Biology subjects

Dolt, K. S.

Publications and source records attributed to Dolt, K. S..

4 recordsLinked to original sources

bioRxiv2025

Genotype-phenotype correlations in Wilms tumor initiation.

Wilms tumor, the most common pediatric kidney cancer, arises from abnormal embryonic kidney development. Therapy resistance and tumor recurrence remain major challenges, likely driven by the presence of Cancer Stem Cells (CSCs). Here, we elucidate the earliest pathogenic events in genetically engineered mouse models exhibiting loss of Wt1 or LIN28B overexpression, two Wilms tumor driver genes. Loss of Wt1 leads to a disturbance of lineage identity of the mutant cells, whereas LIN28B leads to a disturbed transition between uninduced and induced NPC (nephron progenitor cell) state. In both cases the appearance of cells positive for all four Wilms tumor cancer stem cell markers is the result of the tumor initiating mutation. The existence of genotype-phenotype correlations in primary developmental phenotypes and cancer stem cell expression patterns has important implications for therapeutic opportunities and requirements.

developmental biology↗

bioRxiv2023

G51D mutation of the endogenous rat Snca gene disrupts synaptic localisation of α-synuclein priming for Lewy-like pathology

Point mutations in the SNCA gene, encoding -synuclein (Syn), are a known cause of familial Parkinsons disease. The G51D mutation causes early onset neurodegeneration with complex pathology. We used CRISPR/Cas9 in rats to introduce the G51D mutation into the endogenous Snca gene. Co-localisation immunostaining studies with synaptic proteins showed that SynG51D protein is no longer efficiently localised to synapses. Furthermore, biochemical isolation of synaptosomes from rat cortex demonstrated a significant depletion of Syn in SncaG51D/+ and SncaG51D/G51D rats. Unbiased proteomic investigation of the cortex identified significant synaptic dysregulation in SncaG51D/G51D animals. Finally, we compared the propensity for Lewy-like pathology of Snca+/+ and SncaG51D/G51D rats by stereotaxically delivering Syn pre-formed fibrils (PFFs) into the pre-frontal cortex. At an early time-point, 6 weeks post-injection, we observed discrete Lewy-like structures positive for phosphoserine-129-Syn (pS129-Syn) only in SncaG51D/G51D brains. At 26 weeks post-injection of PFFs SncaG51D/G51D brains exhibited intense, discrete pS129-Syn-positive structures, while Snca+/+ brains exhibited diffuse pS129-Syn immunostaining. Quantification of discrete pS129-Syn-positive structures revealed the striatum of SncaG51D/G51D rats had significantly more Lewy-like pathology than Snca+/+ rats. In summary, this novel SncaG51D rat model exhibits molecular characteristics of early synaptic dysfunction and is primed for Syn pathology.

neuroscience↗

bioRxiv2021

Midbrain organoids with an SNCA gene triplication model key features of synucleinopathy

SNCA, the first gene associated with Parkinsons disease, encodes the -synuclein (-syn) protein, the predominant component within pathological inclusions termed Lewy bodies (LBs). The presence of LBs is one of the classical hallmarks found in the brain of patients with Parkinsons disease, and LBs have also been observed in patients with other synucleinopathies. However, the study of -syn pathology in cells has relied largely on two-dimensional culture models, which typically lack the cellular diversity and complex spatial environment found in the brain. Here, to address this gap, we use 3D midbrain organoids (hMOs), differentiated from human induced pluripotent stem cells derived from patients carrying a triplication of the SNCA gene and from CRISPR/Cas9 corrected isogenic control iPSCs. These hMOs recapitulate key features of -syn pathology observed in the brains of patients with synucleinopathies. In particular, we find that SNCA triplication hMOs express elevated levels of -syn and exhibit an age-dependent increase in -syn aggregation, manifested by the presence of both oligomeric and phosphorylated forms of -syn. These phosphorylated -syn aggregates were found in both neurons and glial cells and their time-dependent accumulation correlated with a selective reduction in dopaminergic neuron numbers. Thus, hMOs from patients carrying SNCA gene multiplication can reliably model key pathological features of Parkinsons disease and provide a powerful system to study the pathogenesis of synucleinopathies.

neuroscience↗

bioRxiv2020

Cryopreservation of midbrain dopaminergic neural cells differentiated from human embryonic stem cells

Recent advancements in protocols to differentiate human pluripotent stem cells into midbrain dopaminergic (mDA) neurons has improved the ability to model Parkinsons disease (PD) in a dish, and has provided a scalable source of donor cells for emerging PD cell replacement therapy (CRT). However, to facilitate reproducibility, collaboration, and clinical trials it would be highly beneficial to cryopreserve committed mDA neural precursors cells in a ready-to-use format. In terms of cell manufacturing for PD CRT trials, a cryopreserved transplantation-ready mDA cell product would provide a critical opportunity for quality control, efficacy testing, and safety assessments. To address this challenge, we have compared six (6) different clinical-grade cryopreservation media and different freezing conditions for mDA neural precursor cells differentiated from two human embryonic stem cell (ESC) lines, MasterShef7 and RC17. Significant differences in cell viability were observed at 24h post-thawing, but no differences were observed immediately upon thawing. This highlights the need to check cell viability over the first 24h after thawing, and that viability of freshly thawed cells is insufficient to gauge the success of a cryopreservation protocol. Considerable apoptosis occurs in the first 24h post-thawing, and significant differences between cryopreservation procedures were only revealed during this time period. The presence of ROCK inhibitors improved cell viability at 24h for all conditions tested. A faster cooling rate of 1-2{degrees}C/min was significantly better than 0.5{degrees}C/min for all conditions tested, while rapid thawing at 37{degrees}C was not always superior to slow thawing at 4{degrees}C. Indeed, the optimal cryopreservation and thawing conditions in this study, as determined by 24h post-thaw viability, were cells frozen in PSC Cryopreservation medium at a cooling rate of 1{degrees}C/min and slow thawing at 4{degrees}C. These conditions permitted recovery of 60%-70% live cells at 24h with respect to the starting number of cryopreserved cells. Importantly, cryopreservation of mDA neural precursor cells did not alter their potential to resume differentiation into mDA neurons. HighlightsO_LIFirst systematic comparison of multiple clinical-grade cryopreservation media for human ESC-derived mDA neural precursor cells C_LIO_LIDifferences in cell viability were observed at 24h after thawing, but not immediately upon thawing C_LIO_LICooling rates of 1{degrees}C/min or 2{degrees}C/min were significantly better than 0.5{degrees}C/min for all cryopreservation conditions tested C_LIO_LIA slow thawing condition at 4{degrees}C was significantly better than quick thawing at 37{degrees}C for cells frozen in PSC Cryopreservation medium C_LIO_LICryopreservation of mDA cells does not significantly alter their potential to differentiate into mDA neurons C_LI

cell biology↗