Single-cell transcriptome profiling reveals multicellular ecosystem of nucleus pulposus during degeneration progression

Tu, Ji; Li, Wentian; Yang, Sidong; Yang, Pengyi; Qi, Yan; Wang, Shenyu; Lai, Kaitao; Bai, Xupeng; Wu, Cenhao; Ding, Wenyuan; Cooper-White, Justin J.; Diwan, Ashish D.; Yang, Cao; Yang, Huilin; Zou, Jun

doi:10.1101/2021.05.25.445620

Cited by 2 publications

(2 citation statements)

References 78 publications

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“…Afterwards, the solution was removed into a Bruker-600 1 H-NMR spectrometer. 1 H-NMR spectra of the samples were then analysed and recorded by the spectrometer.…”

Section: H-nmr Spectroscopy Analysis Of Gelmamentioning

confidence: 99%

“…Intervertebral disc (IVD) degeneration is regarded as the primary cause of low back pain (LBP), which constitutes a severe global public health problem [1]. The whole IVD connects the adjacent bony vertebral bodies, and comprises three compartments: nucleus pulposus (NP), annulus Ivyspring International Publisher fibrosus (AF), and cartilaginous endplate (CEP) [2].…”

Section: Introductionmentioning

confidence: 99%

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Spheroid Formation Enhances the Regenerative Capacity of Nucleus Pulposus Cells via Regulating N-CDH and ITGβ1 Interaction

Wang¹,

Wang²,

Zhuo³

et al. 2022

Int. J. Biol. Sci.

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Background: Nucleus pulposus (NP) degeneration is the core pathological change of intervertebral disc (IVD) degenerative diseases, but currently, no effective therapy is available. With the rapid development of biomaterials and tissue engineering in recent years, biomaterial-assisted cell transplantation becomes a promising therapy for IVD degeneration. However, the application is severely limited by the weak biological characteristics of NP cells (NPCs), such as a moderate proliferation ability, weak self-renewal capacity, and minimal extracellular matrix (ECM) synthesis capacity, caused by the current inappropriate cell seeding or grafting methods. Methods: Here, we developed a three-dimensional (3D) spheroidizing culture method to construct NPC spheroids and investigated repair and regeneration potential of these spheroids in vitro and in vivo. The in vitro biological characteristics (including cell viability and proliferation), and in vivo functions (including anti-degeneration potential and ability to induce tissue repair) of NPC spheroids and monolayer-cultured NPCs were compared. Furthermore, an RNA-seq-based transcriptome analysis and a series of function experiments were performed to elucidate the potential mechanisms of their differences that were involved in the tissue regeneration process. Results: NPC spheroids exhibited obviously superior self-renewal and ECM synthesis capacities compared to monolayers of NPCs in vitro. In vivo, NPC spheroids generated more functional ECM components, primarily aggrecan (ACAN) and collagen type II (Col2), and markedly promoted NP regeneration in the disc degeneration model induced by partial NP excision. Additionally, the biological characteristics and functions of NPC spheroids were to some extent regulated by the interaction of N-cadherin (N-CDH) and Integrinβ1 (ITGβ1), two key mechanosensing ECM-receptors expressed on NPCs. Conclusions: The NPC spheroidizing culture method is beneficial for cell renewal and the generation of functional ECM in NP tissue. The molecular mechanism involved in this regeneration process is closely associated with the regulation of the N-CDH and ITGβ1 interaction-mediated ECM homeostesis. Moreover, the strategy of hydrogel-assisted NPC spheroids transplantation may potentially be used in the future treatment of IVD degeneration.

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“…Afterwards, the solution was removed into a Bruker-600 1 H-NMR spectrometer. 1 H-NMR spectra of the samples were then analysed and recorded by the spectrometer.…”

Section: H-nmr Spectroscopy Analysis Of Gelmamentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Spheroid Formation Enhances the Regenerative Capacity of Nucleus Pulposus Cells via Regulating N-CDH and ITGβ1 Interaction

Wang¹,

Wang²,

Zhuo³

et al. 2022

Int. J. Biol. Sci.

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Systematic study of single-cell isolation from musculoskeletal tissues for single-sell sequencing

Gao

Guo

Liu

et al. 2022

BMC Mol and Cell Biol

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Background The single-cell platform provided revolutionary way to study cellular biology. Technologically, a sophistic protocol of isolating qualified single cells would be key to deliver to single-cell platform, which requires high cell viability, high cell yield and low content of cell aggregates or doublets. For musculoskeletal tissues, like bone, cartilage, nucleus pulposus and tendons, as well as their pathological state, which are tense and dense, it’s full of challenge to efficiently and rapidly prepare qualified single-cell suspension. Conventionally, enzymatic dissociation methods were wildly used but lack of quality control. In the present study, we designed the rapid cycling enzymatic processing method using tissue-specific enzyme cocktail to treat different human pathological musculoskeletal tissues, including degenerated nucleus pulposus (NP), ossifying posterior longitudinal ligament (OPLL) and knee articular cartilage (AC) with osteoarthritis aiming to rapidly and efficiently harvest qualified single-cell suspensions for single-cell RNA-sequencing (scRNA-seq). Results We harvested highly qualified single-cell suspensions from NP and OPLL with sufficient cell numbers and high cell viability using the rapid cycling enzymatic processing method, which significantly increased the cell viability compared with the conventional long-time continuous digestion group (P < 0.05). Bioanalyzer trace showed expected cDNA size distribution of the scRNA-seq library and a clear separation of cellular barcodes from background partitions were verified by the barcode-rank plot after sequencing. T-SNE visualization revealed highly heterogeneous cell subsets in NP and OPLL. Unfortunately, we failed to obtain eligible samples from articular cartilage due to low cell viability and excessive cell aggregates and doublets. Conclusions In conclusion, using the rapid cycling enzymatic processing method, we provided thorough protocols for preparing single-cell suspensions from human musculoskeletal tissues, which was timesaving, efficient and protective to cell viability. The strategy would greatly guarantee the cell heterogeneity, which is critical for scRNA-seq data analysis. The protocol to treat human OA articular cartilage should be further improved.

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Single-cell transcriptome profiling reveals multicellular ecosystem of nucleus pulposus during degeneration progression

Cited by 2 publications

References 78 publications

Spheroid Formation Enhances the Regenerative Capacity of Nucleus Pulposus Cells via Regulating N-CDH and ITGβ1 Interaction

Spheroid Formation Enhances the Regenerative Capacity of Nucleus Pulposus Cells via Regulating N-CDH and ITGβ1 Interaction

Systematic study of single-cell isolation from musculoskeletal tissues for single-sell sequencing

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