Células Cultivadas , Técnica del Anticuerpo Fluorescente , Humanos , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Human pluripotent stem cells (hPSCs) can undergo unlimited self-renewal and have the capacity to differentiate into all somatic cell types, and are therefore an ideal source for the generation of cells and tissues for research and therapy. To realize this potential, defined cell culture systems that allow expansion of hPSCs and subsequent controlled differentiation, ideally in an implantable three-dimensional (3D) matrix, are required. Here we mimic spider silk - Nature's high performance material - for the design of chemically defined 2D and 3D matrices for cell culture. The silk matrices do not only allow xeno-free long-term expansion of hPSCs but also differentiation in both 2D and 3D. These results show that biomimetic spider silk matrices enable hPSC culture in a manner that can be applied for experimental and clinical purposes.
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Diferenciación Celular/efectos de los fármacos , Células Madre Pluripotentes/citología , Seda/farmacología , Animales , Adhesión Celular/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Femenino , Humanos , Masculino , Ratones , ArañasRESUMEN
There are limited data available on the effect of a physicochemical microenvironment on mesenchymal stem cell (MSC) differentiation and repopulation of the liver. Therefore, in this study nanofibers have been used to better differentiate and maintain the function and engraftment of differentiating MSCs both in vitro and in vivo. Mouse MSCs were differentiated into early (day 18) and late (day 36) hepatocyte-like cells (HLCs) in the presence or absence of ultraweb nanofibers (nano(+) and nano(-)) and their transplantation for recovery in mice with CCl(4) induced hepatic fibrosis was investigated. In the nano(+) group, hepatocyte markers-ALB and HNF4α- were elevated in a time-dependent manner; however, those were similar levels or slightly decreased in the nano(-) group from day 18 to 36. Ultrastructural studies of the differentiated cells revealed some similarities to hepatocytes. Urea production, secretion of albumin and α-fetoprotein, and metabolic activity of the CYP450 enzymes were significantly increased within in vitro differentiated HLCs on nanofibers at day 36. MSCs, early and late HLCs in both nano(-) and nano(+) culture conditions that were transplanted by an intravenous route caused a decrease in liver fibrosis when engrafted in the recipient liver and were able to differentiate into functional hepatocytes (ALB(+)), except for late HLCs in the nano(-) group. Late HLCs transplanted in the nano(+) group were more effective in rescuing liver failure, enhancing serum ALB, homing transplanted cells and undergoing functional engraftment than the other groups. These results showed that topographic properties of nanofibers enhance differentiation of HLCs from MSCs and maintain their function in long-term culture, which has implications for cell therapies.
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Células de la Médula Ósea/citología , Diferenciación Celular , Hepatocitos/citología , Cirrosis Hepática/terapia , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Nanofibras/química , Animales , Biomarcadores/metabolismo , Tetracloruro de Carbono , Células Cultivadas , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Hepatocitos/metabolismo , Hepatocitos/ultraestructura , Cirrosis Hepática/inducido químicamente , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/ultraestructura , Ratones , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Andamios del Tejido/químicaRESUMEN
BACKGROUND: Orthotropic liver transplantation (OLT) is the final procedure of both end stage and metabolic liver diseases. Hepatocyte transplantation is an alternative for OLT, but the sources of hepatocytes are limited. Bone marrow mesenchymal stem cells (BM-MSCs) can differentiate into hepatocyte-like cells and are a potential alternative source for hepatocytes. We aimed to investigate the differentiation potential of BM-MSCs into hepatocyte-like cells. METHODS: Human BM-MSCs from a healthy donor were cultured and differentiated into hepatocyte-like cells. We investigated the expression of hepatocyte-specific markers in MSC-derived hepatocyte-like cells (MSC-HLC) and evaluated their functionality using metabolic assays. RESULTS: MSC-HLCs expressed hepatocyte-specific markers at both mRNA and protein levels. In addition, the cells had the ability to uptake low density lipoprotein (LDL), clear ammonia, secrete albumin, and store glycogen. MSC-HLCs were transplanted into a familial hypercholesteromia patient. CONCLUSION: Human MSCs can be differentiated into partially functional hepatocyte-like cells. Thus, they could be a potential source for cell therapy in liver disorders.