# Comprehensive Cytogenetic and Genomic Profiling of the Murine AML12 (Alpha Mouse Liver 12) Hepatocyte Cell Line

## TL;DR

This study provides a detailed genetic and functional profile of the AML12 mouse liver cell line, showing it has a complex genome and partial loss of mature liver cell features.

## Contribution

The first integrated cytogenetic, STR, and transcriptomic reference map for the AML12 cell line is presented.

## Key findings

- AML12 cells have a near-tetraploid, highly aneuploid karyotype with structural rearrangements and copy number changes.
- AML12 maintains hepatocyte lineage identity but shows partial de-differentiation and reduced metabolic gene expression.
- A 16-locus STR barcode was developed to authenticate AML12 and distinguish it from other murine cell lines.

## Abstract

What are the main findings?
We present the first integrated cytogenetic, STR-based, and transcriptomic reference map of the widely used murine hepatocyte line AML12. Our findings reveal a near-tetraploid, highly aneuploid karyotype with recurrent structural rearrangements and segmental copy number changes.Bulk RNA-seq, along with conventional and quantitative RT-PCR, Western blotting, immunofluorescence, light microscopy, and ultrastructural analyses, demonstrate that AML12 cells maintain hepatocyte lineage identity but exhibit partial de-differentiation, including ductular/progenitor- and stress-associated transcriptional signatures.

We present the first integrated cytogenetic, STR-based, and transcriptomic reference map of the widely used murine hepatocyte line AML12. Our findings reveal a near-tetraploid, highly aneuploid karyotype with recurrent structural rearrangements and segmental copy number changes.

Bulk RNA-seq, along with conventional and quantitative RT-PCR, Western blotting, immunofluorescence, light microscopy, and ultrastructural analyses, demonstrate that AML12 cells maintain hepatocyte lineage identity but exhibit partial de-differentiation, including ductular/progenitor- and stress-associated transcriptional signatures.

What are the implications of the main findings?
We combined a cytogenetic and STR profile that establishes a practical authentication and quality control framework that supports the reliable use of AML12 across laboratories and aids in detecting clonal drift over time.Overall, AML12 remains a useful and experimentally tractable murine hepatocyte model, particularly for studying hepatocyte signaling, stress responses, and epithelial plasticity at a mechanistic level. However, if fully mature metabolic functions or precise handling of bile acids/xenobiotics are required, our data suggest that AML12 should be used as a heuristic model and complemented with more differentiated systems (such as primary hepatocytes, organoids, or in vivo models). This should ideally be done with condition-dependent optimization and appropriate functional validation.

We combined a cytogenetic and STR profile that establishes a practical authentication and quality control framework that supports the reliable use of AML12 across laboratories and aids in detecting clonal drift over time.

Overall, AML12 remains a useful and experimentally tractable murine hepatocyte model, particularly for studying hepatocyte signaling, stress responses, and epithelial plasticity at a mechanistic level. However, if fully mature metabolic functions or precise handling of bile acids/xenobiotics are required, our data suggest that AML12 should be used as a heuristic model and complemented with more differentiated systems (such as primary hepatocytes, organoids, or in vivo models). This should ideally be done with condition-dependent optimization and appropriate functional validation.

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.