BioProject

Cartilage pellets generated from ectomesenchymal progeny of human pluripotent stem cells (hPSCs) in vitro eventually show signs of commitment of chondrocytes to hypertrophic differentiation. More...

Cartilage pellets generated from ectomesenchymal progeny of human pluripotent stem cells (hPSCs) in vitro eventually show signs of commitment of chondrocytes to hypertrophic differentiation. When transplanted subcutaneously, most of the surviving pellets were fully mineralized by 8 weeks. In contrast, treatment with the adenylyl cyclase activator, forskolin, in vitro resulted in slightly enlarged cartilage pellets containing an increased proportion of proliferating immature chondrocytes that expressed very low levels of hypertrophic/terminally matured chondrocyte-specific genes. Forskolin treatment also enhanced hyaline cartilage formation by reducing type I collagen gene expression and increasing sulfated glycosaminoglycan accumulation in the developed cartilage. Furthermore, forskolin treatment in vitro increased the frequency at which the cartilage pellets maintained unmineralized chondrocytes after subcutaneous transplantation. To elucidate the type of cartilage that forskolin preferentially promotes and potential molecular mechanisms involved in the forskolin-suppression of chondrocyte hypertrophic differentiation in vitro, we performed genome-wide, comparative transcriptomic analyses on the cartilage pellets formed from 3 to 4 independent pellet cultures of ectomesenchymal cells with or without forskolin treatment. RNAs were isolated on day 26-28 and mRNA-sequencing (RNA-seq) analyses were performed. Overall design: Three independent sets of PTB (PDGF+TGFβ3+BMP4) cartilage pellets, and four independent sets of PTBFk (PTB+30 uM forskolin) pellets were prepared from passage 5-8 ectomesenchymal cells (i.e., FSb[FGF2+SB431542]-expanded CD271hiCD73- neural crest-like progeny of H9 hESCs). On day 26-28 of pellet culture, 5 to 6 cartilage pellets were harvested, combined and total RNA was extracted with an RNeasy mini kit (Qiagen). Poly (A)-tailed messenger RNA was enriched using Poly(A)Purist Kit (Ambion, Foster City, CA) before the preparation of the RNA-seq library using an Ultra directional RNA library prep kit for Illumina (New England Biolabs, Ipswich, MA) per manufacturer’s instructions. RNA-seq was performed using the Illumina Nextseq500 with the 150 bp pair-ended running mode. Sequencing reads were aligned against the GRCh37/hg19 reference genome using bowtie2 (Langmead and Salzberg, 2012) with default parameter. Only uniquely mapped reads were used for downstream analysis. HTseq was used to count the read numbers mapped to each gene. DESeq2 (Love et al., 2014) was used to call the significantly differentially expressed genes (fold change ≥2; false discovery rate ≤0.05) between two conditions. The normalized gene counts from DESeq2 was used to do the normalization. Less...