The cell cycle is one of the cell’s fundamental internal clocks. It does much more than count down to the next division. As a cell moves through the cycle, it coordinates changes in gene expression, metabolism, signaling and cellular structure. At the same time, checkpoints integrate information from outside the cell, such as growth factors and mechanical cues, with internal conditions, including nutrient availability and DNA integrity.
Together, these signals help determine whether a cell should divide, when it should do so and whether that division will contribute to tissue maintenance, growth or repair.
Two cells can look remarkably similar from the outside while being in very different biological states. Their positions in the cell cycle can influence how they migrate, respond to stimulation, reorganize their internal structures or commit to a specialized identity. Without knowing the time on this internal clock, researchers may attribute a difference to an experimental treatment when it actually reflects normal cell-cycle progression.
The problem becomes especially important in stem-cell and regenerative medicine research. Here, proliferation, migration, differentiation and functional maturation happen together and continuously influence one another. In a developing cardiac tissue, for example, an increase in size could come from the production of new cells, the growth of existing cells or the proliferation of non-cardiac populations. Even when a cardiomyocyte re-enters the cell cycle, it may divide successfully, form multiple nuclei, or copy its DNA without dividing.
Understanding these processes requires more than identifying whether a cell is cycling. We need to track the cell-cycle clock while simultaneously observing cellular structure and function in the same living sample.
This was the motivation for CALIPERS: Cell-cycle-Aware Live-cell Imaging for Phenotyping Experiments and Regeneration Studies.
CALIPERS is not simply another FUCCI cell-cycle reporter. It is an integrated framework combining a redesigned reporter, human stem-cell models, live microscopy, computational analysis and adaptive image acquisition. Its purpose is to make cell-cycle state a practical coordinate of the phenotype, rather than a separate measurement—or an invisible source of variability.
Freeing up valuable fluorescence channels
Following the cell-cycle clock alongside structure and function presents a practical problem. Most established FUCCI reporters use green and red fluorescent proteins, which are also used by many of the most valuable biosensors.
We redesigned FastFUCCI to create FUCCIplex, replacing its green and red proteins with cyan and far-red alternatives. Nuclei appear cyan during G1, when the cell grows and prepares to copy its DNA, and magenta during S/G2/M, which covers DNA replication and preparation for division. This leaves the green and red channels available for measurements such as actin organization, microtubule dynamics, and calcium activity.
Giving each cell a biological timestamp
These broad phases are useful, but many cellular changes happen continuously. Together with Florian Jug’s laboratory at Human Technopole, we developed FUCCIphase, an open-source analysis tool that converts sequences of FUCCIplex signals into a continuous estimate of cell-cycle progression for every tracked cell.
This gives each cell an internal biological timestamp. Instead of comparing cells only by how long they have been under the microscope, researchers can organize measurements by progression through the cycle. Migration, morphology and lineage histories can then be interpreted in the correct cell-cycle context.
We also used this information to guide the microscope. Detailed three-dimensional imaging can expose cells to damaging levels of light when performed repeatedly. Our FUCCIsmart routine monitors cells using gentler imaging and triggers detailed confocal acquisition only when a cell is approaching mitosis. This captured mitotic events while avoiding much of the unnecessary exposure associated with continuous high-resolution imaging.
From stem cells to developing cardiac tissues
The next challenge was to make CALIPERS work across human induced pluripotent stem-cell models.
Lentiviral delivery offered a rapid way to introduce FUCCIplex into particular cell types or developmental stages. For experiments spanning the entire differentiation process, we created stable reporter lines that maintained the cell-cycle signal as stem cells specialized and assembled into three-dimensional cardiac organoids, or cardioids.
This work was carried out in collaboration with Alessandro Bertero’s laboratory at the University of Turin, which shared its cardiac organoid expertise with our team and led the associated single-cell RNA-sequencing studies.
In the stable cardioid models, CALIPERS followed cells leaving the cycle, changes in cell size and tissue organization, and the later appearance of coordinated calcium activity. These processes are usually measured separately; CALIPERS placed them on a common timeline within the same living tissue.
It also resolved an important ambiguity in cardiac regeneration. A cardiomyocyte entering the cycle does not necessarily produce two new cells. We followed rare cycling cardiomyocytes that completed productive division, but also cells that became multinucleated or copied their DNA without dividing. Monitoring actin organization and calcium activity alongside the cell cycle allowed these different outcomes to be distinguished as they happened.
Looking beyond tissue growth
Finally, we tested whether CALIPERS could improve the evaluation of candidate regenerative interventions.
We examined factors previously linked to cardiomyocyte proliferation or repair. Of the conditions tested after most cardiomyocytes had exited the cycle, FGF2 was the only one that reproducibly increased cardioid growth.
But a larger tissue is not necessarily a regenerated tissue. CALIPERS showed that the response included both more cells (hyperplasia) and larger cells (hypertrophy). Calcium imaging also allowed us to estimate which putative regenerating cells were cardiomyocytes: approximately 40% showed detectable calcium activity.
These results do not establish FGF2 as a regenerative therapy. Instead, they demonstrate how CALIPERS can help triage candidates by asking, within the same living sample: Did cells re-enter the cycle? Were new cells produced? Did existing cells become larger? Were cardiomyocytes involved, and did they remain functional?
Putting the clock back into the phenotype
The central idea is simple: when phenotype is the endpoint, cell-cycle state should be measured explicitly. By tracking a cell’s internal clock alongside its structure and function, CALIPERS reveals not only what the cell is doing, but also where it is in the cell cycle—context that may help explain why it behaves that way.
CALIPERS brought together genome engineering, stem-cell biology, cardiac organoids, microscopy, and computational analysis across our laboratories. We also designed it for broad adoption: researchers can use the complete four-color CALIPERS hiPSC line, add FUCCIplex to any existing GFP/RFP reporter line, or combine a FUCCIplex-only hiPSC line with biosensors of their choice. Across all three routes, the open-source FUCCIphase plugin provides a shared analytical backbone, making cell-cycle-aware phenotyping easier to integrate into existing live-cell imaging workflows.