Long-Term Expandable SOX9+ Chondrogenic Ectomesenchymal Cells from Human Pluripotent Stem Cells

Abstract

SummaryHere we report the successful generation and long-term expansion of SOX9-expressing CD271+PDGFRα+CD73+ chondrogenic ectomesenchymal cells from the PAX3/SOX10/FOXD3-expressing MIXL1−CD271hiPDGFRαloCD73− neural crest-like progeny of human pluripotent stem cells in a chemically defined medium supplemented with Nodal/Activin/transforming growth factorβ (TGFβ) inhibitor and fibroblast growth factor (FGF). When “primed” with TGFβ, such cells efficiently formed translucent cartilage particles, which were completely mineralized in 12 weeks in immunocompromized mice. The ectomesenchymal cells were expandable without loss of chondrogenic potential for at least 16 passages. They maintained normal karyotype for at least 10 passages and expressed genes representing embryonic progenitors (SOX4/12, LIN28A/B), cranial mesenchyme (ALX1/3/4), and chondroprogenitors (SOX9, COL2A1) of neural crest origin (SOX8/9, NGFR, NES). Ectomesenchyme is a source of many craniofacial bone and cartilage structures. The method we describe for obtaining a large quantity of human ectomesenchymal cells will help to model craniofacial disorders in vitro and potentially provide cells for the repair of craniofacial damage.

Step-Wise Chondrogenesis of Human Induced Pluripotent Stem Cells and Purification Via a Reporter Allele Generated by CRISPR-Cas9 Genome Editing

“…Our laboratory and others have demonstrated the utility of CPs as an expandable depot of cells for subsequent chondrogenesis [20,49]. We show that the day 21 purified CPs (GFP high population) maintain improved chondrogenic capacity over the course of 5 passages compared to controls.…”

“…Indeed, the heterogeneity of in vitro chondrogenesis may preclude our understanding of the mechanisms by which genetic variation can impact cartilage development and arthritis progression. Previous attempts to purify iPSC-derived CPs relied on many rounds of sorting multiple surface markers, using reporter constructs that are activated by minimal promoter regions of chondrocyte markers, or physical dissection of self-aggregating chondroprogenitors [20,29,37,49,50]. During chondrocyte differentiation, COL2A1 expression is regulated by concerted coordination of promoter regions, distal and intronic enhancers, and UTRs [51–53].…”

Specification and formation of the neural crest: Perspectives on lineage segregation

“…Recently, the enormous progress in pluripotent stem cell biology has permeated to neural crest studies (Chambers, Mica, Lee, Studer, & Tomishima, ; Fukuta et al, ; Hackland et al, ; Leung et al, ; Mica, Lee, Chambers, Tomishima, & Studer, ; Rada‐Iglesias et al, ; Umeda et al, ). These models, to a great extent, have confirmed the findings of the few human embryological studies (Betters et al, ; O'Rahilly & Müller, ), which remain restricted due to technical and ethical limitations.…”

WNT/β-catenin signaling mediates human neural crest induction via a pre-neural border intermediate

“…We establish a culture system that generates human NC cells validated with markers demonstrated in human embryos (Betters et al, 2010). Furthermore, our protocol demonstrates cost effectiveness, high efficiency and unprecedented speed, and dispenses with the use of serum replacement, heregulin, activin antagonists, and the pre-differentiation (ESC expansion) period employed by previous protocols Fukuta et al, 2014;Menendez et al, 2011;Mica et al, 2013;Umeda et al, 2015). Our study demonstrates a key role for canonical WNT signaling in human NC formation, confirming contributions from bone morphogenetic protein (BMP) and fibroblast growth factor (FGF) pathways and supports an independent origin of NC from PAX6 + neural progenitors.…”