Transcriptomic profiling reveals key early response genes during GDF6-mediated differentiation of human adipose-derived stem cells to nucleus pulposus cells.

Background: Stem 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. Methods: Human 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. Results: The 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. Conclusions: This 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.

Loss of regulation of protein synthesis and turnover underpins an attenuated stress response in senescent human mesenchymal stem cells.

Cells respond to stress by synthesizing chaperone proteins that seek to correct protein misfolding and maintain function. However, abrogation of protein homeostasis is a hallmark of aging, leading to loss of function and the formation of proteotoxic aggregates characteristic of pathology. Consequently, discovering the underlying molecular causes of this deterioration in proteostasis is key to designing effective interventions to disease or to maintaining cell health in regenerative medicine strategies. Here, we examined primary human mesenchymal stem cells, cultured to a point of replicative senescence and subjected to heat shock, as an in vitro model of the aging stress response. Multi -omics analysis showed how homeostasis components were reduced in senescent cells, caused by dysregulation of a functional network of chaperones, thereby limiting proteostatic competence. Time-resolved analysis of the primary response factors, including those regulating heat shock protein 70 kDa (HSPA1A), revealed that regulatory control is essentially translational. Senescent cells have a reduced capacity for chaperone protein translation and misfolded protein (MFP) turnover, driven by downregulation of ribosomal proteins and loss of the E3 ubiquitin ligase CHIP (C-terminus of HSP70 interacting protein) which marks MFPs for degradation. This limits the cell's stress response and subsequent recovery. A kinetic model recapitulated these reduced capacities and predicted an accumulation of MFP, a hypothesis supported by evidence of systematic changes to the proteomic fold state. These results thus establish a specific loss of regulatory capacity at the protein, rather than transcript, level and uncover underlying systematic links between aging and loss of protein homeostasis.

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

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.

Mechanically activated Piezo1 channels of cardiac fibroblasts stimulate p38 mitogen-activated protein kinase activity and interleukin-6 secretion.

Piezo1 is a mechanosensitive cation channel with widespread physiological importance; however, its role in the heart is poorly understood. Cardiac fibroblasts help preserve myocardial integrity and play a key role in regulating its repair and remodeling following stress or injury. Here we investigated Piezo1 expression and function in cultured human and mouse cardiac fibroblasts. RT-PCR experiments confirmed that Piezo1 mRNA in cardiac fibroblasts is expressed at levels similar to those in endothelial cells. The results of a Fura-2 intracellular Ca2+ assay validated Piezo1 as a functional ion channel that is activated by its agonist, Yoda1. Yoda1-induced Ca2+ entry was inhibited by Piezo1 blockers (gadolinium and ruthenium red) and was reduced proportionally by siRNA-mediated Piezo1 knockdown or in murine Piezo1+/- cells. Results from cell-attached patch clamp recordings on human cardiac fibroblasts established that they contain mechanically activated ion channels and that their pressure responses are reduced by Piezo1 knockdown. Investigation of Yoda1 effects on selected remodeling genes indicated that Piezo1 activation increases both mRNA levels and protein secretion of IL-6, a pro-hypertrophic and profibrotic cytokine, in a Piezo1-dependent manner. Moreover, Piezo1 knockdown reduced basal IL-6 expression from cells cultured on softer collagen-coated substrates. Multiplex kinase activity profiling combined with kinase inhibitor experiments and phosphospecific immunoblotting established that Piezo1 activation stimulates IL-6 secretion via the p38 mitogen-activated protein kinase downstream of Ca2+ entry. In summary, cardiac fibroblasts express mechanically activated Piezo1 channels coupled to secretion of the paracrine signaling molecule IL-6. Piezo1 may therefore be important in regulating cardiac remodeling.

Nuclear decoupling is part of a rapid protein-level cellular response to high-intensity mechanical loading.

Studies of cellular mechano-signaling have often utilized static models that do not fully replicate the dynamics of living tissues. Here, we examine the time-dependent response of primary human mesenchymal stem cells (hMSCs) to cyclic tensile strain (CTS). At low-intensity strain (1 h, 4% CTS at 1 Hz), cell characteristics mimic responses to increased substrate stiffness. As the strain regime is intensified (frequency increased to 5 Hz), we characterize rapid establishment of a broad, structured and reversible protein-level response, even as transcription is apparently downregulated. Protein abundance is quantified coincident with changes to protein conformation and post-translational modification (PTM). Furthermore, we characterize changes to the linker of nucleoskeleton and cytoskeleton (LINC) complex that bridges the nuclear envelope, and specifically to levels and PTMs of Sad1/UNC-84 (SUN) domain-containing protein 2 (SUN2). The result of this regulation is to decouple mechano-transmission between the cytoskeleton and the nucleus, thus conferring protection to chromatin.

The consequences of ageing, progeroid syndromes and cellular senescence on mechanotransduction and the nucleus.

The ageing process is a progressive decrease in physiological function, caused by accruement of damage and misregulation in the cells and tissues of an organism. Human ageing has been the focus of much scientific investigation, but studies have been complicated by the variability of the process between subjects and the slow pace at which it occurs. Although the consequences of ageing on cellular biochemical signalling and metabolism have been well studied, the impact on the mechanical properties of cells and the extracellular matrix - and the mechanotransduction pathways that connect the two - have often been overlooked. In this review we will discuss recent advances in the fields of nuclear and cytoskeletal biophysics, and consider this work in the context of ageing. In particular, we will examine the role of the nucleus in cellular mechanotransduction and in 'age-related diseases/phenomena' such as progeria and cellular senescence. Finally, we will discuss the therapeutic options being explored, drawing attention to a new field of medicine termed 'mechano-medicine' that may prove useful in addressing age-related pathology.

An immortalised mesenchymal stem cell line maintains mechano-responsive behaviour and can be used as a reporter of substrate stiffness.

The mechanical environment can influence cell behaviour, including changes to transcriptional and proteomic regulation, morphology and, in the case of stem cells, commitment to lineage. However, current tools for characterizing substrates' mechanical properties, such as atomic force microscopy (AFM), often do not fully recapitulate the length and time scales over which cells 'feel' substrates. Here, we show that an immortalised, clonal line of human mesenchymal stem cells (MSCs) maintains the responsiveness to substrate mechanics observed in primary cells, and can be used as a reporter of stiffness. MSCs were cultured on soft and stiff polyacrylamide hydrogels. In both primary and immortalised MSCs, stiffer substrates promoted increased cell spreading, expression of lamin-A/C and translocation of mechano-sensitive proteins YAP1 and MKL1 to the nucleus. Stiffness was also found to regulate transcriptional markers of lineage. A GFP-YAP/RFP-H2B reporter construct was designed and virally delivered to the immortalised MSCs for in situ detection of substrate stiffness. MSCs with stable expression of the reporter showed GFP-YAP to be colocalised with nuclear RFP-H2B on stiff substrates, enabling development of a cellular reporter of substrate stiffness. This will facilitate mechanical characterisation of new materials developed for applications in tissue engineering and regenerative medicine.

Degenerate intervertebral disc-like pH induces a catabolic mechanoresponse in human nucleus pulposus cells.

Mechanical stimulation is known to influence intervertebral disc (IVD) cell behavior and function, but the effect on disc cells is routinely considered in isolation from other microenvironmental factors. Acidic pH has been shown to be a prominent and detrimental microenvironmental factor present in degenerate IVDs, but its influence on the human disc cell mechanoresponse has never been studied. We investigated the response of agarose-encapsulated human nucleus pulposus (NP) cells to 0.004 MPa, 1.0 Hz and 1 hour of compression (Flexcell FX4000 Compression System) under pH conditions representative of nondegenerate (pH 7.1) and degenerate (pH 6.5) IVDs. Cell viability, extracellular matrix production, and expression of anabolic/anti-catabolic and catabolic genes were assessed. We report that preculture of NP cells in agarose gels was required in order for cells to be mechanoresponsive, and this correlated with increased type VI collagen, α5ß1 integrin, and fibronectin expression. Furthermore, the matrix homeostatic response observed at pH 7.1 (representative of nondegenerate IVDs; increased aggrecan [AGC], tissue inhibitor of metalloproteinases-1 [TIMP1], matrix metalloproteinase-3 [MMP3], a disintegrin and metalloproteinase with thrombospondin motif-5 [ADAMTS5] gene expression) was RGD-integrin dependent, whereas only MMP3 remained mechanoresponsive at pH 6.5, and this was independent of RGD-integrins. Our findings suggest differential mechanotransduction pathways operating for specific genes, with RGD-integrin dependent AGC expression, but not RGD-independent MMP3 expression, inhibited at pH representative of degenerate IVDs (pH 6.5), which could contribute to the catabolic phenotype observed during IVD degeneration. CLINICAL SIGNIFICANCE: Characterizing the influence of the mechanical and chemical intervertebral disc microenvironment on disc cells, particularly in disc degeneration, could help develop future therapeutic strategies for the treatment of discogenic back pain.

Acidic pH promotes intervertebral disc degeneration: Acid-sensing ion channel -3 as a potential therapeutic target.

The aetiology of intervertebral disc (IVD) degeneration remains poorly understood. Painful IVD degeneration is associated with an acidic intradiscal pH but the response of NP cells to this aberrant microenvironmental factor remains to be fully characterised. The aim here was to address the hypothesis that acidic pH, similar to that found in degenerate IVDs, leads to the altered cell/functional phenotype observed during IVD degeneration, and to investigate the involvement of acid-sensing ion channel (ASIC) -3 in the response. Human NP cells were treated with a range of pH, from that of a non-degenerate (pH 7.4 and 7.1) through to mildly degenerate (pH 6.8) and severely degenerate IVD (pH 6.5 and 6.2). Increasing acidity of pH caused a decrease in cell proliferation and viability, a shift towards matrix catabolism and increased expression of proinflammatory cytokines and pain-related factors. Acidic pH resulted in an increase in ASIC-3 expression. Importantly, inhibition of ASIC-3 prevented the acidic pH induced proinflammatory and pain-related phenotype in NP cells. Acidic pH causes a catabolic and degenerate phenotype in NP cells which is inhibited by blocking ASIC-3 activity, suggesting that this may be a useful therapeutic target for treatment of IVD degeneration.

In situ-forming robust chitosan-poly(ethylene glycol) hydrogels prepared by copper-free azide-alkyne click reaction for tissue engineering.

A water-soluble azide-functionalised chitosan was crosslinked with propiolic acid ester-functional poly(ethylene glycol) using copper-free click chemistry. The resultant hydrogel materials were formed within 5-60 min at 37 °C and resulted in mechanically robust materials with tuneable properties such as swelling, mechanical strength and degradation. Importantly, the hydrogels supported mesenchymal stem cell attachment and proliferation and were also non-toxic to encapsulated cells. As such these studies indicate that the hydrogels have potential to be used as injectable biomaterials for tissue engineering.

A microstereolithography resin based on thiol-ene chemistry: towards biocompatible 3D extracellular constructs for tissue engineering.

A new class of degradable aliphatic poly(carbonate) resins for use in microstereolithographic process is described. Using a biologically inert photo-inhibiting dye, exemplar 3-dimensional structures were produced using thiol-ene chemistry via microstereolithography. Fabricated constructs demonstrated good biological compatibility with cells and had tensile properties that render them suitable for use as tissue engineering scaffolds.

Stem cell regeneration of degenerated intervertebral discs: current status (update).

Low back pain, strongly associated with intervertebral disc (IVD) degeneration, affects a large proportion of the population and has major social and economic costs. Current treatments remain inadequate, targeting the symptoms without addressing the underlying cause. As such, efforts are being directed towards development of therapies aimed at alleviating pain through the restoration of IVD function. The potential of cell-based therapies for the treatment of IVD degeneration are being actively explored, with an emphasis on cell/biomaterial tissue engineering. Adult mesenchymal stem cells, capable of differentiating down the discogenic lineage, have shown promise as a suitable cell source for IVD tissue engineering. However, a number of factors, (discussed in this review), remain to be addressed, including development of a differentiation protocol to produce the correct cell phenotype, identification of suitable biomaterials for cell delivery/implantation, and ensuring cell survival and correct function upon implantation into the degenerate IVD.

Integrin - dependent mechanotransduction in mechanically stimulated human annulus fibrosus cells: evidence for an alternative mechanotransduction pathway operating with degeneration.

Intervertebral disc (IVD) cells derived from degenerate tissue respond aberrantly to mechanical stimuli, potentially due to altered mechanotransduction pathways. Elucidation of the altered, or alternative, mechanotransduction pathways operating with degeneration could yield novel targets for the treatment of IVD disease. Our aim here was to investigate the involvement of RGD-recognising integrins and associated signalling molecules in the response to cyclic tensile strain (CTS) of human annulus fibrosus (AF) cells derived from non-degenerate and degenerate IVDs. AF cells from non-degenerate and degenerate human IVDs were cyclically strained with and without function blocking RGD - peptides with 10% strain, 1.0 Hz for 20 minutes using a Flexercell® strain device. QRT-PCR and Western blotting were performed to analyse gene expression of type I collagen and ADAMTS -4, and phosphorylation of focal adhesion kinase (FAK), respectively. The response to 1.0 Hz CTS differed between the two groups of AF cells, with decreased ADAMTS -4 gene expression and decreased type I collagen gene expression post load in AF cells derived from non-degenerate and degenerate IVDs, respectively. Pre-treatment of non-degenerate AF cells with RGD peptides prevented the CTS-induced decrease in ADAMTS -4 gene expression, but caused an increase in expression at 24 hours, a response not observed in degenerate AF cells where RGD pre-treatment failed to inhibit the mechano-response. In addition, FAK phosphorylation increased in CTS stimulated AF cells derived from non-degenerate, but not degenerate IVDs, with RGD pre-treatment inhibiting the CTS - dependent increase in phosphorylated FAK. Our findings suggest that RGD -integrins are involved in the 1.0 Hz CTS - induced mechano-response observed in AF cells derived from non-degenerate, but not degenerate IVDs. This data supports our previous work, suggesting an alternative mechanotransduction pathway may be operating in degenerate AF cells.

Fabrication of 3-dimensional cellular constructs via microstereolithography using a simple, three-component, poly(ethylene glycol) acrylate-based system.

A novel method for the production of inhibitor- and solvent-free resins suitable for three-dimensional (3D) microstereolithography is reported. Using an exemplar poly(ethylene glycol)-based resin, the control of features in the X, Y, and Z planes is demonstrated such that complex structures can be manufactured. Human mesenchymal stem cells cultured on the manufactured scaffolds remained viable during the 7 day assessment period, with proliferation rates comparable to those observed on tissue culture polystyrene. These data suggest that this novel, yet simple, method is suitable for the production of 3D scaffolds for tissue engineering and regenerative medicine applications.

The involvement of interleukin-1 and interleukin-4 in the response of human annulus fibrosus cells to cyclic tensile strain: an altered mechanotransduction pathway with degeneration.

INTRODUCTION: Recent evidence suggests that intervertebral disc (IVD) cells derived from degenerative tissue are unable to respond to physiologically relevant mechanical stimuli in the 'normal' anabolic manner, but instead respond by increasing matrix catabolism. Understanding the nature of the biological processes which allow disc cells to sense and respond to mechanical stimuli (a process termed 'mechanotransduction') is important to ascertain whether these signalling pathways differ with disease. The aim here was to investigate the involvement of interleukin (IL)-1 and IL-4 in the response of annulus fibrosus (AF) cells derived from nondegenerative and degenerative tissue to cyclic tensile strain to determine whether cytokine involvement differed with IVD degeneration. METHODS: AF cells were isolated from nondegenerative and degenerative human IVDs, expanded in monolayers and cyclically strained in the presence or absence of the cytokine inhibitors IL-1 receptor antagonist (IL-1Ra) or IL-4 receptor antibody (IL-4RAb) with 10% strain at 1.0 Hz for 20 minutes using a Flexcell strain device. Total RNA was extracted from the cells at time points of baseline control and 1 or 24 hours poststimulation. Quantitative real-time polymerase chain reaction was used to analyse the gene expression of matrix proteins (aggrecan and type I collagen) and enzymes (matrix metalloproteinase 3 (MMP3) and a disintegrin and metalloproteinase with a thrombospondin type 1 motif 4 (ADAMTS4)). RESULTS: Expression of catabolic genes (MMP3 and ADAMTS4) decreased in AF cells derived from nondegenerative tissue in response to 1.0-Hz stimulation, and this decrease in gene expression was inhibited or increased following pretreatment of cells with IL-1Ra or IL-4RAb respectively. Treatment of AF cells derived from degenerative tissue with an identical stimulus (1.0-Hz) resulted in reduced anabolic gene expression (aggrecan and type I collagen), with IL-1Ra or IL-4RAb pretreatment having no effect. CONCLUSIONS: Both IL-1 and IL-4 are involved in the response of AF cells derived from nondegenerative tissue to 1.0-Hz cyclic tensile strain. Interestingly, the altered response observed at 1.0-Hz in AF cells from degenerative tissue appears to be independent of either cytokine, suggesting an alternative mechanotransduction pathway in operation.

The response of human anulus fibrosus cells to cyclic tensile strain is frequency-dependent and altered with disc degeneration.

OBJECTIVE: Mechanical loads are important for homeostasis of the intervertebral disc (IVD) cell matrix, with physiologic and nonphysiologic loads leading to matrix anabolism and catabolism, respectively. Previous investigations into the effects of load on disc cells have predominantly used animal models, with the limited number of human studies focusing primarily on nucleus pulposus cells. The aim of this study was to examine the effect of cyclic tensile strain (CTS) on human anulus fibrosus (AF) cells to ascertain whether the response was frequency-dependent and to compare AF cells derived from nondegenerated and degenerated tissue samples. METHODS: AF cells were isolated from nondegenerated and degenerated human IVDs, expanded in monolayer, and cyclically strained for 20 minutes, applying 10% strain at a frequency of 1.0 Hz or 0.33 Hz with the use of a Flexcell strain device. Total RNA was extracted from the cells at baseline (control) and at 1, 3, and 24 hours following application of CTS. Real-time quantitative polymerase chain reaction was used to analyze gene expression of matrix proteins (aggrecan, type I collagen, and type II collagen) and enzymes (matrix metalloproteinases [MMPs] 3, 9, 13, and ADAMTS-4). RESULTS: The expression of catabolic genes (MMP-3 and ADAMTS-4) in AF cells derived from nondegenerated tissue decreased in response to 1.0 Hz of CTS, whereas changing the frequency to 0.33 Hz resulted in a shift toward matrix catabolism. Application of 1.0 Hz of CTS reduced anabolic gene expression (aggrecan and type I collagen) in AF cells derived from degenerated tissue, with 0.33 Hz of CTS resulting in increased catabolic gene expression. CONCLUSION: The response of human AF cells to CTS is frequency-dependent and is altered by degeneration.