Biology subjects

Stachelscheid, H.

Publications and source records attributed to Stachelscheid, H..

10 recordsLinked to original sources

bioRxiv2026

A human iPS cell line for ready-to-use human iAstrocytes that support human neurons

Human iPSC-derived neuronal networks are increasingly being employed in basic and applied research to enhance translation. Astrocytes are essential for neuronal network function, but are often not included, or replaced with mouse astrocytes, which compromises translation. Current protocols produce hiPSC-derived astrocytes by stepwise differentiation using small molecules and cytokines, or by forward programming by inducing transcription factors introduced by lentiviral transduction. Here we created a stable, inducible hiPSC line capable of producing iAstrocytes by introducing the transcription factors NFIB and SOX9 into the AAVS1 locus of the BIHi005-A hiPSC line. iAstrocytes induced from this line upregulated astrocytic genes over four weeks in culture, expressed GFAP and S100B and exhibited spontaneous calcium waves and responses to ATP and CPA. In co-cultures, iAstrocytes supported the growth and function of mature iNeuron networks. Pre- and post-synaptic markers and synchronous neuronal activity measured by high-density multi-electrode array recordings and neuronal calcium imaging, appeared by four weeks. The use of iAstrocytes will help to standardize the use of human astrocytes to support human neural networks and enhance translation.

neuroscience↗

bioRxiv2025

StemCNV-check: a pipeline for human pluripotent stem cell (hPSC) genomic integrity control using SNP array data and copy number variant scoring

Human pluripotent stem cells (hPSCs) and other continuously cultured cell lines are prone to acquiring mutations and genomic aberrations over time, even when derived from well-characterized cell banks. To ensure experimental reproducibility and maintain cell line integrity, routine monitoring for genomic abnormalities is essential. Single nucleotide polymorphism (SNP) arrays represent a cost-effective and widely accessible method for detecting copy number variations (CNVs) with genome-wide resolution, making them particularly suitable for quality control (QC) in cell culture systems. Despite the established utility of SNP arrays for CNV detection, there remains a lack of comprehensive, user-friendly software solutions that support end-to-end analysis tailored to hPSC line quality assessment. Existing tools are either limited to discrete analysis steps requiring specialized bioinformatics expertise or are proprietary solutions that do not adequately address the specific needs of cell line monitoring. To bridge this gap, we developed an accessible and integrated analysis pipeline for SNP array-based QC of hPSC lines. The pipeline facilitates all stages of analysis--from raw data processing to the generation of interpretable reports--and includes specialized features such as sample-to-reference comparison, a CNV scoring system according to CNV biological impact, single nucleotide variation (SNV) evaluation and identity verification via SNP genotyping profiles, all tailored to hPSC. We benchmarked the pipeline against established methodologies and implemented strategies to enhance CNV detection reliability through expert-guided improvement process.

cell biology↗

bioRxiv2025

An autologous human iPSC-derived 3D organoid infection model for preclinical testing of antiviral T cells

Immunocompromised patients, such as those undergoing hematopoietic stem cell or solid organ transplantation, are highly susceptible to viral complications. Given the limitations and side effects of available antiviral therapies, adoptive transfer of antiviral T cells offers a promising alternative by restoring immune defense. However, existing models for evaluating antiviral T cell therapies lack physiological relevance, limiting accurate predictions of efficacy and safety. There is a critical need for in vitro human infection platforms that support personalized assessment of therapeutic responses. To address this, we developed antiviral T cell products (TCPs) targeting Influenza A virus (IAV)-infected cells, alongside an autologous human induced pluripotent stem cell (iPSC)-derived 3D lung organoid infection platform. This model recapitulates key immunological responses and is compatible with a new 3D high-throughput, high-content imaging pipeline. Our study provides the first proof-of-concept for assessing T cell-mediated cytotoxicity in a 3D in vitro lung infection model, advancing personalized antiviral immunotherapy development.

immunology↗

bioRxiv2025

SteMClass: A Novel DNA Methylation-Based Classifier for iPSC In Vitro Differentiation States.

Human induced pluripotent stem cells (iPSCs) hold great promise for regenerative medicine, disease modelling, and drug discovery, but most downstream applications require differentiation into specialised cell types not covered by current quality control assays. Here, we present "SteMClass", a proof-of-concept DNA methylation-based classifier that standardises iPSC differentiation state identification across protocols with one test. We curated a reference cohort of 15 iPSC lines differentiated into seven distinct states (n = 97), performed array-based DNA methylation profiling, and trained a random forest model to classify the eight distinct differentiation states. In nested cross-validation, SteMClass achieved a Brier score of 0.018, and on an independent cohort (n = 58) attained 96.5% accuracy (Cohens K = 0.959) with a 3% rejection rate. Applied to external data (n = 249), SteMClass achieved 85.1% overall accuracy (Cohens K = 0.687) with a 12.9% rejection rate. Among classified samples (n = 217), accuracy was 97.7% (Cohens K = 0.93). SteMClass is compatible with all Illumina methylation array versions, and accessible via an interactive web interface that supports classification and exploration of DNA methylation profiles. By providing a harmonised, single-assay framework for iPSC-derived differentiation state characterisation, SteMClass improves reproducibility and comparability across studies, paving the way for robust quality control standards and accelerating clinical translation.

cell biology↗

bioRxiv2025

DTX4 regulates neural progenitor transitions during cortical development

Neural progenitors drive cellular diversification in the neocortex. Prolongation of their proliferative capacity and increased diversity underpins the evolutionary expansion and morphological complexity of the human neocortex. Here, we investigate the mechanisms that regulate maintenance of the highly proliferative early neural progenitor subtypes and transition to subsequent progenitors of limited proliferative capacity during human and murine neocortical development. We identify DTX4, a Deltex family member, as an evolutionarily conserved molecular determinant of neural progenitor identity in the mammalian neocortex. DTX4 sustains the identity of radial glia, a highly proliferative progenitor subtype. Loss of DTX4, on the other hand, is a prerequisite for the generation of intermediate progenitors which possess low proliferative capacity. Perturbing DTX4 expression in human cerebral organoids and in the murine neocortex, alters progenitor composition, thereby inducing changes in neuronal diversity and cortical morphology. Mechanistically, we reveal that DTX4 controls progenitor identity by regulating the length of the cell cycle. Our findings underscore the critical role of cell cycle dynamics and of DTX4 in determining progenitor identity and thereby defining neuronal outcome during mammalian development. Highlights[bullet] The Deltex family member, DTX4, is expressed in human and murine neuroepithelia and radial glial progenitors but is absent in intermediate progenitor cells. [bullet]DTX4 maintains radial glia identity and prevents generation of intermediate progenitors during murine and human development. [bullet]Disruption of DTX4 expression compromises neuronal composition and neocortical architecture [bullet]DTX4 regulates progenitor identity by regulating cell cycle length

neuroscience↗

bioRxiv2024

Dissecting compounded hepatocyte injury in a model of MASLD progression from human induced pluripotent stem cells

Drug discovery for multifactorial diseases like metabolic dysfunction-associated steatotic liver disease (MASLD) remains challenging due to inadequate models and untargeted drug screenings. We combined stem-cell-based modeling with computational drug predictions identifying flavin pathways as therapeutic targets in MASLD. For disease stage-specific discovery, we established a MASLD testing model, compounding metabolic triggers to intensify mitochondrial crisis. In vitro injuries included adipo- and myokines, immune cell co-culture, and genomic risk factors. Benchmarking experiments revealed similarities with advanced human MASLD. To query therapeutic compounds, protein-protein-interaction networks, weighted gene co-expression, and knowledge graph-based analyses independently predicted flavin adenine dinucleotide (FAD) as an anti-MASLD factor. Dysregulated flavoproteomes in vitro and in vivo-in pediatric and adult MASLD patients- supported our flavin network-focused strategy. We established therapeutic FAD concentrations to mitigate metabolic injury and fibro-inflammation in human multicellular liver organoids and other assays. We enhanced therapeutic FAD effects through genetic mitochondrial biogenic augmentation and identified orally available flavo-active compounds--including Aspirin--restoring mitochondrial respiration. Our study demonstrates how integrating stem cell-derived disease modeling with computed drug predictions can expedite therapeutic discovery.

cell biology↗

bioRxiv2023

Spatiotemporal expression of thyroid hormone transporter MCT8 and THRA mRNA in human cerebral organoids recapitulating first trimester cortex development

Thyroid hormones (TH) play critical roles during nervous system development and patients carrying coding variants of MCT8 (Monocarboxylate transporter 8) or THRA (Thyroid hormone receptor alpha) present a spectrum of neurological phenotypes resulting from perturbed local TH action during early brain development. Recently, human cerebral organoids (hCOs) emerged as powerful three-dimensional in vitro tools for disease modelling recapitulating key aspects of early human cerebral cortex development. To begin exploring prospects of this model for thyroid research, we performed a detailed characterization of the spatiotemporal expression of the TH transporter MCT8 and TH receptor THRA in developing hCOs. Immunostaining of hCOs showed MCT8 membrane expression in neuronal progenitor cell types including early neuroepithelial cells, SOX2+/Nestin+ radial glia cells (RGCs), TBR2+ intermediate progenitors and HOPX+ outer RGCs. In addition, we detected robust MCT8 protein expression in CTIP2+ deep layer neurons and at later developmental stages in SATB2+ upper layer neurons. Spatiotemporal SLC16A2 mRNA expression, detected by fluorescent in situ hybridization (FISH), was highly concordant with MCT8 protein expression across cortical cell layers. FISH detected THRA mRNA expression already in neuroepithelium before the onset of neurogenesis and THRA expression was maintained in RGCs of the ventricular zone. Increased THRA expression was later detected in the subventricular zone whereas highest THRA expression was observed in excitatory neurons in peripheral hCO regions. In combination with strong up-regulation of known T3 response genes following short-term T3 treatment of hCOs, these observations show that hCOs provide a promising and experimentally tractable model to probe local TH action during human cortical neurogenesis and eventually to model the consequences of impaired TH function for early cortex development.

neuroscience↗

bioRxiv2020

FRC-QE: A robust and comparable 3D microscopy image quality metric for cleared organoids

Three-dimensional stem-cell-derived organoids are a powerful tool for studying cellular processes in tissue-like structures, enabling in vitro experiments in an organ-specific context. While organoid research has been closely linked to advances in fluorescence microscopy, capturing cellular structures within their global context in an organoid often remains challenging due to the organoids dense structure and opacity. The development of optical clearing methods has provided a solution for fixed organoids but optimizing clearing protocols for a given sample type and staining can be challenging. Importantly, quantitative measures for assessing image quality throughout cleared fluorescent samples are missing. Here, we propose Fourier ring correlation quality estimation (FRC-QE) as a new metric for automated 3D image quality estimation in cleared organoids. We show that FRC-QE robustly captures differences in clearing efficiency within an organoid, across replicates and clearing protocols, as well as for different microscopy modalities. FRC-QE is open-source, written in ImgLib2 and provided as an easy-to-use and macro-scriptable plugin for the popular Fiji software. We therefore envision FRC-QE to fill the gap of providing a reliable quality metric for testing, optimizing and comparing optical clearing methods.

bioinformatics↗

bioRxiv2020

Self-assembly of progenitor cells under the aegis of platelet factors facilitates human skin organoid formation and vascularized wound healing

Stem/progenitor cells can self-organize into organoids modelling tissue function and regeneration. Here we demonstrate that human platelet-derived factors can orchestrate 3D self-assembly of clonally expanded adult skin fibroblasts, keratinocytes and endothelial progenitors forming skin organoids within three days. Organoids showed distinct signaling patterns in response to inflammatory stimuli that clearly differed from separated cell types. Human induced pluripotent stem cell (hiPSC)-derived skin cell progenitors also self-assembled into stratified human skin within two weeks, healing deep wounds of immune-deficient mice. Co-transplantation of endothelial progenitors significantly accelerated vascularization. Mechanistically, platelet-derived extracellular vesicles mediated the platelet-derived trophic effects. Long-term fitness of epidermal cells was accelerated further by keratinocyte growth factor mRNA transfection. No tumorigenesis was observed upon xenografting. This permits novel rapid 3D skin-related pharmaceutical testing opportunities and facilitates development of iPSC-based skin regeneration strategies.

cell biology↗

bioRxiv2020

Extra-hematopoietic immunomodulatory role of the SCID-susceptibility gene DOCK-2 identified by stepwise maturation of human iPSCs into clonogenic mesodermal stromal progenitors

Stromal cells contribute to organ integrity as fibroblasts and to vascular stability as pericytes, in addition to their enigmatic niche function in many tissues. Their inherent immunomodulatory capacity attracted particular attention, initiating numerous clinical trials, particularly testing trophic regeneration and immunomodulation. Key stromal immune functions are still enigmatic. Here we show that dedicator of cytokinesis (DOCK-2) previously described for causing immune cell dysfunction plays a role in extra-hematopoietic immunity by regulating stromal/fibroblast immunomodulatory function. We used three independent strategies including iPSC-derived mesodermal stromal cell (MSC) lineage maturation, severe combined immunodeficiency (SCID) patient-derived cells and CRISPR/Cas9 knockout to support our findings. Human induced pluripotent stem cells (iPSCs) were generated from healthy bone marrow and umbilical cord blood-derived fibroblasts by Sendai virus-mediated transient expression of Yamanaka factors. After mesoderm induction, stromal differentiation was induced by platelet-derived growth factors under animal serum-free conditions. Under feeder-free defined conditions, iPSCs differentiated into expandable and cryo-preservable CD73+/CD105+/Tra-1-81- early iPS-MSCs lacking immunosuppressive potential. Successive maturation was required for reaching the canonical MSC phenotype and immunomodulatory competence over time, while maintaining clonogenicity, comparable to parental MSCs. Sequential RNAseq revealed acquisition of a spectrum of immune-related genes significantly expressed in mature iPS-MSCs and resembling parental MSCs immune gene expression. The DOCK-2 gene attracted our attention because mutations can cause SCID. Interestingly, SCID patient-derived fibroblast lines harboring bi-allelic DOCK-2 mutations showed significantly reduced immunomodulatory capacity compared to non-mutated control fibroblasts. CRISPR/Cas9-mediated DOCK-2 knockout in healthy iPSCs resulted in iPS-MSCs that also displayed reduced immunomodulatory capacity, thus confirming a role of DOCK-2 in stromal immune function. At a mechanistic level, DOCK-2 deficiency resulted in disturbed subcellular localization of CDC42. This provides first evidence for an extra-hematopoietic immunomodulatory role of DOCK-2 in stromal cells, previously considered restricted to hampering immune cell migration/function resulting in SCID. We may speculate that some of the signs and symptoms of persisting immune disease after successful hematopoietic stem cell transplantation in SCID patients could at least in part be due to mutations, like DOCK-2-/-, permissive outside the hematopoietic immune system, as evidenced also by the increased virus infection susceptibility of DOCK-2 deficient fibroblasts.

immunology↗