Craniofacial cartilage differentiation from human neural crest stem cells in 3D cultures

Craniofacial cartilage differentiation from human neural crest stem cells in 3D cultures Birth defects and injuries to the head and face require surgical reconstruction and rehabilitation. It is difficult to reconstruct cartilaginous features (nose, ears) with plastic surgery techniques, and transplanted tissue is often rejected without

immunosuppressants. The ability to make bona fide craniofacial cartilage – cartilage of the head and face – from patient-derived induced-pluripotent stem (iPS) cells to repair these birth defects and injuries has tremendous translational applications, but is not yet possible because we don't know enough about the mechanisms of differentiation that

form this tissue in human beings. Using stem cells is the key to understanding human- specific cell signaling mechanisms that drive differentiation of craniofacial chondrocytes, the cells that make cartilage. We have grown craniofacial cartilage organoids from human neural crest stem cells (NCSCs) derived from embryonic stem cells (hESCs)

and induced-pluripotent stem cells (iPSCs). We have initiated detailed characterization of gene expression at the RNA and protein level in these organoids with an eye towards generating specific hypotheses about mechanisms of differentiation that may be manipulated for recellularization of damaged or defective craniofacial cartilage. Our

strategy involves seeding cells in three dimensional (3D) matrices (hydrogels) together with extracellular matrix (ECM) components that we have identified in organoids. We focus on cells cultured in 3D scaffolds because we hypothesize that craniofacial cartilage generation will be more rapid and reproducible in 3D constructed organoids

than in self-organizing organoids. We hypothesize that, in addition to being components of the structurally resilient matrix that defines cartilage, the ECM components that we identified play a profound cell signaling role in chondrocyte differentiation. A second strategy is to target cell signaling pathways that are hypothesized to play a role in

specifying chondrocyte cell fate. We have identified a number of growth factors and their receptors in two populations of cells that generate cartilage, mesenchyme cells and nascent chondrocytes. We hypothesize that adding these growth factors will activate cell signaling pathways to make cartilage differentiation more rapid and

reproducible in 3D cultures. This project will combine NCSC differentiation with 3D bioprinting with an eye towards scaling up to grow functional, transplantable craniofacial cartilage in the lab. These studies will considerably advance an innovative approach to tissue engineering towards the long-term goal of building a nose, ear, or other

cartilaginous structures of the head and face using human stem cells.