Differentiation and Characterization of Osteoclasts from Human Induced Pluripotent Stem Cells

Introduction.Osteoclasts are a multinucleated cell type commonly used by researchers in areas such as bone disease research, cancer research, tissue engineering, and endoprosthesis research. Nevertheless, osteoclast differentiation can be challenging, since fusion of precursors is necessary. Additionally, differentiation of human osteoclasts from IPSCs requires the use of a variety of biological factors at the right time.

When it comes to human primary cells. CD34+peripheral blood mononuclear cells are currently the most widely used cell type for the differentiation into osteoclasts. However, this approach is limited by the heterogeneity within the CD34+population and their limited expandability.

Human IPSCs present an alternative source for osteoclasts. As they can be propagated indefinitely, they allow for expandability and upscaling of osteoclast production. This allows for differentiation of large numbers of osteoclasts, which facilitates osteoclast research.

Here, our postdoc Alexander Blumke and PhD student Jessica Simon, demonstrate the differentiation of osteoclasts from human IPSCs Passaging IPSCs. Begin passaging IPSCs by removing differentiated regions or regions with many dead IPSC aggregates under the stereomicroscope by using a P-10 or P-20 pipette tip or a cell scraper. Differentiated regions will appear denser and whiter in color.

Wash loose the detached cells with old medium, then wash away any remaining detached cells with PBS twice. Next, add one milliliter of five units per milliliter dispase per well to the well. The edges of the colonies lifting off from the dish can be observed under the stereomicroscope after three to five minutes.

Carefully remove the dispase and add one milliliter of DMEM/F-12 with 15-millimolar HEPES. Use a stem cell passaging tool or a disposable cell lifter to cut the aggregates into small size. Transfer the media with aggregates into a 15-milliliter conical tube using a five-milliliter serological pipette or a P-1000 with wide-bore tip.

Rinse the well with DMEM/F-12 and transfer media to the 15-milliliter tube. Centrifuge the sliced IPSC aggregates at 200 G for three minutes. Remove the supernatant with a Pasteur glass pipette and add two milliliters of human IPSC serum-free medium to dislodge and resuspend the IPSCs using a five-milliliter serological pipette or P-1000 with wide-bore tips.

Aspirate the leftover basal membrane extract from the pre-coated well plates, and add one milliliter of human IPSC serum-free medium to each well of the six-well plate. Transfer IPSCs into new wells of a six-well plate so that the final volume is two milliliters of human IPSC serum-free medium per well using a P-1000 with wide-bore tips. Depending on the IPSC line, split ratios need to be optimized.

Here, a 1:6 split ratio was used. Swirl the well plate to distribute the cells evenly across the plate after the aggregates have been transferred. Embryoid body induction.

Aspirate the old medium from the IPSC cultures and rinse the wells with DPBS. Add half a milliliter of prewarmed to room temperature into each well of a six-well plate and swirl the culture vessel to coat the entire well surface. Incubate the culture vessel at 37 degrees C for five to eight minutes.

Remove the vessel from the incubator, aspirate the solution, and add stage one medium to wells consisting of 50 nanograms per milliliter human bone morphogenic protein 4, 50 nanograms per milliliter human vascular endothelial growth factor 165, 20 nanograms per milliliter human stem cell factor, and 10 micromolar ROCK inhibitor Y-27632. Gently detach cells by rinsing the well with stage one medium. Pull cells into a conical tube.

Add new stage one medium to wells and detach any remaining cells using a cell scraper. After transferring all cells to the tube, centrifuge at 200 G for five minutes at room temperature to pellet the cells. Aspirate and resuspend the cells in a total of two milliliters pre-equilibrated stage one medium.

Count cells using a hemocytometer or automated cell counting device Seed 12, 500 cells per well in a round-bottom ultralow-attachment 96-well plate in 100 microliters of stage one medium. Under the microscope, cells will appear diffusely throughout the solution. Centrifuge the 96-well plates for three minutes at 100 G After centrifuging, the cells should begin to resemble spheroids when viewed under the microscope.

Place the plate in a 37 degrees C incubator. Change half the medium on day one and day two with stage one media. Use a multichannel pipette to dispose old medium into a Petri dish.

After disposing of media from the 96-well plate, check for EBs which might have been accidentally removed. Transfer the accidentally removed EBs in the Petri dish back using a P-1000 pipette Hematopoietic differentiation. Pre-fill wells of a six-well plate with three milliliters of stage two medium, to which two-millimolar ultraglutamine, 55-micromolar beta-mercaptoethanol, 25 nanograms per milliliter, human interleukin-3, and 100 nanograms per milliliter human macrophage colony stimulating factor is added.

Using a P-1000 and wide-bore tip, transfer eight EBs into each well of the six-well plate. After transferring, check by eye are under the stereo microscope that eight EBs are in each well. After five to seven days, a floating cell population comprised of hematopoietic cells should become visible.

The hematopoietic differentiation period can be varied, starting with seven days. Differentiation for 10 days showed a hematopoietic population comprised of large CD45+CD14+and CD11b+subpopulations. After five days of treatment with stage two medium, perform a medium change by carefully removing the old medium and dispensing it into a 50-milliliter conical tube.

Immediately add one milliliter of new stage two medium to the wells. In order to save any floating hematopoietic cells that may already be present, spin down the tube with the old medium at 300 G for five minutes. Add new stage two medium to the tube and resuspend in order to detach cells that might have been transferred with the old medium.

Add two milliliters of new stage two medium with saved cells into each well, which was previously filled with one milliliter of stage two medium. At day 10 of hematopoietic differentiation, floating hematopoietic cells are harvested by collecting them into a 50-milliliter conical tube and can either be frozen back by using 10%DMSO, 50%FBS, and 40%medium, or immediately used to further differentiate cells into osteoclasts. M-CSF maturation and osteoclast differentiation.

Seed cells at 200, 000 cells per centimeter squared and treat for three days with alpha MEM supplemented with 10%FBS and 50 nanograms per milliliter M-CSF. In order to perform functional assays or imaging, detach the MCSF-matured cells by washing with a P-1000 with wide-bore tip. Transfer detached cells with old medium into a tube and spin down at 300 G for five minutes.

Resuspend and dissolve the cell pellet with fresh alpha MEM supplemented with 10%FBS, 50 nanograms per milliliter M-CSF, and 80 nanograms per milliliter RANK ligand. Reseed cells at 200, 000 cells per centimeter squared and differentiate for seven to nine days. Multinucleated osteoclasts typically appear around day five to seven.

Representative results. The composition of the generated hematopoietic cell population can be analyzed using flow cytometry. Here, hematopoietic cells show a large population of CD45+precursor cells.

Cell populations expressing the monocyte markers CD14 and CD11b usually make up a third of the entire population when compared to isotype controls. Terminally differentiated IPSC-derived osteoclast can be viewed under the microscope as TRAP-positive multinucleated cells. Confocal microscopy shows cells staining for cathepsin K.Functional assays such as bone or mineral absorption assays allow for further quantification of osteoclast activity.

Here, resorption pits are visible after removing mature osteoclast with 10%bleach prior to imaging, while M-CSF-matured osteoclast precursors treated without the addition of RANK ligand do not show resorption pits.Conclusion. This protocol enables the robust and reliable differentiation of human osteoclasts and could provide an off-the-shelf solution for bone diseases or diseases involving excess calcification. We use osteoclasts to further engineer them into our RANK cells that use an intracellular signaling domain that can be activated in conjunction with a chemical inducer of dimerization for controlled and osteoprotegerin-independent osteoclast activation.