Biology subjects

Tiklova, K.

Publications and source records attributed to Tiklova, K..

6 recordsLinked to original sources

bioRxiv2026

Efficient murine cardiac phenotyping by combining synchrotron-based phase-contrast micro-CT, histology, immunofluorescence and spatial transcriptomics

1.Congenital heart disease is commonly studied using genetically modified mouse models, but characterization of complex three-dimensional (3D) cardiac anomalies remains technically challenging. Histology of fixed paraffin-embedded samples, typically used for structural and molecular analysis, is limited to two dimensions (2D). Synchrotron radiation-based phase-contrast micro-computed tomography (SRPC-{micro}CT) enables rapid, high-resolution, 3D imaging but had yet to be fully integrated with molecular tissue analysis. Importantly, SRPC-{micro}CT is nondestructive and compatible with paraffin-embedded tissue, potentially allowing integration with downstream molecular analyses such as multiplexed spatial transcriptomic profiling on tissue sections. Here, we present a pipeline combining SRPC-{micro}CT with complementary molecular approaches for rapid and accurate phenotyping of mouse hearts and for investigating disease mechanisms. We demonstrate the successful integration of high-resolution 3D imaging with spatial transcriptomics, fluorescent stainings, and histochemistry. Notably, all 2D modalities were applied sequentially to a single tissue section and registered within the 3D volume. This multimodal framework provides a powerful approach for linking structural and molecular information that is broadly applicable across biomedical research.

developmental biology↗

bioRxiv2024

Sox9 and Nfi transcription factors regulate the timing of neurogenesis and ependymal maturation in dopamine progenitors

Correct timing of neurogenesis is critical for both generating the correct number and subtypes of glia and neurons in the embryo, as well as preventing tumours and the depletion of stem cell pools in the adults. Here we analyse how the midbrain dopamine neuron (mDA) progenitors transition into cell cycle arrest (G0) and begin to mature into ependymal cells. The comparison of mDA progenitors from different embryonic stages revealed the upregulation of Nfi and Sox9 transcription factors during development. Their conditional inactivation in the early embryonic midbrain leads to delayed G0 entry and ependymal maturation, reduced gliogenesis, and increased generation of neurons, including mDA neurons. In contrast, their inactivation in late embryogenesis does not result in mitotic re-entry, suggesting that these factors are necessary for the G0 induction, but not for its maintenance. Our characterisation of mDA-progenitor-derived adult ependymal cells by single-cell RNA sequencing and histology show that they both retain several progenitor features but also secrete neuropeptides and contact neighbouring cells and blood vessels, indicating that these cells may form a novel part of the circumventricular organ system.

developmental biology↗

bioRxiv2023

Parkin is not required to sustain OXPHOS function in adult mammalian tissues

Loss-of-function variants in the PRKN gene encoding the ubiquitin E3 ligase PARKIN cause autosomal recessive early-onset Parkinsons disease (PD). Extensive in vitro and in vivo studies have reported that PARKIN is involved in multiple pathways of mitochondrial quality control, including mitochondrial degradation and biogenesis. However, these findings are surrounded by substantial controversy due to conflicting experimental data. In addition, the existing PARKIN-deficient mouse models have failed to faithfully recapitulate PD phenotypes. Therefore, we have investigated the mitochondrial role of PARKIN during ageing and in response to stress by employing a series of conditional Parkin knockout mice. We report that PARKIN loss does not affect oxidative phosphorylation (OXPHOS) capacity and mitochondrial DNA (mtDNA) levels in the brain, heart, and skeletal muscle of aged mice. We also demonstrate that PARKIN deficiency does not exacerbate the brain defects and the pro-inflammatory phenotype observed in mice carrying high levels of mtDNA mutations. To rule out compensatory mechanisms activated during embryonic development of Parkin-deficient mice, we generated a mouse model where loss of PARKIN was induced in adult dopaminergic (DA) neurons. Surprisingly, also these mice did not show motor impairment or neurodegeneration, and no major transcriptional changes were found in isolated midbrain DA neurons. Finally, we report a patient with compound heterozygous PRKN pathogenic variants that lacks PARKIN and has developed PD. The PARKIN deficiency did not impair OXPHOS activities or induce mitochondrial pathology in skeletal muscle from the patient. Altogether, our results argue that PARKIN is dispensable for OXPHOS function in adult mammalian tissues.

cell biology↗

bioRxiv2023

Single cell-resolution in situ sequencing elucidates spatial dynamics of multiple sclerosis lesion and disease evolution

Multiple sclerosis (MS) is a neurological disease characterised by multifocal lesions and smouldering pathology. While single-cell analyses have provided insights into neuropathology, cellular processes underlying MS remain poorly understood. We modelled the cellular dynamics of MS by examining temporal and regional rates of disease progression in the experimental autoimmune encephalomyelitis (EAE) mouse model. By performing single-cell spatial expression profiling using In situ sequencing, we annotated disease neighbourhoods during lesion evolution and found centrifugal propagation of active lesions. We demonstrated that disease-associated (DA) glia are dynamic and induced independently of lesions, preceding their formation. Single-cell spatial analysis of human archival MS spinal cord confirmed differential distribution of DA-glia, enabled deconvolution of active and inactive lesions into sub-compartments, and identification of new lesion areas. By establishing a spatial resource of mouse and human MS neuropathology at a single-cell resolution, our study unveils the intricate cellular dynamics underlying MS disease evolution.

cell biology↗

bioRxiv2023

Optimizing Xenium In Situ data utility by quality assessment and best practice analysis workflows

The Xenium In Situ platform is a new spatial transcriptomics product commercialized by 10X Genomics capable of mapping hundreds of transcripts in situ at a subcellular resolution. Given the multitude of commercially available spatial transcriptomics technologies, recommendations in choice of platform and analysis guidelines are increasingly important. Herein, we explore eight preview Xenium datasets of the mouse brain and two of human breast cancer by comparing scalability, resolution, data quality, capacities and limitations with eight other spatially resolved transcriptomics technologies. In addition, we benchmarked the performance of multiple open source computational tools when applied to Xenium datasets in tasks including cell segmentation, segmentation-free analysis, selection of spatially variable genes and domain identification, among others. This study serves as the first independent analysis of the performance of Xenium, and provides best-practices and recommendations for analysis of such datasets.

bioinformatics↗

bioRxiv2019

Single Cell Gene Expression Analysis Reveals Human Stem Cell-Derived Graft Composition in a Cell Therapy Model of Parkinson’s Disease

Since the pioneering studies using fetal cell transplants in Parkinsons disease (PD), brain repair by cell replacement has remained a long-standing and realistic goal for the treatment of neurodegenerative disorders including PD. Authentic and functional midbrain dopamine (DA) neurons can now be generated from human pluripotent stem cells (hPSCs) via a floor plate intermediate1,2, and these cell preparations are both safe and functional when transplanted to animal models of PD3. However, although resulting grafts from fetal brain tissue and hPSCs contain large numbers of desired DA neurons, these therapeutic cells are a minor component of the grafts. Moreover, the cellular composition of the graft has remained difficult to assess due to limitations in histological methods that rely on pre-conceived notions concerning cell types. Here, we used single cell RNA sequencing (scRNA-seq) combined with comprehensive histological analyses to characterize intracerebral grafts from ventral midbrain (VM)-patterned human embryonic stem cells (hESCs) and VM fetal tissue after long-term survival and functional maturation in a pre-clinical rat model of PD. The analyses revealed that while both cell preparations gave rise to neurons and astrocytes, oligodendrocytes were only detected in grafts of fetal tissue. On the other hand, a cell type closely resembling a class of newly identified perivascular-like cells was identified as a unique component of hESC-derived grafts. The presence of these cells was confirmed in transplants from three different hESC lines, as well as from iPSCs. Thus, these experiments have addressed one of the major outstanding questions in the field of cell replacement in neurological disease by revealing graft composition and differences between hESC- and fetal cell-derived grafts, which can have important implications for clinical trials.

neuroscience↗