SUMMARY The developmental potential of human pluripotent stem cells suggests that they can produce disease-relevant cell types for biomedical research. However, substantial variation has been reported among pluripotent cell lines, which could affect their utility and clinical safety. Such cell-line-specific differences must be better understood before one can confidently use embryonic stem (ES) or induced pluripotent stem (iPS) cells in translational research. Toward this goal we have established genome-wide reference maps of DNA methylation and gene expression for 20 previously derived human ES lines and 12 human iPS cell lines, and we have measured the in vitro differentiation propensity of these cell lines. This resource enabled us to assess the epigenetic and transcriptional similarity of ES and iPS cells and to predict the differentiation efficiency of individual cell lines. The combination of assays yields a scorecard for quick and comprehensive characterization of pluripotent cell lines.
Human embryonic (ESC) and induced pluripotent stem cells (iPSC) present exciting opportunities for studying development and in vitro disease modeling. However, reported variability in iPSC behavior has called their utility into question. We therefore constituted a test set of 16 iPSCs lines from 7 individuals of varying gender and health status, characterized them extensively for pluripotency, and evaluated their ability to terminally differentiate. Using standardized procedures in two independent laboratories, 13 of the iPSC lines gave rise to functional motor neurons with a range of efficiencies similar to ESCs. Although three iPSC lines were resistant to neural differentiation, early neuralization rescued their performance. Therefore, all lines in the test set passed a stringent test of differentiation capacity despite variations in expression of early pluripotency markers, transgenes and karyotype. This novel iPSC/ESC test set is a robust resource for those interested in the basic biology of stem cells and their applications.
Summary Although many distinct mutations in a variety of genes are known to cause Amyotrophic Lateral Sclerosis (ALS), it remains poorly understood how they selectively impact motor neuron biology and whether they converge on common pathways to cause neuronal degeneration. Here, we have combined reprogramming and stem cell differentiation approaches with genome engineering and RNA sequencing to define the transcriptional and functional changes that are induced in human motor neurons by mutant SOD1. Mutant SOD1 protein induced a transcriptional signature indicative of increased oxidative stress, reduced mitochondrial function, altered sub-cellular transport as well as activation of the ER stress and unfolded protein response pathways. Functional studies demonstrated that these pathways were perturbed in a manner dependent on the SOD1 mutation. Finally, interrogation of stem cell-derived motor neurons produced from ALS patients harboring a repeat expansion in C9orf72 indicates at least a subset of these changes are more broadly conserved in ALS.
Human pluripotent stem cells are a promising source of differentiated cells for developmental studies, cell transplantation, disease modeling, and drug testing. However, their widespread use even for intensely studied cell types like spinal motor neurons is hindered by the long duration and low yields of existing protocols for in vitro differentiation and by the molecular heterogeneity of the populations generated. We report a combination of small molecules that within 3 weeks induce motor neurons at up to 50% abundance and with defined subtype identities of relevance to neurodegenerative disease. Despite their accelerated differentiation, motor neurons expressed combinations of HB9, ISL1 and column-specific markers that mirror those observed in vivo in human fetal spinal cord. They also exhibited spontaneous and induced activity, and projected axons towards muscles when grafted into developing chick spinal cord. Strikingly, this novel protocol preferentially generates motor neurons expressing markers of limb-innervating lateral motor column motor neurons (FOXP1+/LHX3−). Access to high-yield cultures of human limb-innervating motor neuron subtypes will facilitate in-depth study of motor neuron subtype-specific properties, disease modeling, and development of large-scale cell-based screening assays.
Intravital microscopy confirms the release of EVs from gliomas and their uptake into microglia and monocytes/macrophages within the brain. Our studies also support functional effects of GBM-released EVs following uptake into microglia, associated in part with increased miRNA levels, decreased target mRNAs, and encoded proteins, presumably as a means for the tumor to manipulate its environs.
Alzheimer's disease (AD) is defined by the presence of intraneuronal neurofibrillary tangles (NFTs) composed of hyperphosphorylated tau aggregates as well as extracellular amyloid-beta plaques. The presence and spread of tau pathology through the brain is classified by Braak stages and thought to correlate with the progression of AD. Several in vitro and in vivo studies have examined the ability of tau pathology to move from one neuron to the next, suggesting a “prion-like” spread of tau aggregates may be an underlying cause of Braak tau staging in AD. Using the HEK293 TauRD-P301S-CFP/YFP expressing biosensor cells as a highly sensitive and specific tool to identify the presence of seed competent aggregated tau in brain lysate—i.e., tau aggregates that are capable of recruiting and misfolding monomeric tau—, we detected substantial tau seeding levels in the entorhinal cortex from human cases with only very rare NFTs, suggesting that soluble tau aggregates can exist prior to the development of overt tau pathology. We next looked at tau seeding levels in human brains of varying Braak stages along six regions of the Braak Tau Pathway. Tau seeding levels were detected not only in the brain regions impacted by pathology, but also in the subsequent non-pathology containing region along the Braak pathway. These data imply that pathogenic tau aggregates precede overt tau pathology in a manner that is consistent with transneuronal spread of tau aggregates. We then detected tau seeding in frontal white matter tracts and the optic nerve, two brain regions comprised of axons that contain little to no neuronal cell bodies, implying that tau aggregates can indeed traverse along axons. Finally, we isolated cytosolic and synaptosome fractions along the Braak Tau Pathway from brains of varying Braak stages. Phosphorylated and seed competent tau was significantly enriched in the synaptic fraction of brain regions that did not have extensive cellular tau pathology, further suggesting that aggregated tau seeds move through the human brain along synaptically connected neurons. Together, these data provide further evidence that the spread of tau aggregates through the human brain along synaptically connected networks results in the pathogenesis of human Alzheimer's disease.
Objective Recent studies have shown that PET tracer AV-1451 exhibits high binding affinity for paired helical filament (PHF)-tau pathology in Alzheimer’s brains. However, the ability of this ligand to bind to tau lesions in other tauopathies remains controversial. Our goal was to examine the correlation of in vivo and postmortem AV-1451 binding patterns in three autopsy-confirmed non-Alzheimer tauopathy cases. Methods We quantified in vivo retention of [F-18]-AV-1451 and performed autoradiography, [H-3]-AV-1451 binding assays and quantitative tau measurements in postmortem brain samples from two Progressive Supranuclear Palsy (PSP) cases and a MAPT P301L mutation carrier. They all underwent [F-18]-AV-1451 PET imaging prior to death. Results The three subjects exhibited [F-18]-AV-1451 in vivo retention predominantly in basal ganglia and midbrain. Neuropathologic examination confirmed the PSP diagnosis in the first two subjects; the MAPT P301L mutation carrier had an atypical tauopathy characterized by grain-like tau-containing neurites in grey and white matter with heaviest burden in basal ganglia. In all three cases, autoradiography failed to show detectable [F-18]-AV-1451 binding in multiple brain regions examined with the exception of entorhinal cortex (reflecting incidental age-related neurofibrillary tangles) and neuromelanin-containing neurons in the substantia nigra (off-target binding). The lack of a consistent significant correlation between in vivo [F-18]-AV-1541 retention and postmortem in vitro binding and tau measures in these cases suggests that this ligand has low affinity for tau lesions primarily made of straight tau filaments. Interpretation AV-1451 may have limited utility for in vivo selective and reliable detection of tau aggregates in these non-Alzheimer tauopathies.
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