Embryo-scale reverse genetics at single-cell resolution.

The maturation of single-cell transcriptomic technologies has facilitated the generation of comprehensive cellular atlases from whole embryos1-4. A majority of these data, however, has been collected from wild-type embryos without an appreciation for the latent variation that is present in development. Here we present the 'zebrafish single-cell atlas of perturbed embryos': single-cell transcriptomic data from 1,812 individually resolved developing zebrafish embryos, encompassing 19 timepoints, 23 genetic perturbations and a total of 3.2 million cells. The high degree of replication in our study (eight or more embryos per condition) enables us to estimate the variance in cell type abundance organism-wide and to detect perturbation-dependent deviance in cell type composition relative to wild-type embryos. Our approach is sensitive to rare cell types, resolving developmental trajectories and genetic dependencies in the cranial ganglia neurons, a cell population that comprises less than 1% of the embryo. Additionally, time-series profiling of individual mutants identified a group of brachyury-independent cells with strikingly similar transcriptomes to notochord sheath cells, leading to new hypotheses about early origins of the skull. We anticipate that standardized collection of high-resolution, organism-scale single-cell data from large numbers of individual embryos will enable mapping of the genetic dependencies of zebrafish cell types, while also addressing longstanding challenges in developmental genetics, including the cellular and transcriptional plasticity underlying phenotypic diversity across individuals.

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

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.

Construction of a mammalian embryo model from stem cells organized by a morphogen signalling centre.

Generating properly differentiated embryonic structures in vitro from pluripotent stem cells remains a challenge. Here we show that instruction of aggregates of mouse embryonic stem cells with an experimentally engineered morphogen signalling centre, that functions as an organizer, results in the development of embryo-like entities (embryoids). In situ hybridization, immunolabelling, cell tracking and transcriptomic analyses show that these embryoids form the three germ layers through a gastrulation process and that they exhibit a wide range of developmental structures, highly similar to neurula-stage mouse embryos. Embryoids are organized around an axial chordamesoderm, with a dorsal neural plate that displays histological properties similar to the murine embryo neuroepithelium and that folds into a neural tube patterned antero-posteriorly from the posterior midbrain to the tip of the tail. Lateral to the chordamesoderm, embryoids display somitic and intermediate mesoderm, with beating cardiac tissue anteriorly and formation of a vasculature network. Ventrally, embryoids differentiate a primitive gut tube, which is patterned both antero-posteriorly and dorso-ventrally. Altogether, embryoids provide an in vitro model of mammalian embryo that displays extensive development of germ layer derivatives and that promises to be a powerful tool for in vitro studies and disease modelling.

Current Status of the Instructional Cues Provided by Notochordal Cells in Novel Disc Repair Strategies.

Numerous publications over the past 22 years, beginning with a seminal paper by Aguiar et al., have demonstrated the ability of notochordal cell-secreted factors to confer anabolic effects upon intervertebral disc (IVD) cells. Since this seminal paper, other scientific publications have demonstrated that notochordal cells secrete soluble factors that can induce anti-inflammatory, pro-anabolic and anti-cell death effects upon IVD nucleus pulposus (NP) cells in vitro and in vivo, direct human bone marrow-derived mesenchymal stem cells toward an IVD NP-like phenotype and repel neurite ingrowth. More recently these factors have been characterized, identified, and used therapeutically to induce repair upon injured IVDs in small and large pre-clinical animal models. Further, notochordal cell-rich IVD NPs maintain a stable, healthy extracellular matrix whereas notochordal cell-deficient IVDs result in a biomechanically and extracellular matrix defective phenotype. Collectively this accumulating body of evidence indicates that the notochordal cell, the cellular originator of the intervertebral disc holds vital instructional cues to establish, maintain and possibly regenerate the intervertebral disc.

Induction of notochordal differentiation of bone marrow mesenchymal­derived stem cells via the stimulation of notochordal cell­rich nucleus pulposus tissue.

The degeneration of intervertebral disc (IVD) tissue, initiated following the disappearance of notochordal cells (NCs), is characterized by the decreased number of nucleus pulposus (NP) cells (NPCs) and extracellular matrix. Transplanting proper cells into the IVD may sustain cell numbers, resulting in the synthesis of new matrix; this represents a minimally invasive regenerative therapy. However, the lack of cells with a correct phenotype severely hampers the development of regenerative therapy. The present study aimed to investigate whether porcine NC­rich NP tissue stimulates bone marrow­derived mesenchymal stem cell (BM­MSC) differentiation toward NC­like cells, which possess promising regenerative ability, for the treatment of disc degeneration diseases. BM­MSCs were successfully isolated from porcine femurs and tibiae, which expressed CD90 and CD105 markers and did not express CD45. Differentiation induction experiments revealed that the isolated cells had osteogenic and adipogenic differentiation potential. When co­cultured with NC­rich NP tissue, the BM­MSCs successfully differentiated into NC­like cells. Cell morphological analysis revealed that the cells exhibited an altered morphology, from a shuttle­like to a circular one, and the expression of NC marker genes, including brachyury, keratin­8, and keratin­18, was enhanced, and the cells exhibited the ability to generate aggrecan and collagen II. Taken together, the findings of the present study demonstrated that the primarily isolated and cultured BM­MSCs may be stimulated to differentiate into NC­like cells by porcine NC­rich NP explants, potentially providing an ideal cell source for regenerative therapies for disc degeneration diseases.

ß1 Integrin regulates convergent extension in mouse notogenesis, ensures notochord integrity and the morphogenesis of vertebrae and intervertebral discs.

The notochord drives longitudinal growth of the body axis by convergent extension, a highly conserved developmental process that depends on non-canonical Wnt/planar cell polarity (PCP) signaling. However, the role of cell-matrix interactions mediated by integrins in the development of the notochord is unclear. We developed transgenic Cre mice, in which the ß1 integrin gene (Itgb1) is ablated at E8.0 in the notochord only or in the notochord and tail bud. These Itgb1 conditional mutants display misaligned, malformed vertebral bodies, hemi-vertebrae and truncated tails. From early somite stages, the notochord was interrupted and displaced in these mutants. Convergent extension of the notochord was impaired with defective cell movement. Treatment of E7.25 wild-type embryos with anti-ß1 integrin blocking antibodies, to target node pit cells, disrupted asymmetric localization of VANGL2. Our study implicates pivotal roles of ß1 integrin for the establishment of PCP and convergent extension of the developing notochord, its structural integrity and positioning, thereby ensuring development of the nucleus pulposus and the proper alignment of vertebral bodies and intervertebral discs. Failure of this control may contribute to human congenital spine malformations.

Live Confocal Imaging of Zebrafish Notochord Cells Under Mechanical Stress In Vivo.

The zebrafish is a vertebrate model suited to the exploration of cell biology within a whole organism. Hypotheses in cell mechanics can be tested by using the zebrafish notochord as a manipulable experimental system. Here, the methodologies to prepare, label, and simultaneously induce and image mechanical loading on live zebrafish notochord cells via electrical stimulation are described. This approach investigates membrane mechanics in a live, physiological setting and is thus suited for caveola research where observations within the tissues of an intact organism are increasingly relevant. This chapter also aims to introduce fundamental methodologies for the use of zebrafish in "in vivo cell biology."

Morphogenetic mechanisms forming the notochord rod: The turgor pressure-sheath strength model.

The notochord is a defining feature of chordates. During notochord formation in vertebrates and tunicates, notochord cells display dynamic morphogenetic movement, called convergent extension, in which cells intercalate and align at the dorsal midline. However, in cephalochordates, the most basal group of chordates, the notochord is formed without convergent extension. It is simply developed from mesodermal cells at the dorsal midline. This suggests that convergent extension movement of notochord cells is a secondarily acquired developmental attribute in the common ancestor of olfactores (vertebrates + tunicates), and that the chordate ancestor innovated the notochord upon a foundation of morphogenetic mechanisms independent of cell movement. Therefore, this review focuses on biological features specific to notochord cells, which have been well studied using clawed frogs, zebrafish, and tunicates. Attributes of notochord cells, such as vacuolation, membrane trafficking, extracellular matrix formation, and apoptosis, can be understood in terms of two properties: turgor pressure of vacuoles and strength of the notochord sheath. To maintain the straight rod-like structure of the notochord, these parameters must be counterbalanced. In the future, the turgor pressure-sheath strength model, proposed in this review, will be examined in light of quantitative molecular data and mathematical simulations, illuminating the evolutionary origin of the notochord.

Directed Differentiation of Notochord-like and Nucleus Pulposus-like Cells Using Human Pluripotent Stem Cells.

Intervertebral disc degeneration might be amenable to stem cell therapy, but the required cells are scarce. Here, we report the development of a protocol for directed in vitro differentiation of human pluripotent stem cells (hPSCs) into notochord-like and nucleus pulposus (NP)-like cells of the disc. The first step combines enhancement of ACTIVIN/NODAL and WNT and inhibition of BMP pathways. By day 5 of differentiation, hPSC-derived cells express notochordal cell characteristic genes. After activating the TGF-ß pathway for an additional 15 days, qPCR, immunostaining, and transcriptome data show that a wide array of NP markers are expressed. Transcriptomically, the in vitro-derived cells become more like in vivo adolescent human NP cells, driven by a set of influential genes enriched with motifs bound by BRACHYURY and FOXA2, consistent with an NP cell-like identity. Transplantation of these NP-like cells attenuates fibrotic changes in a rat disc injury model of disc degeneration.

NOTO Transcription Factor Directs Human Induced Pluripotent Stem Cell-Derived Mesendoderm Progenitors to a Notochordal Fate.

The founder cells of the Nucleus pulposus, the centre of the intervertebral disc, originate in the embryonic notochord. After birth, mature notochordal cells (NC) are identified as key regulators of disc homeostasis. Better understanding of their biology has great potential in delaying the onset of disc degeneration or as a regenerative-cell source for disc repair. Using human pluripotent stem cells, we developed a two-step method to generate a stable NC-like population with a distinct molecular signature. Time-course analysis of lineage-specific markers shows that WNT pathway activation and transfection of the notochord-related transcription factor NOTO are sufficient to induce high levels of mesendoderm progenitors and favour their commitment toward the notochordal lineage instead of paraxial and lateral mesodermal or endodermal lineages. This study results in the identification of NOTO-regulated genes including some that are found expressed in human healthy disc tissue and highlights NOTO function in coordinating the gene network to human notochord differentiation.

Transforming Growth Factor ß Enhances Tissue Formation by Passaged Nucleus Pulposus Cells In Vitro.

The nucleus pulposus (NP) is composed of NP and notochord cell. It is a paucicellular tissue and if it is to be used as a source of cells for tissue engineering the cell number will have to be expanded by cell passaging. The hypothesis of this study is that passaged NP and notochordal cells grown in three-dimensional (3D) culture in the presence of transforming growth factor ß (TGFß) will show enhanced NP tissue formation compared with cells grown in the absence of this growth factor. Bovine NP cells isolated by sequential enzymatic digestion from caudal intervertebral discs were either placed directly in 3D culture (P0) or serially passaged up to passage 3 (P3) prior to placement in 3D culture. Serial cell passage in monolayer culture led to de-differentiation, increased senescence and oxidative stress and decreases in the gene expression of NP and notochordal associated markers and increases in de-differentiation markers. The NP tissue regeneration capacity of cells in 3D culture decreases with passaging as indicated by diminished tissue thickness and total collagen content when compared with tissues formed by P0 cells. Immunohistochemical studies showed that type II collagen accumulation appeared to decrease. TGFß1 or TGFß3 treatment enhanced the ability of cells at each passage to form tissue, in part by decreasing cell death. However, neither TGFß1 nor TGFß3 were able to restore the notochordal phenotype. Although TGFß1/3 recovered NP tissue formation by passaged cells, to generate NP in vitro that resembles the native tissue will require identification of conditions facilitating retention of notochordal cell differentiation. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:438-449, 2020.

Differential Response of Bovine Mature Nucleus Pulposus and Notochordal Cells to Hydrostatic Pressure and Glucose Restriction.

OBJECTIVE: The nucleus pulposus of the human intervertebral disc contains 2 cell types: notochordal (NC) and mature nucleus pulposus (MNP) cells. NC cell loss is associated with disc degeneration and this process may be initiated by mechanical stress and/or nutrient deprivation. This study aimed to investigate the functional responses of NC and MNP cells to hydrostatic pressures and glucose restriction. DESIGN: Bovine MNP and NC cells were cultured in 3-dimensional alginate beads under low (0.4-0.8 MPa) and high (1.6-2.4 MPa) dynamic pressure for 24 hours. Cells were cultured in either physiological (5.5 mM) glucose media or glucose-restriction (0.55 mM) media. Finally, the combined effect of glucose restriction and high pressure was examined. RESULTS: Cell viability and notochordal phenotypic markers were not significantly altered in response to pressure or glucose restriction. MNP cells responded to low pressure with an increase in glycosaminoglycan (GAG) production while high pressure significantly decreased ACAN gene expression compared with atmospheric controls. NC cells showed no response in matrix gene expression or GAG production with either loading regime. Glucose restriction decreased NC cell TIMP-1 expression but had no effect on MNP cells. The combination of glucose restriction and high pressure only affected MNP cell gene expression, with decreased ACAN, Col2α1, and ADAMTS-5 expression. CONCLUSION: This study shows that NC cells are more resistant to acute mechanical stresses than MNP cells and provides a strong rationale for future studies to further our understanding the role of NC cells within the disc, and the effects of long-term exposure to physical stresses.

Iterative and Complex Asymmetric Divisions Control Cell Volume Differences in Ciona Notochord Tapering.

The notochord of the invertebrate chordate Ciona forms a tapered rod at tailbud stages consisting of only 40 cylindrical cells in a single-file column. This tapered shape involves differences in notochord cell volume along the anterior-posterior axis. Here, we quantify sibling cell volume asymmetry throughout the developing notochord and find that there are distinctive patterns of unequal cleavage in all 4 bilateral pairs of A-line primary notochord founder cells and also in the B-line-derived secondary notochord founder cells. A quantitative model confirms that the observed patterns of unequal cleavage are sufficient to explain all the anterior-posterior variation in notochord cell volume. Many examples are known of cells that divide asymmetrically to give daughter cells of different size and fate. Here, by contrast, a series of subtle but iterative and finely patterned asymmetric divisions controls the shape of an entire organ. Quantitative 3D analysis of cell shape and spindle positioning allows us to infer multiple cellular mechanisms driving these unequal cleavages, including polarized displacements of the mitotic spindle, contributions from the shape of the mother cell, and late changes occurring between anaphase and abscission that potentially involve differential cortical contractility. We infer differential use of these mechanisms between different notochord blastomeres and also between different rounds of cell division. These results demonstrate a new role for asymmetric division in directly shaping a developing organ and point toward complex underlying mechanisms.

Comprehensive single-cell transcriptome lineages of a proto-vertebrate.

Ascidian embryos highlight the importance of cell lineages in animal development. As simple proto-vertebrates, they also provide insights into the evolutionary origins of cell types such as cranial placodes and neural crest cells. Here we have determined single-cell transcriptomes for more than 90,000 cells that span the entirety of development-from the onset of gastrulation to swimming tadpoles-in Ciona intestinalis. Owing to the small numbers of cells in ascidian embryos, this represents an average of over 12-fold coverage for every cell at every stage of development. We used single-cell transcriptome trajectories to construct virtual cell-lineage maps and provisional gene networks for 41 neural subtypes that comprise the larval nervous system. We summarize several applications of these datasets, including annotating the synaptome of swimming tadpoles and tracing the evolutionary origin of cell types such as the vertebrate telencephalon.

A histocytological and radiological overview of the natural history of intervertebral disk: from embryonic formation to age-related degeneration.

PURPOSE: To elucidate the natural history of intervertebral disk (IVD) and characterize its embryonic beginnings and age-related degeneration. METHODS: Coronal sections of embryonic (E13.5-neonatal) and postnatal (4-60-week-old) Sprague-Dawley rat IVD were stained by a series of histological stainings (hematoxylin and eosin, Alcian blue, Picrosirius red, Masson, Periodic acid-Schiff). Growth kinetics within embryonic IVD were evaluated by immunohistochemical staining of Ki67 and proliferating cell nuclear antigen. Postnatal maturation and degeneration of IVD were visualized on radiology by X-ray, CT, and MR imaging. RESULTS: During the formation of rat IVD, inner annulus fibrosus (AF) and cartilaginous endplate (CEP) shared similar cell density, extracellular matrix, and potential of growth kinetics; notochord provided increased and enlarged cytoplasmic vacuoles to generate nucleus pulposus (NP), part of which was retained within CEP. Postnatally, vacuolated notochord cells were reduced by devacuolation, while chondrocytic NP cells increased; cartilaginous layers of CEP were narrowed by vertebrae growth and secondary ossification; fibrotic portion of AF decreased as cartilaginous matrix accumulated and infiltrated outward. In aged and degenerated IVD, large longitudinal fissures were detected near the boundaries between inner and outer AF, whereas both reduced cellularity and accumulated cell clusters were evident within the dehydrated NP; only part of these histocytological changes could be reported on radiology. CONCLUSIONS: By showing that the natural history of IVD is orchestrated by a dynamic histocytological regulation, our study may facilitate better understanding of the developmental defects, cellular heterogeneity, age-related degenerative mechanisms, and biological regeneration of IVD. These slides can be retrieved under Electronic Supplementary Material.

Notochordal Cell Matrix As a Therapeutic Agent for Intervertebral Disc Regeneration.

Notochordal cells (NCs) reside in the core of the healthy disc and produce soluble factors that can stimulate nucleus pulposus cells (NPCs). These NC-derived factors may be applied in intervertebral disc regeneration for treatment of low-back pain. However, identification of the active soluble factors is challenging. Therefore a novel approach to directly use porcine NC-rich NP matrix (NCM) is introduced. We explored porcine NCM's anabolic effects on bovine NPCs harvested from caudal discs of adolescent and adult (2-2.5 vs. 4-6 year old) cows. NC-conditioned medium (NCCM) and NCM were produced from porcine NC-rich NP tissue. Bovine NPCs were cultured in alginate beads for 4 weeks in base medium (BM), NCCM, and NCM to investigate NCM's regenerative potential. Porcine NCM increased glycosaminoglycan (GAG) content of both adolescent and adult bovine NPCs. This was through increased proliferation of adolescent bovine NPCs, whereas in adult bovine NPCs, it was mostly through increased GAG production per NPC. Furthermore, adolescent bovine NPCs were cultured in BM and porcine NCM treated with interleukin (IL)-1ß to investigate NCM's potential in an inflammatory environment. Addition of IL-1ß enhanced IL1ß and CXCL8 (IL8) gene expression, while NCM diminished IL1ß gene expression. IL-1ß reduced GAG and DNA content, but the addition of NCM relative to BM improved GAG and DNA content. Altogether, porcine NCM exerts bovine NPC-age dependent effects, and NCM's anabolic effect on adult NPCs is stronger compared with NCCM. Furthermore, porcine NCM induced an anabolic response of bovine NPCs in an inflammatory environment and may have anti-inflammatory properties. Therefore, NCM has potential in a regenerative therapy for disc degeneration, and warrants additional in vivo studies.

Ascidian notochord elongation.

The elongation of embryo and tissue is a key morphogenetic event in embryogenesis and organogenesis. Notochord, a typical chordate organ, undergoes elongation to perform its regulatory roles and to form the structural support in the embryo. Notochord elongation is morphologically similar across all chordates, but ascidian has evolved distinct molecular and cellular processes. Here, we summarize the current understanding of ascidian notochord elongation. We divide the process into three phases and discuss the underlying molecular mechanisms in each phase. In the first phase, the notochord converges and extends through invagination and mediolateral intercalation, and partially elongates to form a single diameter cell column along the anterior-posterior axis. In the second phase, a cytokinesis-like actomyosin ring is constructed at the equator of each cell and drives notochord to elongate approximately two-fold. The molecular composition and architecture of the ascidian notochord contractile ring are similar to that of the cytokinetic ring. However, the notochord contractile ring does not impose cell division but only drives cell elongation followed by disassembly. We discuss the self-organizing property of the circumferential actomyosin ring, and why it disassembles when certain notochord length is achieved. The similar ring structures are also present in the elongation process of other organs in evolutionarily divergent animals such as Drosophila and C. elegans. We hereby propose that actomyosin ring-based circumferential contraction is a common mechanism adopted in diverse systems to drive embryo and tissue elongation. In the third phase, the notochord experiences tubulogenesis and the endothelial-like cells crawl bi-directionally on the notochord sheath to further lengthen the notochord. In this review, we also discuss extracellular matrix proteins, notochord sheath, and surrounding tissues that may contribute to notochord integrity and morphogenesis.

Regeneration of the acorn worm pygochord with the implication for its convergent evolution with the notochord.

The origin of the notochord is a central issue in chordate evolution. This study examined the development of the acorn worm pygochord, a putative homologue of the notochord. Because the pygochord differentiates only after metamorphosis, the developmental was followed process by inducing regeneration after artificial amputation in Ptychodera flava. It was found that although the regeneration of the posterior part of the body did not proceed via formation of an obvious regeneration bud, pygochord regeneration was observed within a few weeks, possibly via trans-differentiation of endoderm cells. The expression of the fibrillary collagen gene (Fcol) and elav in the pygochord during regeneration was detected. This indicates that pygochord cells are not part of gut epithelial cells, but that they differentiated as a distinct cell type. Our gene expression analyses do not provide supporting evidence for the homology between the pygochord and notochord, but rather favored the convergent evolution between them.

Transcriptionally dynamic progenitor populations organised around a stable niche drive axial patterning.

The elongating mouse anteroposterior axis is supplied by progenitors with distinct tissue fates. It is not known whether these progenitors confer anteroposterior pattern to the embryo. We have analysed the progenitor population transcriptomes in the mouse primitive streak and tail bud throughout axial elongation. Transcriptomic signatures distinguish three known progenitor types (neuromesodermal, lateral/paraxial mesoderm and notochord progenitors; NMPs, LPMPs and NotoPs). Both NMP and LPMP transcriptomes change extensively over time. In particular, NMPs upregulate Wnt, Fgf and Notch signalling components, and many Hox genes as progenitors transit from production of the trunk to the tail and expand in number. In contrast, the transcriptome of NotoPs is stable throughout axial elongation and they are required for normal axis elongation. These results suggest that NotoPs act as a progenitor niche whereas anteroposterior patterning originates within NMPs and LPMPs.

Large Cytoplasmic Vacuoles within Notochordal Nucleus Pulposus Cells: A Possible Regulator of Intracellular Pressure That Shapes the Cytoskeleton and Controls Proliferation.

Degeneration of the intervertebral disc, which is closely associated with the loss of vacuolated notochordal nucleus pulposus cells (NNPC), remains a major cause of lower-back pain and motor deficiency. Being the most defining characteristic of NNPC, large cytoplasmic vacuoles not only modulate the cytoskeleton and shape cell morphology but they also respond to the disc microenvironment and regulate the biological behavior of vacuolated cells as a potent reporter of the histocytological changes that occur at the beginning of disc aging and degeneration. Here we hypothesize a model in which large cytoplasmic vacuoles primarily function to maintain a reasonable intracellular pressure (Pv) that facilitates NNPC in resisting the extracellular mechanical loading (Pe), part of which is absorbed by the extracellular matrix (Pm), forming the equation Pe = Pm + Pv. By mimicking a situation of contact-induced growth inhibition, the crowded cytoplasmic vacuoles slow down the proliferation of NNPC and restrain the generation of nonvacuolated chondrocytic nucleus pulposus cells (CNPC), whereas increased mechanical loading (↑Pe) alters cytoskeletons and breaches cytoplasmic vacuoles, which in turn weakens the vacuoles-mediated proliferation check, increases the generation of CNPC that accumulates fibrocartilaginous matrix, and rebalances the increased loading with elevated Pm (↑Pm) and lowered Pv (↓Pv), equating to ↑Pe = ↑Pm + ↓Pv. By depicting the biological function and the disappearance of the cytoplasmic vacuoles, our model highlights a mechanical exhaustion of the notochordal cell resources, which might help to elucidate the histocytological changes that initiate disc aging and degeneration.