fspasqualini.bsky.social
@fspasqualini.bsky.social
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
July 8, 2025 at 9:36 AM
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
July 8, 2025 at 9:36 AM
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...
@alleninstitute.org Cell Collection: the largest repository of GFP/RFP-based hiPSC reporter lines www.allencell.org/cell-catalog...
July 8, 2025 at 9:36 AM
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...
@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!
July 8, 2025 at 9:36 AM
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
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
July 8, 2025 at 9:36 AM
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
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.
July 8, 2025 at 9:36 AM
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.
And works with organoids and light sheet microscopy as well!
January 23, 2025 at 8:02 PM
And works with organoids and light sheet microscopy as well!
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.
January 17, 2025 at 4:14 PM
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.
January 17, 2025 at 4:14 PM
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.
January 17, 2025 at 4:14 PM
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.
What’s in the gel? We used fish gelatin crosslinked with microbial transglutaminase, enabling precise control over stiffness and maintaining compatibility with liquid handling robots. Results? Physiological stiffness for cell culture.
January 17, 2025 at 4:14 PM
What’s in the gel? We used fish gelatin crosslinked with microbial transglutaminase, enabling precise control over stiffness and maintaining compatibility with liquid handling robots. Results? Physiological stiffness for cell culture.
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.
January 17, 2025 at 4:14 PM
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.
Why HYDRA? Traditional high-throughput screening (HTS) platforms rely on stiff substrates (plastic/glass) that poorly mimic tissue environments. Our solution combines physiological stiffness with standard imaging capabilities to improve preclinical drug testing accuracy.
January 17, 2025 at 4:14 PM
Why HYDRA? Traditional high-throughput screening (HTS) platforms rely on stiff substrates (plastic/glass) that poorly mimic tissue environments. Our solution combines physiological stiffness with standard imaging capabilities to improve preclinical drug testing accuracy.
And, of course, looking is only half the fun. We can quantify changes in cell structure and function before and after cell proliferation, multinucleation, and endoreplication!
January 17, 2025 at 4:09 PM
And, of course, looking is only half the fun. We can quantify changes in cell structure and function before and after cell proliferation, multinucleation, and endoreplication!
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?
Isn't it awesome to look at cell structure, function, and CC progression in the same movie?
January 17, 2025 at 4:09 PM
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?
Isn't it awesome to look at cell structure, function, and CC progression in the same movie?
This ability might be relevant in drug testing, e.g., in cardio-oncology. A cancer drug like nocodazole kills cycling cells by arresting spindle dynamics and altering their CC. But what does that do to hiPSC-CM in G0? In our experiments, it alters their ability to cycle calcium!
January 17, 2025 at 4:09 PM
This ability might be relevant in drug testing, e.g., in cardio-oncology. A cancer drug like nocodazole kills cycling cells by arresting spindle dynamics and altering their CC. But what does that do to hiPSC-CM in G0? In our experiments, it alters their ability to cycle calcium!
To turn OFF Fucciplex in hiPSC-CM, we expressed it under the EF1a promoter in hiPSCs. Then, we worked with
Ale Bertero in Turin to perform scRNAseq in cardiac organoids to show progressive downregulation along the cardiac myocyte lineage.
Ale Bertero in Turin to perform scRNAseq in cardiac organoids to show progressive downregulation along the cardiac myocyte lineage.
January 17, 2025 at 4:09 PM
To turn OFF Fucciplex in hiPSC-CM, we expressed it under the EF1a promoter in hiPSCs. Then, we worked with
Ale Bertero in Turin to perform scRNAseq in cardiac organoids to show progressive downregulation along the cardiac myocyte lineage.
Ale Bertero in Turin to perform scRNAseq in cardiac organoids to show progressive downregulation along the cardiac myocyte lineage.
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.
January 17, 2025 at 4:09 PM
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.
Stem cells differentiate in most cells, including terminally differentiated ones (CC phase: G0). EG cardiomyocytes stay in G0 but can proliferate for regeneration or perform non-canonical CC events such as multinucleation/endoreplication for maturation. Can we track those?
January 17, 2025 at 4:09 PM
Stem cells differentiate in most cells, including terminally differentiated ones (CC phase: G0). EG cardiomyocytes stay in G0 but can proliferate for regeneration or perform non-canonical CC events such as multinucleation/endoreplication for maturation. Can we track those?
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).
Bruce Conklin's GCaMP6f-WTC11 hiPSCs reference line (parental line of the @alleninstitute.bsky.social Cell Collection).
January 17, 2025 at 4:09 PM
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).
Bruce Conklin's GCaMP6f-WTC11 hiPSCs reference line (parental line of the @alleninstitute.bsky.social Cell Collection).
check out these beautiful spindles!
January 17, 2025 at 4:09 PM
check out these beautiful spindles!
Alternatively, we can focus on S/G2/M events where mTurquoise2 is not expressed. For example, CALIPERS combines FUCCIplex and FUCCIphase to maximize confocal spindle imaging yield while minimizing phototoxicity in a 4-color HaCaT reporter line (w/ GFP tagged to b-tubulin)
January 17, 2025 at 4:09 PM
Alternatively, we can focus on S/G2/M events where mTurquoise2 is not expressed. For example, CALIPERS combines FUCCIplex and FUCCIphase to maximize confocal spindle imaging yield while minimizing phototoxicity in a 4-color HaCaT reporter line (w/ GFP tagged to b-tubulin)
But, hey, mTurquoise2 and GFP spectrally overlap! How can we multiplex four signals? So glad you asked! We can use a GFP-based sensor for a fast, functional signal so the slower CC bleedthrough can be removed.
January 17, 2025 at 4:09 PM
But, hey, mTurquoise2 and GFP spectrally overlap! How can we multiplex four signals? So glad you asked! We can use a GFP-based sensor for a fast, functional signal so the slower CC bleedthrough can be removed.
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.
@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.
January 17, 2025 at 4:09 PM
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.
@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).
January 17, 2025 at 4:09 PM
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).