Aspects of zone-like identity and holotomographic tracking of human stem cell-derived liver sinusoidal endothelial cells

Mikel Amirola-Martinez; Thomas Combriat; Katharina Ferencevic; Ingrid Wilhelmsen; Andrea Dalmao-Fernandez; Petter Angell Olsen; Justyna Stokowiec; Aleksandra Aizenshtadt; Stefan Krauss

doi:10.3389/fcell.2025.1528991

Aspects of zone-like identity and holotomographic tracking of human stem cell-derived liver sinusoidal endothelial cells

Front Cell Dev Biol. 2025 Oct 17:13:1528991. doi: 10.3389/fcell.2025.1528991. eCollection 2025.

Authors

Mikel Amirola-Martinez^{1

2}, Thomas Combriat^{1

3}, Katharina Ferencevic¹, Ingrid Wilhelmsen^{1

2}, Andrea Dalmao-Fernandez^{1

4}, Petter Angell Olsen^{1

2}, Justyna Stokowiec¹, Aleksandra Aizenshtadt^#^{1

2}, Stefan Krauss^#^{1

2}

Affiliations

¹ Hybrid Technology Hub, Institute of Basic Medical Research, University of Oslo, Oslo, Norway.
² Department of Immunology and Transfusion Medicine, Oslo University Hospital, Oslo, Norway.
³ Njord Center, Department of Physics, University of Oslo, Oslo, Norway.
⁴ Section for Pharmacology and Pharmaceutical Biosciences, Department of Pharmacy, University of Oslo, Oslo, Norway.

^# Contributed equally.

Abstract

Introduction: Liver sinusoidal endothelial cells (LSEC) are specialized endothelial cells with unique metabolic and barrier functions adapted to the needs of the liver sinusoid. LSECs are highly sensitive to their environment, and this fragile nature causes challenges in analyzing their phenotype under in vitro conditions.

Methods: In this work, we first differentiated LSEC-like cells (scLSECs) from two human pluripotent stem cell lines and characterized them by a panel of qPCR markers, immunohistochemistry, substrate oxidation for energy metabolism, scavenger function, and nitric oxide secretion. We then introduced holotomography, a technique that allows to recover quantitative and three-dimensional information about the refractive indexes of cell components, as a tool to image and track scLSEC in vitro in a minimally intrusive, label-free manner.

Results and discussion: Holotomography and developed machine learning-based algorithm for image processing allowed us to describe and monitor changes in intracellular pore-like structures over time. Finally, we tested the possibility of inducing aspects of zone-specific LSEC phenotype and metabolism using culture-based treatments, which resulted in modest shifts in marker expression and metabolic activity. The presented strategy provides an advanced tool kit for investigating liver endothelial cells.

Keywords: LSEC; fenestration; holotomography (HT); liver zonation; pluripotent stem cells (PSC).