Quantitative high‐content/high‐throughput microscopy analysis of lipid droplets in subject‐specific adipogenesis models

Abstract

Neutral lipids packed in lipid droplets (LDs) are essential as a source of fuel for organisms, and specialized storing cells, the adipocytes, provide a buffer for energy variations. Many modern-society-disorders are connected with excess accumulation or deficiency of LDs in adipose tissue. Intracellular LD number and size distribution reflect the tissue conditions, while the associated mechanisms and genes rs are still poorly understood. Large-scale genetic screens using human in vitro differentiated primary adipocytes require cell samples donated from many patients. The heterogeneity appearing between donors highlighted the need for high-throughput methods robust to individual variations. Previous image analysis algorithms failed to handle individual LDs, but focused on averages, hiding population heterogeneity. We present a new high-content analysis (HCA) technique for analysis of fat cell metabolism using data from a large-scale RNAi screen including images of more than 500 k in vitro differentiated adipocytes from three donors. The RNAi-based suppression of Perilipin 1 (PLIN1), a protein involved in the adipocyte lipid metabolism, served as a positive control, while cells treated with randomized RNA served as negative controls. We validate our segmentation by comparing our results to those of previously published methods: We also evaluate the discriminative power of different morphological features describing LD size distribution. Classification of cells as containing few large or many small LDs followed by calculating the percentage of cells in each class proved to discriminate the positive PLIN1-suppressed phenotype from the untreated negative control with an area under the receiver operating characteristic curve of 0.98. The results suggest that this HCA method offers improved segmentation and classification accuracy, and can, thus, be utilized to quantify changes in LD metabolism in response to treatment in many cell models relevant to a variety of diseases. © 2017 International Society for Advancement of Cytometry.

PEDF regulates plasticity of a novel lipid–MTOC axis in prostate cancer-associated fibroblasts

“…In this study, we have demonstrated that human prostatederived CAFs store more neutral lipid in LDs than normal prostate fibroblasts (NFs). Each cell type had a unique LD distribution pattern and size range, which could impact signaling (Welte and Gould, 2017;Bombrun et al, 2017). Normal fibroblasts had a distinct concentric ring of neutral lipids residing close to the nucleus, whereas LDs in CAFs were dispersed throughout the cytoplasm.…”

Tributyltin chloride (TBT) induces RXRA down-regulation and lipid accumulation in human liver cells

“…Image analysis was performed using CellProfiler (www.cellprofiler.org) pipelines (example shown in S1B Fig) for nuclear, cellular and lipid droplets segmentation and measurement extraction. For lipid droplet analysis the following steps were used: images were background subtracted using the rolling ball method; Smooth, IdentifyPrimaryObjects (generates nuclear mask based on smoothed DAPI labeling, its intensity and size), IdentifySecondaryObjects (generates the “cell” mask based on the lipid staining and distance from the nuclear mask–similar to [22]), IdentifyPrimaryObjects (generates the lipid droplet mask by analyzing the Lipidtox stain images using Otsu thresholding and a size filter), IdentifyTertiaryObjects (used to determine the “cytoplasm”, subtracts the nuclear mask from the cell mask), and RelateObjects (relates each lipid droplet mask to the corresponding cytoplasm mask). A much simpler pipeline was used for nuclear protein ( i .…”

Adipocytes role in the bone marrow niche

“…These techniques will further reveal the complex nature of the mechanisms by which extrinsic factors regulate signal transduction and cell-fate decisions of hematopoietic stem cells. In addition to studies in genetic mouse models, transcriptomic and single cell analysis after fluorescence Communication to the Editor activated cell sorting of live cells represent fundamental tools that will help us understand the roles of adipocytes within the bone marrow microenvironment (81)(82)(83)(84). In spite of powerful experimental mouse models providing a proof of concept for adipocytes biology in the bone marrow, we are still lacking direct demonstration of adipocytes roles in the human bone marrow.…”