Dataset Information

Effect of Conditioned Medium from Human Umbilical Cord-Derived Mesenchymal Stromal Cells on Rejuvenation of Nucleus Pulposus Derived Stem/Progenitor Cells from Degenerated Intervertebral Disc.

ABSTRACT:

Background and objectives

Mesenchymal stromal cells (MSCs)-based treatment for degeneration of intervertebral disc (IVD) has been proposed recently. We here addressed whether MSC secreted factors can rejuvenate nucleus pulposus- derived stem/progenitor cells from degenerated disc (D-NPSCs) in vitro.

Methods and results

We analyzed the expression of MSCs and NP cell specific surface markers, pluripotency related genes, multilineage potential and cell proliferative capacity of D-NPSCs upon incubation with the conditioned medium which was collected from the umbilical cord derived MSCs (UCMSCs). Our results indicated that the conditioned medium restore the stemness of D-NPSCs by up-regulating the expression level of CD29 and CD105, pluripotency related genes OCT4 and Nanog, and NP progenitor marker Tie2. The increased stemness was accompanied by promoted cell proliferative capacity and improved osteogenic and chondrogenic differentiation potential.

Conclusions

Our findings suggested that the UCMSCs derived conditioned medium might be used to rejuvenate the degenerated NP stem/progenitor cells.

SUBMITTER: Zeng X

PROVIDER: S-EPMC7378895 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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Publications

Effect of Conditioned Medium from Human Umbilical Cord-Derived Mesenchymal Stromal Cells on Rejuvenation of Nucleus Pulposus Derived Stem/Progenitor Cells from Degenerated Intervertebral Disc.

Zeng Xiaoli X Lin Jinhua J Wu Hao H Yu Jiayue J Tu Mei M Cheang Lek Hang LH Zhang Jiaqing J

International journal of stem cells 20200701 2

<h4>Background and objectives</h4>Mesenchymal stromal cells (MSCs)-based treatment for degeneration of intervertebral disc (IVD) has been proposed recently. We here addressed whether MSC secreted factors can rejuvenate nucleus pulposus- derived stem/progenitor cells from degenerated disc (D-NPSCs) in vitro.<h4>Methods and results</h4>We analyzed the expression of MSCs and NP cell specific surface markers, pluripotency related genes, multilineage potential and cell proliferative capacity of D-NPS ...[more]

PMID: 32587132

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Project description:BackgroundIntervertebral disc degeneration (IVDD) is a major contributor to spinal disorders. Previous studies have indicated that the infiltration of immunocytes, specifically macrophages, plays a crucial role in the advancement of IVDD. Exosomes (exo) are believed to play a significant role in intercellular communication. This study aims to investigate the role of exosomes derived from degenerated nucleus pulposus (dNPc) in the process of macrophages M1 polarization.MethodsNucleus pulposus (NP) tissue and nucleus pulposus cells (NPc) were collected from patients with intervertebral disc degeneration (IVDD) and idiopathic scoliosis. Immunohistochemistry analysis was performed to determine the number of M1 macrophages in NP tissue. Subsequently, exosomes derived from degenerated NP cells (dNPc-exo) and non-degenerated NP cells (nNPc-exo) were collected and co-cultured with M0 macrophages, which were induced from THP-1 cells. The M1 phenotype was assessed using western blot, flow cytometry, immunofluorescence staining, and qRT-PCR. RNA-sequencing analysis was conducted to examine the expression levels of microRNAs in the dNPc-exo and nNPc-exo groups, and qRT-PCR was performed to investigate the effect pf different microRNA to induce macrophage polarization. Furthermore, western blot and qRT-PCR were employed to demonstrate the regulatory effect of microRNAs carried by dNPc-exo on downstream target signaling pathways in macrophages. Finally, an animal model of IVDD was utilized to investigate the impact of dNPc-exo on inducing M1 polarization of macrophages and its role in the IVDD process.ResultsIn this study, we observed an increase in the number of M1 macrophages as the intervertebral disc (IVD) degraded. Additionally, we discovered that dNPc releases exosomes (dNPc-exo) could promote the polarization of macrophages towards the M1 phenotype. Notably, through RNA-sequencing analysis of dNPc-exo and nNPc-exo groups, we identified miR-27a-3p as a highly expressed miRNA in the dNPc-exo group, which significantly influences the induction of M1 polarization of macrophages. And then, we discovered that dNPc-exo has the ability to transport miR-27a-3p and target the PPARγ/NFκB/PI3K/AKT signaling pathway, thereby influencing the M1 polarization of macrophages. We conducted experiments using rat model of IVDD and observed that the exosomes carrying miR-27a-3p actually induced the M1 polarization of macrophages and exacerbated the degradation of IVD.ConclusionIn conclusion, our findings highlight the significant role of dNPc-exo in IVDD process and provide a basis for further investigation into the mechanism of IVDD and the potential of exosome-based therapy.

Project description:Introduction: An in vitro model that appropriately recapitulates the degenerative disc disease (DDD) microenvironment is needed to explore clinically relevant cell-based therapeutic strategies for early-stage degenerative disc disease. We developed an advanced 3D nucleus pulposus (NP) microtissues (µT) model generated with cells isolated from human degenerating NP tissue (Pfirrmann grade: 2-3), which were exposed to hypoxia, low glucose, acidity and low-grade inflammation. This model was then used to test the performance of nasal chondrocytes (NC) suspension or spheroids (NCS) after pre-conditioning with drugs known to exert anti-inflammatory or anabolic activities. Methods: NPµTs were formed by i) spheroids generated with NP cells (NPS) alone or in combination with ii) NCS or iii) NC suspension and cultured in healthy or degenerative disc disease condition. Anti-inflammatory and anabolic drugs (amiloride, celecoxib, metformin, IL-1Ra, GDF-5) were used for pre-conditioning of NC/NCS. The effects of pre-conditioning were tested in 2D, 3D, and degenerative NPµT model. Histological, biochemical, and gene expression analysis were performed to assess matrix content (glycosaminoglycans, type I and II collagen), production and release of inflammatory/catabolic factors (IL-6, IL-8, MMP-3, MMP-13) and cell viability (cleaved caspase 3). Results: The degenerative NPµT contained less glycosaminoglycans, collagens, and released higher levels of IL-8 compared to the healthy NPµT. In the degenerative NPµT, NCS performed superior compared to NC cell suspension but still showed lower viability. Among the different compounds tested, only IL-1Ra pre-conditioning inhibited the expression of inflammatory/catabolic mediators and promoted glycosaminoglycan accumulation in NC/NCS in DDD microenvironment. In degenerative NPµT model, preconditioning of NCS with IL-1Ra also provided superior anti-inflammatory/catabolic activity compared to non-preconditioned NCS. Conclusion: The degenerative NPµT model is suitable to study the responses of therapeutic cells to microenvironment mimicking early-stage degenerative disc disease. In particular, we showed that NC in spheroidal organization as compared to NC cell suspension exhibited superior regenerative performance and that IL-1Ra pre-conditioning of NCS could further improve their ability to counteract inflammation/catabolism and support new matrix production within harsh degenerative disc disease microenvironment. Studies in an orthotopic in vivo model are necessary to assess the clinical relevance of our findings in the context of IVD repair.

Project description:BACKGROUND: Notochordal cells (NC) remain in the focus of research for regenerative therapy for the degenerated intervertebral disc (IVD) due to their progenitor status. Recent findings suggested their regenerative action on more mature disc cells, presumably by the secretion of specific factors, which has been described as notochordal cell conditioned medium (NCCM). The aim of this study was to determine NC culture conditions (2D/3D, fetal calf serum, oxygen level) that lead to significant IVD cell activation in an indirect co-culture system under normoxia and hypoxia (2% oxygen). METHODS: Porcine NC was kept in 2D monolayer and in 3D alginate bead culture to identify a suitable culture system for these cells. To test stimulating effects of NC, co-cultures of NC and bovine derived coccygeal IVD cells were conducted in a 1:1 ratio with no direct cell contact between NC and bovine nucleus pulposus cell (NPC) or annulus fibrosus cells (AFC) in 3D alginate beads under normoxia and hypoxia (2%) for 7 and 14 days. As a positive control, NPC and AFC were stimulated with NC-derived conditioned medium (NCCM). Cell activity, glycosaminoglycan (GAG) content, DNA content and relative gene expression was measured. Mass spectrometry analysis of the NCCM was conducted. RESULTS: We provide evidence by flow cytometry that monolayer culture is not favorable for NC culture with respect to maintaining NC phenotype. In 3D alginate culture, NC activated NPC either in indirect co-culture or by addition of NCCM as indicated by the gene expression ratio of aggrecan/collagen type 2. This effect was strongest with 10% fetal calf serum and under hypoxia. Conversely, AFC seemed unresponsive to co-culture with pNC or to the NCCM. Further, the results showed that hypoxia led to decelerated metabolic activity, but did not lead to a significant change in the GAG/DNA ratio. Mass spectrometry identified connective tissue growth factor (CTGF, syn. CCN2) in the NCCM. CONCLUSIONS: Our results confirm the requirement to culture NC in 3D to best maintain their phenotype, preferentially in hypoxia and with the supplementation of FCS in the culture media. Despite these advancements, the ideal culture condition remains to be identified.

Project description:Conditioned medium derived from notochordal cell-rich nucleus pulposus tissue (NCCM) was previously shown to have a stimulatory effect on bone marrow stromal cells (BMSCs) and nucleus pulposus cells (NPCs) individually, in mixed species in vitro cell models. The objective of the current study was to assess the stimulatory effect of NCCM on NPCs in a homologous canine in vitro model and to investigate whether combined stimulation with NCCM and addition of BMSCs provides a synergistic stimulatory effect.BMSCs and NPCs were harvested from chondrodystrophic dogs with confirmed early intervertebral disc (IVD) degeneration. NCCM was produced from NP tissue of nonchondrodystrophic dogs with healthy IVDs. BMSCs or NPCs alone (3×10(6) cells/mL) and NPCs+BMSCs (6×10(6) cells/mL; mixed 1:1) were cultured for 4 weeks in 1.2% alginate beads under base medium (BM), NCCM, or with addition of 10?ng/mL transforming growth factor-?1 (TGF-?1) as a positive control. Beads were assessed for glycosaminoglycan (GAG) and DNA contents by biochemical assays, GAG deposition by Alcian blue staining, and gene expression (aggrecan, versican, collagen 1 and 2, SOX9, A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), and matrix metalloproteinase 13 [MMP13]) with real-time quantitative RT-PCR.NCCM increased NPC proliferation, proteoglycan production, and expression of genes associated with a healthy NP-like phenotype. BMSCs also showed increased proteoglycan production under NCCM, but these effects were not observed at the gene level. Combined stimulation of NPCs with NCCM and coculturing with BMSCs did not result in increased proteoglycan content compared to stimulation with NCCM alone.NCCM stimulates matrix production by both NPCs and BMSCs and directs NPCs toward a healthier phenotype. NCCM is therefore promising for IVD regeneration and identification of the bioactive components will be helpful to further develop this approach. In the current study, no synergistic effect of adding BMSCs was observed.

Dataset Information

Effect of Conditioned Medium from Human Umbilical Cord-Derived Mesenchymal Stromal Cells on Rejuvenation of Nucleus Pulposus Derived Stem/Progenitor Cells from Degenerated Intervertebral Disc.

Background and objectives

Methods and results

Conclusions

Publications

Effect of Conditioned Medium from Human Umbilical Cord-Derived Mesenchymal Stromal Cells on Rejuvenation of Nucleus Pulposus Derived Stem/Progenitor Cells from Degenerated Intervertebral Disc.

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