Derivation of notochordal cells from human embryonic stem cells reveals unique regulatory networks by single cell-transcriptomics.

Intervertebral disc degeneration (IDD) is a public health dilemma as it is associated with low back and neck pain, a frequent reason for patients to visit the physician. During IDD, nucleus pulposus (NP), the central compartment of intervertebral disc (IVD) undergo degeneration. Stem cells have been adopted as a promising biological source to regenerate the IVD and restore its function. Here, we describe a simple, two-step differentiation strategy using a cocktail of four factors (LDN, AGN, FGF, and CHIR) for efficient derivation of notochordal cells from human embryonic stem cells (hESCs). We employed a CRISPR/Cas9 based genome-editing approach to knock-in the mCherry reporter vector upstream of the 3' untranslated region of the Noto gene in H9-hESCs and monitored notochordal cell differentiation. Our data show that treatment of H9-hESCs with the above-mentioned four factors for 6 days successfully resulted in notochordal cells. These cells were characterized by morphology, immunostaining, and gene and protein expression analyses for established notochordal cell markers including FoxA2, SHH, and Brachyury. Additionally, pan-genomic high-throughput single cell RNA-sequencing revealed an efficient and robust notochordal differentiation. We further identified a key regulatory network consisting of eight candidate genes encoding transcription factors including PAX6, GDF3, FOXD3, TDGF1, and SOX5, which are considered as potential drivers of notochordal differentiation. This is the first single cell transcriptomic analysis of notochordal cells derived from hESCs. The ability to efficiently obtain notochordal cells from pluripotent stem cells provides an additional tool to develop new cell-based therapies for the treatment of IDD.

Single-cell RNA-seq identifies unique transcriptional landscapes of human nucleus pulposus and annulus fibrosus cells.

Intervertebral disc (IVD) disease (IDD) is a complex, multifactorial disease. While various aspects of IDD progression have been reported, the underlying molecular pathways and transcriptional networks that govern the maintenance of healthy nucleus pulposus (NP) and annulus fibrosus (AF) have not been fully elucidated. We defined the transcriptome map of healthy human IVD by performing single-cell RNA-sequencing (scRNA-seq) in primary AF and NP cells isolated from non-degenerated lumbar disc. Our systematic and comprehensive analyses revealed distinct genetic architecture of human NP and AF compartments and identified 2,196 differentially expressed genes. Gene enrichment analysis showed that SFRP1, BIRC5, CYTL1, ESM1 and CCNB2 genes were highly expressed in the AF cells; whereas, COL2A1, DSC3, COL9A3, COL11A1, and ANGPTL7 were mostly expressed in the NP cells. Further, functional annotation clustering analysis revealed the enrichment of receptor signaling pathways genes in AF cells, while NP cells showed high expression of genes related to the protein synthesis machinery. Subsequent interaction network analysis revealed a structured network of extracellular matrix genes in NP compartments. Our regulatory network analysis identified FOXM1 and KDM4E as signature transcription factor of AF and NP respectively, which might be involved in the regulation of core genes of AF and NP transcriptome.