A novel and efficient method for culturing mouse nucleus pulposus cells

Kushioka, Junichi; Kaito, Takashi; Chijimatsu, Ryota; Okada, Rintaro; Ishiguro, Hiroyuki; Bal, Zeynep; Kodama, Joe; Takenaka, Shota; Makino, Takahiro; Sakai, Yusuke; Yoshikawa, Hideki

doi:10.1016/j.spinee.2019.04.005

Cited by 12 publications

(10 citation statements)

References 35 publications

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“…[42][43][44] In this study, the combination of fibronectin coating with the addition of FGF-2 into the culture media, as previously described with the culture of murine NPCs, should overcome this limitation of "stemness" potential loss. 45 This expansion protocol, by still keeping the cells in a mimicking microenvironment after previously cultured in 3D (alginate beads), will maintain the cells with a phenotype and gene expression close to cells in their native tissue. Briefly, even at the end of the second phase of expansion,…”

Section: Discussionmentioning

confidence: 99%

The effects of 3D culture on the expansion and maintenance of nucleus pulposus progenitor cell multipotency

et al. 2020

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Introduction: Low back pain (LBP) is a global health concern. Increasing evidence implicates intervertebral disk (IVD) degeneration as a major contributor. In this respect, tissue-specific progenitors may play a crucial role in tissue regeneration, as these cells are perfectly adapted to their niche. Recently, a novel progenitor cell population was described in the nucleus pulposus (NP) that is positive for Tie2 marker. These cells have self-renewal capacity and in vitro multipotency potential. However, extremely low numbers of the NP progenitors limit the feasibility of cell therapy strategies. Objective: Here, we studied the influence of the culture method and of the microenvironment on the proliferation rate and the differentiation potential of human NP progenitors in vitro. Method: Cells were obtained from human NP tissue from trauma patients. Briefly, the NP tissue cells were cultured in two-dimensional (2D) (monolayer) or threedimensional (3D) (alginate beads) conditions. After 1 week, cells from 2D or 3D culture were expanded on fibronectin-coated flasks. Subsequently, expanded NP cells were then characterized by cytometry and tri-lineage differentiation, which was analyzed by qPCR and histology. Moreover, experiments using Tie2 + and Tie2 − NP cells were also performed. Results: The present study aims to demonstrate that 3D expansion of NP cells better preserves the Tie2 + cell populations and increases the chondrogenic and osteogenic differentiation potential compared to 2D expansion. Moreover, the cell sorting experiments reveal that only Tie2 + cells were able to maintain the pluripotent gene expression if cultured in 3D within alginate beads. Therefore, our results highly suggest that the maintenance of the cell's multipotency is mainly, but not exclusively, due to the higher presence of Tie2 + cells due to 3D culture. Conclusion: This project not only might have a scientific impact by evaluating the influence of a two-step expansion protocol on the functionality of NP progenitors, but it could also lead to an innovative clinical approach.

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Section: Discussionmentioning

confidence: 99%

The effects of 3D culture on the expansion and maintenance of nucleus pulposus progenitor cell multipotency

et al. 2020

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“…Male C57BL6J mice (age, 10–12 weeks) were purchased from Oriental Yeast Co., Ltd. (Tokyo, Japan). mNPCs were cultured as described previously 25 . With this method, NP cells from five mice were required to prepare one set of NP cells to perform in vitro experiments.…”

Section: Methodsmentioning

confidence: 99%

“…Two-dimensional micromass culture was performed as described previously 17 , 25 . Briefly, a high concentration of mNPCs or hNPCs was cultured in micromass (1 × 10 5 cells/10 μL) in chondrogenic basal medium (DMEM with 1% ITS [insulin, transferrin, selenium, Corning Inc., Corning, NY], 50 μg/mL ascorbic acid [Sigma-Aldrich], 40 μg/mL L-proline [Fujifilm Wako], 1% FBS, and 1% A/A) supplemented with vehicle or TD-198946 (1 nM to 1 μM) for 7 days.…”

Section: Methodsmentioning

confidence: 99%

The small compound, TD-198946, protects against intervertebral degeneration by enhancing glycosaminoglycan synthesis in nucleus pulposus cells

Kushioka

Kaito

Chijimatsu

et al. 2020

Sci Rep

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Degeneration of the nucleus pulposus (NP) might serve as a trigger for intervertebral disc degeneration (IDD). A recent drug screening study revealed that the thienoindazole derivative, TD-198946, is a novel drug for the treatment of osteoarthritis. Because of the environmental and functional similarities between articular cartilage and intervertebral disc, TD-198946 is expected to prevent IDD. Herein, we sought to evaluate the effects of TD-198946 on IDD. TD-198946 enhanced glycosaminoglycan (GAG) production and the related genes in mouse NP cells and human NP cells (hNPCs). Further, Kyoto Encyclopedia of Genes and Genomes pathway analysis using the mRNA sequence of hNPCs suggested that the mechanism of action of TD-198946 primarily occurred via the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway. The Akt inhibitor suppressed the enhancement of GAG production induced by TD-198946. The effects of TD-198946 on IDD at two different time points (immediate treatment model, immediately after the puncture; latent treatment model, 2 weeks after the puncture) were investigated using a mouse tail-disc puncture model. At both time points, TD-198946 prevented a loss in disc height. Histological analysis also demonstrated the preservation of the NP structures. TD-198946 exhibited therapeutic effects on IDD by enhancing GAG production via PI3K/Akt signaling.

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“…The cell passage was done at 1:3 ratio till it reaches 80%-90% confluence. After three passages, the NPMSCs were used for further studies (Kushioka et al, 2019).…”

Section: Isolation and Culture Of Npmscsmentioning

confidence: 99%

Elucidating the role of cell‐mediated inflammatory cytokines on allogeneic mouse‐derived nucleus pulposus mesenchymal stem cells

Chinnarasu¹,

Rani²

2021

J. Food Biochem.

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In this present study, our aim was to evaluate the cell-mediated specific anti-donor antibody and its associated inflammatory cytokine secretion along with its succeeding effects on Nucleus pulposus-derived mesenchymal stem cells (NPMSCs). Tissue from the NP compartment of 12 normal mice was isolated, expanded in cell culture, and the cell phenotypes were confirmed by flow cytometry. Multipotent differentiation and its specific gene expression analysis were confirmed by reverse transcriptase PCR. T and B cells were monitored for both donor and recipient mouse and further analysis of anti-donor antibody secretion was confirmed by lymphocyte crossmatch.In conjunction with anti-donor-specific antibody analysis, the associated inflammatory cytokine expression was analyzed by ELISA. In co-culture, cell-mediated antibody secretion was elevated in T and B cells positive mouse group, when compared to control mouse group. Allogeneic-derived donor NPMSCs were found to be stimulated the secretion of pro-inflammatory cytokines and the level of pro-inflammatory cytokines showed reduced expression in control mouse serum. In co-culture group the concentration of the cell-mediated pro-and anti-inflammatory cytokines found to be increased. Practical applicationsMesenchymal stem cell exhibit good regeneration capacity for many types of disease, and the mechanism belongs to regeneration is not clear. In intervertebral disc, the nucleus pulposus-derived mesenchymal stem cells showed a better regeneration capacity. On the contrary, the NP cells-based therapy, the Mesenchymal stem cells showed expanded anabolic and reduced catabolic activity together with induced anti-inflammatory effect. In this study, the T & B cells were used to evaluate the antidonor antibody secretion and also to study how it stimulates the production of antidonor antibodies against the donor cells. Finally, it was found that T & B cells lead the synthesis of inflammatory cytokines are IL-1, IL-6, and TNFα. From this study, the results proved that the cell-mediated pro-and anti-inflammatory cytokines to be monitored in allogeneic stem cells-based therapy of intervertebral disc degeneration.

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A novel and efficient method for culturing mouse nucleus pulposus cells

Cited by 12 publications

References 35 publications

The effects of 3D culture on the expansion and maintenance of nucleus pulposus progenitor cell multipotency

The effects of 3D culture on the expansion and maintenance of nucleus pulposus progenitor cell multipotency

The small compound, TD-198946, protects against intervertebral degeneration by enhancing glycosaminoglycan synthesis in nucleus pulposus cells

Elucidating the role of cell‐mediated inflammatory cytokines on allogeneic mouse‐derived nucleus pulposus mesenchymal stem cells

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