Notochordal Differentiation and Integrative Transcriptomic Analysis Using Human Pluripotent Stem Cells

Progressive loss of nucleus pulposus cells (NPCs) is associated with the onset of intervertebral disc degeneration (IDD). Transplantation of NPCs, derived from human pluripotent stem cells including ESC/iPSCs, may offer a novel therapy for IDD. Nonetheless notochordal differentiation and NPC generation from human ESC/iPSC are poorly understood. We developed a three-step protocol to directly differentiate ESC/iPSC towards mesodermal, then notochordal and finally NPCs. Our results showed that notochordal-like cells (NCCs) were successfully derived from the first two-steps of the protocol. Furthermore, these cells could be differentiated into NPCs. These NPCs expressed the tyrosine kinase receptor Tie2 (Tie2), disialoganglioside 2 (GD2), Collagen II and Aggrecan. Genome-wide transcriptomic analyses by sequencing (RNA-seq) revealed the expression of a wide array of extra-cellular matrix (ECM) genes, up-stream regulators and pathways. Cross-comparison of our RNA-seq profiles with human body-derived NPC data confirmed the differentiated products were more similar to NPCs than ESC/iPSCs. Transplantation of NPCs effectively attenuated disc injury in a rat model of IDD. We utilized CRISPR/Cas9 to seamlessly knock in an enhanced fluorescent protein (GFP) to the loci of the Noto gene in ESCs for NCC generation. Our study achieved effective notochordal differentiation and transcriptomic insight into the use of human ESC/iPSCs.