Biology subjects

Maksour, S.

Publications and source records attributed to Maksour, S..

8 recordsLinked to original sources

bioRxiv2026

TriPort tangential flow filtration: a lab-assembled system for EV isolation across diverse cell-culture sources

Tangential flow filtration (TFF) is an effective method for scalable EV isolation. Here, we describe and validate a reference protocol for TriPort-TFF, a laboratory-assembled TFF system featuring a three- port reservoir design enabling sample circulation, retentate return, and diafiltration. Constructed from individually sourced, commercially available components with detailed assembly and operating parameters, TriPort-TFF can be built at a lower cost than commercially integrated TFF platforms. We evaluated the system using conditioned media (CM) from human MDM cultured with EV-depleted human serum, human cerebral organoids in serum-free medium, and mouse BMDM cultured with EV-depleted foetal bovine serum (FBS) unstimulated or LPS-stimulated. The system reproducibly isolated EVs from all three sources, and biological replicates of BMDM showed distinct proteomic clusters between LPS-stimulated and unstimulated conditions. The requirement for subsequent size-exclusion chromatography (SEC) after TFF depended on the sample serum-presence. In serum-containing CM, TFF alone co-concentrated serum proteins alongside EVs, subsequent SEC improved the particle-to- protein ratio, reduced inter-replicate variability and improved proteomic depth. TFF alone in serum- free organoid CM already yielded higher particle-to-protein ratios than TFF-only preparations from serum-containing CM, with SEC further improving this ratio. TriPort-TFF offers an accessible system for EV-isolation that can be combined with SEC for diverse sample types.

cell biology↗

bioRxiv2025

Optimizing the In Vitro Neuronal Microenvironment to Mitigate Phototoxicity in Live-cell Imaging

Long-term imaging formats are ideal for capturing dynamic neuronal network formation in vitro, yet fluorescent techniques are often constrained by the impact of phototoxicity on cell survival. Here we present a live-imaging protocol that was optimised via quantitative analysis of 3 target culturing conditions on neuromorphological health: extracellular matrix (human- versus murine-derived laminin), culture media (Neurobasal versus Brainphys Imaging media), and seeding density (1x105 versus 2x105 cells/cm2). A cortical neuron reporter line was differentiated from human embryonic stem cells by transduction of Neurogenin-2 and green fluorescent protein, then fluorescently imaged in 8 different microenvironments daily for 33 days. Alongside viability analysis by PrestoBlue assay and gene quantification by digital polymerase chain reaction, an automated image analysis pipeline was developed to characterise network morphology and organisation over time. Brainphys Imaging medium was observed to support neuron viability, outgrowth, and self-organisation to a greater extent than Neurobasal medium with either laminin type, while the combination of Neurobasal medium and human laminin reduced cell survival. Further, a higher seeding density fostered somata clustering, but did not significantly extend viability compared to low density. These findings suggest a synergistic relationship between species-specific laminin and culture media in phototoxic environments, which is positively mediated by light-protective compounds found in Brainphys Imaging medium.

cell biology↗

bioRxiv2025

THE EFFECT OF SERTRALINE AND VOLUNTARY EXERCISE DURING PREGNANCY ON LITTER CHARACTERISTICS AND POSTNATAL AFFECTIVE BEHAVIOUR IN RAT DAMS

IntroductionSertraline is the frontline pharmacotherapy for the treatment of depression and anxiety during pregnancy. However, there is little evidence regarding the effects of sertraline on maternal behaviour or the maternal brain. Furthermore, the efficacy of non-pharmacological approaches to treatment in pregnancy, such as exercise, are unclear. Therefore, the aim of this study was to examine the effects of sertraline and exercise during pregnancy on maternal postnatal depressive-like, anxiety-like and associated behaviours, as well as litter characteristics, in a rat model of depression. We also investigated the effects of these treatments on the maternal brain, focusing initially on DNA methylation and glutamatergic markers, which have been implicated in depression. MethodTwenty-four female Wistar-Kyoto (WKY; strain that models depression and anxiety) rats were divided into three groups: 1. WKY-Sertraline; 2. WKY-Exercise, 3. WKY-Vehicle; Six female Wistar rats were included as controls. Rats were treated with sertraline (10mg/kg) or vehicle (33% propylene glycol) twice/day, from gestational day (GD) 1 to postnatal day (PN) 14. The WKY-Exercise group were provided access to a running wheel during pregnancy for 3 hours/day from GD1-18. Dam and litter characteristics, as well as pup ultrasonic vocalisations (USVs), were measured. Dams underwent behavioural testing at 5-weeks postnatal to assess depressive-, anxiety- and cognitive-like behaviours. Gene expression of DNA methylation markers (Dnmt1, Dnmt3a) and glutamate receptors (Grin1, Grin2a, Grin2b) were measured in the prefrontal cortex (PFC), using RT-qPCR. ResultThe WKY-Sertraline group gained 39% less weight in their first pregnancy week compared to all other groups (p<0.05) and produced smaller litters compared to Wistar controls (-43%; p=0.003) and WKY-Exercise (-38%; p=0.012), and WKY-Sertraline pups had slightly smaller brain weights (p=0.031 compared to WKY-Vehicle). The WKY-Exercise pups produced increased number of USVs at PN7 compared to WKY-Vehicle, with no treatment differences at PN14. The WKY strain however, did showed reduced average mean amplitude of calls and reduced average duration of calls at PN7 compared to WIS (p<0.01) and reduced number of calls at PN14 (p<0.01). Maternal sertraline treatment did not significantly affect dam behavioural measures, all maternal cortical gene expression. The WKY-Exercise group however showed reduced anxiety-like behaviours, spending more time in the open arms (620%; p=0.027) and less time in the closed arms (-22%; p=0.047) of the elevated plus maze (EPM) compared to WKY-Vehicle, and more time in the centre of the open field test (OFT) compared to WKY-Vehicle (132%; p=0.057). Furthermore, WKY-Exercise dams showed a 64% increase in Dnmt3a mRNA levels in the PFC compared to WKY-Vehicle (p=0.019). ConclusionVoluntary exercise during pregnancy in the WKY rat model, reduced postnatal anxiety-like behaviour in the dam. This was accompanied by elevated DNMT3a gene expression in the PFC, suggesting this region may be sensitive to DNA methylation changes following maternal exercise. In contrast, maternal sertraline did not impact these behaviours or genes. Maintaining sertraline treatment beyond PN14, may have resulted in broader effects on dam behaviour, which should be explored further. Maternal sertraline did appear to have some adverse effects on the in utero environment, evidenced by smaller litters, with slightly smaller pup brain weights, which should be investigated further. Our findings suggest a long-term beneficial effect of exercise during pregnancy and supports future studies examining the effects of exercise in antenatal depression in the human population.

neuroscience↗

bioRxiv2024

Distinct molecular and functional properties of human induced-proprioceptor and low-threshold mechanoreceptor neurons

Sensing mechanical stimuli is crucial for the function of internal and external tissues, such as the skin and muscles. Much of our understanding of mechanosensory physiology relies on rodent studies, which may not directly translate to humans. To address the knowledge gap in human mechanosensation, we developed distinct populations of human mechanosensory neuronal subtypes from human pluripotent stem cells (hPSC). By inducing co-expression of NGN2/RUNX3 or NGN2/SHOX2 in hPSC-derived migrating neural crest cells we directed their specification to proprioceptor and low-threshold mechanoreceptor neuronal subtypes, respectively. The induced neurons exhibited transcriptional profiles consistent with mechanosensory neurons and displayed functional responses to mechanical stimuli, such as stretch and submicrometer probe indentation to the soma. Notably, each subtype displayed unique mechanical thresholds and desensitization properties akin to proprioceptors and low-threshold mechanoreceptors and both induced neuronal subtypes fired action potentials in response to minute mechanical stimuli, predominantly relying on PIEZO2 for mechanosensory function. Collectively, this study provides a foundational model for exploring human neuronal mechanosensory biology.

neuroscience↗

bioRxiv2024

Alzheimer's disease induced neurons bearing PSEN1 mutations exhibit reduced excitability

Alzheimers disease (AD) is a devastating neurodegenerative condition that affects memory and cognition, characterized by neuronal loss and currently lacking a cure. Mutations in PSEN1 (Presenilin 1) are among the most common causes of early-onset familial AD (fAD). While changes in neuronal excitability are believed to be early indicators of AD progression, the link between PSEN1 mutations and neuronal excitability remains to be fully elucidated. This study examined induced pluripotent stem cell (iPSC)-derived NGN2 induced neurons (iNs) from fAD patients with PSEN1 mutations S290C or A246E, alongside CRISPR-corrected isogenic cell lines, to investigate early changes in excitability. Electrophysiological profiling revealed reduced excitability in both PSEN1 mutant iNs compared to their isogenic controls. Neurons bearing S290C and A246E mutations exhibited divergent passive membrane properties compared to isogenic controls, suggesting distinct effects of PSEN1 mutations on neuronal excitability. Additionally, both PSEN1 backgrounds exhibited higher current density of voltage-gated potassium (Kv) channels relative to their isogenic iNs, while displaying comparable voltage-gated sodium (Nav) channel current density. This suggests that the Nav/Kv imbalance contributes to impaired neuronal firing in fAD iNs. Deciphering these early cellular and molecular changes in AD is crucial for understanding the disease pathogenesis.

cell biology↗

bioRxiv2024

REST and RCOR genes display distinct expression profiles in neurons and astrocytes using 2D and 3D human pluripotent stem cell models

Repressor element-1 silencing transcription factor (REST) is a transcriptional repressor involved in neurodevelopment and neuroprotection. REST forms a complex with the REST corepressors, CoREST1, CoREST2, or CoREST3 (encoded by RCOR1, RCOR2, and RCOR3, respectively). Emerging evidence suggests that the CoREST family can target unique genes independently of REST, in various neural and glial cell types during different developmental stages. However, there is limited knowledge regarding the expression and function of the CoREST family in human neurodevelopment. To address this gap, we employed 2D and 3D human pluripotent stem cell (hPSC) models to investigate REST and RCOR gene expression levels. Our study revealed a significant increase in RCOR3 expression in glutamatergic cortical and GABAergic ventral forebrain neurons, as well as mature functional NGN2-induced neurons. Additionally, a simplified astrocyte transdifferentiation protocol resulted in a significant decrease in RCOR2 expression following differentiation. REST expression was notably reduced in mature neurons and cerebral organoids, along with RCOR2 in the latter. In summary, our findings provide the first insights into the cell-type-specific expression patterns of RCOR genes in human neuronal and glial differentiation. Specifically, RCOR3 expression increases in neurons, while RCOR2 levels decrease in astrocytes. The dynamic expression patterns of REST and RCOR genes during hPSC neuronal and glial differentiation underscore the potential distinct roles played by REST and CoREST proteins in regulating the development of these cell types in humans. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=138 SRC="FIGDIR/small/584254v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@7be30borg.highwire.dtl.DTLVardef@1771388org.highwire.dtl.DTLVardef@a376aforg.highwire.dtl.DTLVardef@1c4dc83_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIREST and RCOR genes display cell-type specific expression patterns in neural cells C_LIO_LIRCOR3 (encodes CoREST3) is upregulated during neuronal and astrocyte differentiation C_LIO_LIRCOR2 (encodes CoREST2) is downregulated during differentiation of astrocytes C_LIO_LIEvidence of potential cell-type specific functions of the CoREST family C_LI

neuroscience↗

bioRxiv2023

Parsing the effect of co-culture with brain organoids on Diffuse Intrinsic Pontine Glioma (DIPG) using quantitative proteomics

Diffuse Intrinsic Pontine Gliomas (DIPGs) are deadly brain cancers in children for which there is currently no effective treatment. This can partly be attributed to preclinical models that lack essential elements of the in vivo tissue environment, resulting in treatments that appear promising preclinically, but fail to result in effective cures. Recently developed co-culture models combining stem cell-derived brain organoids with brain cancer cells provide tissue dimensionality and a human-relevant tissue-like microenvironment. As these models are technically challenging and time consuming it is imperative to establish whether interaction with the organoid influences DIPG biology and thus warrants their use. To address this question, we cultured DIPG cells with cortical organoids. We created "mosaic" co-cultures enriched for tumour cell-neuronal cell interactions versus "assembloid" co-cultures enriched for tumour cell-tumour cell interactions. Sequential window acquisition of all theoretical mass spectra (SWATH-MS) was used to analyse the proteomes of DIPG fractions isolated by flow-assisted cell sorting. Control proteomes from DIPG spheroids were compared with DIPG cells isolated from mosaic and assembloid co-cultures. This revealed that tumour cell adhesion was reduced, and DNA synthesis and replication were increased, in DIPG cells under either co-culture condition. By contrast, the mosaic co-culture was associated with pathways implicated in dendrite growth. We propose that co-culture with brain organoids is a valuable tool to parse the contribution of the brain microenvironment to DIPG tumour biology.

cancer biology↗

bioRxiv2020

Identification of repurposable cytoprotective drugs for Vanishing White Matter Disease

Vanishing white matter disease (VWMD) is a rare leukodystrophy involving loss of function mutations of the guanine exchange factor eIF2B and typically presenting with juvenile onset. We aimed to identify repurposable FDA approved drugs in an in vitro drug screen using patient-derived fibroblasts and induced pluripotent stem cell (iPSC)-derived astrocytes. Dysregulated GADD34 and CHOP were identified in patient fibroblasts and iPSC-derived astrocytes under proteasomal stress conditions. A drug screen from a 2400 FDA approved drug library with EIF2B5 disease patient fibroblasts identified 113 anti-inflammatory drugs as a major class of hits with cytoprotective effects. A panel of potential candidate drugs including berberine, deflazacort, ursodiol, zileuton, guanabenz and Anavex 2-73, and preclinical ISRIB, increased cell survival of MG132-stressed EIF2B2 and EIF2B5 disease VWMD astrocytes, and were further investigated for their effect on the integrated stress response and mitochondrial stress. ISRIB but not other drugs significantly affected eIF2 phosphorylation and GADD34 expression. Ursodiol demonstrated capacity to reduce complex I subunit upregulation, ameliorate oxidative stress, loss of mitochondrial membrane potential and upregulation of eIF2B subunits in VWMD astrocytes, highlighting its potential as a cytoprotective compound for VWMD.

neuroscience↗