GSE131065
bulk RNA-seq of human nucleusus pulposus from scoliosis patients
Intervertebral disc degeneration (IDD) leads to low back pain and disability globally. Progressive loss of nucleus pulposus cells (NPCs) are associated with the onset of IDD. Cell-based therapy has been shown the promising for many diseases, including the IDD in preclinical studies. However, the limited availability of human NPCs has hurdled such application for IDD. This study aimed to define strategies to derive NPCs from human ESC/iPSC. Human ESC3, ESC9 and IMR-90-iPSC were used for a three-step protocol to direct differentiation toward mesodermal, subsequently notochordal and finally NPCs. Our results showed that notochordal-like cells (NCCs) were successfully derived from the first two-step of protocol confirmed by immunofluorescence staining of Noto, Brachyury (T) and Foxa2. RT-PCR results showed that the expression of Noto, T and Foxa2 was the highest at day 5 after differentiation. Furthermore, these NCCs can be differentiated into NPCs via the final step of the protocol. These NPCs expressed the tyrosine kinase receptor Tie2 (Tie2), disialoganglioside 2 (GD2), collagen II and aggrecan. Genome-wide transcriptomic analyses by sequencing (RNA-seq) further reveals 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 public NPC data confirms the differentiated products are much closer to NPCs than are ESC/iPSC. Co-culturing NPCs with Light2 cell line, a luciferase-based reporter responsive to sonic hedgehog (Shh) secreted protein, showed NPCs synthesized Shh. Transplantation of NPCs effectively attenuated the spinal injury in a puncture-induced disc degeneration (PDD) model in rat. Furthermore, we utilized CRISPR/Cas9 technology to seamlessly knock in an enhanced fluorescent protein (GFP) to the loci of the noto gene in ESC9. Our study demonstrates that NPCs could be induced from human ESC/iPSC. This study provides a cell source for the development of novel strategy for IDD diseases. Overall design: We sequenced four NP from three scoliosis individuals