By tracking rare cycling hiPSC-derived cardiomyocytes, we can monitor cell proliferation (useful in regeneration) and multinucleation and endoreplication (useful for assessing maturation) as well as structure and function integration #CCAwarePhenotyping #CCAwareRegenerativeStudies #MethodInACellLine

New light-sheet capture of 4-color hiPSC-cardioids reveals compaction before calcium transients initiation. 90 % exit cycle by d4.5 → maturation on cue. #CCAwarePhenotyping #MethodInACellLine

Safe-harbor genome editing ensures expression across differentiated lineages, and WTC-11 genetic background means full compatibility with the
@alleninstitute.org Cell Collection: the largest repository of GFP/RFP-based hiPSC reporter lines www.allencell.org/cell-catalog...

WTC11-based hiPSCs reporter lines. We provide a 4-color CALIPERS hiPSC line with sensors for actin, calcium, and the cell cycle, as well as a blank canvas line to add your favorite sensors. CALIPERS is a #MethodInACellLine!

Phase-locked motility
Each nucleus gets a %-cell-cycle score that rides with its mask and trajectory. Compare shape or speed by cycle progress, not frame time. Watch HaCaTs sprint through physical space and cell cycle (CC) phases! #PhaseLockedMotility #CCAwarePhenotyping

Proposed solution: To overcome this, we integrated a spectrally re-engineered FUCCI variant, open-source analysis software, and four-color human stem cell reporter lines into CALIPERS: a method for Cell-cycle-Aware Live-cell Imaging in Phenotyping and Regeneration Studies.

HYDRA supports fluorescence microscopy, enabling cell-cycle and cytoskeletal analyses with FUCCIplex sensors using CALIPERS (biorxiv.org/content/10.1...). From cell proliferation to mitotic spindle dynamics, this method works with cutting-edge imaging and image-analysis modalities.

Validated for drug testing: Using epithelial cells and holographic imaging, HYDRA reveals distinct cell responses to nocodazole & paclitaxel. Confluency analysis showed the importance of biomimetic substrates.

HYDRA is scalable! From 96-well to 384-well plates, we demonstrated consistent quality. Automated fabrication ensures reproducible thickness and flatness, with >90% successful wells in HTS plates.

HYDRA prevents meniscus formation during hydrogel casting, achieving flat, planar layers. Using robotic liquid handling, we dispense & re-aspirate hydrogel precursors to create tunable 10-50 μm gel layers.

Finally, for regeneration studies, we need a 4-color CALIPERS hiPSC reporter line! We genome-edited CAG-FUCCIplex in the safe harbor site human Rosa26.

Isn't it awesome to look at cell structure, function, and CC progression in the same movie?

To accommodate cases in which we prefer to free spectral channels in terminally differentiated hiPSC-CMs, we let FUCCIplex turn OFF in mature hiPSC-CMs. For example, this let us image microtubule dynamics with the contractile apparatus.

Then, we extended CALIPERS to human stem cells by expressing FUCCIplex and LifeAct in
Bruce Conklin's GCaMP6f-WTC11 hiPSCs reference line (parental line of the @alleninstitute.bsky.social Cell Collection).

check out these beautiful spindles!

With
@florianjug.bsky.social's team, we developed an open-source FUCCIphase plugin to calculate CC phase percentages, thus enabling CC-aware morphology/motility analyses in live cells.

FUCCIplex replaces GFP/RFP with iRFP & mTurquoise2, allowing multiplexed imaging of CC with standard fluorescence microscopes. See it in action tracking HaCaT cells across 40 hours (LifeAct in grey).