Notochordal Cell-Based Treatment Strategies and Their Potential in Intervertebral Disc Regeneration

Abstract

Chronic low back pain is the number one cause of years lived with disability. In about 40% of patients, chronic lower back pain is related to intervertebral disc (IVD) degeneration. The standard-of-care focuses on symptomatic relief, while surgery is the last resort. Emerging therapeutic strategies target the underlying cause of IVD degeneration and increasingly focus on the relatively overlooked notochordal cells (NCs). NCs are derived from the notochord and once the notochord regresses they remain in the core of the developing IVD, the nucleus pulposus. The large vacuolated NCs rapidly decline after birth and are replaced by the smaller nucleus pulposus cells with maturation, ageing, and degeneration. Here, we provide an update on the journey of NCs and discuss the cell markers and tools that can be used to study their fate and regenerative capacity. We review the therapeutic potential of NCs for the treatment of IVD-related lower back pain and outline important future directions in this area. Promising studies indicate that NCs and their secretome exerts regenerative effects, via increased proliferation, extracellular matrix production, and anti-inflammatory effects. Reports on NC-like cells derived from embryonic- or induced pluripotent-stem cells claim to have successfully generated NC-like cells but did not compare them with native NCs for phenotypic markers or in terms of their regenerative capacity. Altogether, this is an emerging and active field of research with exciting possibilities. NC-based studies demonstrate that cues from developmental biology can pave the path for future clinical therapies focused on regenerating the diseased IVD.

Keywords: Notochordal cell; cell therapeutic potential; conditioned media (CM); extracellular matrix (ECM); intervertebral disc – degeneration; low back pain; secretome.

FIGURE 1

Representation of the healthy and degenerated human intervertebral disc (IVD) at macroscopic and magnetic resonance imaging typically used to visualize the IVD. Human healthy and degenerated L4-L5 IVDs are employed, a location commonly affected in chronic low back pain. The core of the IVD [nucleus pulposus, NP (*)] is constrained by the two endplates and the annulus fibrosus [AF(#)]. A healthy IVD has a gel-like NP with high water and glycosaminoglycan content (white on the T2 weighted sequence). IVD degeneration starts with loss of glycosaminoglycan and water within the NP (black on T2w sequence). Images by courtesy of Prof. Joachim Wilke.

FIGURE 2

Schematic representation of intervertebral disc (IVD) maturation and degeneration during human life. During IVD maturation, a transition in nucleus pulposus (NP) cell phenotype occurs from large, vacuolated notochordal cells (NCs) to smaller, non-vacuolated nucleus pulposus cells (NPCs). Humans have lost (almost) all their NCs before adulthood. In immature human NPs (e.g,. fetuses and babies), vacuolated NCs (arrowhead), transitional cells with some small vacuoles remaining (*), and non-vacuolated NPCs (arrow) or only NPCs are present (dependent on the fetal stage/age of the donor). In maturated human IVDs (e.g., individuals >10 years), only NPCs are present. Degenerated IVDs contain single NPCs, or small or large clusters of NPCs (as a reactive reparative response during moderate/severe degeneration).

FIGURE 3

Histologic images to exemplify the terminology of embryonic notochord cells (eNCs), vacuolated notochordal cells (NCs), non-vacuolated nucleus pulposus cells (NPCs), and cells with a transitional phenotype (with some small vacuoles remaining). In E11.5 and E12.5 murine fetuses, the notochord is a rod-like structure with tightly packed small non-vacuolated eNCs confined by the basement membrane sheath (Alcian Blue). In mice, the appearance of small intracytoplasmic vacuoles (arrow) are an indication of eNC into NC transition observed from E12.5. By E14.5, the regression of the notochord is almost complete and the intervertebral discand vertebral bodies are being formed (Alcian Blue). At this stage, NCs exhibit large intracytoplasmic vacuoles. In contrast, a 10-week postconceptional human fetus (Safranin O/Alcian Blue), the notochord is pinching into NP tissue containing NCs, and the rest of the IVD (annulus fibrosus (AF) and vertebras) are being formed. The human NC, transitional cell type, and NPC (Safranin O/Fast Green) pictures were obtained from a 22-week postconceptional human fetus. Human NC generation image by courtesy of Wilson Chan and Vivian Tam; murine images by courtesy of Maeva Dutilleul. E, embryonic day; IVD, intervertebral disc; VB, vertebral body.

FIGURE 4

High level differences in embryonic and postnatal cell transitions within the nucleus pulposus (NP). Cellular morphological transitions of the cells in the NP and associated interspecies variation in species commonly used as models to study NC (pathobiology) are provided. Large animal models, including sheep, goats and cows lose their NCs rapidly after birth (Alini et al., 2008). *, Authors have evidence that pigs keep their NCs until at least 2 years of age. CD, chondrodystrophic; E, embryonic day; eNC, embryonic notochord cell; IVD, intervertebral disc; NC, notochordal cell; NCD, non-chondrodystrophic; NP, nucleus pulposus; NPC, nucleus pulposus cell.

FIGURE 5

The therapeutic potential of notochordal cells (NCs) has been studied in several ways: either in co-culture with (degenerated) nucleus pulposus cells (NPCs) or by NC-conditioned medium (NCCM). NCCM contains all (bioactive) factors that NCs have secreted, and has been shown to exert regenerative effects on for instance canine and human NPCs (species that suffer from clinical IVD disease). In NCCM, several proteins (growth factors) have been identified, besides extracellular vesicles (EVs). The recent focus of several research groups has shifted from NCCM towards NC-derived extracellular matrix (ECM). This NC-derived ECM (both non-decellularized and decellularized) has been shown to exert regenerative effects, even in in vivo canine and rabbit studies. Species that are most commonly used to generate NCCM or NC-derived ECM are small rodents (mice, rats), rabbits, pigs, non-chondrodystrophic dogs. In limited studies, human NC-rich tissues have been employed derived from fetal IVD or from diseased children.

FIGURE 6

Overview of the number of studies (1999–2021) on the regenerative potential of Notochordal cells. A trend is observed in time from mainly coculture- and NCCM-based studies towards NC-derived matrix and NC-like cell generation studies. Details of these studies are provided in Supplementary Materials S1, S2 and the main text. NCCM, Notochordal cell-conditioned medium; NC, Notochordal cell; ES, embryonic stem cell; iPSC, induced pluripotent stem cells.

FIGURE 7

Overview of the proteins that were identified in human, canine, and/or porcine NCCM and murine, human and bovine fetal NC-rich matrisome. The Upset plot depicts the number of proteins identified in the specific study and relates to the number of proteins that overlap with other studies. Intersection size indicates the number of proteins identified in each specific comparison. The set size indicates the number of proteins identified in the specific study. The heatmap depicts the overlap of proteins between the different studies. The main functions of the discovered proteins are provided in a Venn diagram. Purmessur et al. (2011) and Gantenbein et al. (2014) identified proteins in porcine NCCM, while Erwin et al. (2006) and Matta et al. (2017) used canine NCCM. In the study of Bach and de Vries et al. (2016), only proteins that were identified in both human, canine, and porcine NCCM were included in this analysis. Rajasekaran et al. (2020), Rajasekaran et al. (2020b) identified proteins in human fetal matrisome, Caldeira et al. (2017) in bovine fetal matrisome, and Kudelko et al. (2021) in murine matrisome. The study by Tam et al. (2020) was not included in this comparative proteomic overview focusing on NC-rich tissues, since it used NPC-containing adolescent human NP tissue.