A Two-Step Strategy that Combines Epigenetic Modification and Biomechanical Cues to Generate Mammalian Pluripotent Cells

The protocol shown in this video for the first time combines the use of chemical epigenetic erasing with biomechanical cues to induce and maintain pluripotency in adult terminally differentiated cells. The main strength of this protocol is that it does not require transgenic or viral vectors. Moreover, it is robust, highly reproducible, and flexible.

This technique does not require the use of gene transfection, and therefore represents a notable technological advance for translational medicine and cell therapy, while supporting long-term culture of high plasticity cells. Demonstrating the procedure will be Georgia Pennarossa, an assistant professor, and Teresina De Iorio, a PhD student from my laboratory. Begin by preparing fresh one-millimolar 5-aza-CR stock solution.

Weigh 2.44 milligrams of 5-aza-CR and dissolve it in 10 milliliters of DMEM with high glucose. Re-suspend the powder by vortexing and sterilize the solution with a 0.22-micrometer filter. Prepare 5-aza-CR working solution by diluting one microliter of the stock solution in one milliliter of fibroblast culture medium.

Trypsinize the cells as described in the text manuscript and dislodge them with gentle pipetting. Then collect the cell suspension and transfer it into a conical tube. Count cells using a counting chamber under an optical microscope.

Centrifuge the cell suspension at 150 times G for five minutes. Then, remove the supernatant and resuspend the pellet to obtain 40, 000 cells in 30 microliters of fibroblast culture medium supplemented with one-micromolar 5-aza-CR. Fill a 35-millimeter Petri dish with PTFE powder to produce a bed and dispense the 30 microliters of cells onto the powder bed.

Gently rotate the Petri dish in a circular motion to ensure that PTFE powder entirely covers the surface of the liquid drop to form a liquid marble microbioreactor. Pick up the liquid marble microbioreactor using a 1000-microliter pipette tip that has been cut to accommodate the diameter of the marble. Plate the microbioreactor onto a clean bacteriology Petri dish to stabilize it.

Then, transfer the microbioreactor from the Petri dish into a 96-well plate. Slowly add 100 microliters of media from the margin of the well. The microbioreactor should float on top of the media.

Incubate the liquid marble microbioreactor for 18 hours at 37 degrees Celsius in a 5%carbon dioxide incubator. After the incubation, collect the liquid marble microbioreactor using a 1000-microliter pipette tip and place it in a new 35-millimeter bacteriology Petri dish. Use a needle to puncture the liquid marble and break it.

Recover the formed spheroids with a 200-microliter pipette tip that has been cut at the edge under a stereomicroscope. To wash off 5-aza-CR residuals, transfer the organoids into a Petri dish containing ESC medium. Prepare a new 35-millimeter Petri dish containing the PTFE powder bed and dispense a single organoid in a 30-microliter droplet of ESC culture medium onto the powder using a 200-microliter cut pipette tip.

Gently rotate the dish in a circular motion to form a new liquid marble microbioreactor and transfer it into a well of a 96-well plate. Add 100 microliters of media from the margin of the well to slowly bathe the marble. Culture the liquid marble microbioreactors at 37 degrees Celsius and 5%carbon dioxide.

Morphological analyses show that after an 18-hour incubation with the demethylating agent 5-aza-CR, fibroblasts from three mammalian species encapsulated in PTFE microbioreactors aggregated and formed 3D spherical structures with a uniform size geometry. Under 3D conditions, 86.31%of encapsulated cells remarkably modified their phenotype. In contrast, post-5-aza-CR cells cultured in 2D standard conditions were considerably smaller in size, with granulated nuclei and retained a monolayer distribution.

The morphological changes were accompanied by the onset of pluripotency-related gene expression, both in 3D and 2D post-5-aza-CR cells. Transcription was observed for POU class five homeobox one, NANOG homeobox, ZFP42 zinc finger protein, and sex-determining region Ybox2, which are absent in untreated fibroblasts. A significant upregulation of the 10-11 translocation 2 epithelial cell adhesion molecule and cadherin 1 genes was also observed in 3D post-5-aza-CR cells compared to cells cultured in 2D standard plastic dishes.

In parallel, downregulation of the fibroblast-specific marker Thy1 cell surface antigen was detected in both 3D and 2D post-5-aza-CR cells. A significant decrease of methylation levels in both 3D and 2D post-5-aza-CR cells was confirmed with ELISA. DNA methylation levels were significantly lower in 3D post-5-aza-CR cells compared to the cells cultured in 2D conditions.

Furthermore, 3D post-5-aza-CR cells retained the acquired 3D spherical structure, high expression levels of pluripotency-related genes, and low DNA methylation levels for 28 days, at which point culture was arrested. The microbioreactors can also be used to maintain optimal conditions for progenitor cell cultures, as well as to boost terminal differentiation efficiency.