RESUMEN
Cytomegalovirus (CMV), a type of herpes virus, is the predominant cause of congenital anomalies due to intrauterine infections in humans. Adverse outcomes related to intrauterine infections with human cytomegalovirus (HCMV) vary widely, depending on factors such as fetal infection timing, infection route, and viral virulence. The precise mechanism underlying HCMV susceptibility remains unclear. In this study, we compared the susceptibility of neonatal human dermal fibroblast cells (NHDFCs) and human induced pluripotent stem cells (hiPSCs) derived from NHDFCs, which are genetically identical to HCMV, using immunostaining, microarray, in situ hybridization, quantitative PCR, and scanning electron microscopy. These cells were previously used to compare CMV susceptibility, but the underlying mechanisms were not fully elucidated. HCMV susceptibility of hiPSCs was significantly lower in the earliest phase. No shared gene ontologies were observed immediately post-infection between the two cell types using microarray analysis. Early-stage expression of HCMV antigens and the HCMV genome was minimal in immunostaining and in in situ hybridization in hiPSCs. This strongly suggests that HCMV does not readily bind to hiPSC surfaces. Scanning electron microscopy performed using the NanoSuit method confirmed the scarcity of HCMV particles on hiPSC surfaces. The zeta potential and charge mapping of the charged surface in NHDFCs and hiPSCs exhibited minimal differences when assessed using zeta potential analyzer and scanning ion conductance microscopy; however, the expression of heparan sulfate (HS) was significantly lower in hiPSCs compared with that in NHDFCs. Thus, HS expression could be a primary determinant of HCMV resistance in hiPSCs at the attachment level. IMPORTANCE: Numerous factors such as attachment, virus particle entry, transcription, and virus particle egress can affect viral susceptibility. Since 1984, pluripotent cells are known to be CMV resistant; however, the exact mechanism underlying this resistance remains elusive. Some researchers suggest inhibition in the initial phase of HCMV binding, while others have suggested the possibility of a sufficient amount of HCMV entering the cells to establish latency. This study demonstrates that HCMV particles rarely attach to the surfaces of hiPSCs. This is not due to limitations in the electrostatic interactions between the surface of hiPSCs and HCMV particles, but due to HS expression. Therefore, HS expression should be recognized as a key factor in determining the susceptibility of HCMV in congenital infection in vitro and in vivo. In the future, drugs targeting HS may become crucial for the treatment of congenital CMV infections. Thus, further research in this area is warranted.
Asunto(s)
Infecciones por Citomegalovirus , Citomegalovirus , Heparitina Sulfato , Células Madre Pluripotentes Inducidas , Humanos , Recién Nacido , Membrana Celular/química , Membrana Celular/metabolismo , Citomegalovirus/fisiología , Heparitina Sulfato/análisis , Heparitina Sulfato/metabolismo , Infecciones por Herpesviridae , Células Madre Pluripotentes Inducidas/química , Células Madre Pluripotentes Inducidas/metabolismo , Fibroblastos/química , Fibroblastos/metabolismo , Fibroblastos/virología , Piel/citologíaRESUMEN
BACKGROUND: Air pollution is recognized as an emerging environmental risk factor for neurological diseases. Large-scale epidemiological studies associate traffic-related particulate matter (PM) with impaired cognitive functions and increased incidence of neurodegenerative diseases such as Alzheimer's disease. Inhaled components of PM may directly invade the brain via the olfactory route, or act through peripheral system responses resulting in inflammation and oxidative stress in the brain. Microglia are the immune cells of the brain implicated in the progression of neurodegenerative diseases. However, it remains unknown how PM affects live human microglia. RESULTS: Here we show that two different PMs derived from exhausts of cars running on EN590 diesel or compressed natural gas (CNG) alter the function of human microglia-like cells in vitro. We exposed human induced pluripotent stem cell (iPSC)-derived microglia-like cells (iMGLs) to traffic related PMs and explored their functional responses. Lower concentrations of PMs ranging between 10 and 100 µg ml-1 increased microglial survival whereas higher concentrations became toxic over time. Both tested pollutants impaired microglial phagocytosis and increased secretion of a few proinflammatory cytokines with distinct patterns, compared to lipopolysaccharide induced responses. iMGLs showed pollutant dependent responses to production of reactive oxygen species (ROS) with CNG inducing and EN590 reducing ROS production. CONCLUSIONS: Our study indicates that traffic-related air pollutants alter the function of human microglia and warrant further studies to determine whether these changes contribute to adverse effects in the brain and on cognition over time. This study demonstrates human iPSC-microglia as a valuable tool to study functional microglial responses to environmental agents.
Asunto(s)
Células Madre Pluripotentes Inducidas , Enfermedades Neurodegenerativas , Humanos , Material Particulado/toxicidad , Material Particulado/análisis , Microglía/química , Células Madre Pluripotentes Inducidas/química , Automóviles , Especies Reactivas de Oxígeno , Emisiones de Vehículos/toxicidad , Emisiones de Vehículos/análisisRESUMEN
Here, we present a standardized, "off-the-shelf" proteomics pipeline working in a single 96-well plate to achieve deep coverage of cellular proteomes with high throughput and scalability. This integrated pipeline streamlines a fully automated sample preparation platform, a data-independent acquisition (DIA) coupled with high-field asymmetric waveform ion mobility spectrometer (FAIMS) interface, and an optimized library-free DIA database search strategy. Our systematic evaluation of FAIMS-DIA showing single compensation voltage (CV) at -35 V not only yields the deepest proteome coverage but also best correlates with DIA without FAIMS. Our in-depth comparison of direct-DIA database search engines shows that Spectronaut outperforms others, providing the highest quantifiable proteins. Next, we apply three common DIA strategies in characterizing human induced pluripotent stem cell (iPSC)-derived neurons and show single-shot mass spectrometry (MS) using single-CV (-35 V)-FAIMS-DIA results in >9,000 quantifiable proteins with <10% missing values, as well as superior reproducibility and accuracy compared with other existing DIA methods.
Asunto(s)
Células Madre Pluripotentes Inducidas , Proteómica , Humanos , Proteómica/métodos , Espectrometría de Masas en Tándem/métodos , Reproducibilidad de los Resultados , Células Madre Pluripotentes Inducidas/química , Proteoma/análisisRESUMEN
Understanding how excitable cells work in health and disease and how that behavior can be altered by small molecules or genetic manipulation is important. Genetically encoded calcium indicators (GECIs) with multiple emission windows can be combined (e.g., for simultaneous observation of distinct subcellular events) or used in extended applications with other light-dependent actuators in excitable cells (e.g., combining genetically encoded optogenetic control with spectrally compatible calcium indicators). Such approaches have been used in primary or stem cell-derived neurons, cardiomyocytes, and pancreatic beta-cells. However, it has been challenging to increase the throughput, or duration of observation, of such approaches due to limitations of the instruments, analysis software, indicator performance, and gene delivery efficiency. Here, a high-performance green GECI, mNeonGreen-GECO (mNG-GECO), and red-shifted GECI, K-GECO, is combined with optogenetic control to achieve all-optical control and visualization of cellular activity in a high-throughput imaging format using a High-Content Imaging System. Applications demonstrating cardiotoxicity testing and phenotypic drug screening with healthy and patient-derived iPSC-CMs are shown. In addition, multi-parametric assessments using combinations of spectral and calcium affinity indicator variants (NIR-GECO, LAR-GECO, and mtGCEPIA or Orai1-G-GECO) are restricted to different cellular compartments are also demonstrated in the iPSC-CM model.
Asunto(s)
Calcio , Células Madre Pluripotentes Inducidas , Calcio/análisis , Evaluación Preclínica de Medicamentos , Humanos , Indicadores y Reactivos , Células Madre Pluripotentes Inducidas/química , Miocitos Cardíacos/química , OptogenéticaRESUMEN
Taxane-induced peripheral neuropathy (TIPN) is a devastating survivorship issue for many cancer patients. In addition to its impact on quality of life, this toxicity may lead to dose reductions or treatment discontinuation, adversely impacting survival outcomes and leading to health disparities in African Americans (AA). Our lab has previously identified deleterious mutations in SET-Binding Factor 2 (SBF2) that significantly associated with severe TIPN in AA patients. Here, we demonstrate the impact of SBF2 on taxane-induced neuronal damage using an ex vivo model of SBF2 knockdown of induced pluripotent stem cell-derived sensory neurons. Knockdown of SBF2 exacerbated paclitaxel changes to cell viability and neurite outgrowth while attenuating paclitaxel-induced sodium current inhibition. Our studies identified paclitaxel-induced expression changes specific to mature sensory neurons and revealed candidate genes involved in the exacerbation of paclitaxel-induced phenotypes accompanying SBF2 knockdown. Overall, these findings provide ex vivo support for the impact of SBF2 on the development of TIPN and shed light on the potential pathways involved.
Asunto(s)
Paclitaxel/efectos adversos , Enfermedades del Sistema Nervioso Periférico/genética , Polimorfismo de Nucleótido Simple , Proteínas Tirosina Fosfatasas no Receptoras/genética , Células Receptoras Sensoriales/citología , Negro o Afroamericano/genética , Supervivencia Celular/efectos de los fármacos , Progresión de la Enfermedad , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Células Madre Pluripotentes Inducidas/química , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Paclitaxel/farmacología , Enfermedades del Sistema Nervioso Periférico/inducido químicamente , Enfermedades del Sistema Nervioso Periférico/etnología , Calidad de Vida , Células Receptoras Sensoriales/química , Células Receptoras Sensoriales/efectos de los fármacos , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Población Blanca/genéticaRESUMEN
Dynamic and temporally specific gene regulatory changes may underlie unexplained genetic associations with complex disease. During a dynamic process such as cellular differentiation, the overall cell type composition of a tissue (or an in vitro culture) and the gene regulatory profile of each cell can both experience significant changes over time. To identify these dynamic effects in high resolution, we collected single-cell RNA-sequencing data over a differentiation time course from induced pluripotent stem cells to cardiomyocytes, sampled at 7 unique time points in 19 human cell lines. We employed a flexible approach to map dynamic eQTLs whose effects vary significantly over the course of bifurcating differentiation trajectories, including many whose effects are specific to one of these two lineages. Our study design allowed us to distinguish true dynamic eQTLs affecting a specific cell lineage from expression changes driven by potentially non-genetic differences between cell lines such as cell composition. Additionally, we used the cell type profiles learned from single-cell data to deconvolve and re-analyze data from matched bulk RNA-seq samples. Using this approach, we were able to identify a large number of novel dynamic eQTLs in single cell data while also attributing dynamic effects in bulk to a particular lineage. Overall, we found that using single cell data to uncover dynamic eQTLs can provide new insight into the gene regulatory changes that occur among heterogeneous cell types during cardiomyocyte differentiation.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Células Madre Pluripotentes Inducidas/citología , Miocitos Cardíacos/citología , Análisis de la Célula Individual/métodos , Técnicas de Cultivo de Célula , Diferenciación Celular , Línea Celular , Linaje de la Célula , Regulación de la Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/química , Miocitos Cardíacos/química , RNA-SeqRESUMEN
We intend to identify marker genes with differential gene expression (DEG) and RGC subtypes in cultures of human-induced pluripotent stem cell (iPSC)-derived retinal ganglion cells. Single-cell sequencing was performed on mature and functional iPSC-RGCs at day 40 using Chromium Single Cell 3' V3 protocols (10X Genomics). Sequencing libraries were run on Illumina Novaseq to generate 150 PE reads. Demultiplexed FASTQ files were mapped to the hg38 reference genome using the STAR package, and cluster analyses were performed using a cell ranger and BBrowser2 software. QC analysis was performed by removing the reads corresponding to ribosomal and mitochondrial genes, as well as cells that had less than 1X mean absolute deviation (MAD), resulting in 4705 cells that were used for further analyses. Cells were separated into clusters based on the gene expression normalization via PCA and TSNE analyses using the Seurat tool and/or Louvain clustering when using BBrowser2 software. DEG analysis identified subsets of RGCs with markers like MAP2, RBPMS, TUJ1, BRN3A, SOX4, TUBB3, SNCG, PAX6 and NRN1 in iPSC-RGCs. Differential expression analysis between separate clusters identified significant DEG transcripts associated with cell cycle, neuron regulatory networks, protein kinases, calcium signaling, growth factor hormones, and homeobox transcription factors. Further cluster refinement identified RGC diversity and subtype specification within iPSC-RGCs. DEGs can be used as biomarkers for RGC subtype classification, which will allow screening model systems that represent a spectrum of diseases with RGC pathology.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Redes Reguladoras de Genes , Células Ganglionares de la Retina/citología , Análisis de la Célula Individual/métodos , Diferenciación Celular , Células Cultivadas , Mapeo Cromosómico , Citometría de Flujo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Células Madre Pluripotentes Inducidas/química , Células Madre Pluripotentes Inducidas/citología , Células Ganglionares de la Retina/química , Análisis de Secuencia de ARN/métodosAsunto(s)
Azitromicina/toxicidad , Cardiotoxinas/toxicidad , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Cardiotoxicidad , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Humanos , Células Madre Pluripotentes Inducidas/química , Células Madre Pluripotentes Inducidas/metabolismo , Miocitos Cardíacos/química , Miocitos Cardíacos/metabolismoRESUMEN
Idiopathic pulmonary fibrosis (PF) is a type of chronic lung disease. Here, we investigated the effect of induced pluripotent stem cell (iPSC)-derived exosomes (iPSC-exosomes) on M2-type macrophages which play a critical role in pulmonary fibrosis. Exosomes were purified from the conditioned medium of iPSCs. Mice models of pulmonary fibrosis were established by intratracheal instillation with 5 mg/kg bleomycin. Thereafter, the histopathological changes and collagen deposition were detected by HE and masson staining. Meanwhile the level of M2-type macrophages was elevated by immunofluorescence staining with F4/80 and Arg-1. Luciferase reporter assay was conducted to verify the binding of miR-302a-3p to ten-eleven translocation 1 (TET1). Our results showed that, after treatment with iPSC-exosomes, the pulmonary fibrosis induced by bleomycin was relieved, with less collagen deposition. In addition, the increased M2-type macrophages in PF mice were reduced upon treatment with iPSC-exosomes. Moreover, we found that the iPSC-exosomes showed higher level of miR-302a-3p. Interestingly, the level of miR-302a-3p in the lungs of PF mice was increased upon treatment with iPSC-exosomes. Furthermore, we verified that TET1 was a direct target of miR-302a-3p. Up-regulation of miR-302a-3p or TET1 silencing repressed M2-type macrophages. Down-regulation of miR-302a-3p abolished the beneficial effects of iPSC-exosomes on pulmonary fibrosis. Collectively, our study revealed that iPSC-exosomes delivered miR-302a-3p to suppress the M2-type macrophages via targeting TET1, thus mitigating pulmonary fibrosis. This study indicates that iPSC-exosomes may become a potential therapeutic agent for pulmonary fibrosis.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Exosomas/metabolismo , Células Madre Pluripotentes Inducidas/química , Macrófagos/metabolismo , MicroARNs/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Fibrosis Pulmonar/tratamiento farmacológico , Fibrosis Pulmonar/genética , Animales , Bleomicina/toxicidad , Modelos Animales de Enfermedad , Embrión de Mamíferos/citología , Exosomas/química , Fibroblastos/química , Fibroblastos/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , MicroARNs/genética , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/patología , Células RAW 264.7 , Transducción de SeñalRESUMEN
Transcription factor (TF)-induced reprogramming of somatic cells across lineages and to induced pluripotent stem cells (iPSCs) has revealed a remarkable plasticity of differentiated cells and presents great opportunities for generating clinically relevant cell types for disease modeling and regenerative medicine. The understanding of iPSC reprogramming provides insights into the mechanisms that safeguard somatic cell identity, drive epigenetic reprogramming, and underlie cell fate specification in vivo. The combinatorial action of TFs has emerged as the key mechanism for the direct and indirect effects of reprogramming factors that induce the remodelling of the enhancer landscape. The interplay of TFs in iPSC reprogramming also yields trophectoderm- and extraembryonic endoderm-like cell populations, uncovering an intriguing plasticity of cell states and opening new avenues for exploring cell fate decisions during early embryogenesis.
Asunto(s)
Reprogramación Celular , Células Madre Pluripotentes Inducidas/química , Factores de Transcripción/metabolismo , Animales , Desarrollo Embrionario , Epigénesis Genética , Humanos , Células Madre Pluripotentes Inducidas/metabolismoRESUMEN
In the United States, approximately one million individuals are hospitalized every year for arrhythmias, making arrhythmias one of the top causes of healthcare expenditures. Mexiletine is currently used as an antiarrhythmic drug but has limitations. The purpose of this work was to use normal and Long QT syndrome Type 3 (LQTS3) patient-derived human induced pluripotent stem cell (iPSC)-derived cardiomyocytes to identify an analog of mexiletine with superior drug-like properties. Compared to racemic mexiletine, medicinal chemistry optimization of substituted racemic pyridyl phenyl mexiletine analogs resulted in a more potent sodium channel inhibitor with greater selectivity for the sodium over the potassium channel and for late over peak sodium current.
Asunto(s)
Trastorno del Sistema de Conducción Cardíaco/patología , Células Madre Pluripotentes Inducidas/química , Síndrome de QT Prolongado/patología , Mexiletine/farmacología , Miocitos Cardíacos/patología , Canal de Sodio Activado por Voltaje NAV1.5/metabolismo , Piridinas/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Mexiletine/química , Estructura Molecular , Piridinas/química , Relación Estructura-ActividadRESUMEN
Cellular therapies based on induced pluripotent stem cells (iPSCs) come out of age and an increasing number of clinical trials applying iPSC-based transplants are ongoing or in preparation. Recent studies, however, demonstrated a high number of small-scale mutations in iPSCs. Although the mutational load in iPSCs seems to be largely derived from their parental cells, it is still unknown whether reprogramming may enrich for individual mutations that could lead to loss of functionality and tumor formation from iPSC derivatives. 30 hiPSC lines were analyzed by whole exome sequencing. High accuracy amplicon sequencing showed that all analyzed small-scale variants pre-existed in their parental cells and that individual mutations present in small subpopulations of parental cells become enriched among hiPSC clones during reprogramming. Among those, putatively actionable driver mutations affect genes related to cell-cycle control, cell death, and pluripotency and may confer a selective advantage during reprogramming. Finally, a short hairpin RNA (shRNA)-based experimental approach was applied to provide additional evidence for the individual impact of such genes on the reprogramming efficiency. In conclusion, we show that enriched mutations in curated onco- and tumor suppressor genes may account for an increased tumor risk and impact the clinical value of patient-derived hiPSCs.
Asunto(s)
Células Clonales/citología , Secuenciación del Exoma/métodos , Células Madre Pluripotentes Inducidas/citología , Mutación , Neoplasias/genética , Anciano , Ciclo Celular , Muerte Celular , Diferenciación Celular , Línea Celular , Células Cultivadas , Reprogramación Celular , Células Clonales/química , Células Endoteliales de la Vena Umbilical Humana , Humanos , Células Madre Pluripotentes Inducidas/química , Neoplasias/patologíaRESUMEN
Induced pluripotent stem cell (iPSC) technology enables the generation of human neurons in vitro, which contain the precise genome of the cell donor, therefore permitting the generation of disease models from individuals with a disease-associated genotype of interest. This approach has been extensively used to model inherited forms of Alzheimer's disease and frontotemporal dementia. The combination of iPSC-derived neuronal models with targeted mass spectrometry analysis has provided unprecedented insights into the regulation of specific proteins in human neuronal physiology and pathology. For example enabling investigations into tau and APP/Aß, specifically: protein isoform expression, relative levels of cleavage fragments, aggregated species and functionally critical post-translational modifications. The use of mass spectrometry has enabled a determination of how closely iPSC-derived models recapitulate disease profiles observed in the human brain. This review will highlight the progress to date in studies using iPSCs and mass spectrometry to model Alzheimer's disease and dementia. We go on to convey our optimism, as studies in the near future will make use of this precedent, together with novel techniques such as genome editing and stable isotope labelling, to provide real progress towards an in depth understanding of early neurodegenerative processes and development of novel therapeutic agents.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/análisis , Demencia/metabolismo , Células Madre Pluripotentes Inducidas/química , Espectrometría de Masas/métodos , Proteínas tau/análisis , Animales , Modelos Animales de Enfermedad , HumanosRESUMEN
Spinocerebellar ataxia type 7 (SCA7) is an inherited neurodegenerative disease caused by a polyglutamine repeat expansion in the ATXN7 gene. Patients with this disease suffer from a degeneration of their cerebellar Purkinje neurons and retinal photoreceptors that result in a progressive ataxia and loss of vision. As with many neurodegenerative diseases, studies of pathogenesis have been hindered by a lack of disease-relevant models. To this end, we have generated induced pluripotent stem cells (iPSCs) from a cohort of SCA7 patients in South Africa. First, we differentiated the SCA7 affected iPSCs into neurons which showed evidence of a transcriptional phenotype affecting components of STAGA (ATXN7 and KAT2A) and the heat shock protein pathway (DNAJA1 and HSP70). We then performed electrophysiology on the SCA7 iPSC-derived neurons and found that these cells show features of functional aberrations. Lastly, we were able to differentiate the SCA7 iPSCs into retinal photoreceptors that also showed similar transcriptional aberrations to the SCA7 neurons. Our findings give technical insights on how iPSC-derived neurons and photoreceptors can be derived from SCA7 patients and demonstrate that these cells express molecular and electrophysiological differences that may be indicative of impaired neuronal health. We hope that these findings will contribute towards the ongoing efforts to establish the cell-derived models of neurodegenerative diseases that are needed to develop patient-specific treatments.
Asunto(s)
Marcadores Genéticos , Células Madre Pluripotentes Inducidas/citología , Neuronas/fisiología , Retina/fisiología , Ataxias Espinocerebelosas/fisiopatología , Ataxina-7/genética , Diferenciación Celular , Células Cultivadas , Reprogramación Celular , Fenómenos Electrofisiológicos , Regulación de la Expresión Génica , Proteínas del Choque Térmico HSP40/genética , Proteínas HSP70 de Choque Térmico/genética , Histona Acetiltransferasas/genética , Humanos , Células Madre Pluripotentes Inducidas/química , Modelos Biológicos , Neuronas/química , Neuronas/citología , Cultivo Primario de Células , Retina/química , Retina/citología , Sudáfrica , Ataxias Espinocerebelosas/genética , Ataxias Espinocerebelosas/patologíaRESUMEN
OCT4 (also known as Oct3 and Oct3/4), which is encoded by Pou5f1, is expressed in early embryonic cells and plays an important role in early development, pluripotency maintenance, and self-renewal of embryonic stem cells. It also regulates the reprogramming of somatic cells into induced pluripotent stem cells. Several OCT4-binding proteins, including SOX2 and NANOG, reportedly regulate gene transcription in stem cells. An increasing number of evidence suggests that not only gene transcription but also post-translational modifications of OCT4 play a pivotal role in regulating the expression and activity of OCT4. For instance, ubiquitination and sumoylation have been reported to regulate OCT4 protein stability. In addition, the phosphorylation of Ser347 in OCT4 also stabilizes the OCT4 protein level. Recently, we identified KAP1 as an OCT4-binding protein and reported the KAP1-mediated regulation of OCT4 protein stability. KAP1 overexpression led to an increased proliferation of mouse embryonic stem cells and promoted the reprogramming of somatic cells resulting in induced pluripotent stem cells. In this review, we discuss how the protein stability and function of OCT4 are regulated by protein-protein interaction in stem cells.
Asunto(s)
Células Madre Embrionarias/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/química , Factor 3 de Transcripción de Unión a Octámeros/genética , Animales , Células Madre Embrionarias/química , Regulación de la Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/química , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Unión Proteica , Estabilidad Proteica , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismoRESUMEN
Chronic spinal cord injury (SCI) is a devastating medical condition. In the acute phase after injury, there is cell loss resulting in chronic axonal damage and loss of sensory and motor function including loss of oligodendrocytes that results in demyelination of axons and further dysfunction. In the chronic phase, the inhibitory environment within the lesion including the glial scar can arrest axonal growth and regeneration and can also potentially affect transplanted cells. We hypothesized that glial scar ablation (GSA) along with cell transplantation may be required as a combinatorial therapy to achieve functional recovery, and therefore we proposed to examine the survival and fate of human induced pluripotent stem cell (iPSC) derived pre-oligodendrocyte progenitor cells (pre-OPCs) transplanted in a model of chronic SCI, whether this was affected by GSA, and whether this combination of treatments would result in functional recovery. In this study, chronically injured athymic nude (ATN) rats were allocated to one of three treatment groups: GSA only, pre-OPCs only, or GSA+pre-OPCs. We found that human iPSC derived pre-OPCs were multi-potent and retained the ability to differentiate into mainly oligodendrocytes or neurons when transplanted into the chronically injured spinal cords of rats. Twelve weeks after cell transplantation, we observed that more of the transplanted cells differentiated into oligodendrocytes when the glial scar was ablated compared with no GSA. Further, we also observed that a higher percentage of transplanted cells differentiated into V2a interneurons and motor neurons in the pre-OPCs only group when compared with GSA+pre-OPCs. This suggests that the local environment created by ablation of the glial scar may have a significant effect on the fate of cells transplanted into the injury site.
Asunto(s)
Gliosis/terapia , Neuronas Motoras/fisiología , Células Precursoras de Oligodendrocitos/fisiología , Oligodendroglía/fisiología , Traumatismos de la Médula Espinal/terapia , Trasplante de Células Madre/métodos , Animales , Células Cultivadas , Femenino , Colorantes Fluorescentes/administración & dosificación , Gliosis/patología , Humanos , Células Madre Pluripotentes Inducidas/química , Células Madre Pluripotentes Inducidas/fisiología , Células Madre Pluripotentes Inducidas/trasplante , Neuronas Motoras/química , Células Precursoras de Oligodendrocitos/química , Células Precursoras de Oligodendrocitos/trasplante , Oligodendroglía/química , Ratas , Rosa Bengala/administración & dosificación , Traumatismos de la Médula Espinal/patología , Vértebras Torácicas/lesionesRESUMEN
Astrocytes, a highly heterogeneous population of glial cells, serve as essential regulators of brain development and homeostasis. The heterogeneity of astrocyte populations underlies the diversity in their functions. In addition to the typical mammalian astrocyte architecture, the cerebral cortex of humans exhibits a radial distribution of interlaminar astrocytes in the supragranular layers. These primate-specific interlaminar astrocytes are located in the superficial layer and project long processes traversing multiple layers of the cerebral cortex. However, due to the lack of accessible experimental models, their functional properties and their role in regulating neuronal circuits remain unclear. Here we modeled human interlaminar astrocytes in humanized glial chimeric mice by engrafting astrocytes differentiated from human-induced pluripotent stem cells into the mouse cortex. This model provides a novel platform for understanding neuron-glial interaction and its alterations in neurological diseases.
Asunto(s)
Astrocitos/química , Astrocitos/fisiología , Corteza Cerebral/química , Corteza Cerebral/fisiología , Células Madre Pluripotentes Inducidas/química , Células Madre Pluripotentes Inducidas/fisiología , Adolescente , Animales , Células Cultivadas , Corteza Cerebral/citología , Femenino , Humanos , Masculino , Ratones , Ratones de la Cepa 129 , Ratones TransgénicosRESUMEN
Neuregulin protein 1 (NRG1) is a large (> 60-amino-acid) natural peptide ligand for the ErbB protein family members HER3 and HER4. We developed an agonistic antibody modality, termed antibody ligand mimetics (ALM), by incorporating complex ligand agonists such as NRG1 into an antibody scaffold. We optimized the linker and ligand length to achieve native ligand activity in HEK293 cells and cardiomyocytes derived from induced pluripotent stem cells (iPSCs) and used a monomeric Fc-ligand fusion platform to steer the ligand specificity toward HER4-dominant agonism. With the help of selectivity engineering, these enhanced ALM molecules can provide an antibody scaffold with increased receptor specificity and the potential to greatly improve the pharmacokinetics, stability, and downstream developability profiles from the natural ligand approach. This ligand mimetic design and optimization approach can be expanded to apply to other cardiovascular disease targets and emerging therapeutic areas, providing differentiated drug molecules with increased specificity and extended half-life.
Asunto(s)
Anticuerpos Monoclonales/química , Neurregulina-1/química , Receptor ErbB-4/agonistas , Anticuerpos Monoclonales/metabolismo , Células HEK293 , Humanos , Células Madre Pluripotentes Inducidas/química , Células Madre Pluripotentes Inducidas/metabolismo , Cinética , Ligandos , Miocitos Cardíacos/química , Miocitos Cardíacos/metabolismo , Neurregulina-1/metabolismo , Unión Proteica , Receptor ErbB-4/metabolismo , Transducción de SeñalRESUMEN
Down syndrome (DS) is a congenital disorder caused by trisomy 21 (T21). It is associated with cognitive impairment, muscle hypotonia, heart defects, and other clinical anomalies. At the same time, individuals with Down syndrome have lower prevalence of solid tumor formation. To gain new insights into aberrant DS development during early stages of mesoderm formation and its possible connection to lower solid tumor prevalence, we developed the first model of two types of DS iPSC-derived stromal cells. Utilizing bioinformatic and functional analyses, we identified over 100 genes with coordinated expression among mesodermal and endothelial cell types. The most significantly down-regulated processes in DS mesodermal progenitors were associated with decreased stromal progenitor performance related to connective tissue organization as well as muscle development and functionality. The differentially expressed genes included cytoskeleton-related genes (actin and myosin), ECM genes (Collagens, Galectin-1, Fibronectin, Heparan Sulfate, LOX, FAK1), cell cycle genes (USP16, S1P complexes), and DNA damage repair genes. For DS endothelial cells, our analysis revealed most down-regulated genes associated with cellular response to external stimuli, cell migration, and immune response (inflammation-based). Together with functional assays, these results suggest an impairment in mesodermal development capacity during early stages, which likely translates into connective tissue impairment in DS patients. We further determined that, despite differences in functional processes and characteristics, a significant number of differentially regulated genes involved in tumorigenesis were expressed in a highly coordinated manner across endothelial and mesodermal cells. These findings strongly suggest that microRNAs (miR-24-4, miR-21), cytoskeleton remodeling, response to stimuli, and inflammation can impact resistance to tumorigenesis in DS patients. Furthermore, we also show that endothelial cell functionality is impaired, and when combined with angiogenic inhibition, it can provide another mechanism for decreased solid tumor development. We propose that the same processes, which specify the basis of connective tissue impairment observed in DS patients, potentially impart a resistance to cancer by hindering tumor progression and metastasis. We further establish that cancer-related genes on Chromosome 21 are up-regulated, while genome-wide cancer-related genes are down-regulated. These results suggest that trisomy 21 induces a modified regulation and compensation of many biochemical pathways across the genome. Such downstream interactions may contribute toward promoting tumor resistant mechanisms.
Asunto(s)
Síndrome de Down/genética , Células Madre Pluripotentes Inducidas/citología , MicroARNs/genética , Neoplasias/genética , Células del Estroma/citología , Movimiento Celular , Proliferación Celular , Células Cultivadas , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Células Madre Pluripotentes Inducidas/química , Desarrollo Musculoesquelético , Análisis de Secuencia de ARN , Células del Estroma/químicaRESUMEN
To evaluate the effects of thermosensitive hydrogels loaded with human-induced pluripotent stem cells transfected with the growth differentiation factor-5 (GDF5-hiPSCs) on rat intervertebral disc regeneration. GDF5-hiPSCs were cocultured with rat nucleus pulposus (NP) cells in vitro. Real-time PCR and western blot were used to determine the differentiation of hiPSCs. Rat caudal intervertebral discs were punctured using a needle under X-ray, and groups of coccygeal (Co) discs were subject to various treatments: Puncture group (Co6/7, punctured without treatment); Hydrogel group (Co7/8, 2 µl of hydrogel injected without cells); GDF5-hiPSCs + Hydrogel group (Co8/9, 2 µl of GDF5-hiPSCs-loaded hydrogel injected); and Normal control (Co5/6). X-ray, MRI, and histological evaluations were performed at 1, 2, and 3 months after cell transplantation and relative changes in the disc height index (DHI%) and voxel count were calculated and compared. GDF5-hiPSCs were successfully differentiated to a chondrogenic linage after cocultured with rat NP cells. In terms of X-ray, MRI, and HE staining scores, the GDF5-hiPSCs + Hydrogel group was significantly superior to the Puncture and Hydrogel groups (p < .05). Compared with the Normal group, the MRI-based voxel count of the GDF5-hiPSCs + Hydrogel group was significantly lower at 1, 2, and 3 months after cell transplantation (p < .05). However, there were no significant differences in histological scores at 1 and 2 months after cell transplantation compared with the Normal group (p > .05). In conclusion, thermosensitive hydrogel-encapsulated hiPSCs overexpressing the GDF5 gene ameliorated intervertebral disc degeneration.