Dataset Information

Minimal Sustainability of Dedifferentiation by ROCK Inhibitor on Rat Nucleus Pulposus Cells In Vitro.

ABSTRACT:

Introduction

Intervertebral disc degeneration is strongly associated with low back pain. Cell transplantation has been extensively studied as a treatment option for intervertebral disc degeneration. It is often necessary to perform cell culture prior to cell transplantation; however, during cell expansion, the cells tend to dedifferentiate and lose their potency. Although the ability to suppress dedifferentiation by ROCK inhibitor (ROCKi) has recently been reported for chondrocytes, its effects on nucleus pulposus cells are still largely unknown.

Methods

Rat nucleus pulposus cells were cultured with or without the addition of ROCKi (Y-27632), and cell proliferation; CD24 positivity; expression of SOX9, COL2A1, Aggrecan, and COL1A1; and cell redifferentiation ability in pellet culture were evaluated.

Results

Although the addition of ROCKi tended to slightly increase the cell proliferative capacity, no significant differences were observed between treated and untreated conditions. The addition of ROCKi showed a trend of minimally increased COL2A1, ACAN, and SOX9 expression. Increases in COL1A1 expression was slightly suppressed by ROCKi. In pellet culture, strong increase in type II collagen deposition was observed by the addition of ROCKi. The addition of ROCKi did not significantly change the levels of CD24 positivity. The supplementation of ROCKi did not significantly enhance nucleus pulposus cell marker expression during monolayer expansion. However, ROCKi addition did result in an increased type II collagen deposition in 3D pellet culture.

Conclusions

Taken together, the results suggest a minimal effect by ROCKi on nucleus pulposus cell phenotype maintenance.

SUBMITTER: Nukaga T

PROVIDER: S-EPMC6834460 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Minimal Sustainability of Dedifferentiation by ROCK Inhibitor on Rat Nucleus Pulposus Cells In Vitro.

Nukaga Tadashi T Sakai Daisuke D Schol Jordy J Suyama Kaori K Nakai Tomoko T Hiyama Akihiko A Watanabe Masahiko M

Spine surgery and related research 20190816 4

<h4>Introduction</h4>Intervertebral disc degeneration is strongly associated with low back pain. Cell transplantation has been extensively studied as a treatment option for intervertebral disc degeneration. It is often necessary to perform cell culture prior to cell transplantation; however, during cell expansion, the cells tend to dedifferentiate and lose their potency. Although the ability to suppress dedifferentiation by ROCK inhibitor (ROCKi) has recently been reported for chondrocytes, its ...[more]

PMID: 31768460

Similar Datasets

Project description:Low back pain is a clinically highly relevant musculoskeletal burden and is associated with inflammatory as well as degenerative processes of the intervertebral disc. However, the pathophysiology and cellular pathways contributing to this devastating condition are still poorly understood. Based on previous evidence, we hypothesize that tissue renin-angiotensin system (tRAS) components, including the SARS-CoV-2 entry receptor angiotensin-converting enzyme 2 (ACE2), are present in human nucleus pulposus (NP) cells and associated with inflammatory and degenerative processes. Experiments were performed with NP cells from four human donors. The existence of angiotensin II, angiotensin II type 1 receptor (AGTR1), AGTR2, MAS-receptor (MasR), and ACE2 in human NP cells was validated with immunofluorescent staining and gene expression analysis. Hereafter, the cell viability was assessed after adding agonists and antagonists of the target receptors as well as angiotensin II in different concentrations for up to 48 h of exposure. A TNF-α-induced inflammatory in vitro model was employed to assess the impact of angiotensin II addition and the stimulation or inhibition of the tRAS receptors on inflammation, tissue remodeling, expression of tRAS markers, and the release of nitric oxide (NO) into the medium. Furthermore, protein levels of IL-6, IL-8, IL-10, and intracellular as well as secreted angiotensin II were assessed after exposing the cells to the substances, and inducible nitric oxide synthase (iNOS) levels were evaluated by utilizing Western blot. The existence of tRAS receptors and angiotensin II were validated in human NP cells. The addition of angiotensin II only showed a mild impact on gene expression markers. However, there was a significant increase in NO secreted by the cells. The gene expression ratios of pro-inflammatory/anti-inflammatory cytokines IL-6/IL-10, IL-8/IL-10, and TNF-α/IL-10 were positively correlated with the AGTR1/AGTR2 and AGTR1/MAS1 ratios, respectively. The stimulation of the AGTR2 MAS-receptor and the inhibition of the AGTR1 receptor revealed beneficial effects on the gene expression of inflammatory and tissue remodeling markers. This finding was also present at the protein level. The current data showed that tRAS components are expressed in human NP cells and are associated with inflammatory and degenerative processes. Further characterization of the associated pathways is warranted. The findings indicate that tRAS modulation might be a novel therapeutic approach to intervertebral disc disease.

Project description:BackgroundThe mechanisms underlying M2 macrophage polarization induced by nucleus pulposus (NP) cells are unclear. The effects that M2-polarized macrophages have on NP cells are also controversial.MethodsTranscriptome sequencing was performed to detect the gene change profiles between NP cells from ruptured intervertebral disc (IVD) and normal IVD. The main difference on biological activities between the two cell groups were analyzed by GO analysis and KEGG analysis. Virus transduction, flow cytometry, immunofluorescence, RT-PCR, western blot, CCK-8, TUNEL staining, and AO/EB staining were performed to explore the interactions between NP cells and RAW264.7 macrophages. Statistics were performed using SPSS26.Results801 upregulated and 276 downregulated genes were identified in NP cells from ruptured IVD in mouse models. According to GO and KEGG analysis, we found that the differentially expressed genes (DEG) were dominantly enriched in inflammatory response, extracellular matrix degradation, blood vessel morphogenesis, immune effector process, ossification, chemokine signaling pathway, macrophage activation, etc. CX3CL1 was one of the top 20% DEG, and we confirmed that both NP tissue and cells expressed remarkably higher level of CX3CL1 in mouse models (p < 0.001∗). Besides, we further revealed that both the recombinant CX3CL1 and NP cells remarkably induced M2 polarization of RAW264.7 (p < 0.001∗), respectively, while this effect was significantly reversed by si-CX3CL1 or JMS-17-2 (p < 0.001∗). Furthermore, we found that M2 macrophages significantly decreased the apoptosis rate (p < 0.001∗) and the catabolic gene levels (p < 0.001∗) of NP cells, while increased the viability, proliferation as well as the anabolic gene levels of NP cells (p < 0.01∗).ConclusionsVia regulating CX3CL1/CX3CR1 pathway, NP cells can induce the M2 macrophage polarization. M2 polarized macrophages can further promote NP cell viability, proliferation, and anabolism, while inhibit NP cell apoptosis and catabolism.

Project description:The angiopoietin-1 receptor (Tie2) marks specific nucleus pulposus (NP) progenitor cells, shows a rapid decline during aging and intervertebral disc degeneration, and has thus sparked interest in its utilization as a regenerative agent against disc degeneration. However, the challenge of maintaining and expanding these progenitor cells in vitro has been a significant hurdle. In this study, we investigated the potential of laminin-511 to sustain Tie2+ NP progenitor cells in vitro. We isolated cells from human NP tissue (n = 5) and cultured them for 6 days on either standard (Non-coat) or iMatrix-511 (laminin-511 product)-coated (Lami-coat) dishes. We assessed these cells for their proliferative capacity, activation of Erk1/2 and Akt pathways, as well as the expression of cell surface markers such as Tie2, GD2, and CD24. To gauge their regenerative potential, we examined their extracellular matrix (ECM) production capacity (intracellular type II collagen (Col2) and proteoglycans (PG)) and their ability to form spherical colonies within methylcellulose hydrogels. Lami-coat significantly enhanced cell proliferation rates and increased Tie2 expression, resulting in a 7.9-fold increase in Tie2-expressing cell yields. Moreover, the overall proportion of cells positive for Tie2 also increased 2.7-fold. Notably, the Col2 positivity rate was significantly higher on laminin-coated plates (Non-coat: 10.24% (±1.7%) versus Lami-coat: 26.2% (±7.5%), p = 0.010), and the ability to form spherical colonies also showed a significant improvement (Non-coat: 40.7 (±8.8)/1000 cells versus Lami-coat: 70.53 (±18.0)/1000 cells, p = 0.016). These findings demonstrate that Lami-coat enhances the potential of NP cells, as indicated by improved colony formation and proliferative characteristics. This highlights the potential of laminin-coating in maintaining the NP progenitor cell phenotype in culture, thereby supporting their translation into prospective clinical cell-transplantation products.

Dataset Information

Minimal Sustainability of Dedifferentiation by ROCK Inhibitor on Rat Nucleus Pulposus Cells In Vitro.

Introduction

Methods

Results

Conclusions

Publications

Minimal Sustainability of Dedifferentiation by ROCK Inhibitor on Rat Nucleus Pulposus Cells In Vitro.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets