iPSC Technology and Bone Regeneration

Bone is a calcified tissue with multiple functions. It allows movement by providing attachment points for skeletal muscle, supports haematopoiesis, stores minerals and protects soft tissues and organs. Additionally, bone has the unique capacity to regenerate upon damage without scar formation or loss of function.

Bone fracture repair recapitulates embryonic endochondral bone formation. This process is characterized by the formation of a cartilaginous template (soft callus) that precedes bone formation. This template is considered to be one of the most important tissue intermediates during endochondral ossification as it provides a structural framework for osteogenesis and it allows recruitment of osteogenic cells and blood vessels.

Despite this regenerative capacity, bone (fracture) healing is often impaired due to local and congenital factors. 10% of all fractures are healing poorly or result in a non-union. Bone tissue engineering aims to provide a solution by combining stem cell biology with engineering science. Typically, skeletal stem cells are combined with growth factors and seeded on scaffolds to induce bone formation. However, current therapeutic strategies are being hampered by the lack of robust and reproducible cell populations, which display favourable differentiation and proliferative properties. Fortunately, cellular reprogramming has provided an alternative and powerful strategy for the derivation of robust cell populations for medicinal research and clinical therapies.

Cellular reprogramming is a strategy in which cells can be converted to alternative cell types through the forced expression of transcription factors that control cell fate. Activation of these factors can lead to the reintroduction of embryonic pluripotency in adult terminally differentiated cell types thus resulting in the derivation of induced pluripotent stem cells. These stem cells can then be further specialized into skeletal cells for bone regeneration (indirect target cell derivation). However, instead of reacquiring pluripotency, cellular reprogramming strategies can be adapted whereby the cell of choice can be directly obtained through the forced expression of cell type specific transcription factors (direct cell reprogramming). Both strategies have been explored within this PhD project and different skeletal cell types have been derived for bone augmentation.

Murine skin fibroblast cells were directly reprogrammed to cartilage cells (chondrocytes) by using Sox9, KLF4 and cMYC. Two chondrogenic cells lines were derived through either constitutive or doxycycline inducible expression of these transcription factors. Although both cell lines displayed in vitro chondrogenic differentiation capacities, cells reprogrammed with inducible transcription factors were able to undergo hypertrophic maturation and could induce bone formation.

To translate these findings towards human cells, (induced) pluripotent stem cells were differentiated towards cartilaginous aggregates. Subsequently upon hypertrophic stimulation, soft callus-like tissue could be obtained. When implanted, these tissue intermediates allowed progressive healing of critical size long bone defects. These results support the concept of developmental tissue engineering strategies for bone regeneration.

In the last part of this PhD, it has been demonstrated that embryonic limb progenitors could be in vitro expanded. When implanted, these cells spontaneously

gave rise to endochondral ossification. To achieve potential clinical translation, culture and differentiation conditions have been established which allowed us to derive these progenitors from induced pluripotent stem cells. These experiments further highlight the promise of cellular reprogramming for the creation of skeletal progenitor cells for novel bone tissue engineering strategies.

Collectively, the findings presented within this dissertation are not only contributing to the in depth knowledge of skeletal tissue regeneration and skeletal stem cell biology, but also demonstrate the potential of cellular reprogramming for the derivation of novel (stem) cell populations capable of triggering bone repair.