This study presents a comprehensive genetic characterization of the H9c2 cell line, a widely used model for cardiac myoblast research. We established a short tandem repeat (STR) profile for H9c2 that is useful to confirm the identity and stability of the cell line. Additionally, we prepared H9c2 metaphase chromosomes and performed karyotyping and molecular cytogenetics to further investigate chromosomal characteristics. The genetic analysis showed that H9c2 cells exhibit chromosomal instability, which may impact experimental reproducibility and data interpretation. Next-generation sequencing (NGS) was performed to analyze the transcriptome, revealing gene expression patterns relevant to cardiac biology. Western blot analysis further validated the expression levels of selected cardiac genes identified through NGS. Additionally, Phalloidin staining was used to visualize cytoskeletal organization, highlighting the morphological features of these cardiac myoblasts. Our findings collectively support that H9c2 cells are a reliable model for studying cardiac myoblast biology, despite some genetic alterations identified resembling sarcoma cells. The list of genes identified through NGS analysis, coupled with our comprehensive genetic analysis, will serve as a valuable resource for future studies utilizing this cell line in cardiovascular medicine.

The 25 kDa glycoprotein lipocalin-2 (LCN2) is widely expressed and has diverse functions, ranging from physiological to pathophysiological processes. In the liver, LCN2 is primarily associated with inflammatory processes and is considered a potential biomarker in metabolic disorders. However, a significant challenge is the absence of a suitable human in vitro model for studying LCN2 and its associated signaling pathways. Therefore, we have successfully generated patient-derived liver organoids of both male and female origin, providing a novel in vitro model for LCN2 research. Our data show that the self-renewing organoids mimic essential architectural features of hepatocytes, as demonstrated by electron microscopy and F-actin staining. Consistent with the expression profile observed in liver tissue, the isolated 3D organoids exhibit minimal endogenous LCN2 levels. Next, the LCN2 expression was studied at the protein and mRNA levels under inflammatory conditions by treating the organoids with various cytokines and lipopolysaccharides (LPS). Our results show that LCN2 expression is significantly upregulated by IL-1β and TNF-α in an NF-κB-dependent manner, but remains unchanged with IL-6 or LPS. In conclusion, we have established human patient-derived liver organoids as a valuable model for investigating LCN2 signaling mechanisms. This study lays the foundation for future research on the role of LCN2 in liver pathologies, aiding in disease progression understanding and facilitating patient-specific treatment predictions.

The immortal murine hepatic stellate cell line Col-GFP HSC was comprehensively characterized using genetic and molecular approaches. Short tandem repeat (STR) profiling and karyotyping combined with multiplex fluorescence in situ hybridization (M-FISH) confirmed the identity of the cell line and revealed no contamination. Col-GFP HSCs showed a near tetraploid karyotype. Additionally, next-generation sequencing (NGS) data, quantitative reverse transcription PCR, and Western blot analyses demonstrated robust expression of genes and proteins associated with hepatic stellate cells, including those involved in extracellular matrix remodeling and fibrogenic pathways. Phalloidin staining revealed filamentous actin patterns characteristic of stellate cells, providing additional support for their cytoskeletal organization and functional status. These findings provide strong evidence that the Col-GFP HSC cell line originates from hepatic stellate cells and can serve as a reliable in vitro model to study stellate cell biology and related pathophysiological processes.

Metabolic dysfunction-associated steatotic liver disease (MASLD) and its consequences represent a growing global health burden that urgently requires physiologically relevant in vitro models beyond conventional 2D culture systems. In this study, we report the successful establishment of 45 patient-derived liver organoid lines. These organoids were generated from healthy, steatotic and cirrhotic tissues collected from 207 liver surgeries at RWTH University Hospital Aachen, with an initiation success rate of 82%. The organoids were propagated for at least six passages using an optimized protocol. Multiplex immunofluorescence analysis revealed highly proliferative structures with approximately 40% Ki-67-positive cells expressing hepatocyte (Albumin and HNF4α) and cholangiocyte (CK19) markers. Intermittent LGR5 staining suggested the presence of liver progenitor cell features. Quantitative PCR results confirmed variable HNF4α expression, indicating inter-patient heterogeneity in differentiation status. Time-lapse imaging combined with mathematical modeling uncovered a biphasic growth dynamic with an initial linear expansion in the first 15 h, followed by exponential growth (doubling time ≈ 20.6 h) between 30 and 72 h. Overall, our workflow produced genetically and phenotypically stable liver organoids that recapitulate essential features of various hepatic conditions. This provides a solid foundation for disease modeling, potential drug testing, and quantitative systems biology.

The murine Alpha Mouse Liver 12 (AML12) cell line, established over four decades ago, is one of the most commonly used non-transformed hepatocyte models in basic and pre-clinical liver research. Despite its widespread use, a comprehensive and current molecular characterization has been lacking. In this study, we combined cytogenetics with high-resolution genomic technologies to establish a detailed genetic reference profile of AML12. Inverted DAPI banding and multicolor fluorescence in situ hybridization (m-FISH) revealed a complex yet stable, near-tetraploid karyotype featuring double X-chromosome deletions [del(X)(A3)×2], a recurrent derivative chromosome der(3)t(2;3)(A2;H4), biallelic deletions of 17D1, two dicentric chromosomes dic(X;17), and multiple whole-chromosome gains (e.g., +1, +6, +15, +19×4) and losses (e.g., −4, −12, −16, −18). Multicolor banding (mcb) further pinpointed cryptic inversions on chromosomes 7 and 11. Copy number imbalances were visualized as in silico array comparative genomic hybridization (aCGH)-style profiles inferred from these metaphase-based assays, and no independent array- or sequencing-based copy number variation (CNV) experiment was performed in this study. Short tandem repeat (STR) profiling created a unique 16-locus authentication barcode that unambiguously distinguishes AML12 from other murine cell lines in public databases. Bulk RNA sequencing (RNA-seq) further demonstrated a transcriptional profile in AML12 cells that is indicative of hepatocyte origin while also revealing partial de-differentiation and reduced expression of selected urea cycle, gluconeogenic, and xenobiotic-metabolizing transcripts, consistent with limited mature hepatocyte functions. These functional inferences are likely based on gene expression patterns rather than on direct physiological assays. In summary, our study provides (i) the first integrated cytogenetic, STR, and next-generation sequencing dataset for AML12, (ii) a practical authentication panel for routine laboratory use, and (iii) reference information that will enhance the interpretation, reproducibility, and translational relevance of future studies using this versatile hepatocyte model.

Caspase-8 and the NF-κB essential modulator (NEMO, also referred to as IKKγ) play critical roles in controlling TNF-α-induced cell death and survival in hepatocytes. The aim of this study was to generate a hepatocyte-derived cell line in which the Casp8 and Nemo genes can be conditionally inactivated simultaneously to investigate the significance of the corresponding signaling pathways. To this end, we induced hepatocellular carcinoma in Casp8^f/fNemo^f/f mice using diethylnitrosamine and established an immortalized hepatoma cell line from explanted liver tumors, which is subsequently referred to as ΔCN60. ΔCN60 cells still retain floxed Casp8 and Nemo alleles, allowing for efficient Cre-mediated deletion to generate Casp8^ΔNemo^Δ derivatives. Loss of both Caspase-8 and NEMO inhibits cell proliferation, increases the expression of tumor and progenitor markers (AFP, CD133), reduces albumin expression, and blocks TNF-α-induced NF-κB p65 nuclear translocation. ΔCN60 Casp8^ΔNemo^Δ cells display altered sensitivity to prolonged TNF-α exposure, suggesting a potential shift in necroptosis-associated signaling pathways, although necroptotic cell death was not directly demonstrated. ΔCN60 serves as a versatile hepatoma model for investigating Caspase-8/NEMO-dependent TNF-α signaling and hepatocyte plasticity and may help prioritize future in vivo experiments by enabling selected mechanistic questions to be addressed first in vitro.