Biology subjects

Wolvetang, E. J.

Publications and source records attributed to Wolvetang, E. J..

15 recordsLinked to original sources

bioRxiv2026

Oligodendrocyte Enriched Brain Organoids Reveal Impaired Oligodendroglial Maturation and Altered Neural Network Activity in Down Syndrome

Individuals with Down syndrome (DS) display developmental delay, intellectual disability, premature brain ageing, and an increased risk of Alzheimer-like neurodegeneration. Although the neuropathology of the postnatal and adult DS brain has been widely described, it remains unclear how trisomy 21 alters early human neural and glial development. Here, we used human oligodendrocyte enriched brain organoids derived from trisomic and euploid iPSCs to define the cellular, functional, and molecular consequences of trisomy 21 during early brain development. Trisomic organoids exhibited an early growth delay, reduced oligodendroglial specification, and impaired oligodendrocyte maturation, resulting in decreased myelination. These defects were accompanied by increased astroglial output and delayed neuronal maturation. At the functional level, trisomic organoids showed elevated spontaneous network activity, but failed to mount normal coordinated responses to pharmacological stimulation, consistent with abnormal neural circuit development. Bulk RNA sequencing revealed the strongest transcriptomic dysregulation occurs at early neural and glial specification. Together, these findings show that trisomy 21 disrupts early developmental stages and establish oligodendrocyte enriched brain organoids as a human model to investigate the developmental origins of white matter and network dysfunction in DS.

cell biology↗

bioRxiv2025

Interaction of Gut-Microbial Amyloids with Endogenous Amyloids can Drives Microglial Hyperactivity and Neuroinflammation in Alzheimers Disease

Gut bacteria have emerged as silent drivers in the pathology of Alzheimers disease (AD). They also make amyloids with structure analogue to pathological amyloids and have potential to cross-seed and propagate in a prion-like manner. AD is characterised by the accumulation of mature extracellular Amyloid-{beta} (A{beta}) plaques which are surrounded by inflammatory microglia. We report that exposure to interspecies microbial amyloids of FapC (fimbriae) and CsgA (curli) from opportunistic gut pathogens Pseudomonas aeruginosa and Escherichia coli hyperactivates microglia against A{beta} fibrils. Microbial amyloids and A{beta} fibrils converge in phagocytic compartments through subsequent internalization, not observed with A{beta} fibrils alone. This convergence promotes pro-inflammatory microglia with a defective proteome similar to those observed in AD brains. The resulting clusters develop a pro-inflammatory, indigestible interactome that is eventually regurgitated, inducing progressive degeneration in bystander neurons and ultimately leading to cognitive decline. Collectively, these findings provide compelling evidence that microbial amyloids can trigger progressive AD pathology through microglia-driven neuroinflammation.

cell biology↗

bioRxiv2024

Unraveling the Role of MIXL1 Activation in Endoderm Differentiation of Isogenic Human Induced Pluripotent Stem Cells

Human induced pluripotent stem cells (hiPSC) possess the ability to differentiate into a multitude of cell and tissue types but display heterogeneous propensity of differentiation into specific lineage. Characterization of the transcriptome of eleven hiPSC lines showed that activation of MIXL1 at the early stage of stem cell differentiation correlated with higher efficacy in generating definitive endoderm and advancing differentiation and maturation of endoderm derivatives. Enforced expression of MIXL1 in the endoderm-inefficient hiPSCs enhanced the propensity of endoderm differentiation, suggesting that modulation of key drivers of lineage differentiation can re-wire hiPSC to the desired lineage propensity to generate the requisite stem cell products.

developmental biology↗

bioRxiv2024

Ataxia Telangiectasia patient-derived neuronal and brain organoid models reveal mitochondrial dysfunction and oxidative stress

Ataxia Telangiectasia (AT) is a rare genetic disorder caused by mutations in the ATM gene and is characterized by oxidative stress, premature ageing, and progressive neurodegeneration of the cerebellum. The molecular mechanisms driving the neurological defects AT remain unclear, mainly due to lack of human neuronal models. Here, we use AT patient-derived pluripotent stem cells (iPSCs) and iPSC-derived neurons and brain organoids to comprehensively explore mitochondrial dysfunction, oxidative stress, and senescence phenotypes. We identified mislocalisation of mitochondria, a prevailing reduction in mitochondrial membrane potential, and increased oxidative stress in AT patient-derived iPSC and neuronal cultures that was restored by ATM gene correction. Cortical brain organoids from AT patients also display extensive oxidative stress, increased levels of senescence, and impaired neuronal function that could be counteracted with antioxidant treatment. Transcriptomic analysis identified disruptions in regulatory networks related to mitochondrial function and maintenance, including alterations in the PARP/SIRT signalling axis and dysregulation of key mitophagy and mitochondrial fission-fusion processes. Our study reveals that progressive mitochondrial dysfunction and aberrant ROS production are hallmarks of AT, and lead us to conclude that ATM is a master regulator of mitochondrial homeostasis.

cell biology↗

bioRxiv2023

Rapamycin mitigates Valproic Acid-induced teratogenicity in human and animal models by suppressing AP-1-mediated senescence

Valproic acid (VPA) is an effective and widely used anti-seizure medication but is teratogenic when used during pregnancy, affecting brain and spinal cord development for reasons that remain largely unclear. Here we designed a genetic recombinase-based SOX10 reporter system in human pluripotent stem cells that enables tracking and lineage tracing of Neural Crest cells (NCCs) in a human organoid model of the developing neural tube. We found that VPA induces extensive cellular senescence and promotes mesenchymal differentiation of human NCCs at the expense of neural lineages. We next show that the clinically-approved drug, Rapamycin, inhibits AP1-mediated senescence and restores aberrant NCC differentiation trajectory in human organoids exposed to VPA. Notably, in vivo validation in developing zebrafish highlighted the therapeutic promise of this approach. Collectively our data identifies a novel mechanism for VPA-associated neurodevelopmental teratogenicity and a potential pharmacological preventative strategy. The results exemplify the power of genetically modified human stem cell-derived organoid models for drug discovery and safety testing.

neuroscience↗

bioRxiv2023

Choroid plexus defects in Down syndrome brain organoids enhance neurotropism of SARS-CoV-2

Why individuals with Down Syndrome (DS, trisomy 21) are particularly susceptible to SARS CoV-2 induced neuropathology remains largely unclear. Since the choroid plexus (CP) performs important barrier and immune-interface functions, secretes the cerebrospinal fluid and strongly expresses the ACE2 receptor and the chromosome 21 encoded TMPRSS2 protease, we hypothesized that the CP could play a role in establishing SARS-CoV-2 infection in the brain. To investigate the role of the choroid plexus in SARS-CoV-2 central nervous system infection in DS, we established a new type of brain organoid from DS and isogenic euploid control iPSC that consists of a core of appropriately patterned functional cortical neuronal cell types that is surrounded by a patent and functional choroid plexus (CPCOs). Remarkably, DS-CPCOs not only recapitulated abnormal features of DS cortical development but also revealed defects in ciliogenesis and epithelial cell polarity of the developing choroid plexus. We next demonstrate that the choroid plexus layer facilitates SARS-CoV-2 replication and infection of cortical neuronal cells, and that this is increased in DS-CPCOs. We further show that inhibition of TMPRSS2 and Furin activity inhibits SARS-CoV-2 replication in DS CPCOs to the level observed in euploid organoids. We conclude that CPCOs are a useful model for dissecting the role of the choroid plexus in euploid and DS forebrain development and enables screening for therapeutics that can inhibit SARS-CoV-2 induced neuro-pathogenesis.

neuroscience↗

bioRxiv2023

Substrate stiffness facilitates improved induced pluripotent stem cell production through modulation of both early and late phases of cell reprogramming

Cell reprogramming involves time-intensive, costly processes that ultimately produce low numbers of reprogrammed cells of variable quality. By screening a range of polyacrylamide hydrogels (pAAm gels) of varying stiffness (1 kPA - 1.3 MPa) we found that a gel of medium stiffness significantly increases the overall number of reprogrammed cells by up to ten-fold with accelerated reprogramming kinetics, as compared to the standard Tissue Culture PolyStyrene (TCPS)-based protocol. We observe that though the gel improves both early and late phases of reprogramming, improvement in the late (reprogramming prone population maturation) phase is more pronounced and produces iPSCs having different characteristics and lower remnant transgene expression than those produced on TCPS. Comparative RNA-Seq analyses coupled with experimental validation reveals that modulation of Bone Morphogenic Protein (BMP) signalling by a novel reprogramming regulator, Phactr3, upregulated in the gel at an earliest time-point without the influence of transcription factors used for reprogramming, plays a crucial role in the improvement in the early reprogramming kinetics and overall reprogramming outcomes. This study provides new insights into the mechanism via which substrate stiffness modulates reprogramming kinetics and iPSC quality outcomes, opening new avenues for producing higher numbers of quality iPSCs or other reprogrammed cells at shorter timescales.

bioengineering↗

bioRxiv2023

Senolytic therapy alleviates physiological human brain aging and COVID-19 neuropathology.

Aging is the primary risk factor for most neurodegenerative diseases, and recently coronavirus disease 2019 (COVID-19) has been associated with severe neurological manifestations that can eventually impact neurodegenerative conditions in the long-term. The progressive accumulation of senescent cells in vivo strongly contributes to brain aging and neurodegenerative co-morbidities but the impact of virus-induced senescence in the aetiology of neuropathologies is unknown. Here, we show that senescent cells accumulate in physiologically aged brain organoids of human origin and that senolytic treatment reduces inflammation and cellular senescence; for which we found that combined treatment with the senolytic drugs dasatinib and quercetin rejuvenates transcriptomic human brain aging clocks. We further interrogated brain frontal cortex regions in postmortem patients who succumbed to severe COVID-19 and observed increased accumulation of senescent cells as compared to age-matched control brains from non-COVID-affected individuals. Moreover, we show that exposure of human brain organoids to SARS-CoV-2 evoked cellular senescence, and that spatial transcriptomic sequencing of virus-induced senescent cells identified a unique SARS-CoV-2 variant-specific inflammatory signature that is different from endogenous naturally-emerging senescent cells. Importantly, following SARS-CoV-2 infection of human brain organoids, treatment with senolytics blocked viral retention and prevented the emergence of senescent corticothalamic and GABAergic neurons. Furthermore, we demonstrate in human ACE2 overexpressing mice that senolytic treatment ameliorates COVID-19 brain pathology following infection with SARS-CoV-2. In vivo treatment with senolytics improved SARS-CoV-2 clinical phenotype and survival, alleviated brain senescence and reactive astrogliosis, promoted survival of dopaminergic neurons, and reduced viral and senescence-associated secretory phenotype gene expression in the brain. Collectively, our findings demonstrate SARS-CoV-2 can trigger cellular senescence in the brain, and that senolytic therapy mitigates senescence-driven brain aging and multiple neuropathological sequelae caused by neurotropic viruses, including SARS-CoV-2.

neuroscience↗

bioRxiv2022

Thyroid hormone and ALK5 inhibitor improve maturation of human pluripotent stem cell derived hepatocytes

Hepatocytes derived from human pluripotent stem cells (PSCs) hold great promise for modeling human liver disease, in vitro hepatotoxicity testing, and future cellular therapy. However, current protocols generate hepatocyte-like cells (HLCs) that resemble fetal hepatocytes, and thus do not accurately recapitulate the molecular identity and functions of the adult liver. To address this, we compared the transcriptomes of human fetal and adult liver to PSC-derived HLCs during progressive stages of in vitro differentiation. This revealed that during the final stages of in vitro differentiation the hepatic transcription factors HNF4A and CEBPA were sub-optimally expressed. Computational analyses predicted that ALK5i II (TGF-{beta} receptor inhibitor) and thyroid hormone (T3) would be able to rectify this and improve HLC maturation. We next show that application of these molecules during hepatocyte differentiation indeed increases CEBPA and HNF4A mRNA and protein expression, and that these HLCs show enhanced albumin secretion, a 25-fold increase in CYP3A4 activity, and 10 to 100-fold increased expression of mature hepatic markers. We demonstrate that this improved maturation is effective across different cell lines and HLC differentiation protocols, and exemplifies that our approach provides a tractable template for identifying and targeting additional factors that that will fully mature human liver cells from human pluripotent stem cells.

developmental biology↗

bioRxiv2022

Deconstructing replicative senescence heterogeneity of human mesenchymal stem cells at single cell resolution reveals therapeutically targetable senescent cell sub-populations

Cellular senescence is characterised by a state of permanent cell cycle arrest. It is accompanied by often variable release of the so-called senescence-associated secretory phenotype (SASP) factors, and occurs in response to a variety of triggers such as persistent DNA damage, telomere dysfunction, or oncogene activation. While cellular senescence is a recognised driver of organismal ageing, the extent of heterogeneity within and between different senescent cell populations remains largely unclear. Elucidating the drivers and extent of variability in cellular senescence states is important for discovering novel targeted seno-therapeutics and for overcoming cell expansion constraints in the cell therapy industry. Here we combine cell biological and single cell RNA-sequencing approaches to investigate heterogeneity of replicative senescence in human ESC-derived mesenchymal stem cells (esMSCs) as MSCs are the cell type of choice for the majority of current stem cell therapies and senescence of MSC is a recognized driver of organismal ageing. Our data identify three senescent subpopulations in the senescing esMSC population that differ in SASP, oncogene expression, and escape from senescence. Uncovering and defining this heterogeneity of senescence states in cultured human esMSCs allowed us to identify potential drug targets that may delay the emergence of senescent MSCs in vitro and perhaps in vivo in the future.

bioinformatics↗

bioRxiv2020

Self-Organizing 3D Human Choroid Plexus-Ventricle-Cortical Organoids

Both the choroid plexus (CP) and the cortex are derived from the rostral neural tube during early embryonic development. In addition to producing CSF, the CP secretes essential factors that orchestrate cortical development and later neurogenesis. Previous brain modeling efforts with human pluripotent stem cells (hPSCs) generated either cortical or CP tissues in 3D culture. Here, we used hPSC-derived neuroectodermal cells, the building blocks of the anterior body, to simultaneously generate CP that forms ventricles and cortical cells in organoids (CVCOs), which can be maintained as 3D organoid cultures. Large scale culture revealed reproducibility of the protocol independent of cell lines, clones or batches. CVCOs contain mature and functional CP that projects multiple cilia into the ventricle-like fluid filled cysts and is in direct contact with appropriately patterned cortical cells. CVCOs thus recapitulate key features of developing forebrain structures observed in in vivo and constitute a useful for dissecting the role of CP in human forebrain development in health and disease.

neuroscience↗

bioRxiv2020

Conserved and divergent features of DNA methylation in embryonic stem cell-derived neurons

DNA methylation functions in genome regulation and is implicated in neuronal maturation. Early post-natal accumulation of atypical non-CG methylation (mCH) occurs in neurons of mice and humans, but its precise function remains unknown. Here we investigate mCH deposition in neurons derived from mouse ES-cells in vitro and in cultured primary mouse neurons. We find that both acquire comparable levels of mCH over a similar period as in vivo. In vitro mCH deposition occurs concurrently with a transient increase in Dnmt3a expression, is preceded by expression of the post-mitotic neuronal marker Rbfox3 (NeuN) and is enriched at the nuclear lamina. Despite these similarities, whole genome bisulfite sequencing reveals that mCH patterning in mESC-derived neurons partially differs from in vivo. mESC-derived neurons therefore represent a valuable model system for analyzing the mechanisms and functional consequences of correct and aberrantly deposited CG and non-CG methylation in neuronal maturation.

genomics↗

bioRxiv2019

Use of human iPSCs and kidney organoids to develop a cysteamine/mTOR inhibition combination therapy to treat cystinosis

Cystinosis is a lysosomal storage disease caused by mutations in CTNS, encoding a cystine transporter, and in its severest form is characterized by cystine accumulation, renal proximal tubule dysfunction and kidney failure. Cystinosis is treated with the cystine-depleting drug cysteamine, however this only slows progression of the disease and there is an urgent need for better treatments. Here, we have generated and characterized the first human induced pluripotent stem cell (iPSC) and kidney organoid models of cystinosis. These models exhibit elevated cystine and cysteine levels, enlarged lysosomes and a block in basal autophagy flux. Cysteamine treatment ameliorates this phenotype except for the basal autophagy flux defect. We found that treatment with Everolimus, an inhibitor of the mTOR pathway, reduces the number of large lysosomes and activates autophagy but does not rescue the cystine/cysteine loading defect. However, dual treatment of cystinotic iPSCs or kidney organoids with cysteamine and Everolimus corrects all of the observed phenotypes indicating that a combination therapy has therapeutic potential to improve the treatment of cystinosis.

pathology↗

bioRxiv2018

Platypus Induced Pluripotent Stem Cells: the Unique Pluripotency Signature of a Monotreme

Summary StatementThe generation and transcriptome analysis of the first induced pluripotent stem cells from the platypus reveals SOX2 has been a key driver of the expanded pluripotency regulatory network in placental mammals.\n\nABSTRACTThe mechanisms by which pluripotency has evolved remain unclear. To gain insight into the evolution of mammalian pluripotency we have generated induced pluripotent stem cells (piPSCs) from the platypus. Deep sequencing of the piPSC transcriptome revealed that piPSCs robustly express the core eutherian pluripotency factors OCT4, SOX2 and NANOG. Given the more extensive role of SOX3 over SOX2 in avian pluripotency, our data indicate that between 315 million years and 166 million years ago primitive mammals replaced the role of SOX3 in the vertebrate pluripotency network with SOX2. DAX1/NR0B1 is not expressed in piPSCs and an analysis of the platypus DAX1 promoter revealed the absence of a proximal SOX2-binding DNA motif known to be critical for DAX1 expression in eutherian pluripotent stem cells, suggesting that the acquisition of SOX2 responsiveness by DAX1 has facilitated its recruitment into the pluripotency network of eutherians. We further show that the expression ratio of X chromosomes to autosomes (X1-5 X1-5:AA) is approximately equal to 1 indicating that there is no upregulation of X-linked genes and that there is no preference for silencing of maternal or paternal alleles (ie imprinting).

cell biology↗

bioRxiv2017

A simple bioreactor-based method to generate kidney organoids from pluripotent stem cells

Kidney organoids generated from human pluripotent stem cells have the potential to revolutionize how kidney development and injury are studied. Current protocols are technically complex and suffer from poor reproducibility and high reagent costs restricting scalability. To overcome these issues, we have established a simple, inexpensive and robust method to grow kidney organoids in bulk from human induced pluripotent stem cells. Our organoids develop tubular structures by day (d) 8 and show optimal tissue morphology at d14. A comparison with fetal human kidney suggests that d14 organoid renal structures most closely resemble capillary loop stage nephrons. We show that deletion of HNF1B, a transcription factor linked to congenital kidney defects, interferes with tubulogenesis, validating our experimental system for studying renal developmental biology. Taken together, our protocol provides a fast, efficient and cost-effective method for generating large quantities of human fetal kidney tissue, enabling the study of normal and aberrant human renal development.

developmental biology↗