iPSCs hold great promise for reconstruction of composite tissue defects in the craniofacial region as they represent a powerful tool capable of regenerating skin, muscle, nerve, and bone deficiencies found in both congenital and acquired disorders (). Importantly, these cells are not subject to the more restrictive lineage differentiation pathways noted with mesenchymal cells and still offer the same potential for development of patient-specific therapies. A number of studies have begun to evaluate the capacity of iPSCs for tissue engineering purposes, and as the safety profile improves, translation into the clinical realm has become increasingly possible.
Figure 2 iPSCs can generate numerous tissues necessary for craniofacial reconstruction including skin, muscle, nerve, and bone. Complex tissue deficiencies from congenital malformations, traumatic defects, and tumor resection may be addressed through cellular-based (more ...)
The first iPSCs were generated using embryonic and adult fibroblasts and many studies have since demonstrated potential application for this technology in the treatment of skin disorders and wound healing. iPSCs have been shown to be capable of differentiation along multiple cutaneous lineages including both keratinocytes and melanocytes. Through sequential application of retinoic acid and bone morphogenetic protein (BMP)-4 to iPSCs cultured on collagen IV-coated plates, in vitro
keratinocyte cultures can be established.35
Furthermore, by subsequently seeding iPSC-derived keratinocytes onto type I collagen matrices, three dimensional skin equivalents have been generated exhibiting a multilayered epidermis with an outer cornified surface.36
In mice, iPSCs have been shown to be capable of reconstituting normal skin with a fully differentiated epidermis, hair follicles, and sebaceous glands.35
Epidermal melanocytes have also been generated in vitro
from iPSCs through the supplementation of culture medium with Wnt3a, stem cell factor, and endothelin-3. On gene analysis, multiple melanocyte markers could be readily detected after seven weeks, and by transmission electron microscopy, melanosomes could be observed in the pigmented cells.37
Together, these studies thus highlight the potential for iPSCs to generate functional, patient-specific skin equivalents that may be employed to treat both a variety of skin disorders and situations involving large skin deficits.
The diverse capacity of iPSCs to generate a variety of different tissues has also been underscored by multiple studies showing the ability for these cells to form skeletal muscle. Investigators have described the growth of iPSCs on low attachment culture plates in the presence of horse serum and β-mercaptoethanol to result in development of embryoid bodies which give rise to contractile spindle fibers.38-39
Interestingly, these fibers functioned spontaneously, stained positively for myosin heavy-chain, and on electron microscopy were found to demonstrate characteristic sarcomere features including Z-lines, I-bands, A-bands, and H-bands.38
More importantly, preliminary animal studies have shown iPSC-derived muscle cells to engraft and sustain their myogenic lineage differentiation following injection.39
And when these cells were implanted into a damaged tibialis anterior muscle in mice, significantly increased isometric tetanic force could be detected.40
Such studies suggest a role for iPSCs in the treatment of injured or deficient muscle. Whether from congenital or acquired etiologies, regenerative strategies using iPSCs may one day offer a treatment option for patients with insufficient functional skeletal muscle in the craniofacial region.
Relative to these potential applications for iPSCs however, neural differentiation represents one of the largest areas of interest for use of these pluripotent cells. In light of the limited available therapeutic modalities to manage damaged or degenerative neural conditions, this is one particular field where iPSCs hold significant promise. As already mentioned, protocols have been developed to generate iPSC-derived neurospheres capable of subsequent tri-lineage differentiation into neurons, astrocytes, and oligodendrocytes.31
Studies have reprogrammed human dermal fibroblasts to give rise to cells which demonstrate electrophysiological characteristics similar to functional neurons. Robust resting membrane potentials, large fast tetrodotoxin-sensitive action potentials, and voltage-gated sodium currents have all been described in these differentiated cells.41
The in vivo
utility of iPSC-derived neurons has also been investigated in animal studies. In particular, Wernig and colleagues employed Sonic Hedgehog and FGF8 to further differentiate neurons into a midbrain dopamine-producing subtype.42
Subsequent implantation into a rat model of Parkinson's disease demonstrated successful synaptic integration. In addition, marked improvement was noted in the behavior of rats receiving iPSC treatment.42
Given these findings, future approaches employing iPSCs may hold substantial impact for various neurological disorders. Several phase I/II clinical trials utilizing ESCs are already underway, and as iPSCs offer the potential advantage for patient-specific therapy, one can imagine the development of novel, functional, and safer treatment options for patients with craniofacial nerve deficits or other debilitating neurologic diseases.
Finally, with craniofacial reconstruction, large skeletal defects can be among the most difficult for surgeons to address. Current strategies including autologous bone grafting or use of allogeneic / alloplastic materials are often associated with donor site morbidity and a variety of complications. In their stead, iPSCs represent a potent building block for regenerative strategies aimed at forming novel bone. In vitro
differentiation of iPSCs along mesenchymal lineages has been thoroughly demonstrated, with standard osteogenic differentiation medium containing β-glycerophosphate and ascorbic acid being capable of promoting osteogenesis in these cells.43
Furthermore, Tashiro et al. found transduction of iPSCs with Runx2 to further accelerate this process. And when iPSCs have been implanted into various skeletal defects in mice, de novo
bone formation has been reported. In combination with silk scaffolds and enamel matrix derivatives, iPSCs were found to promote enhanced alveolar bone regeneration, formation of both cementum and periodontal ligaments.44
Using Special AT-rich sequence-binding protein 2 transduced iPSCs, Ye and colleagues were also able to demonstrate significantly increased bone formation in critical-sized calvarial defects.45
Interestingly, no teratomas were noted in animals receiving this genetically manipulated iPSC. Therefore, these studies confirm the potential for iPSCs in bone regenerative strategies. Future studies will undoubtedly look to further promote this osteogenic capacity through guided differentiation while simultaneously enhancing their safety profile by limiting risk of teratoma formation.