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LC-MS of Craniofacial cartilage organoids from ESCs

ABSTRACT: Knowledge of cell signaling pathways that drive human neural crest differentiation into craniofacial chondrocytes is incomplete, yet essential for using stem cells to regenerate craniomaxillofacial structures. To accelerate translational progress, we developed a differentiation protocol that generated self-organizing craniofacial cartilage organoids from human embryonic stem cell-derived neural crest stem cells. Histological staining of cartilage organoids revealed tissue architecture and staining typical of elastic cartilage. Protein and post-translational modification (PTM) mass spectrometry and snRNASeq data showed that chondrocyte organoids expressed robust levels of cartilage extracellular matrix (ECM) components: many collagens, aggrecan, perlecan, proteoglycans, and elastic fibers. We identified two populations of chondroprogenitor cells, mesenchyme cells and nascent chondrocytes and the growth factors involved in paracrine signaling between them. We show that ECM components secreted by chondrocytes not only create a structurally resilient matrix that defines cartilage, but also play a pivotal autocrine cell signaling role to determine chondrocyte fate.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Stem Cell, Cell Culture

SUBMITTER: TYLER LEVY

LAB HEAD: Mark Grimes

PROVIDER: PXD050668 | Pride | 2024-04-04

REPOSITORIES: Pride

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Project description:Background: Human pluripotent stem cells (hPSCs) give rise to chondrogenic activity in culture. Such activity was found in mesodermal and neural crest like progeny of hPSCs, and readily expanded as SOX9+ msenchymal cells in a medium containing FGF2. However, once expanded, the type of chondrocytes they perfer to form is growth plate-ike transient chondrocytes that are destined to form bone. However, we have recently developed a method to generate GDF5+ mesenchymal cells from such SOX9+ cells which ehibit activity to preferentially form articular-like permanent chondrocytes. The aim of the present prospective follow-up study was to reveal the cell-type and developmental origin of the GDF5+ cells as well as the SOX9+ cells. Methods: Paraxial mesoderm was generated from human PSCs, purified by FACS, and expanded to generate SOX9+ chondroprogenitors. Then, the SOX9+ cells were further differentiated into joint progenitor-like GDF5+ chondroprogenitors. These two chondroprogenitors were subjcted to bulk RNA-seq. Results: Based on the transcriptomic data, we examined differentially expressed genes, cell-type deconvolution analysis, and cell-type correlation analysis. The results suggested that GDF5+ cells might contain a teno/ligamento-genic potential (e.g., SCX+ cells), while SOX9+ cells were related to (neural crest-derived) ectomesenchymal chondroprogenitors. The followup biological analyses indicated as follows. The fast-growing SOX9+ cells formed cartilage that expressed chondrocyte hypertrophy markers and was readily mineralized after ectopic transplantation. In contrast, the slowly-growing GDF5+ cells were derived from SOX9+ cells. They formed cartilage that tended to express low to no chondrocyte hypertrophy markers, while expressing PRG4, a marker of embryonic articular chondrocytes, and to remain largely unmineralized in vivo. Conclusions: Our results imply that GDF5+ cells specified to generate permanent-like cartilage are likely to emerge in association with the commitment of SOX9+ cells to the tendon/ligament lineage.

Project description:The tongue is a muscular organ in the vertebrate oral cavity that performs complex functions in daily life, including feeding and phonetic articulation. The tongue consists of mesenchyme cells of two distinct origins: the muscle cells are derived from occipital somites whereas the tendons and other connective tissues derived from the cranial neural crest. Cranial neural crest cells are important for the initiation of tongue swelling and proper patterning of intrinsic and extrinsic tongue muscle groups. However, little is known regarding the molecular and cellular mechanisms of tongue morphogenesis. We show that the odd-skipped related 1 (Osr1) transcription factor exhibits dynamic expression in the tongue mesenchyme during early tongue development. Tissue-specific inactivation of Osr1 in the early neural crest cells resulted in ectopic cartilage formation in the mouse tongue. We show that Sox9, the master regulator of chondrocyte differentiation, is initially widely expressed in the neural crest derived mesenchyme in the tongue and subsequently down-regulated concomitant by up-regulation of Osr1 expression. Osr1 mutant embryos exhibit persistent expression of Sox9 and chondrocyte differentiation from the neural crest derived tongue mesenchyme. Further biochemical analyses indicate that Osr1 may directly suppresses Sox9 gene expression in the tongue mesenchyme. These data reveal a novel mechanism in suppression of chondrogenic fate during tongue development. Remarkably, the ectopic cartilage in the Osr1 mutant mice resembles the entoglossal cartilage naturally develops in the avian tongue. These results suggest that modulation of expression of Osr1 may underline the evolutionary divergence in tongue cartilage formation. RNAs were isolated from microdissected E12 embryonic mouse tongue of Osr1f/-;Wnt1cre and control littermates and characterized by RNAseq E12 mouse embryonic tongues were micro-dissceted, 3 pairs of control and mutant samples were pooled for the RNA extraction

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LC-MS of Craniofacial cartilage organoids from ESCs

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