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We discuss how to diversify the reference iPSC line concept. We highlight workflows for generating diverse iPSC lines. We ask whether reference lines can act as inclusive sources of human diversity for use in benchmarking controls for disease models or drug screens, or as clinical grade lines for cell therapies.
MOTIVATION: Ribosome dynamics are vital in the process of protein expression. Current methods rely on ribosome profiling (Ribo-seq), RNA-seq profiles, and full genomic context. This restricts their use in de novo sequence design, like messenger RNA (mRNA) vaccines. Simulation-only approaches like the Totally Asymmetric Simple Exclusion Process (TASEP) oversimplify translation by focusing solely on codon elongation times. RESULTS: We present seq2ribo, a hybrid simulation and machine learning framework that predicts ribosome A-site locations using only an mRNA sequence as input. Our method first employs a novel structure-aware TASEP (sTASEP), which models translation using a comprehensive set of fitted parameters that include codon wait times and structural features, such as local angles, base-pairing, and discrete positional buckets. The ribosome locations generated by sTASEP are then processed by a polisher model, which learns to refine the simulated ribosome distributions. seq2ribo provides high-fidelity predictions of ribosome locations across diverse cell types (iPSC, HEK293, LCL, and RPE-1), significantly outperforming baselines. seq2ribo is the first method to achieve meaningful positional correlation with observed ribosome profiles from sequence alone, reaching transcript-level Pearson correlations up to 0.920 and within-transcript shape correlations up to 0.186, where all baselines yield near-zero values on these metrics. seq2ribo also reduces elementwise error by up to 37.7% relative to the sequence-only Translatomer baseline. By adding a task-specific head, seq2ribo achieves Pearson correlations up to 0.732 with experimental translation efficiency (TE) across several cell lines, and up to 0.903 with measured protein expression. By operating from sequence alone, seq2ribo provides a new tool for synthetic biology, enabling the rational design and optimization of mRNA sequences without the need for expression-level data or genomic context. AVAILABILITY: seq2ribo is available at https://github.com/Kingsford-Group/seq2ribo.
MOTIVATION: Ribosome dynamics are vital in the process of protein expression. Current methods rely on ribosome profiling (Ribo-seq), RNA-seq profiles, and full genomic context. This restricts their use in de novo sequence design, like messenger RNA (mRNA) vaccines. Simulation-only approaches like the Totally Asymmetric Simple Exclusion Process (TASEP) oversimplify translation by focusing solely on codon elongation times. RESULTS: We present seq2ribo, a hybrid simulation and machine learning framework that predicts ribosome A-site locations using only an mRNA sequence as input. Our method first employs a novel structure-aware TASEP (sTASEP), which models translation using a comprehensive set of fitted parameters that include codon wait times and structural features, such as local angles, base-pairing, and discrete positional buckets. The ribosome locations generated by sTASEP are then processed by a polisher model, which learns to refine the simulated ribosome distributions. seq2ribo provides high-fidelity predictions of ribosome locations across diverse cell types (iPSC, HEK293, LCL, and RPE-1), significantly outperforming baselines. seq2ribo is the first method to achieve meaningful positional correlation with observed ribosome profiles from sequence alone, reaching transcript-level Pearson correlations up to 0.920 and within-transcript shape correlations up to 0.186, where all baselines yield near-zero values on these metrics. seq2ribo also reduces elementwise error by up to 37.7% relative to the sequence-only Translatomer baseline. By adding a task-specific head, seq2ribo achieves Pearson correlations up to 0.732 with experimental translation efficiency (TE) across several cell lines, and up to 0.903 with measured protein expression. By operating from sequence alone, seq2ribo provides a new tool for synthetic biology, enabling the rational design and optimization of mRNA sequences without the need for expression-level data or genomic context.
The stratum griseum superficiale (SGS) of the superior colliculus contains a high concentration of the recently described GABA(C) receptor. In a previous study, it was postulated that activation of these receptors on inhibitory interneurons functions to disinhibit projection cells that relay visual information to the thalamus and brainstem. To test this model, we used in vitro whole-cell patch-clamp methods to measure effects of GABA and muscimol on EPSCs and IPSCs evoked in rat SGS by electrical optic layer stimulation. The neurons were filled with biocytin for later morphological characterization. As expected, bath applications of GABA and muscimol always strongly depressed evoked PSCs at concentrations of >100 and >1 micrometer, respectively. However, at lower agonist concentrations, which most likely activate GABA(C) but not GABA(A) receptors, effects were not uniform. Evoked responses were suppressed by both agonists in 48% of the neurons, whereas the remaining cells exhibited enhanced responses with increased evoked EPSCs, decreased evoked IPSCs, or both types of change. Most morphologically identified cells with suppressed responses (14 of 17 cells) had morphological characteristics of putative GABAergic interneurons, whereas almost all cells with enhanced responses (8 of 10 cells) had morphological characteristics of projection cells. Finally, all effects of GABA and muscimol at low concentrations were blocked by (1,2,5,6-tetrahydropyridine-4-yl) methylphosphinic acid, a specific GABA(C) receptor antagonist, but not by the specific GABA(A) receptor antagonist bicuculline. Taken together, these results indicate that in SGS, GABA(C) receptors are predominantly expressed by GABAergic neurons and that activation of these receptors leads to disinhibition of SGS projection cells.
Mutual synaptic interactions between GABAergic interneurons are thought to be of critical importance for the generation of network oscillations and for temporal encoding of information in the hippocampus. However, the functional properties of synaptic transmission between hippocampal interneurons are largely unknown. We have made paired recordings from basket cells (BCs) in the dentate gyrus of rat hippocampal slices, followed by correlated light and electron microscopical analysis. Unitary GABA(A) receptor-mediated IPSCs at BC-BC synapses recorded at the soma showed a fast rise and decay, with a mean decay time constant of 2.5 +/- 0.2 msec (32 degrees C). Synaptic transmission at BC-BC synapses showed paired-pulse depression (PPD) (32 +/- 5% for 10 msec interpulse intervals) and multiple-pulse depression during repetitive stimulation. Detailed passive cable model simulations based on somatodendritic morphology and localization of synaptic contacts further indicated that the conductance change at the postsynaptic site was even faster, decaying with a mean time constant of 1.8 +/- 0.6 msec. Sequential triple recordings revealed that the decay time course of IPSCs at BC-BC synapses was approximately twofold faster than that at BC-granule cell synapses, whereas the extent of PPD was comparable. To examine the consequences of the fast postsynaptic conductance change for the generation of oscillatory activity, we developed a computational model of an interneuron network. The model showed robust oscillations at frequencies >60 Hz if the excitatory drive was sufficiently large. Thus the fast conductance change at interneuron-interneuron synapses may promote the generation of high-frequency oscillations observed in the dentate gyrus in vivo.
Tumor organoids are important tools for cancer research, but current models have drawbacks that limit their applications for predicting response to therapy. Here, we developed a fast, efficient, and complex culture system (IPTO, individualized patient tumor organoid) that accurately recapitulates the cellular and molecular pathology of human brain tumors. Patient-derived tumor explants were cultured in induced pluripotent stem cell (iPSC)-derived cerebral organoids, thus enabling culture of a wide range of human tumors in the central nervous system (CNS), including adult, pediatric, and metastatic brain cancers. Histopathological, genomic, epigenomic, and single-cell RNA sequencing (scRNA-seq) analyses demonstrated that the IPTO model recapitulates cellular heterogeneity and molecular features of original tumors. Crucially, we showed that the IPTO model predicts patient-specific drug responses, including resistance mechanisms, in a prospective patient cohort. Collectively, the IPTO model represents a major breakthrough in preclinical modeling of human cancers, which provides a path toward personalized cancer therapy.
The GGGGCC hexanucleotide repeat expansion (HRE) within the C9orf72 gene constitutes the leading genetic driver of amyotrophic lateral sclerosis (ALS). This fatal neurodegenerative disorder is characterized by the systematic loss of both the upper and lower motor neurons across both the central and peripheral nervous systems. This work describes the successful reprogramming of two human induced pluripotent stem cell (iPSC) lines originating from two independent ALS patients, both of whom carry a C9orf72 HRE mutation. Validation of the two established iPSC lines confirmed the expression of pluripotency markers, normal karyotypes, and successful trilineage differentiation. Consequently, these lines provide a robust in vitro platform to model ALS and study C9orf72-mediated disease mechanisms.
Developmental upregulation of the GABAA receptor alpha1 subunit causes a faster decay of GABAergic inhibitory postsynaptic currents (IPSCs) in the visual cortex around the time of eye opening. In alpha1 deficient mice, a juvenile type of GABAA receptors is retained during maturation. As a result the decay time of the IPSCs is longer in alpha1-/- mice than in WT mice during the whole life span of the mice. Hence they form a valuable mouse model for studies on cellular aspects of neuronal network functioning. Using voltage sensitive dye imaging methods, we monitored the spatiotemporal excitation patterning in visual cortex slices upon local stimulation of the network. We found that in the alpha1-/- mice, the ability of the network to fire synchronously at gamma-frequencies (20-50 Hz) is diminished. This finding indicates that early onset of GABA synapse maturation is required for the normal neuronal network function in the maturating visual cortex.
Loss of filaggrin (FLG) function impairs skin barrier formation and contributes to common inflammatory skin diseases. In this study, we established a FLG knockout human induced pluripotent stem cell (iPSC) line based on KOLF2.1 J using CRISPR/Cas12a (Cpf1)-mediated genome editing. A guide RNA targeting exon 2 introduced a homozygous mutation, which was confirmed by sequencing. The edited cells maintained typical pluripotent stem cell morphology, expressed key undifferentiated markers, and retained the ability to differentiate into all three germ layers. Karyotype and copy number variation (CNV) analyses confirmed genomic stability and parental origin; the cells were free of mycoplasma. This cell line enables studies of FLG-associated skin biology and pathology.
Duplication 15q (dup15q) syndrome is a leading genetic cause of autism spectrum disorder, offering a key model for studying autism-related mechanisms. Using single-cell and single-nucleus RNA sequencing of cortical organoids from dup15q patient-derived iPSCs and post-mortem brain samples, we identify increased glycolysis, disrupted layer-specific marker expression, and aberrant morphology in deep-layer neurons during fetal-stage organoid development. In adolescent-adult postmortem brains, upper-layer neurons exhibit heightened transcriptional burden related to synaptic signaling, a pattern shared with idiopathic autism. Using spatial transcriptomics, we confirm these cell-type-specific disruptions in brain tissue. By gene co-expression network analysis, we reveal disease-associated modules that are well preserved between postmortem and organoid samples, suggesting metabolic dysregulation that may lead to altered neuron projection, synaptic dysfunction, and neuron hyperexcitability in dup15q syndrome.
Cortical dysplasia has a strong association with epilepsy in humans, but the underlying mechanisms for this are poorly understood. In utero irradiation of rats produces diffuse cortical dysplasia and neuronal heterotopia in the neocortex and hippocampus. Using in vitro neocortical slices, whole-cell patch-clamp recordings were obtained from pyramidal neurons in dysplastic cortex and control neocortex. Spontaneous IPSCs were reduced in amplitude (35%) and frequency (70%) in pyramidal cells from dysplastic cortex. Miniature IPSCs were reduced in frequency (66%) in dysplastic cortex. Two additional measures of cortical inhibition, monosynaptic evoked IPSCs and paired pulse depression of evoked EPSCs, were also impaired in dysplastic cortex. Spontaneous EPSCs were increased in amplitude (42%) and frequency (77%) in dysplastic cortex, but miniature EPSCs were not different between the two groups. These data demonstrate significant physiological impairment in inhibitory synaptic transmission in experimental cortical dysplasia. This supports previous immunohistochemical findings in this model and observations in humans of a reduction in the density of inhibitory interneurons in dysplastic cortex.
Hypoxia induces depression of excitatory postsynaptic currents (EPSCs) and inhibitory postsynaptic currents (IPSCs) in CA1 neurons of the hippocampus. The effect of antagonists that act at the A1 adenosine receptor on hypoxia-induced depression of EPSCs and IPSCs were examined in hippocampal slices with the patch clamp technique (whole-cell configuration). The A1 receptor antagonists 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) (200 nM) and 8-phenyltheophilline (8-PT) (10 microM) significantly prevented depression of EPSCs by hypoxia but failed to protect IPSCs. This result suggests that the hypoxia-induced depression of the EPSC involves the activation of adenosine receptors (possibly of the A1 subtype), whereas depression of the IPSC results from a different mechanism.
LEMD2 is an inner nuclear membrane protein. The pathogenic LEMD2 variant (NM_181336.4: c.38 T > G, p.L13R) has been associated with an inherited cardiomyopathy characterized by left ventricular dysfunction and severe arrhythmias. To gain more insights into this disease and investigate the role of LEMD2 more broadly, LEMD2 p.L13R knock-in (LEMD2-KI; UKWCHFi001-B-1) and knock-out (LEMD2-KO; UKWCHFi001-B-2) iPSC lines were generated in a healthy control iPSC (UKWCHFi001-B) line using CRISPR/Cas9 gene editing. Both new iPSC lines retained pluripotency, normal karyotypes, and differentiation potential, whereby expression of LEMD2 was successfully disrupted in LEMD2-KO cells. Thus, these lines provide a suitable model for analyzing LEMD2-associated diseases.
Recent experimental and modeling results demonstrated that surviving mossy cells in the dentate gyrus play key roles in the generation of network hyperexcitability. Here we examined if mossy cells exhibit long-term plasticity in the posttraumatic, hyperexcitable dentate gyrus. Mossy cells 1 wk after fluid percussion head injury did not show alterations in their current-firing frequency (I-F) and current-membrane voltage (I-V) relationships. In spite of the unchanged I-F and I-V curves, mossy cells showed extensive modifications in Na(+), K(+) and h-currents, indicating the coordinated nature of these opposing modifications. Computational experiments in a realistic large-scale model of the dentate gyrus demonstrated that individually, these perturbations could significantly affect network activity. Synaptic inputs also displayed systematic, opposing modifications. Miniature excitatory postsynaptic current (EPSC) amplitudes were decreased, whereas miniature inhibitory postsynaptic current (IPSC) amplitudes were increased as expected from a homeostatic response to network hyperexcitability. In addition, opposing alterations in miniature and spontaneous synaptic event frequencies and amplitudes were observed for both EPSCs and IPSCs. Despite extensive changes in synaptic inputs, cannabinoid-mediated depolarization-induced suppression of inhibition was not altered in posttraumatic mossy cells. These data demonstrate that many intrinsic and synaptic properties of mossy cells undergo highly specific, long-term alterations after traumatic brain injury. The systematic nature of such extensive and opposing alterations suggests that single-cell properties are significantly influenced by homeostatic mechanisms in hyperexcitable circuits.
The pilocarpine model of temporal lobe epilepsy was used to study the time-dependent changes in dentate gyrus circuitry after seizures. Seizures caused a decrease in mu- and delta-opioid receptor immunoreactive (MOR-IR and DOR-IR, respectively) neurons in the hilus and MOR-IR neurons in the granule cell layer. Additionally, diffuse DOR-IR, MOR-IR, and GABA immunoreactivities (GABA-IR) were increased in the inner molecular layer. Using the in vitro hippocampal slice preparation to study the physiological consequences of the anatomical changes, we found that the disinhibitory effects of the mu-opioid receptor agonist [D-Ala2, MePhe4,Gly-(ol)5]-enkephalin (DAMGO) and the GABAA receptor antagonist bicuculline were greatly depressed 5-13 d after pilocarpine injection but returned to control levels within 6 weeks. The amplitudes of monosynaptic evoked IPSCs and the effects of DAMGO on this parameter were also slightly decreased 5-13 d after pilocarpine injection but significantly increased at 6 weeks. DAMGO significantly decreased the mean amplitude of spontaneous IPSCs (sIPSCs) at 6 weeks after pilocarpine injection but not in controls. The delta-opioid receptor agonist [D-Pen2,5]-enkephalin (DPDPE) principally inhibited excitatory transmission in saline-treated animals without affecting either sIPSCs or evoked IPSCs. The DPDPE-induced inhibition of excitatory transmission became more pronounced at 6 weeks after pilocarpine injection. These results illustrate the anatomical reorganization and functional changes in dentate gyrus circuitry evident in an animal model of temporal lobe epilepsy and provide evidence of compensatory changes after trauma to the hippocampal formation.
Blocking inhibition provides one of the most common experimental means of triggering epileptic activity in hippocampus and neocortex. However, it has proved much more difficult to show that chronic models of epilepsies are due to disinhibition. One problem is knowing how much inhibition needs to be blocked to provide a sufficient mechanism for epileptic activity. We have found that inhibitory (GABAA) transmission, estimated from evoked monosynaptic IPSCs, must be reduced to 17% of their control amplitude (by 4-7 microM bicuculline) before hippocampal slices generate all-or-none epileptic discharges. Similar estimates of inhibition in chronic epileptic foci induced by intrahippocampal injection of tetanus toxin showed that monosynaptic IPSCs dropped to 10% of control in the injected hippocampus during the first 2 weeks after injection. At all other stages of the active epileptic foci in the two hippocampi the reduction in IPSCs was not alone sufficient for epileptic activity; at 4-6 weeks IPSCs were normal despite continued epileptic activity. One likely mechanism for the late epileptic activity is a reduction of either the intrinsic excitability, or the synaptic excitation, of inhibitory interneurons so they fail to be recruited normally. Alternative mechanisms include the formation of new excitatory connections, as found at modest levels in the dentate gyrus. Several mechanisms may play a part in chronic foci such as those induced by tetanus toxin, either acting together, or sequentially during the progression of the epileptic focus.
Rat hypothalamic neurons and endocrine cells from the intermediate lobe of the pituitary were grown in dissociated coculture. Neurons positively stained with an antibody against glutamate decarboxylase established apparent contacts with the alpha-melanocyte-stimulating hormone-positive endocrine cells. These sites of contact were intensely labeled with an antibody against the synaptic protein synapsin I and displayed ultrastructural features characteristic of synapses. Using patch-clamp recordings, we have demonstrated that these contacts correspond to functional GABAergic synapses. The synaptic currents were blocked reversibly by bicuculline (5 microM) and SR95531 (5 microM), two competitive antagonists of the GABAA receptor. At a holding potential of -60 mV, spontaneously occurring IPSCs (s-IPSCs) had small amplitudes (10-100 pA), whereas electrically evoked IPSCs (ee-IPSCs) had amplitudes up to 1 nA. The rise times of both types of IPSCs were fast ( < or = 1 msec), and their decaying phases were fitted in most cases with a single exponential function (time constant 50 msec). The amplitude distribution of s-IPSCs did not reveal clear, equally spaced peaks and was little affected by tetrodotoxin, suggesting that most s-IPSCs were miniature IPSCs. Reduction of extracellular calcium concentration to 0.3 mM induced a marked decrease in s-IPSC frequency and revealed a single amplitude peak at 10 pA, suggesting that a single quantum of GABA activates 8-10 GABAA channels. Thus, our preparation might be an interesting model to study different aspects of synapse formation between a central neuron and its target as well as the fundamental mechanisms of synaptic transmission at central synapses.
In order to determine whether changes in synaptic inhibition are involved in chronic models of epilepsy, it is necessary to understand the factors which determine the kinetics of fast gamma-aminobutyric acid (GABA)ergic inhibition. For this purpose, we analyzed the decaying phase of isolated inhibitory postsynaptic currents (IPSC) in rats CA1 pyramidal cells. Reduction of GABA release (by reducing [Ca2+]o or paired-pulse stimulation) or blockade of GABA uptake (with tiagabine) led to the conclusion that small changes in the amount of GABA available for postsynaptic binding have little effect on the peak amplitude, but have marked effect on the duration of the IPSC. We then studied isolated GABAA receptor-mediated inhibition in area CA1 of the El mouse strain, which is genetically predisposed to epilepsy. Results were compared with the non-epileptogenic mother strain, ddY. Inhibitory postsynaptic potentials (IPSPs) in El mice (IPSPEl) were not significantly different in amplitude of those from ddY mice (IPSPddY). However, the rise-time and duration of IPSPEl were respectively about 25% and 50% shorter than those of IPSPddY. With appropriate pharmacological manipulation of GABA release or uptake, IPSPEl could be made to resemble the IPSPddY and vice versa. It is concluded that the synaptic release of GABA in area CA1 of the El mouse is decreased compared to that of the ddY mouse.