Comparison of osteoclast diferentiation protocols from human induced pluripotent stem cells of diferent tissue origins

Background Ever since their discovery, induced pluripotent stem cells (iPSCs) have been extensively diferentiated into a large variety of cell types. However, a limited amount of work has been dedicated to diferentiating iPSCs into osteoclasts. While several diferentiation protocols have been published, it remains unclear which protocols
or diferentiation methods are preferable regarding the diferentiation of osteoclasts.
Methods In this study, we compared the osteoclastogenesis capacity of a peripheral blood mononuclear cell
(PBMC)-derived iPSC line to a fbroblast-derived iPSC line in conjunction with either embryoid body-based or monolayer-based diferentiation strategies. Both cell lines and diferentiation protocols were investigated regarding their
ability to generate osteoclasts and their inherent robustness and ease of use. The ability of both cell lines to remain undiferentiated while propagating using a feeder-free system was assessed using alkaline phosphatase staining. This was followed by evaluating mesodermal diferentiation and the characterization of hematopoietic progenitor cells using fow cytometry. Finally, osteoclast yield and functionality based on resorptive activity, Cathepsin K and tartrateresistant acid phosphatase (TRAP) expression were assessed. The results were validated using qRT-PCR throughout the diferentiation stages.
Results Embryoid body-based diferentiation yielded CD45+, CD14+, CD11b+ subpopulations which in turn differentiated into osteoclasts which demonstrated TRAP positivity, Cathepsin K expression and mineral resorptive
capabilities. This was regardless of which iPSC line was used. Monolayer-based diferentiation yielded lower quantities
of hematopoietic cells that were mostly CD34+ and did not subsequently diferentiate into osteoclasts.
Conclusions The outcome of this study demonstrates the successful diferentiation of osteoclasts from iPSCs in conjunction with the embryoid-based diferentiation method, while the monolayer-based method did not yield osteoclasts. No diferences were observed regarding osteoclast diferentiation between the PBMC and fbroblast-derived
iPSC lines.