Chondrocyte‐like nested cells in the aged intervertebral disc are late‐stage nucleus pulposus cells

Abstract Aging is a major risk factor of intervertebral disc degeneration and a leading cause of back pain. Pathological changes associated with disc degeneration include the absence of large, vacuolated and reticular‐shaped nucleus pulposus cells, and appearance of smaller cells nested in lacunae. These small nested cells are conventionally described as chondrocyte‐like cells; however, their origin in the intervertebral disc is unknown. Here, using a genetic mouse model and a fate mapping strategy, we have found that the chondrocyte‐like cells in degenerating intervertebral discs are, in fact, nucleus pulposus cells. With aging, the nucleus pulposus cells fuse their cell membranes to form the nested lacunae. Next, we characterized the expression of sonic hedgehog (SHH), crucial for the maintenance of nucleus pulposus cells, and found that as intervertebral discs age and degenerate, expression of SHH and its target Brachyury is gradually lost. The results indicate that the chondrocyte‐like phenotype represents a terminal stage of differentiation preceding loss of nucleus pulposus cells and disc collapse.

Establishing the identity of CLCs, and how they appear in degenerated discs, will help design therapeutics targeted towards them.
Hence, in this study, using a genetic mouse model we lineage-traced NP cells to determine whether they differentiate into CLCs with age.
Previous studies showed appearance of CLCs between one and two years of age in mouse lumbar discs (Winkler, Mahoney, Sinner, Wylie, & Dahia, 2014). Here, we timestamped the appearance of CLCs in mouse lumbar discs between 14 and 28 months (M) of age ( Figure 1 and Table S1). At 14M, the NP cells were reticular but compact; the AF was organized in thin layers; and a clear NP/AF boundary was observed (Figure 1a-c',m and n). By 18M, nested CLCs were observed, and the AF layers were disorganized (Figure 1d-f, m and n). By 20M, the disc was hypocellular with outer AF bulging outwards (black arrow), and the inner AF moving into the NP space (blue arrow, Figure 1g Histopathological scoring indicates that the L5-S1 discs are the most affected ( Figure 1m and n, Tables S2 and S3) and the disc content changed from reticular NP to CLC with aging (Figures 1o and 1 and   Table S4). These age-related changes are similar to those reported in human lumbar discs Weiler et al., 2010). As CLCs were seen at 16 to 18M of age, we chose this time point for lineage tracing studies.
However, as the NP cells were pooled from various discs and may represent NP cells with different phenotypes, qPCR results only show changes in gene expression associated with age, and not with NP phenotype. The results indicate that, although a subset of NP cells continue to express some of its unique molecular markers as they trans-differentiate into a CLC phenotype, the expression of these markers is dramatically reduced, and expression of key signals including SHH was restricted within the lacunae.
Overall, these observations indicate that NP cells trans-differentiate into smaller cells that fuse together to form a nest or syncytium with aging. Furthermore, this differentiation is associated with a decline in expression of key developmental molecules Shh and Bra.
However, the precise mechanism of this differentiation remains uncertain. Previously, blockade of SHH signalling and its conditional targeting in neonatal mouse lumbar discs resulted in loss of BRA expression and change in NP cell phenotype from reticular to small round cells (Dahia et al., 2012). Recently, we observed that SHH and BRA expression reduced more rapidly and by 12 weeks of age in the cranial sacral discs of mouse spines. This, in turn, was associated with a change in NP cell phenotype from reticular-shaped cells to collapsed and rounder cells (Bonavita et al., 2018). Further, conditional activation of hedgehog (Hh) signalling in a subset of NP cells of 12-week-old mice increased BRA expression in all NP cells and reversed the phenotype of NP cells from small and round back to reticular, indicating that in the sacral discs differentiation of NP cells is regulated by SHH signalling. It is likely that decline in SHH expression with physiological aging in the lumbar disc, and restriction of signalling potential within a subset of lacunae, results in loss of Hh response, which drives the differentiation of NP cells into a CLC like phenotype. It is also likely that elevated levels of inflammatory cytokines during normal aging and disc degeneration (reviewed by [Risbud & Shapiro, 2014]) play a role in differentiation of NP cells into CLCs. Future studies aimed at elucidating the molecular mechanism regulating progression of healthy and larger NP cells into their terminally differentiated state will provide a platform for research aimed at preventing disc aging and curing back pain.

ACK N OWLED G M ENTS
The TROMA-III antibody against Krt19 was developed by Kemler, R. was obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242. We thank Dr.
Means, Vanderbilt University, for kindly providing the Krt19CreERT allele and Dr. Joyner, Memorial Sloan Kettering, for providing the ShhLacZ allele. The MTA for the T-nGFP allele was obtained from RIKEN, Japan, and the mice were kindly provided by Dr.

CO N FLI C T O F I NTE R E S T
None declared.

AUTH O R CO NTR I B UTI O N S
CLD conceived and designed the study, interpreted the data and supervised the project. SM, RP, PP and CLD carried out the experiments and generated the data. CLD, SM, PP and TJA analysed the data. CLD and SM wrote the manuscript. All authors reviewed the manuscript and gave their final approval for submission.