Three-dimensional culture model to study the biology of vacuolated notochordal cells from mouse nucleus pulposus explants

Abstract

Intervertebral disc degeneration (IDD) involves cellular changes in the nucleus pulposus (NP) characterised by a decline of the large vacuolated notochordal cells (vNCs) and a rise of smaller vacuole-free mature chondrocyte-like NP cells. An increasing number of studies demonstrate that notochordal cells (NCs) exert disease-modifying effects, establishing that NC-secreted factors are essential for the maintenance of a healthy intervertebral disc (IVD). However, understanding the role of the NCs is hampered by a restricted reserve of native cells and the lack of robust ex vivo cell model. A precise dissection enabled the isolation of NP cells from 4 d post-natal stage mouse spines and their culture into self-organised micromasses. The maintenance of cells’ phenotypic characteristics was demonstrated by the presence of intracytoplasmic vacuoles and the immuno-colocalisation of the NC-markers (brachyury; SOX9) after 9 d of culture both in hypoxic and normoxic conditions. A significant increase of the size of the micromass was observed under hypoxia, consistent with a higher level of Ki-67+ immunostained proliferative cells. Furthermore, several proteins of interest for the study of vNCs phenotype (CD44; caveolin-1; aquaporin 2; patched-1) were successfully detected at the plasma membrane of NP-cells cultured in micromasses under hypoxic condition. IHC was performed on mouse IVD sections as control staining. An innovative 3D culture model of vNCs derived from mouse postnatal NP is proposed, allowing future ex vivo exploration of their basic biology and of the signalling pathways involved in IVD homeostasis that may be relevant for disc repair.

In vitro modelling of anterior primitive streak patterning with human pluripotent stem cells identifies the path to notochord progenitors

“…These two clusters revealed a clear APS signature with the expression of genes associated with the APS in mice including MIXL1 (Hart et al, 2002), LHX1 (Costello et al, 2015), EOMES (Costello et al, 2011), GSC, CDX2, FOXA2 (Burtscher and Lickert, 2009), KLF5 (Aksoy et al, 2014) as well as the NMP-associated gene NKX1-2 (Albors et al, 2018). Encouragingly we also found a peak of expression of SLIT2 and CAV1 at 36h, recently reported as markers of human embryonic notochord (Paillat et al, 2023; Warin et al, 2024). However, NMPs and NotoPs associated genes decreased over time indicating that axial progenitors failed to emerge.…”

Recommendations for intervertebral disc notochordal cell investigation: From isolation to characterization

“…Here, we describe potential markers which can be useful to investigate key phenotypic and functional features of NCs, and whilst these are not all specific to NC phenotype, together they form a picture of cell status (Table 2). 13,17,19,32–34,42,44,71,75,91–125 Cells that reside within the NP experience dynamic changes of gene transcription profiles throughout postnatal cell proliferation, differentiation, IVD growth, aging, and degeneration. The recent review by Bach et al, 15 discussed potential markers which can be utilized to aid in the characterization of eNCs, NCs to NPCs highlighting key issues with identification of NC/NP‐specific markers and differential results between studies.…”

“…Recent micromass cultures have also been investigated for a simple and reproducible 3D culture model of NCs, to study basic biology and function, isolated from mouse immature NP tissue 97 . Culture conditions were optimized to enable growth of self‐organized micromasses of NCs in suspension.…”

In vitro and in vivo models define a molecular signature reference for human embryonic notochordal cells

“…Interestingly, some have been previously implicated in the regulation of notochord formation in various animal models (mouse, zebrafish, Xenopus, chicken) or the maintenance of IVD homeostasis in humans, dogs or bovine. 15 , 25 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 80 , 81 , 82 From this analysis, no lysosome-related organelles, nor vacuole markers were found except for CAV1 (manually curated gene list from the following ref. 72 , 83 , 84 is available on the Gitlink ).…”

“…This in vitro model generates embryonic NLC in an immature state (i.e., embryonic state) and does not support vacuole appearance within 7 days of differentiation. 76 …”