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PubMed2025

Proteomics of Duchenne Muscular Dystrophy Patient iPSC-Derived Skeletal Muscle Cells Reveal Differential Expression of Cytoskeletal and Extracellular Matrix Proteins.

Proteomics of dystrophic muscle samples is limited by the amount of protein that can be extracted from patient biopsies. Cells and tissues derived from patient-derived induced pluripotent stem cells (iPSCs) can be an expandable alternative source. We have patterned iPSCs from three Duchenne muscular dystrophy (DMD) patient lines into skeletal muscle cells using a two-dimensional as well as our three-dimensional organoid differentiation system. Probes with sufficient protein amounts could be extracted and prepared for mass spectrometry. In total, 3007 proteins in 2D and 2709 proteins in 3D were detected in DMD patient probes. A total of 83 proteins in 2D and 338 proteins in 3D can be described as differentially expressed between DMD and control patient probes in a post hoc test. We have identified and we propose Myosin-9, Collagen 18A, Tropomyosin 1, BASP1, RUVBL1, and NCAM1 as proteins specifically altered in their expression in DMD for further investigation. Proteomics of skeletal muscle organoids resulted in greater consistency of results between cell lines in comparison to the two-dimensional myogenic differentiation protocol.

Humans↗
PubMed1999

Junctional versus extrajunctional glycine and GABA(A) receptor-mediated IPSCs in identified lamina I neurons of the adult rat spinal cord.

Colocalization of GABA and glycine in synaptic terminals of the superficial dorsal horn raises the question of their relative contribution to inhibition of different classes of neurons in this area. To address this issue, miniature IPSCs (mIPSCs) mediated via GABA(A) receptors (GABA(A)Rs) and glycine receptors (GlyRs) were recorded from identified laminae I-II neurons in adult rat spinal cord slices. GABA(A)R-mediated mIPSCs had similar amplitude and rise times, but significantly slower decay kinetics than GlyR-mediated mIPSCs. Lamina I neurons appeared to receive almost exclusively GlyR-mediated mIPSCs, even after application of hypertonic solutions. Yet, all neurons responded to exogenous applications of both GABA and glycine, indicating that they expressed both GABA(A)Rs and GlyRs. Given that virtually all glycinergic interneurons also contain GABA, the possibility was examined that GABA(A)Rs may be located extrasynaptically in lamina I neurons. A slow GABA(A)R-mediated component was revealed in large, but not minimally evoked monosynaptic IPSCs. Administration of the benzodiazepine flunitrazepam unmasked a GABA(A)R component to most mIPSCs, suggesting that both transmitters were released from the same vesicle. The isolated GABA(A)R component of these mIPSCs had rising kinetics 10 times slower than that of the GlyR component (or of GABA(A)R mIPSCs in lamina II). The slow GABA(A)R components were prolonged by GABA uptake blockers. It is concluded that, whereas GABA and glycine are likely released from the same vesicle of transmitter in lamina I, GABA(A)Rs appear to be located extrasynaptically. Thus, glycine mediates most of the tonic inhibition at these synapses. This differential distribution of GABA(A)Rs and GlyRs confers distinct functional properties to inhibition mediated by these two transmitters in lamina I.

2-Amino-5-phosphonovalerate↗
PubMed1997

Slow kinetics of miniature IPSCs during early postnatal development in granule cells of the dentate gyrus.

Whole-cell patch-clamp recordings were used to investigate the properties of GABAA receptor-mediated postsynaptic currents during development in dentate gyrus granule cells from neonatal [postnatal day 0 (P0)] to adult rats in brain slices. The frequency of miniature IPSCs (mIPSCs) was low at birth and increased progressively with age. The mIPSCs of all ages could be satisfactorily fitted with the sum of a single exponential rise and single exponential decay. From P0 to P14, both the rise time and the decay time constants were significantly longer than in the adult. The mIPSC rise and decay kinetics did not change during the first 2 postnatal weeks, but during the third week the kinetics sped up and by P21 attained adult values. In contrast, the amplitude of the mIPSCs did not change during development. The synaptic GABAA receptors in immature and adult cells showed differential sensitivity to modulators. The subunit-specific benzodiazepine agonist zolpidem increased the decay time constant of the IPSCs of immature granule cells with a reduced potency compared with the adult. Furthermore, zinc decreased the amplitude and decay time constant of mIPSCs from developing granule cells, whereas it had no effect on mIPSCs in adult neurons. The results reveal for the first time that until the end of the second postnatal week the synaptic GABAA receptor-mediated currents in dentate granule cells display slower rise and decay kinetics but similar amplitudes compared with adult, resulting in a net decrease in synaptic charge transfer during development.

Aging↗
PubMed2026

Biallelic rescue of CTG18.1 in two Fuchs endothelial corneal dystrophy-derived iPSC lines (SCTCi047-A-2, SCTCi046-A-2) following a two-step gene editing strategy.

Fuchs endothelial corneal dystrophy (FECD) is an age-related condition distinguished by the degeneration of the corneal endothelium. An intronic CTG18.1 repeat in the transcription factor 4 (TCF4) gene has been associated with a 78-fold increased risk of developing the disease when at least one copy of the CTG18.1 expands above 50 repeats. Employing patient-derived material, we applied a dual CRISPR/Cas9-mediated editing approach to rescue the expansion. Combining non-homologous end-joining (NHEJ) and homologous direct repair (HDR) events, we generated two FECD-derived +/+(CTG)8 induced pluripotent stem cell (iPSC) lines, which were then successfully characterized, providing relevant isogenic controls for disease-modelling purposes.

Humans↗
PubMed2026

Dual CRISPR/Cas9 correction of compound heterozygous MARS2 mutations in the iPSC line ISMMSi060-A from a patient with COXPD25.

We previously described the induced pluripotent stem cell (iPSC) line ISMMSi060-A derived from a patient with Combined Oxidative Phosphorylation Deficiency 25 (COXPD25) carrying compound heterozygous pathogenic variants in the mitochondrial methionyl-tRNA synthetase gene, MARS2. Here, we report the generation of the isogenic control line ISMMSi060-A-1 by CRISPR/Cas9-mediated correction of the MARS2 variants c.424C>T (p.Arg142Trp) and c.550C>T (p.Gln184*). The corrected line retained normal morphology, pluripotency, genomic integrity, and differentiation capacity, providing a valuable resource to study MARS2-related mitochondrial dysfunction and therapeutic strategies for COXPD25.

Humans↗
PubMed2026

Generation of an NKX2-1-EGFP reporter iPSC line with inducible Cas9 for lung progenitor cell tracing.

NK2 homeobox 1 (NKX2-1), a master regulator robustly expressed in lung, thyroid, and forebrain, is indispensable for specifying lung epithelial fate and serves as a definitive marker of lung progenitors. Here, we generated a human induced Pluripotent Stem Cell (iPSC) line harboring a doxycycline (dox)-inducible Cas9 and an NKX2-1-EGFP-puro reporter via CRISPR/Cas9-mediated homology-directed repair. This dual-function line combines inducible genome editing with real-time tracing of early lung progenitors, enabling their prospective isolation and screening for stage-specific maturation regulators. Therefore, this engineered iCas9-NKX2-1 EGFP line is a key resource for dissecting human lung development, modeling pulmonary disease, and advancing regenerative therapies.

Humans↗
PubMed1999

Developmental study of miniature IPSCs of CA3 hippocampal cells: modulation by midazolam.

Maturation of GABAA/benzodiazepine receptors is associated with changes in their subunit composition. We have investigated whether these changes are accompanied by a developmental modification in the kinetic properties of miniature IPSCs (mIPSCs) and sensitivity to midazolam, a benzodiazepine agonist. In the presence of TTX (10 microM) and excitatory amino acid antagonists, AP5 (50 microM) and CNQX (50 microM), we whole-cell recorded mIPSCs in CA3 cells of hippocampal slices of adult and young (4-8 days) rats. mIPSCs were mediated by GABAA receptors as they were suppressed by bicuculline (10 microM). In both the adult and young rats, mIPSCs were similar in amplitude and kinetic properties. However, the mIPSCs frequency markedly increased with age from 4+/-3 Hz in the young rats to 20+/-9 Hz in the adult rats. In both age groups, midazolam (0.01 microM(-10) microM) and pentobarbital (30 microM) did not affect the amplitude, frequency and rise time of the mIPSCs but they increased to a similar extent their decay time constant. The current responses to isoguvacine, a GABAA agonist, were potentiated by 0.1 microM midazolam in both age groups. It is concluded that in immature and adult rats, synaptic GABAA receptors of CA3 were not different in their kinetic properties and sensitivity to midazolam.

2-Amino-5-phosphonovalerate↗
PubMed2003

Amiloride inhibition of glycinergic miniature IPSCs in mechanically dissociated rat spinal neurons.

The effect of amiloride on glycinergic transmission in mechanically dissociated rat spinal dorsal horn neurons was examined with the use of whole-cell patch-clamp recording. Amiloride reversibly reduced both the frequency and amplitude of spontaneous glycinergic miniature IPSCs (mIPSCs) and its inhibitory effect on glycinergic mIPSCs persisted in either Ca2+-free or Na+-free external solutions while it disappeared in K+-free external solution. Analysis of the relationship between mIPSCs amplitude and frequency at various holding potentials shows that amiloride inhibition of glycinergic mIPSC frequency could result indirectly from its inhibition of amplitude.

Amiloride↗
PubMed2026

ARID5A RNA-binding coordinates microglial defense and ferroptosis in iPSC-derived models.

RNA-binding proteins (RBPs) are key regulators of gene expression that shape cellular function in health and disease. However, the roles of RBPs in immune cells within the central nervous system (CNS) remain poorly understood. Here, we identify ARID5A as an RBP highly expressed in microglia and uncover its RNA-mediated regulatory functions using integrated multi-omics analyses of its RNA, DNA, and protein interactions. ARID5A regulates the splicing and translation of its RNA targets, many of which are integral to lysosomal, immune, and iron metabolism pathways. We confirm the functional relevance of this ARID5A-dependent RNA regulatory network by demonstrating that ARID5A modulates lysosomal activity, cytokine secretion, iron accumulation, and ferroptosis in iPSC-derived microglia. We further demonstrate that knockdown of microglial ARID5A reduces neuronal ferroptosis in co-cultures, underscoring the interconnected nature of these pathways. Moreover, in microglia harboring the TREM2-T66M mutation, ARID5A depletion restores dysregulated lysosomal and metabolic functions. Our results highlight the importance of protein-RNA interactions in regulating microglial cell biology.

Microglia↗
PubMed2025

Mapping key mitochondrial genes in Alzheimer's disease through human tissue and iPSC derived neurons.

Alzheimer's disease (AD) is a progressive neurodegenerative condition that has become a global health challenge due to an aging world population and no available effective treatment. Mitochondrial dysfunction plays a crucial role in the development of AD due to its critical role in neuronal survival and function. However, the specific mitochondrial genes and pathways involved in AD pathogenesis remain poorly defined. In this study, we incorporated seven AD human postmortem and three AD iPSC-derived neurons (iNs) gene expression datasets to identify mitochondria-related Differentially Expressed Genes (mitoDEGs) between AD and control. The Gene Ontology (GO) analysis is conducted to investigate the AD biological mechanisms, and a random forest model is developed to assess how well the key mitoDEGs differentiate AD and control groups. Through our analysis, we identified fourteen key mitochondria related genes that show significant dysregulation in both postmortem brain tissues and iNs derived from AD patients. These genes have strong connections to oxidative stress, indicating mitochondrial dysfunction plays a crucial role in Alzheimer's disease pathology. Our study identified the key genes and pathways as promising targets for future research and therapeutic interventions, highlighting the importance of mitigating oxidative stress and restoring mitochondrial function in AD.

Humans↗
PubMed2001

Slow IPSC kinetics, low levels of alpha1 subunit expression and paired-pulse depression are distinct properties of neonatal inhibitory GABAergic synaptic connections in the mouse superior colliculus.

Remodelling of visual maps in the superior colliculus (SC) depends on neuronal activity. Synaptic inhibition could contribute to this process because spontaneous spike discharge in the SC was modulated by GABA(A) receptor activation at postnatal days (P) 1-3. To investigate the functional capacity of GABAergic synaptic transmission at this early stage of development, whole-cell patch-clamp recordings were made from wide field neurons (WFNs) in horizontal slices comprising the superficial grey layer of the SC. Focal stimulation in the vicinity of WFNs evoked tetrodotoxin-sensitive stimulus-locked inhibitory postsynaptic currents (eIPSCs). The failure rate of eIPSCs was low ( approximately 0.2), and the maximal amplitude of evoked unitary eIPSCs exceeded the amplitude of average miniature IPSCs (mIPSCs) by a factor of 4-5, suggesting that action potential-mediated GABA release was more effective than spontaneous release. Some of the properties of GABAergic synaptic transmission in the neonatal SC were age-specific. In contrast with eIPSCs in the more mature SC at P20-22, neonatal eIPSCs decayed more slowly, preferentially fluctuated in duration, not amplitude, and mostly lacked temporal summation, due to depression at shorter intervals. The paired-pulse ratio (eIPSC2 : eIPSC1) was inversely related to the duration of eIPSCs. PCR analysis showed, in addition, that the ratio of alpha1 : alpha3 subunit expression was lower in the neonatal SC. Together, these results suggest that, at a young age, efficacy of GABAergic synaptic transmission is primarily constrained by the slow kinetics and the saturation of postsynaptic GABA(A) receptors.

Action Potentials↗
PubMed2000

Altering the concentration of GABA in the synaptic cleft potentiates miniature IPSCs in rat occipital cortex.

We have tested the effect of dextran (40 kDa, 5%) on miniature IPSCs (mIPSCs) recorded in layer V cortical pyramidal cells. This compound increases the amplitude of mIPSCs at room and physiological temperatures by 15%, leaving their duration unaffected at room temperature and slightly increased at physiological temperature. The amplitude increase is attributable to an increase in the number of receptors bound by GABA during synaptic transmission, as shown by the occlusion between the effects of dextran and zolpidem on mIPSC amplitude at room temperature. As dextran presumably enhances the concentration and dwell time of GABA in the synaptic cleft, these results demonstrate that the postsynaptic GABAA receptors are not saturated at room and physiological temperatures.

Animals↗
PubMed2003

Developmental differences in the sensitivity of hippocampal GABAA receptor-mediated IPSCS to ethanol.

BACKGROUND: Ethanol consumption by juveniles and adolescents is a public health problem of massive proportions. Recent studies have indicated that adolescents may be less sedated by ethanol than adults, and may thus be able to consume more ethanol, putting them at greater risk for ethanol addiction and other negative consequences of ethanol use. However, the mechanisms underlying this developmental difference are unknown. One contributing factor may be GABAA receptor-mediated inhibition, which is known to produce sedation. Ethanol is known to enhance this inhibitory process. METHODS: Using whole cell recording, we tested the response of GABAA receptor-mediated IPSCs in brain slices of rat hippocampus from animals representing three developmental stages: juvenile, early adolescent, and adult. RESULTS: We found significantly greater ethanol-induced enhancement of GABA-mediated IPSPs in cells from adult animals compared to those from juvenile or adolescent animals. CONCLUSIONS: We conclude that the sensitivity of GABAA receptor-mediated inhibitory processes to ethanol increases with development from the juvenile period to adulthood.

Animals↗
PubMed2025

Multi-omic phenotyping of iPSC-derived neurons harboring the MAPT V337M mutation reveals tau hypophosphorylation and perturbed axon morphology pathways.

Tau aggregation is a hallmark of several neurodegenerative diseases, including Alzheimer's disease and frontotemporal dementia. There are disease-causing variants of the tau-encoding gene, MAPT, and the presence of tau aggregates is highly correlated with disease progression. However, the molecular mechanisms linking pathological tau to neuronal dysfunction are not well understood. This is in part due to an incomplete understanding of the normal functions of tau in development and aging, and how the associated molecular and cellular processes change in the context of causal disease variants of tau. To address these questions in an unbiased manner, we conducted multi-omic characterization of iPSC-derived neurons harboring the MAPT V337M mutation or MAPT knockdown. RNA-seq and phosphoproteomics revealed that both V337M mutation and tau knockdown perturbed levels of transcripts and phosphorylation of proteins related to axonogenesis or axon morphology. Surprisingly, we found that neurons with V337M tau had much lower tau phosphorylation than neurons with WT tau. Functional genomics screens uncovered regulators of tau phosphorylation in neurons and found that factors involved in axonogenesis modified tau phosphorylation in both MAPT WT and MAPT V337M neurons. Intriguingly, the p38 MAPK pathway specifically modified tau phosphorylation in MAPT V337M neurons. We propose that V337M tau perturbs tau phosphorylation and axon morphology pathways that are relevant to the normal function of tau, which could contribute to previously reported cognitive changes in preclinical MAPT variant carriers.

Journal Article↗
PubMed2025

A xeno-free protocol for rapid differentiation of human iPSC-derived microglia from the KOLF2.1J reference line.

We present a detailed, xeno-free protocol for the rapid differentiation of human induced pluripotent stem cells (hiPSCs) into microglia using the well-characterized KOLF2.1J reference line. This system employs doxycycline-inducible expression of six transcription factors (6-TF), stably integrated into the CLYBL safe harbor locus, to drive uniform microglial differentiation within two weeks. Adapted from Dräger et al. (1), our protocol includes key optimizations for KOLF2.1J, including culture on Laminin-521 to support xeno-free conditions. The resulting i-Microglia exhibit hallmark features of mature microglia, including expression of P2RY12, loss of the pluripotency marker SSEA4, phagocytic activity, and upregulation of immune markers (e.g., CD80, CD83) upon LPS stimulation. We also demonstrate compatibility with co-culture systems using iPSC-derived neurons. Additionally, we describe a modification of the line to include a constitutive mCherry reporter integrated into the SH4-2 safe harbor locus, enabling fluorescent tracking of microglia in mixed cultures or in vivo. This protocol provides a reproducible and scalable platform for generating functional human microglia from a widely used hiPSC line, supporting applications in disease modeling, neuroinflammation research, and therapeutic screening.

Journal Article↗
PubMed1995

Different mechanisms for use-dependent depression of two GABAA-mediated IPSCs in rat hippocampus.

1. The mechanisms involved in the use-dependent depression of GABAA,fast and GABAA,slow, two GABAA-mediated IPSCs in the rat hippocampal slice preparation, were investigated by observing the effects of paired-pulse depression and of baclofen and CGP 35348 on monosynaptic inhibitory currents recorded from CA1 pyramidal neurons. 2. The second of a pair of evoked responses that consisted of both inhibitory components was depressed and decayed more rapidly compared to the first at an interpulse interval (IpI) of 200 ms. This effect was due to a decrease in the amplitude of GABAA,slow, with no effect on the time constant of decay or on the amplitude or time constant of GABAA,fast. 3. The time course of paired-pulse depression of both components at IpIs ranging from 5 to 2560 ms was compared. GABAA,slow was depressed maximally by 55% at IpIs of 80-160 ms. GABAA,fast was depressed maximally by 38% at 5 ms, and recovered exponentially with a time constant of 130 ms. 4. GABAA,slow was more sensitive than GABAA,fast to depression by baclofen. GABAA,slow was susceptible to complete block, with an ED50 of approximately 200 nM for (+/-)-baclofen and 100 nM for the active enantiomer, (R)-(+)-baclofen. GABAA,fast was blocked by only 50% by the highest concentrations of baclofen tested (10-100 microM (R)-(+)-baclofen), with an ED50 of approximately 2 microM for (+/-)-baclofen and 1 microM for (R)-(+)-baclofen. Paired-pulse depression of GABAA,fast was not occluded by 10 or 100 microM (R)-(+)-baclofen. 5. The GABAB antagonist CGP 35348 (0.4-1 mM), prevented paired-pulse depression of GABAA,slow at IpIs of 160 to 200 ms, and reversed the depression of GABAA,fast by baclofen, but had no effect on paired-pulse depression of GABAA,fast at IpIs of 20 to 40 ms. 6. It is concluded that use-dependent depression of GABAA,slow, but not GABAA,fast, is mediated by a presynaptic GABAB receptor. It is speculated that use-dependent depression of GABAA,fast, which occurs only over a much faster time scale, may be due to rapid postsynaptic GABAA receptor desensitization.

Animals↗
PubMed1996

Noradrenaline induces IPSCs in rat medial septal/diagonal band neurons: involvement of septohippocampal GABAergic neurons.

1. The physiological and pharmacological actions of noradrenaline (NA) on neurons of the medial septum and diagonal band of Broca (MSDB) were examined using extracellular, intracellular and whole-cell patch-clamp recordings in an in vitro rat brain slice preparation. 2. In current- and voltage-clamp recordings with KCl- or potassium gluconate-containing electrodes, bath-applied NA increased the number of tetrodoxin- and bicuculline-sensitive synaptic events in > 80% of cholinergic- and GABA-type neurons tested. The NA-induced synaptic activity originated from GABAergic neurons located within the MSDB itself, as a similar effect occurred in brain slices in which the MSDB had been surgically isolated from neighbouring structures. 3. In antidromic studies, NA dose-dependently increased firing in a subpopulation of septohippocampal neurons with fast conducting fibres (mean conduction velocity, 1.78 +/- 0.10 m s-1; presumably GABAergic). The NA excitation was mimicked by the alpha 1-agonist phenylephrine (PE) and blocked by the alpha 1-antagonists prazosin and WB-4101, suggesting the presence of alpha 1-receptors on septohippocampal GABAergic neurons. 4. Similarly, in whole-cell recordings in both cholinergic- and non-cholinergic-type MSDB neurons, prazosin blocked the effects of NA and PE mimicked the effects of NA by inducing IPSCs with a similar amplitude distribution. 5. Consistent with the above findings, GABA-type neurons that responded directly to NA and PE with a prazosin-sensitive inward current were found within the MSDB. 6. In conclusion, NA, via alpha 1-adrenoceptors, excites MSDB septohippocampal GABAergic neurons and influences both septal and septohippocampal circuitry.

Animals↗
PubMed2003

Methylmercury differentially affects GABA(A) receptor-mediated spontaneous IPSCs in Purkinje and granule cells of rat cerebellar slices.

Using whole-cell recording techniques we compared effects of the environmental cerebellar neurotoxicant methylmercury (MeHg) on spontaneous IPSCs (sIPSCs) of both Purkinje and granule cells in cerebellar slices of the rat. In Purkinje cells, bath application of 10, 20 or 100 microM MeHg initially increased then suppressed the frequency of sIPSCs to zero. In granule cells, the initial increase in frequency was not observed in approximately 50% of cells examined, but suppression of sIPSCs by MeHg occurred in every cell tested. For both cells, time to onset of effects of MeHg was inversely related to the concentration; moreover, the pattern of changes in mIPSCs induced by MeHg in the presence of tetrodotoxin was similar to that in sIPSCs. For ea ch concentration of MeHg, it took 2-3 times longer to block sIPSCs in Purkinje cells than it did in granule cells. MeHg also initially increased then decreased amplitudes of sIPSCs to block in both cells; again the response was more variable in granule cells. In most Purkinje and some granule cells, MeHg induced a giant, slow inward current during the late stages of exposure. Appearance of this current appeared to be MeHg concentration dependent, and the direction of current flow was reversed by changing the holding potentials. Reduction of the [Cl-] in the internal solution caused inwardly directed, but not outwardly directed giant currents to disappear, suggesting that this current is a Cl(-)-mediated response. However, bicuculline and picrotoxin failed to block it. MeHg apparently acts at both presynaptic and postsynaptic sites to alter GABA(A) receptor-mediated inhibitory synaptic transmission. GABA(A) receptors in granule cells appear to be more sensitive to block by MeHg than are those in Purkinje cells, although the general patterns of effects on the two cells are similar.

Animals↗