Spatiotemporal single-cell phenotyping in living 3D skin organoids.

Abstract

Continuous monitoring of cell states in living tissues remains elusive despite the explosion of single cell technologies. Here, we use organelle-specific environment-sensitive probes (ESPs) combined with hyperspectral imaging and a dedicated quantitative analysis pipeline to spatially and temporally track keratinocyte cell states in living 3D skin organoids. Our technique, named ESPRESSO (Environmental Sensors Profiling Relayed by Subcellular Structures and Organelles) combines hyperspectral imaging and phasor unmixing, enabling imaging of up to 6 ESPs (targeting chromatin, mitochondria, lysosomes, tubulin, Golgi apparatus and lipid droplets) at the same time with a single laser excitation. The quantification of morphological (e.g., number, size, organization) and functional (e.g., membrane potential, pH, polarity) characteristics of the organelles and subcellular structures allows us to identify the cell state at the single-cell level and is applicable to living cells in 2D and 3D. Cell state and cell-cell interactions are tightly regulated in space and time to determine tissue function. However, the spatiotemporal regulation of cell states and their interactions at the systems level during skin development and dysfunction remains poorly understood. Determination of cell states is routinely achieved by high-dimensional methods such as single-cell RNA sequencing (scRNA-seq), which yields the transcriptional profile at the single cell level of cell cultures and tissue. However, processing of the sample is required with consequent loss of spatial information. Genetic modification to include fluorescent tagging of a biomarker of interest can sometimes be used for living samples, although this approach is limited to observing a few parameters at one time and it may not be applicable to primary cells due to difficulty in transfection and early loss of proliferative capacity. Here, we demonstrate the capability of ESPRESSO to characterize keratinocyte differentiation as a function of space and time in living 3D skin organoid.