Differentiation of notochordal-like cells from human pluripotent stem cells

Abstract

Exhaustion of notochordal-like cells (NCCs) plays an essential role in the development of intervertebral disc degeneration (IDD). Cell-based therapy has emerged as a novel strategy of regenerative medicine for many tissues and organs, including IDD. Human pluripotent stem cells (ESC/iPSCs) offer the possibility of generating individual-specific NCCs. This study was to define strategies to derive NCCs from human ESC/iPSC. hESC7 and hESC9 were used for a two-step protocol to differentiate into NCCs. Activin A was used in step 1 for 3 days. Activin A, DKK1, Noggin, FGF2 and AGN193109 were used in step 2 for further 5days. Immunofluorescence staining was performed after differentiation. Our results showed that NCCs were successfully given rise from above protocol induced differentiating human ESC confirmed by immunofluorescence staining of Noto, Brachyury and Foxa2. The differentiation efficiency was only around 5%. Moreover, PI3-kinase inhibitor didn’t enhance the differentiation efficiency of NCCs from human ESC. Co-culturing NCCs with Light2 cell line, a luciferase-based reporter responsive to Hedgehog (Hh) secreted protein by NCCs, was examined. Further differentiation of NCCs into nucleus pulpous progenitor cells (NPCs) was detected by expression of Tie2 (Tie2) and disialoganglioside 2 (GD2). Notochordal-like cells could be induced from human pluripotent stem cells through regulation of retinoic acid, BMP and Wnt signaling. Moreover, NCCs differentiation from human pluripotent stem cells is functional in Shh signaling and NPCs differentiation.