Biology subjects

Lelos, M.

Publications and source records attributed to Lelos, M..

3 recordsLinked to original sources

bioRxiv2024

Exploring the Epigenetic Influences on the Differentiation Capacity of Human iPSCs Derived from the Lateral Ganglionic Eminence: Implications for Striatal Medium Spiny Neuron Differentiation and Authenticity

Human pluripotent stem cells (hPSCs) are increasingly used to model human disease and as donor cells for regenerative medicine. However, the fidelity of hPSC-derived cell types remains a major concern, particularly when these cells are intended to replicate complex or region-specific subtypes, such as those required to explore and treat neurological diseases. Medium spiny neurons (MSNs), the principal projection neurons of the striatum, are one such target cell type relevant to disorders such as Huntingtons disease. While protocols for generating hPSC-derived MSNs (hPSC-MSNs) exist, the extent to which these cells faithfully recapitulate their genuine counterparts is unclear. Here, we generated isogenic human induced pluripotent stem cells (hiPSCs) from striatal (LGE) and non-neural (fibroblast) fetal tissues, and differentiated them into MSN-like cells alongside a naive human embryonic stem cell (hESC) line. Using DNA methylation profiling and single-cell RNA sequencing, we systematically compared the epigenetic and transcriptional features of these hPSC-MSNs to authentic fetal MSNs. Our findings reveal persistent epigenetic signatures inherited from the tissue of origin, which influence differentiation outcomes. While LGE-derived hiPSCs retained elements of a striatal-biased methylome and yielded MSN-like cells with enhanced similarity to authentic MSNs, all hPSC-MSNs remained epigenetically and transcriptionally distinct from genuine MSNs and we identified clusters of hPSC-derived cells with aberrant or incomplete phenotypes. These results demonstrate that even isogenic hiPSC lines exhibit variable differentiation potential due to residual epigenetic memory and protocol compatibility. We highlight the need for refined protocols and rigorous benchmarking of hPSC-derived models, particularly for regionally specified neuronal subtypes. Our study underscores the complex relationship between epigenetic status, cell lineage, protocol adaptation, and differentiation outcome. Paper SummaryHuman pluripotent stem cells (hPSCs) are widely used to study otherwise inaccessible human cell phenotypes. However, ensuring the molecular authenticity of hPSC-derived cell types remains critical, as differences between hPSC-derived cells and their native counterparts may impact the validity of these models. Here, medium spiny neurons (MSNs; relevant for studying basal ganglia function and disorders such as Huntingtons disease), serve as a valuable prototype for evaluating the fidelity of hPSC-derived cell types. This study generated human induced pluripotent stem cells (hiPSCs) from developing fetal striatal tissues and fibroblasts, differentiating them into MSN-like cells alongside a human embryonic stem cell (hESC) line. Using single-cell RNA sequencing and DNA methylation analysis, we compared these hPSC-derived MSNs to authentic fetal MSNs. Our findings reveal a significant epigenetic gap between hPSC-derived and authentic MSNs, suggesting that hPSC-MSNs do not acquire a complete and normal striatal epigenome. Additionally, while genetic expression of hPSC-MSNs was striatal-like, it was not equivalent, indicating abberant cells and a failure to reproduce an authentic phoenotype. This study provides insights into the challenges of achieving molecular authenticity in hPSC-derived cells and underscores the need for rigorous evaluation to enhance their utility in research and medicine.

cell biology↗

bioRxiv2024

Cas9 nickase-mediated contraction of CAG/CTG repeatsat multiple disease loci

Expanded CAG/CTG repeats cause over 15 different diseases that all remain without a disease-modifying treatment. Because repeat length accounts for most of the variation in disease severity, contracting them presents an attractive therapeutic avenue. Here, we show that the CRISPR-Cas9 nickase targeted to CAG/CTG repeats leads to efficient contractions in Huntingtons disease patient-derived neurons and astrocytes, and in myotonic dystrophy type 1 patient-derived neurons. The approach is allele-selective and free of detectable off-target mutations. Striatal injection of the Cas9 nickase in a mouse model for Huntingtons disease using adeno-associated viral vectors led to contractions in over half the infected cells. Upon injection, we observed a reduction in the number of inclusion bodies, improved transcriptome, and ameliorated locomotion. The effects were greater than expected from the contractions induced and suggest that non-cell autonomous mechanisms may be involved. Our results provide the proof-of-concept that correction of CAG/CTG repeats can improve Huntingtons disease phenotypes in vivo. One sentence summaryThe Cas9 nickase contracts CAG/CTG repeats at multiple disease loci in patient-derived cells and improves molecular and behavioral phenotypes in HD.

neuroscience↗

bioRxiv2023

Differences in white matter detected by ex vivo 9.4T MRI are associated with axonal changes in the R6/1 model of Huntington's Disease

White matter (WM) volume loss has been reported in people with Huntingtons disease (HD), but the cellular basis of this deficit remains to be elucidated. To address this, we assessed ex vivo WM microstructure in the transgenic R6/1 mouse model of HD with magnetic resonance imaging (MRI) and studied the neurobiological basis of the MRI brain signals with histological and electron microscopy analyses in a separate cohort of age- and sex-matched mice. Differences in the macromolecular proton fraction (MPF) from quantitative magnetization transfer (qMT) as a proxy myelin measure, and the intra-axonal signal fraction (FR) from the composite hindered and restricted model of diffusion (CHARMED) as a proxy marker of axon density, were assessed alongside diffusion tensor imaging (DTI) parameters. A tractometry approach was employed to inspect region-specific differences across the corpus callosum (CC). Furthermore, voxel-based morphometry (VBM) and tract-based spatial statistics (TBSS) were used to explore brain-wise WM macro- and microstructure abnormalities. To gain insight into disease-associated impairments in attentional and visuospatial processing, a third cohort of age-matched mice was assessed with the 5-choice serial reaction time task (5-CSRTT). We report cognitive impairments in R6/1 mice and, by evaluating MRI and light and electron microscopy results, we show that this HD mouse model presents disruptions in axonal morphology (i.e. less complex, thinner axons) and organization (i.e. more densely packed axons). Furthermore, we show that, at least early in disease progression, R6/1 mice present a reduction in the expression or content of myelin-associated proteins without significant alterations in the structure of myelin sheaths. Finally, our findings indicate that neuroinflammation-driven glial and axonal swelling might also affect this mouse model early in disease progression. Crucially, we demonstrate the potential of FR, an in vivo estimate of axon density, as a novel MRI biomarker of HD-associated changes in WM microstructure.

neuroscience↗