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Regenerative Response of Degenerate Human Nucleus Pulposus Cells to GDF6 Stimulation.

ABSTRACT: Growth differentiation factor (GDF) family members have been implicated in the development and maintenance of healthy nucleus pulposus (NP) tissue, making them promising therapeutic candidates for treatment of intervertebral disc (IVD) degeneration and associated back pain. GDF6 has been shown to promote discogenic differentiation of mesenchymal stem cells, but its effect on NP cells remains largely unknown. Our aim was to investigate GDF6 signalling in adult human NP cells derived from degenerate tissue and determine the signal transduction pathways critical for GDF6-mediated phenotypic changes and tissue homeostatic mechanisms. This study demonstrates maintained expression of GDF6 receptors in human NP and annulus fibrosus (AF) cells across a range of degeneration grades at gene and protein level. We observed an anabolic response in NP cells treated with recombinant GDF6 (increased expression of matrix and NP-phenotypic markers; increased glycosaminoglycan production; no change in catabolic enzyme expression), and identified the signalling pathways involved in these responses (SMAD1/5/8 and ERK1/2 phosphorylation, validated by blocking studies). These findings suggest that GDF6 promotes a healthy disc tissue phenotype in degenerate NP cells through SMAD-dependent and -independent (ERK1/2) mechanisms, which is important for development of GDF6 therapeutic strategies for treatment of degenerate discs.

SUBMITTER: Hodgkinson T

PROVIDER: S-EPMC7582366 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Regenerative Response of Degenerate Human Nucleus Pulposus Cells to GDF6 Stimulation.

Hodgkinson Tom T Gilbert Hamish T J HTJ Pandya Tej T Diwan Ashish D AD Hoyland Judith A JA Richardson Stephen M SM

International journal of molecular sciences 20200927 19

Growth differentiation factor (GDF) family members have been implicated in the development and maintenance of healthy nucleus pulposus (NP) tissue, making them promising therapeutic candidates for treatment of intervertebral disc (IVD) degeneration and associated back pain. GDF6 has been shown to promote discogenic differentiation of mesenchymal stem cells, but its effect on NP cells remains largely unknown. Our aim was to investigate GDF6 signalling in adult human NP cells derived from degenera ...[more]

PMID: 32992671

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Project description:BackgroundStem cell-based therapies show promise as a means of repairing the degenerate intervertebral disc, with growth factors often used alongside cells to help direct differentiation toward a nucleus pulposus (NP)-like phenotype. We previously demonstrated adipose-derived stem cell (ASC) differentiation with GDF6 as optimal for generating NP-like cells through evaluating end-stage differentiation parameters. Here we conducted a time-resolved transcriptomic characterization of ASCs response to GDF6 stimulation to understand the early drivers of differentiation to NP-like cells.MethodsHuman ASCs were treated with recombinant human GDF6 for 2, 6, and 12 h. RNA sequencing and detailed bioinformatic analysis were used to assess differential gene expression, gene ontology (GO), and transcription factor involvement during early differentiation. Quantitative polymerase chain reaction (qPCR) was used to validate RNA sequencing findings and inhibitors used to interrogate Smad and Erk signaling pathways, as well as identify primary and secondary response genes.ResultsThe transcriptomic response of ASCs to GDF6 stimulation was time-resolved and highly structured, with "cell differentiation" "developmental processes," and "response to stimulus" identified as key biological process GO terms. The transcription factor ERG1 was identified as a key early response gene. Temporal cluster analysis of differentiation genes identified positive regulation NP cell differentiation, as well as inhibition of osteogenesis and adipogenesis. A role for Smad and Erk signaling in the regulation of GDF6-induced early gene expression response was observed and both primary and secondary response genes were identified.ConclusionsThis study identifies a multifactorial early gene response that contributes to lineage commitment, with the identification of a number of potentially useful early markers of differentiation of ASCs to NP cells. This detailed insight into the molecular processes in response to GDF6 stimulation of ASCs is important for the development of an efficient and efficacious cell-based therapy for intervertebral disc degeneration-associated back pain.

Project description:Cell-based regenerative approaches have been suggested as primary or adjuvant procedures for the treatment of degenerated intervertebral disc (IVD) diseases. Our aim was to evaluate the regenerative and immunogenic properties of mildly and severely degenerated cervical nucleus pulposus (NP) cells with regard to cell isolation, proliferation and differentiation, as well as to cell surface markers and co-cultures with autologous or allogeneic peripheral blood mononuclear cells (PBMC) including changes in their immunogenic properties after 3-dimensional (3D)-culture. Tissue from the NP compartment of 10 patients with mild or severe grades of IVD degeneration was collected. Cells were isolated, expanded with and without basic fibroblast growth factor and cultured in 3D fibrin/poly (lactic-co-glycolic) acid transplants for 21 days. Real-time reverse-transcription polymerase chain reaction (RT-PCR) showed the expression of characteristic NP markers ACAN, COL1A1 and COL2A1 in 2D- and 3D-culture with degeneration- and culture-dependent differences. In a 5,6-carboxyfluorescein diacetate N-succinimidyl ester-based proliferation assay, NP cells in monolayer, regardless of their grade of degeneration, did not provoke a significant proliferation response in T cells, natural killer (NK) cells or B cells, not only with donor PBMC, but also with allogeneic PBMC. In conjunction with low inflammatory cytokine expression, analyzed by Cytometric Bead Array and fluorescence-activated cell sorting (FACS), a low immunogenicity can be assumed, facilitating possible therapeutic approaches. In 3D-culture, however, we found elevated immune cell proliferation levels, and there was a general trend to higher responses for NP cells from severely degenerated IVD tissue. This emphasizes the importance of considering the specific immunological alterations when including biomaterials in a therapeutic concept. The overall expression of Fas receptor, found on cultured NP cells, could have disadvantageous implications on their potential therapeutic applications because they could be the targets of cytotoxic T-cell activity acting by Fas ligand-induced apoptosis.

Project description:Study designIsolated human nucleus pulposus (hNP) cells from degenerated intervertebral disc were incubated under hydrostatic pressure and evaluated for regenerative potential.ObjectivesTo characterize metabolic turnover in hNP cells isolated from degenerated intervertebral discs classified by Pfirrmann grade under physiologically relevant hydrostatic pressure at high osmolality in vitro.Summary of background dataWe demonstrated that bovine caudal nucleus pulposus cells isolated from healthy cows produced more extracellular matrix under cyclic hydrostatic pressure followed by constant pressure (mimicking physiological intradiscal pressure in humans) than under no pressure in vitro. We assessed the effects of pressure on human degenerated cells isolated under the same regimen of pressure used for bovine cells.MethodshNP cells isolated from discarded tissue classified as Pfirrmann Grade 2-3 (n=13: age, 46.7±14.0) and Grade 4 (n=13: age, 53.0±11.5) were incubated under cyclic hydrostatic pressure at 0.2-0.7 MPa, 0.5 Hz for 2 days followed by constant pressure at 0.3 MPa for 1 day, repeated twice over 6 days. The gene expression and immunohistology of matrix molecules, catabolic and anti-catabolic proteins were evaluated.ResultsAggrecan and collagen type II expression was significantly more upregulated under HP in Grade 2-3 than in Grade 4 tissues (both, P<0.01). Linear regression analysis showed a positive correlation between matrix metalloproteinase 13 and tissue inhibitor for metalloproteinase 2 expression in Grade 2-3, while a negative correlation was found in Grade 4 (P<0.05). Immunohistological staining revealed the activation of a mechanoreceptor, transient receptor potential vanilloid 4, under HP.ConclusionsResident cells in mild-moderate degenerated disc classified as Pfirrmann grade 2-3 have the potential to promote extracellular matrix production and maintain adequate cell viability under physiological spinal loading.RelevanceThis study explored the potential of degenerated remnant NP cells under physiological environment, possibly leading to establishing strategies for IVD regeneration.

Project description: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.

Project description:STUDY DESIGN:Development of an in vitro model for assessing the anti-inflammatory efficacies of naringin (Nar) and naringenin (NG). PURPOSE:To evaluate the efficacy of natural flavonoids as therapeutic drugs against anti-inflammatory processes in the nucleus pulposus (NP) cells using in-vitro and in-silico methods. OVERVIEW OF LITERATURE:Intervertebral disc (IVD) disease is a common cause of low back pain. Chronic inflammation and degeneration play a significant role in its etiopathology. Thus, a better understanding of anti-inflammatory agents and their role in IVD degeneration and pro-inflammatory cytokines expression is necessary for pain management and regeneration in IVD. METHODS:We performed primary cell culture of NP cells; immunocytochemistry; gene expression studies of cytokines, metalloproteases, extracellular proteins, and apoptotic markers using quantitative polymerase chain reaction and reverse transcription-polymerase chain reaction (RT-PCR); cytotoxicity assay (MTT); and molecular docking studies using AutoDock 4.2 software (Molecular Graphics Laboratory, La Jolla, CA, USA) to confirm the binding mode of proteins and synthesized complexes. We calculated the mean±standard deviation values and performed analysis of variance and t-test using SPSS ver. 17.0 (SPSS, Inc., Chicago, IL, USA). RESULTS:Molecular docking showed that both Nar and NG bind to the selected genes of interest. Semi-quantitative RT-PCR analysis reveals differential gene expression of collagen (COL)9A1, COL9A2, COL9A3, COL11A2, COMT (catechol-O-methyltransferase), and THBS2 (thrombospondin 2); up regulation of ACAN (aggrecan), COL1A1, COL11A1, interleukin (IL)6, IL10, IL18R1, IL18RAP, metalloprotease (MMP)2, MMP3, MMP9, ADAMTS5 (a disintegrin and metalloproteinase with thrombospondin motifs 5), IGF1R (insulin-like growth factor type 1 receptor), SPARC (secreted protein acidic and cysteine rich), PARK2 (parkin), VDR (vitamin D receptor), and BCL2 (B-cell lymphoma 2); down regulation of IL1A, CASP3 (caspase 3), and nine genes with predetermined concentrations of Nar and NG. CONCLUSIONS:The present study evaluated the anti-inflammatory and regenerative efficiencies of Nar and NG in degenerated human NP cells. Altered gene expressions of cytokines, metalloproteases, extracellular proteins, apoptotic genes were dose responsive. The molecular docking (in silico) studies showed effective binding of these native ligands (Nar and NG) with genes identified as potent inhibitors of inflammation. Thus, these natural flavonoids could serve as anti-inflammatory agents in the treatment of low back pain and sciatica.

Project description:BACKGROUND:Intervertebral disc (IVD) degeneration is characterized by an early decrease in cellularity of the nucleus pulposus (NP) region, and associated extracellular matrix changes, reduced hydration, and progressive degeneration. Cell-based IVD therapy has emerged as an area of great interest, with studies reporting regenerative potential for many cell sources, including autologous or allogeneic chondrocytes, primary IVD cells, and stem cells. Few approaches, however, have clear strategies to promote the NP phenotype, in part due to a limited knowledge of the defined markers and differentiation protocols for this lineage. Here, we developed a new protocol for the efficient differentiation of human induced pluripotent stem cells (hiPSCs) into NP-like cells in vitro. This differentiation strategy derives from our knowledge of the embryonic notochordal lineage of NP cells as well as strategies used to support healthy NP cell phenotypes for primary cells in vitro. METHODS:An NP-genic phenotype of hiPSCs was promoted in undifferentiated hiPSCs using a stepwise, directed differentiation toward mesodermal, and subsequently notochordal, lineages via chemically defined medium and growth factor supplementation. Fluorescent cell imaging was used to test for pluripotency markers in undifferentiated cells. RT-PCR was used to test for potential cell lineages at the early stage of differentiation. Cells were checked for NP differentiation using immunohistochemistry and histological staining at the end of differentiation. To enrich notochordal progenitor cells, hiPSCs were transduced using lentivirus containing reporter constructs for transcription factor brachyury (T) promoter and green fluorescent protein (GFP) fluorescence, and then sorted on T expression based on GFP intensity by flow cytometry. RESULTS:Periods of pellet culture following initial induction were shown to promote the vacuolated NP cell morphology and NP surface marker expression, including CD24, LM?5, and Basp1. Enrichment of brachyury (T) positive cells using fluorescence-activated cell sorting was shown to further enhance the differentiation efficiency of NP-like cells. CONCLUSIONS:The ability to efficiently differentiate human iPSCs toward NP-like cells may provide insights into the processes of NP cell differentiation and provide a cell source for the development of new therapies for IVD diseases.

Dataset Information

Regenerative Response of Degenerate Human Nucleus Pulposus Cells to GDF6 Stimulation.

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Regenerative Response of Degenerate Human Nucleus Pulposus Cells to GDF6 Stimulation.

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