Application of Live Mitochondria Staining in Cell-Sorting to Purify Hepatocytes Derived from Human Induced Pluripotent Stem Cells

The scope of our research is to purify human induced pluripotent stem cell-derived hepatocytes using a cell sorter without genetic modification. We aim to address the issue of the tumorigenic risk associated with residual undifferentiated cells among those differentiated from human iP cells. One of the current experimental challenges is a low cell viability due to the mechanical stress of sorting.

We are developing a new purification method that utilizes the unique metabolism features of hepatocytes for large-scale purification. One non-cell sorting method for hepatic progenitor cells cannot obtain pure hepatocytes. Another one for mature hepatocytes can only treat a small fraction of hepatocytes because of the limited expression of the marker protein.

In contrast, our approach can obtain hepatocytes at different maturation levels with a higher E.Human iP cell-derived hepatocytes are extremely immature and show more reduced hepatocyte macro expression after purification. Our future goal is to enhance hepatocyte modulation using organoid technology and feed these advancements to evolve regenerative medicine and drug discovery. To begin, coat each well of a six-well plate with one milliliter of basement membrane matrix and incubate at 37 degrees Celsius for one hour.

Remove the coated matrix from the plate and add human induced pluripotent stem cells prepared in AK02N medium supplemented with 10 micromolar ROCK inhibitor. Incubate the cells at 37 degrees Celsius and 5%carbon dioxide for three days. After three days on day zero of the initiating hepatic differentiation, wash the cells once with RPMI 1640 medium.

Next, to initiate endoderm differentiation, add RPMI B-27 GlutaMAX supplemented with three to six micromolar CHIR-99021 and 100 nanograms-per-milliliter activin-A into the plate. Incubate the cells at 37 degrees Celsius and 5%carbon dioxide for 24 hours. On day one, replace the existing medium with RPMI B-27 GlutaMAX supplemented with 50 nanograms per milliliter of activin-A.

Incubate the cells at 37 degrees Celsius and 5%carbon dioxide for 24 hours. On day two, to induce hepatic progenitor differentiation, replace the existing medium with RPMI B-27 GlutaMAX medium supplemented with 1%dimethyl sulfoxide, and continue incubation for up to five days. On day seven, to initiate hepatocyte maturation, switch to hepatocyte maturation medium and incubate for 20 days at 37 degrees Celsius and 5%carbon dioxide.

On day 27, human induced pluripotent stem cells displayed polygonal cell shapes and rounded nuclei, characteristics of hepatocytes. After 27 days of initiating hepatic differentiation of human induced pluripotent stem cells, wash the cells with two milliliters of PBS per well. Prepare the enzyme solution in ADS buffer using the given components.

Add one milliliter of solution per well of a six-well plate. Place the plate at 37 degrees Celsius on a rotating shaker for about two hours to detach the cells from the plate. Confirm the cell detachment under a microscope.

Then pipette the cell suspension to disperse into single cells and collect them in a 50-milliliter tube containing the hepatocyte maturation medium. Centrifuge the cell suspension at 200G for five minutes at 18 degrees Celsius. And using an aspirator, remove the supernatant.

To stain the mitochondria of cells, resuspend the pellet in five milliliters of hepatocyte maturation medium containing 100 nanomolar TMRM. Incubate the cells for 30 minutes at 37 degrees Celsius while protecting them from light. Again, centrifuge the cell suspension and resuspend the pellet in one to five milliliters of cold ADS buffer containing 2%FBS.

After cell counting, aliquot 500, 000 cells to a 1.5-milliliter tube and label it as the no-primary-antibody control. Centrifuge the remaining cell suspension at 200G for five minutes at four degrees Celsius, and remove the supernatant. Add 100 microliters of diluted primary antibody per one million cells.

Transfer the cell suspension to a 1.5-milliliter tube and incubate on ice for 50 minutes. After incubation, centrifuge the cell suspension and wash the cells twice with cold ADS buffer containing 2%FBS. Now, add 100 microliters of diluted secondary antibody per one million cells to the primary antibody stained or unstained cells, and incubate for 30 minutes on ice.

Again, centrifuge the cell suspension and wash the cells twice with cold ADS buffer containing 2%FBS. After resuspending the cells in ADS buffer, filter them through a snap-cap cell strainer and place the tube on ice. To begin, set up the fluorescence-activated cell sorting or, FACS machine, for cell sorting following the manufacturer's instructions.

Place the sample tube on the FACS machine and load it. Gate TMRM-high and ALCAM-negative population in the P1 region to sort non-hepatocytes. Then gate the TMRM-high and LCAM-positive population in the P2 region to sort hepatocytes.

Collect sorted cells in 15-milliliter collection tubes containing hepatocyte maturation medium. After sorting, remove the collection tube from the FACS machine. Centrifuge the sorted cell suspension at 200G for five minutes at 18 degrees Celsius.

Remove the supernatant and resuspend the pellet in two milliliters of hepatocyte maturation medium with 10 micromolar ROCK inhibitor and 20 nanomolar cyclosporine A.Seed the cells on mitomycin C-treated mouse embryonic fibroblasts with hepatocyte maturation medium in a six-well plate. Culture the cells in a 37 degree Celsius carbon dioxide incubator for five days before immunostaining for hepatocyte purity test. FACS analysis conducted on day 27 of hepatic differentiation revealed TMRM-high and ALCAM-positive population.

The immunohistochemical staining of P1 and P2 cells cultured on mouse embryonic fibroblasts after sorting is shown. A purity test by counting albumin-positive cells showed 0%of P1, and approximately 97%of P2 cells were hepatocyte-like cells.