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.

Effect of Peptide-Polymer Host-Guest Electrostatic Interactions on Self-Assembling Peptide Hydrogels Structural and Mechanical Properties and Polymer Diffusivity.

Peptide-based supramolecular hydrogels are an attractive class of soft materials for biomedical applications when biocompatibility is a key requirement as they exploit the physical self-assembly of short self-assembling peptides avoiding the need for chemical cross-linking. Based on the knowledge developed through our previous work, we designed two novel peptides, E(FKFE)2 and K(FEFK)2, that form transparent hydrogels at pH 7. We characterized the phase behavior of these peptides and showed the clear link that exists between the charge carried by the peptides and the physical state of the samples. We subsequently demonstrate the cytocompatibility of the hydrogel and its suitability for 3D cell culture using 3T3 fibroblasts and human mesenchymal stem cells. We then loaded the hydrogels with two polymers, poly-l-lysine and dextran. When polymer and peptide fibers carry opposite charges, the size of the elemental fibril formed decreases, while the overall level of fiber aggregation and fiber bundle formation increases. This overall network topology change, and increase in cross-link stability and density, leads to an overall increase in the hydrogel mechanical properties and stability, i.e., resistance to swelling when placed in excess media. Finally, we investigate the diffusion of the polymers out of the hydrogels and show how electrostatic interactions can be used to control the release of large molecules. The work clearly shows how polymers can be used to tailor the properties of peptide hydrogels through guided intermolecular interactions and demonstrates the potential of these new soft hydrogels for use in the biomedical field in particular for delivery or large molecular payloads and cells as well as scaffolds for 3D cell culture.

Characterization of the Biophysical Properties and Cell Adhesion Interactions of Marine Invertebrate Collagen from Rhizostoma pulmo.

Collagen is the most ubiquitous biomacromolecule found in the animal kingdom and is commonly used as a biomaterial in regenerative medicine therapies and biomedical research. The collagens used in these applications are typically derived from mammalian sources which poses sociological issues due to widespread religious constraints, rising ethical concern over animal rights and the continuous risk of zoonotic disease transmission. These issues have led to increasing research into alternative collagen sources, of which marine collagens, in particular from jellyfish, have emerged as a promising resource. This study provides a characterization of the biophysical properties and cell adhesion interactions of collagen derived from the jellyfish Rhizostoma pulmo (JCol). Circular dichroism spectroscopy and atomic force microscopy were used to observe the triple-helical conformation and fibrillar morphology of JCol. Heparin-affinity chromatography was also used to demonstrate the ability of JCol to bind to immobilized heparin. Cell adhesion assays using integrin blocking antibodies and HT-1080 human fibrosarcoma cells revealed that adhesion to JCol is primarily performed via ß1 integrins, with the exception of α2ß1 integrin. It was also shown that heparan sulfate binding plays a much greater role in fibroblast and mesenchymal stromal cell adhesion to JCol than for type I mammalian collagen (rat tail collagen). Overall, this study highlights the similarities and differences between collagens from mammalian and jellyfish origins, which should be considered when utilizing alternative collagen sources for biomedical research.

Collagen-like Osteoclast-Associated Receptor (OSCAR)-Binding Motifs Show a Co-Stimulatory Effect on Osteoclastogenesis in a Peptide Hydrogel System.

Osteoclastogenesis, one of the dynamic pathways underlying bone remodelling, is a complex process that includes many stages. This complexity, while offering a wealth of therapeutic opportunities, represents a substantial challenge in unravelling the underlying mechanisms. As such, there is a high demand for robust model systems to understand osteoclastogenesis. Hydrogels seeded with osteoclast precursors and decorated with peptides or proteins mimicking bone's extracellular matrix could provide a useful synthetic tool to study pre-osteoclast-matrix interactions and their effect on osteoclastogenesis. For instance, fibrillar collagens have been shown to provide a co-stimulatory pathway for osteoclastogenesis through interaction with the osteoclast-associated receptor (OSCAR), a regulator of osteoclastogenesis expressed on the surface of pre-osteoclast cells. Based on this rationale, here we design two OSCAR-binding peptides and one recombinant OSCAR-binding protein, and we combine them with peptide-based hydrogels to study their effect on osteoclastogenesis. The OSCAR-binding peptides adopt the collagen triple-helical conformation and interact with OSCAR, as shown by circular dichroism spectropolarimetry and surface plasmon resonance. Furthermore, they have a positive effect on osteoclastogenesis, as demonstrated by appropriate gene expression and tartrate-resistant acid phosphatase staining typical of osteoclast formation. Combination of the OSCAR-binding peptides or the OSCAR-binding recombinant protein with peptide-based hydrogels enhances osteoclast differentiation when compared to the non-modified hydrogels, as demonstrated by multi-nucleation and by F-actin staining showing a characteristic osteoclast-like morphology. We envisage that these hydrogels could be used as a platform to study osteoclastogenesis and, in particular, to investigate the effect of costimulatory pathways involving OSCAR.

Importance of Matrix Cues on Intervertebral Disc Development, Degeneration, and Regeneration.

Back pain is one of the leading causes of disability worldwide and is frequently caused by degeneration of the intervertebral discs. The discs' development, homeostasis, and degeneration are driven by a complex series of biochemical and physical extracellular matrix cues produced by and transmitted to native cells. Thus, understanding the roles of different cues is essential for designing effective cellular and regenerative therapies. Omics technologies have helped identify many new matrix cues; however, comparatively few matrix molecules have thus far been incorporated into tissue engineered models. These include collagen type I and type II, laminins, glycosaminoglycans, and their biomimetic analogues. Modern biofabrication techniques, such as 3D bioprinting, are also enabling the spatial patterning of matrix molecules and growth factors to direct regional effects. These techniques should now be applied to biochemically, physically, and structurally relevant disc models incorporating disc and stem cells to investigate the drivers of healthy cell phenotype and differentiation. Such research will inform the development of efficacious regenerative therapies and improved clinical outcomes.

Urocortin-1 Is Chondroprotective in Response to Acute Cartilage Injury via Modulation of Piezo1.

Post-traumatic OA (PTOA) is often triggered by injurious, high-impact loading events which result in rapid, excessive chondrocyte cell death and a phenotypic shift in residual cells toward a more catabolic state. As such, the identification of a disease-modifying OA drug (DMOAD) that can protect chondrocytes from death following impact injury, and thereby prevent cartilage degradation and progression to PTOA, would offer a novel intervention. We have previously shown that urocortin-1 (Ucn) is an essential endogenous pro-survival factor that protects chondrocytes from OA-associated pro-apoptotic stimuli. Here, using a drop tower PTOA-induction model, we demonstrate the extent of Ucn's chondroprotective role in cartilage explants exposed to excessive impact load. Using pathway-specific agonists and antagonists, we show that Ucn acts to block load-induced intracellular calcium accumulation through blockade of the non-selective cation channel Piezo1 rather than TRPV4. This protective effect is mediated primarily through the Ucn receptor CRF-R1 rather than CRF-R2. Crucially, we demonstrate that the chondroprotective effect of Ucn is maintained whether it is applied pre-impact or post-impact, highlighting the potential of Ucn as a novel DMOAD for the prevention of injurious impact overload-induced PTOA.

Multiscale Regulation of the Intervertebral Disc: Achievements in Experimental, In Silico, and Regenerative Research.

Intervertebral disc (IVD) degeneration is a major risk factor of low back pain. It is defined by a progressive loss of the IVD structure and functionality, leading to severe impairments with restricted treatment options due to the highly demanding mechanical exposure of the IVD. Degenerative changes in the IVD usually increase with age but at an accelerated rate in some individuals. To understand the initiation and progression of this disease, it is crucial to identify key top-down and bottom-up regulations' processes, across the cell, tissue, and organ levels, in health and disease. Owing to unremitting investigation of experimental research, the comprehension of detailed cell signaling pathways and their effect on matrix turnover significantly rose. Likewise, in silico research substantially contributed to a holistic understanding of spatiotemporal effects and complex, multifactorial interactions within the IVD. Together with important achievements in the research of biomaterials, manifold promising approaches for regenerative treatment options were presented over the last years. This review provides an integrative analysis of the current knowledge about (1) the multiscale function and regulation of the IVD in health and disease, (2) the possible regenerative strategies, and (3) the in silico models that shall eventually support the development of advanced therapies.

Using Stereochemistry to Control Mechanical Properties in Thiol-Yne Click-Hydrogels.

The stereochemistry of polymers has a profound impact on their mechanical properties. While this has been observed in thermoplastics, studies on how stereochemistry affects the bulk properties of swollen networks, such as hydrogels, are limited. Typically, changing the stiffness of a hydrogel is achieved at the cost of changing another parameter, that in turn affects the physical properties of the material and ultimately influences the cellular response. Herein, we report that by manipulating the stereochemistry of a double bond, formed in situ during gelation, materials with diverse mechanical properties but comparable physical properties can be obtained. Click-hydrogels that possess a high % trans content are stiffer than their high % cis analogues by almost a factor of 3. Human mesenchymal stem cells acted as a substrate stiffness cell reporter demonstrating the potential of these platforms to study mechanotransduction without the influence of other external factors.

BayesENproteomics: Bayesian Elastic Nets for Quantification of Peptidoforms in Complex Samples.

Multivariate regression modelling provides a statistically powerful means of quantifying the effects of a given treatment while compensating for sources of variation and noise, such as variability between human donors and the behavior of different peptides during mass spectrometry. However, methods to quantify endogenous post-translational modifications (PTMs) are typically reliant on summary statistical methods that fail to consider sources of variability such as changes in the levels of the parent protein. Here, we compare three multivariate regression methods, including a novel Bayesian elastic net algorithm (BayesENproteomics) that enables assessment of relative protein abundances while also quantifying identified PTMs for each protein. We tested the ability of these methods to accurately quantify expression of proteins in a mixed-species benchmark experiment and to quantify synthetic PTMs induced by stable isotope labelling. Finally, we extended our regression pipeline to calculate fold changes at the pathway level, providing a complement to commonly used enrichment analysis. Our results show that BayesENproteomics can quantify changes to protein levels across a broad dynamic range while also accurately quantifying PTM and pathway-level fold changes.

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.

Photoresponsive Hydrogels with Photoswitchable Stiffness: Emerging Platforms to Study Temporal Aspects of Mesenchymal Stem Cell Responses to Extracellular Stiffness Regulation.

An extensive number of cell-matrix interaction studies have identified matrix stiffness as a potent regulator of cellular properties and behaviours. Perhaps most notably, matrix stiffness has been demonstrated to regulate mesenchymal stem cell (MSC) phenotype and lineage commitment. Given the therapeutic potential for MSCs in regenerative medicine, significant efforts have been made to understand the molecular mechanisms involved in stiffness regulation. These efforts have predominantly focused on using stiffness-defined polyacrylamide (PA) hydrogels to culture cells in 2D and have enabled elucidation of a number of mechano-sensitive signalling pathways. However, despite proving to be a valuable tool, these stiffness-defined hydrogels do not reflect the dynamic nature of living tissues, which are subject to continuous remodelling during processes such as development, ageing, disease and regeneration. Therefore, in order to study temporal aspects of stiffness regulation, researchers have developed and exploited novel hydrogel substrates with in situ tuneable stiffness. In particular, photoresponsive hydrogels with photoswitchable stiffness are emerging as exciting platforms to study MSC stiffness regulation. This chapter provides an introduction to the use of PA hydrogel substrates, the molecular mechanisms of mechanotransduction currently under investigation and the development of these emerging photoresponsive hydrogel platforms.

Mesenchymal stem cells in regenerative medicine: Focus on articular cartilage and intervertebral disc regeneration.

Musculoskeletal disorders represent a major cause of disability and morbidity globally and result in enormous costs for health and social care systems. Development of cell-based therapies is rapidly proliferating in a number of disease areas, including musculoskeletal disorders. Novel biological therapies that can effectively treat joint and spine degeneration are high priorities in regenerative medicine. Mesenchymal stem cells (MSCs) isolated from bone marrow (BM-MSCs), adipose tissue (AD-MSCs) and umbilical cord (UC-MSCs) show considerable promise for use in cartilage and intervertebral disc (IVD) repair. This review article focuses on stem cell-based therapeutics for cartilage and IVD repair in the context of the rising global burden of musculoskeletal disorders. We discuss the biology MSCs and chondroprogenitor cells and specifically focus on umbilical cord/Wharton's jelly derived MSCs and examine their potential for regenerative applications. We also summarize key components of the molecular machinery and signaling pathways responsible for the control of chondrogenesis and explore biomimetic scaffolds and biomaterials for articular cartilage and IVD regeneration. This review explores the exciting opportunities afforded by MSCs and discusses the challenges associated with cartilage and IVD repair and regeneration. There are still many technical challenges associated with isolating, expanding, differentiating, and pre-conditioning MSCs for subsequent implantation into degenerate joints and the spine. However, the prospect of combining biomaterials and cell-based therapies that incorporate chondrocytes, chondroprogenitors and MSCs leads to the optimistic view that interdisciplinary approaches will lead to significant breakthroughs in regenerating musculoskeletal tissues, such as the joint and the spine in the near future.

EGR1 controls divergent cellular responses of distinctive nucleus pulposus cell types.

BACKGROUND: Immediate early genes (IEGs) encode transcription factors which serve as first line response modules to altered conditions and mediate appropriate cell responses. The immediate early response gene EGR1 is involved in physiological adaptation of numerous different cell types. We have previously shown a role for EGR1 in controlling processes supporting chondrogenic differentiation. We recently established a unique set of phenotypically distinct cell lines from the human nucleus pulposus (NP). Extensive characterization showed that these NP cellular subtypes represented progenitor-like cell types and more functionally mature cells. METHODS: To further understanding of cellular heterogeneity in the NP, we analyzed the response of these cell subtypes to anabolic and catabolic factors. Here, we test the hypothesis that physiological responses of distinct NP cell types are mediated by EGR1 and reflect specification of cell function using an RNA interference-based experimental approach. RESULTS: We show that distinct NP cell types rapidly induce EGR1 exposure to either growth factors or inflammatory cytokines. In addition, we show that mRNA profiles induced in response to anabolic or catabolic conditions are cell type specific: the more mature NP cell type produced a strong and more specialized transcriptional response to IL-1ß than the NP progenitor cells and aspects of this response were controlled by EGR1. CONCLUSIONS: Our current findings provide important substantiation of differential functionality among NP cellular subtypes. Additionally, the data shows that early transcriptional programming initiated by EGR1 is essentially restrained by the cells' epigenome as it was determined during development and differentiation. These studies begin to define functional distinctions among cells of the NP and will ultimately contribute to defining functional phenotypes within the adult intervertebral disc.

Simultaneous orthogonal dual-click approach to tough, in-situ-forming hydrogels for cell encapsulation.

The use of tough hydrogels as biomaterials is limited as a consequence of time-consuming fabrication techniques, toxic starting materials, and large strain hysteresis under deformation. Herein, we report the simultaneous application of nucleophilic thiol-yne and inverse electron-demand Diels-Alder additions to independently create two interpenetrating networks in a simple one-step procedure. The resultant hydrogels display compressive stresses of 14-15 MPa at 98% compression without fracture or hysteresis upon repeated load. The hydrogel networks can be spatially and temporally postfunctionalized via radical thiylation and/or inverse electron-demand Diels-Alder addition to residual functional groups within the network. Furthermore, gelation occurs rapidly under physiological conditions, enabling encapsulation of human cells.

An understanding of intervertebral disc development, maturation and cell phenotype provides clues to direct cell-based tissue regeneration therapies for disc degeneration.

Cell-based regenerative medicine therapies have been proposed for repairing the degenerated intervertebral disc (a major cause of back pain). However, for this approach to be successful, it is essential to characterise the phenotype of its native cells to guarantee that implanted cells differentiate and maintain the correct phenotype to ensure appropriate cell and tissue function. While recent studies have increased our knowledge of the human nucleus pulposus (NP) cell phenotype, their ontogeny is still unclear. The expression of notochordal markers by a subpopulation of adult NP cells suggests that, contrary to previous reports, notochord-derived cells are retained in the adult NP, possibly coexisting with a second population of cells originating from the annulus fibrosus or endplate. It is not known, however, how these two cell populations interact and their specific role(s) in disc homeostasis and disease. In particular, notochordal cells are proposed to display both anabolic and protective roles; therefore, they may be the ideal cells to repair the degenerate disc. Thus, understanding the ontogeny of the adult NP cells is paramount, as it will inform the medical and scientific communities as to the ideal phenotype to implant into the degenerate disc and the specific pathways involved in stem cell differentiation towards such a phenotype.

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.

Progenitor-like cells contributing to cellular heterogeneity in the nucleus pulposus are lost in intervertebral disc degeneration.

The nucleus pulposus (NP) in the intervertebral disc (IVD) arises from embryonic notochord. Loss of notochordal-like cells in humans correlates with onset of IVD degeneration, suggesting that they are critical for healthy NP homeostasis and function. Comparative transcriptomic analyses identified expression of progenitor-associated genes (GREM1, KRT18, and TAGLN) in the young mouse and non-degenerated human NP, with TAGLN expression reducing with aging. Lineage tracing using Tagln-CreERt2 mice identified peripherally located proliferative NP (PeriNP) cells in developing and postnatal NP that provide a continuous supply of cells to the entire NP. PeriNP cells were diminished in aged mice and absent in puncture-induced degenerated discs. Single-cell transcriptomes of postnatal Tagln-CreERt2 IVD cells indicate enrichment for TGF-ß signaling in Tagln descendant NP sub-populations. Notochord-specific removal of TGF-ß/BMP mediator Smad4 results in loss of Tagln+ cells and abnormal NP morphologies. We propose Tagln+ PeriNP cells are potential progenitors crucial for NP homeostasis.

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.

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.