National Institute of Standards and Technology

A 3D Subcellular Map of Human Retinal Pigment Epithelium Using Quantitative Confocal Fluorescent Microscopy

Purpose: Quantify 3D Characteristics of Human Retinal Pigment Epithelium and Their Relevant Biological Functions to Several Degenerative Retinopathies

Paper Title: A quantitative 3D Subcellular Map of Human Retinal Pigment Epithelium Discovers Non-stochastic Cell State Transition in Establishment of Apical/Basal Polarity

Abstract: The retinal pigment epithelium (RPE) is a highly specialized monolayer that forms a selective barrier between the subretinal and the choroidal spaces. During development RPE cells polarize perpendicular to the plane of the monolayer such that cellular organelles attain specific location and volume in the cell. This allows the cell to differentially interact with overlying photoreceptors and underlying choriocapillaris. When RPE polarity is disrupted, retinal homeostasis is altered leading to degeneration.
In this study, we used high-content imaging, image segmentation, phenotypic quantification techniques, and mathematical modeling to develop statistically robust quantitative 3-dimensional (3D) maps of subcellular organelles and structures during the establishment of RPE apical/basal polarity. We provide a holistic description of RPE cell phenotypic states at four different timepoints of maturation.
The establishment and maintenance of this polarized phenotype are crucial to the health of the retina-blood barrier. Disruption of RPE polarity is linked to several degenerative retinopathies, such as age-related macular degeneration (AMD), proliferative retinopathy and diabetic retinopathy.
In the RPE, chemically enhancing ciliogenesis with Prostaglandin E2 (PGE2) was shown to promote the expression of RPE maturation and polarity markers, while using the small molecule Hedgehog Pathway Inhibitor 4 (HPI4) caused a reduced expression of the same markers and disrupted ciliary protein trafficking. We used cells expressing a fluorescent tag conjugated to

ACTB (actin filaments)
CETN2 (centrioles)
CTNNB1 (adherens junctions)
DSP (desmosomes)
FBL (nucleolus)
GJA1 (gap junctions)
LAMP1 (lysosomes)
LC3B (autophagosomes)
LMNB1 (nuclear envelope)
MYH10 (actomyosin bundles)
RAB5 (endosomes)
SEC61B (ER)
SLC25A17 (peroxisomes)
ST6GAL1 (Golgi apparatus)
TOM20 (mitochondria)
Z01 (TJP1 tight junction protein)

Experimental Overview

iRPE culture plate — Project layout. (A) Immature iRPE cells from 16 lines, each expressing a protein tagged with a fluorophore, were grown for four weeks in the presence of PGE2, a primary cilium enhancer, or HPI4, an inhibitor of primary cilium trafficking, to promote or inhibit cell apical/basal polarity. (B) Immature iRPE were seeded in glass-bottom 96-well plates. Seven days after cell seeding, PGE2 or HPI4 treatment was started to respectively promote or prevent cell polarization. Once a week a plate was fixed in 4 % PFA to preserve RPE cell states at different timepoints of RPE polarizations. The first timepoint (week 1) represents a baseline since treatment with PGE2 and HPI4 started after the first collection. Cells were stained with phalloidin-AF555 and Hoechst 33342 to highlight cells and nuclei borders. Automated high-content imaging was performed with a spinning-disk microscope to collect 3D stacks. An artificial intelligence algorithm was trained to segment RPE cells and nuclei borders that were used as reference to report organelles and structures’ location. Each tagged organelle was segmented with a unique combination of model-based segmentation algorithms. The quality of segmentation was visually inspected by two experts to select the best parameters for the algorithms. Organelle location and morphometry were calculated for each condition to generate reference maps representative of cell states at different timepoints of polarization.

View Example: Deep Zoom view of Retinal Pigment Epithelium (RPE) cell images at the week 3:

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Dataset description

Each folder is a separate imaging run. Every run corresponds to a row of a 96-well plate. In every row only one organelle of interest is labelled (channel 01 -wavelength 488). The other channels (C02, C03, C04) correspond to reference cellular structures and never change (ex: C03 always corresponds to the actin cytoskeleton).
For every row:
Column 02 to 06 – Cells treated with PGE2
Columns 07 to 11 – Cells treated with HPI4
Folder name convention: Ex: 190102-Pol-Inhibitor-P1-W2-LAMP1_20190102_101748
Date - Experiment name - Plate # (1 to 5)- Week # (1 to 6 ) -Organelle of interest_ Date_Time
File name convention:
Ex: P1-W2-LAMP1_B02_T0001F001L01A01Z01C02.tif
Plate # (1 to 5)- Week # (1 to 6)- Organelle of interest_Well # (02 to 11)_
T # (Time point number, never changes)
F # (Fields of view, 6 per each well, no tile overlap, distributed around the well)
L # (Time line number, never changes)
A # (Related to acquisition, it tells which channels have been acquired at the same time. It goes from 1 to 4.)
A01 – Channel 02 and 04
A02 – Channel 01
A03 – Channel 03
A04 – Channel 05
Z # (Slice number in the Z plane, from 01 to 27. The interval between slices is 0.5um)
C # (Channel, from 01 to 05.)
C01 – wavelength 488 – Organelle of interest
C02 – wavelength 405 – Nuclear DNA
C03 – wavelength 561 – Actin cytoskeleton
C04 – wavelength 640 – Cell membrane
C05 – Brightfield
Images from slice Z01 to Z27 have been taken for all channels except for Brightfield (C05). Only one image has been taken for brightfield.
Dark and shading correction has been performed on the fly.
The reference images can be found in every folder. Ex: “OTF_DC_sCMOS #1_CAM1.tif” and “OTF_SC_BP445-45_60xW_M01_CH02.tif”
Geometric correction has been performed on the fly using “Nearest Neighbor Interpolation”.
The reference file can be found in every folder: OTF_geometry_parameter.xml
Other files containing measurement details (ex: scale bar: 0.21666667 um) can be found in every folder.

Exceptions

The folder names are:

190115-DAO-Pol-Inihb_20190115_104740
190122O-Pol-Inihbitor-P1-W5
190123-Pol-Inihbitor-P1-W6_20190123_173506

Inside these folders, multiple subfolders are present, each corresponding to a different row of a 96-well plate (i.e., a different organelle of interest). These subfolders are labeled with the date and time of acquisition. Only from the measurement file (.mes) can information about the row (or organelle of interest) be retrieved.

Credit

A DOI has been minted for this data record: https://doi.org/10.18434/mds2-3529

If you use this data then, please, cite the DOI and the appropriate publication:

A Quantitative 3D Subcellular Map of Human Retinal Pigment Epithelium Discovers Non-stochastic Cell State Transition in Establishment of Apical/Basal Polarity: Davide Ortolan, Pushkar Sathe, Andrei Volkov, Dominik Reichert, Sheldon Sebastian, Arvydas Maminishkis, Nicholas J. Shaub, Bengt Ljungquist, Devika Bose, Jorge Ferrari, Nyusha Lin, Gianluca Pegoraro, Carl G. Simon Jr., Ruchi Sharma, Peter Bajcsy, Kapil Bharti
(under review)