WG Rossi

Environmental pollution in a dish: Modeling pollution-induced effects on taste and nutrient sensing using tongue and stomach organoids (EpiDish)

This project explores how environmental pollution affects human health by compromising sensory functions, particularly taste perception and nutrient sensing. Air- and foodborne pollutants may disrupt taste and nutrient uptake, potentially impacting calorie intake, appetite regulation, and overall health. To investigate this, the study will combine mechanistic cell-based experiments, sensory trials, and epidemiological studies without relying on animal models. Using human induced pluripotent stem cells (iPSCs), researchers will develop tongue and stomach organoids to model human sensory and digestive functions. These organoids will be exposed to pollutants such as traffic-related particulate matter and heavy metals to assess changes in taste perception, receptor expression, and nutrient uptake. Insights gained from these in-vitro experiments will be applied to a large-scale epidemiological study (NaKo cohort) to explore how urban air pollution affects dietary choices across different age groups. Ultimately, this research aims to enhance our understanding of the impact of pollution on taste and nutrient sensing, promote healthier eating, and improve public health outcomes. The project is funded by the Leibniz Association (competitive procedure). Cooperation partners are the working groups Schikowski, Schins and Staerk, as well as the Leibniz Institute for Food Systems Biology (LSB) in Munich.

Phenotypic variability due to genetic buffering: The role of environmental factors

Genetic factors play a key role in disease development and aging, with Mendelian inheritance patterns representing extremes on a spectrum of genetic variation. However, the effects of harmful genetic mutations can often be mitigated by processes like genetic buffering, which involves mechanisms such as genetic compensation, transcriptional adaptation, and phenotypic plasticity. Though some of these processes are thought to be non-genetic, the influence of environmental factors on genetic buffering and its role in environmentally-induced aging remains poorly understood. In this project, we aim to explore how environmental factors, like air pollutants and food contaminants, affect the expression of modifier genes involved in genetic buffering. Using iPSCs and mutants modeling diseases like Duchenne muscular dystrophy (DMD) and ACTB-associated syndromes, we will expose cells to low-toxicity environmental factors and analyze their impact on modifier gene expression. We hypothesize that these factors, potentially through oxidative stress, will alter gene expression patterns and modulate genetic buffering. We will examine these changes at the epigenetic and transcriptomic level and screen small molecules that may influence environmentally-sensitive modifiers. This work, funded by the DFG and in collaboration with the Haarmann-Stemmann group, aims to provide critical insights into how environmental exposures affect genetic buffering mechanisms.

Small molecules that enhance prime editing in therapeutic applications

Genome engineering, a transformative biomedical technology, has gained popularity for disease treatment. While current techniques enable straightforward genome manipulation, achieving precise mutations, especially in in vivo translational applications, remains challenging for both efficiency and safety. Common methods inducing DNA double strand breaks (DSBs) often result in undesired outcomes. CRISPR/Cas-mediated genome editing, specifically base editing (BE) and prime editing (PE), offer alternatives. BE faces challenges such as unintentional deamination and bystander mutations. PE, a newer technique, writes genetic information without DSBs, but its efficiency needs improvement. The lab is focused on identifying small molecules that enhance PE efficiency, identifying candidates for further evaluation in clinically relevant models, aiming to advance gene therapy tools for correcting deleterious mutations in patients. The project is supported by AFM Téléthon.

Quality control of hiPSCs

This project aims to enhance the quality control of human induced pluripotent stem cells (iPSCs), building on previous work that identified new markers for assessing iPSC pluripotency and differentiation potential using long-read nanopore sequencing. The prior study developed “hiPSCore”, a machine learning-based scoring system that accurately classifies pluripotent and differentiated cell states, streamlining the evaluation process and reducing subjectivity. The current project is a direct follow-up, seeking to further improve the quality control standards of iPSCs by refining and validating these novel markers, ultimately enhancing the reliability and clinical utility of iPSCs for research and therapeutic applications. The project is funded by NRW.

Understanding risks in gene editing: Analyzing prime editing outcomes and large deletions compared to CRISPR/Cas9 in iPSCs

Genome engineering technologies present challenges in achieving precise and safe genetic modifications, which are critical for translational medicine. While CRISPR/Cas9 can result in unintended off-target effects, prime editing (PE) is more precise. For both, the occurrence and extent of large deletions at on-target sites remain less understood. In this project, we aim to systematically investigate the on-target effects of CRISPR/Cas9 and prime editing in therapeutically relevant induced pluripotent stem cells (iPSCs). By designing and transfecting guide RNAs and prime editing guide RNAs (pegRNAs) into both male and female iPSC lines, we will generate targeted edits. Fluorescence-Activated Cell Sorting (FACS) will be employed to isolate successfully edited cells. Preliminary analysis of editing efficiency and locus integrity will be conducted using digital PCR (dPCR). Comprehensive sequencing using both short-read Illumina (for small indels) and long-read Nanopore platforms (for large deletions up to 10kb) will be performed to thoroughly examine on-target editing outcomes, including large deletions and structural variants. Additionally, we will refine our previously developed software, CRISPRnano, to improve its capability in analyzing prime editing outcomes, particularly focusing on better alignment, visualization, and detection of unintended indels. This research will provide critical insights into the on-target safety profiles of CRISPR/Cas9 and prime editing, contributing to the development of more accurate and reliable genome editing tools for research and therapeutic applications. The project is funded by the DFG.

Assessing epilepsy in brain organoid models of mitochondrial diseases

Mitochondrial diseases are a group of genetic disorders impairing mitochondrial respiration. Leigh syndrome (LS) and polymerase gamma (POLG)-related pathology are among the most common mitochondrial diseases in which epilepsy can occur. The link between mitochondrial failure and seizure generation is unclear and assumed to be dependent on calcium homoeostasis, reactive oxygen species, or plasma membrane potential. To shed light on the mechanisms underlying seizures in mitochondrial diseases and possibly uncover modulating strategies, we aim to develop a new approach for assessing the epileptic components of mitochondrial diseases. In this collaborative project, we will combine synergistic expertise in stem cell engineering, brain organoid modeling, and cellular electrophysiology. We will utilize patient-derived and genetically engineered induced pluripotent stem cells (iPSCs) for LS and POLG-related pathology to generate human cortical brain organoids to study epileptic aspects using high-density multi-electrode array technology and single-cell electrophysiology. This model platform may provide insights into the underlying mechanisms and possible modulatory avenues for seizures occurring in patients. This project is conducted together with Alessandro Prigione and Nico Melzer from the Heinrich Heine University Düsseldorf. It is funded by the Research Commission of the Medical Faculty of the Heinrich Heine University Düsseldorf (FoKo grant).