Craniofacial cartilage organoids from human embryonic stem cells via a neural crest cell intermediate

Abstract

Severe birth defects or major injuries to the face require surgical reconstruction and rehabilitation. The ability to make bona fide craniofacial cartilage - cartilage of the head and face - from patient-derived induced pluripotent stem cells (iPSCs) to repair these birth defects and injuries has tremendous translational applications, but is not yet possible. The neural crest is the normal developmental pathway for craniofacial cartilage, however, the knowledge of cell signaling pathways that drive neural crest differentiation into craniofacial chondrocytes is limited. Here we describe a differentiation protocol that generated self-organizing craniofacial cartilage organoids from human embryonic stem cells (hESCs) and IPSCs through a neural crest stem cell (NCSC) intermediate. Histological staining of cartilage organoids revealed tissue architecture typical of hyaline cartilage. Organoids were composed of rounded aggregates of glassy, gray matrix that contained scattered small nuclei in lacunae. Mass spectrometry shows that the organoids express robust levels of cartilage markers including aggrecan, perlecan, proteoglycans, and many collagens. Organoids expressed markers indicative of neural crest lineage, as well as growth factors that are candidates for chondrocyte differentiation factors. The data suggest that chondrocyte differentiation is initiated by autocrine loops driven by a combination of secreted growth factors that bind to chondrocyte receptors. Craniofacial cartilage organoids were continuously cultured for one year, reaching up to one centimeter in diameter. The ability to grow craniofacial cartilage from NCSCs provides insights into the cell signaling mechanisms of differentiation into craniofacial cartilage, which lays the groundwork for understanding mechanistic origins of congenital craniofacial anomalies and repairing cartilaginous structures of the head and face.

Electron cryo-tomography reveals the subcellular architecture of growing axons in human brain organoids

“…Furthermore, a declination of this approach could take advantage of the organoid system to generate defined cell types difficult to isolate from primary samples. For example, one could place neural crest organoids ( Foltz et al, 2021 ) in close proximity to EM grids to capture neural crest cells and study their ultrastructure during migration and differentiation into different neural crest cell derivatives. Such an approach, in combination with thinning of the cells by cryo-focussed ion beam milling ( Mahamid et al, 2016 ), would provide a way to perform cryo-EM studies on physiologically relevant migratory cell types.…”

Cartilage organoids for cartilage development and cartilage-associated disease modeling

“…Intriguingly, recent breakthroughs have uncovered “mini-joint” models comprising of multicellular components and extracellular matrices of joint cartilage for potential realization of novel disease-modifying strategies for personalized therapeutics of cartilage-associated diseases ( Delplace et al, 2021 ; Abraham et al, 2022 ). A recent striking study has developed a novel differentiation protocol that generated self-organizing craniofacial cartilage organoids from human embryonic stem cells via a neural crest cell intermediate ( Foltz et al, 2021 ).…”

Cartilage organoids and osteoarthritis research: a narrative review

“…Lauren et al differentiated ESCs into neural crest stem cells and then further differentiated and self-assembled into craniofacial cartilage organoids (Foltz et al, 2021). Mass spectrometry analysis showed that cartilage organoids highly expressed cartilage marker proteins, providing new insights for studying craniofacial cartilage development and cell signaling (Foltz et al, 2021). However, the ethical consideration over separating ESCs has limited its application.…”