Human notochordal cell transcriptome unveils potential regulators of cell function in the developing intervertebral disc

Ricardo Rodrigues-Pinto,Leo A H Zeef,Judith A Hoyland,Lizzy Ward,Andrew Berry,Karen Piper Hanley,Neil Hanley,Matthew Humphreys,Stephen M Richardson

doi:10.1038/s41598-018-31172-4

Abstract

The adult nucleus pulposus originates from the embryonic notochord, but loss of notochordal cells with skeletal maturity in humans is thought to contribute to the onset of intervertebral disc degeneration. Thus, defining the phenotype of human embryonic/fetal notochordal cells is essential for understanding their roles and for development of novel therapies. However, a detailed transcriptomic profiling of human notochordal cells has never been achieved. In this study, the notochord-specific marker CD24 was used to specifically label and isolate (using FACS) notochordal cells from human embryonic and fetal spines (7.5–14 weeks post-conception). Microarray analysis and qPCR validation identified CD24, STMN2, RTN1, PRPH, CXCL12, IGF1, MAP1B, ISL1, CLDN1 and THBS2 as notochord-specific markers. Expression of these markers was confirmed in nucleus pulposus cells from aged and degenerate discs. Ingenuity pathway analysis revealed molecules involved in inhibition of vascularisation (WISP2, Noggin and EDN2) and inflammation (IL1-RN) to be master regulators of notochordal genes. Importantly, this study has, for the first time, defined the human notochordal cell transcriptome and suggests inhibition of inflammation and vascularisation may be key roles for notochordal cells during intervertebral disc development. The molecules and pathways identified in this study have potential for use in developing strategies to retard/prevent disc degeneration, or regenerate tissue.

Highlights

To enable the development of novel biological or cell-based therapies for disc degeneration it is essential to characterise the pathways and processes involved in IVD development, maturation and degeneration
While studies have been undertaken using animal models[11,12,13,14,15,16,17,18], to date the human notochordal cell phenotype has not been characterised in detail and this lack of understanding of human notochordal cell phenotype and biology is a major limitation in the field
In a pivotal study using human embryonic and fetal spines, we have recently shown that the developing nucleus pulposus (NP) is composed of large vacuolated notochordal cells and that keratin (KRT) 8, KRT18, KRT19 are uniquely expressed by notochordal cells at all spine levels investigated at all stages studied (Carnegie Stage 10 (equivalent to 3.5 weeks post-conception (WPC)) to 18 WPC), with CD24 being uniquely expressed at all stages except 3.5 WPC19

Summary

Introduction

To enable the development of novel biological or cell-based therapies for disc degeneration it is essential to characterise the pathways and processes involved in IVD development, maturation and degeneration. Fetal and juvenile human IVD the nucleus pulposus (NP) is populated by large vacuolated notochordal cells, the adult disc is populated by small non-vacuolated chondrocyte-like cells (reviewed in[5]). Understanding the phenotype of notochordal cells and their potential regulatory molecules will help identify factors important in maintaining healthy disc homeostasis which may be exploited in the development of novel biological/regenerative therapies. While studies have been undertaken using animal models[11,12,13,14,15,16,17,18], to date the human notochordal cell phenotype has not been characterised in detail and this lack of understanding of human notochordal cell phenotype and biology is a major limitation in the field. The unique expression of these markers makes them suitable for use in identification and isolation of notochordal cells from human embryos and foetuses and CD24, being a cell-surface marker, allows for the isolation of viable notochordal cells

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