The species-specific regenerative effects of notochordal cell-conditioned medium on chondrocyte-like cells derived from degenerated human intervertebral discs

During intervertebral disc (IVD) maturation, the main cell type shifts from notochordal cells (NCs) to chondrocytelike cells (CLCs). NCs secrete factors with regenerative potential, making them an interesting focus for regenerative treatments. During initial development, these strategies preferably employ non-human donors due to easy availability of their NC-rich nucleus pulposus (NP) tissue. To increase the success of translating these strategies for clinical application, this study aimed to delineate whether NC-secreted factors of different species have a regenerative effect on human CLCs. Human, canine and porcine NC-rich NP tissue and NC-conditioned medium (NCCM) were analysed biochemically and histologically. Human CLC micro-aggregates from degenerated IVDs were cultured in human, canine or porcine NCCM. Collagen, glycosaminoglycan (GAG) and DNA content was determined and histology was performed. Canine and porcine NPs were richer in NCs than human NPs. Human NPs contained the highest collagen content, whereas the DNA and GAG content of canine NPs was significantly higher than that of human or porcine NPs. NCCM from all species significantly increased the DNA and GAG content of the human CLC micro-aggregates. Porcine and canine NCCM were significantly more potent than human NCCM in inducing GAG deposition, whereas only human NCCM induced collagen type II production. Secreted factors from human, canine and porcine NC-rich NPs exerted regenerative effects on human CLCs, indicating a cross-species effect. Bioactive compound(s) are present in NCCM of different species that may reverse human IVD degeneration, supporting further research into strategies based on NC-technology employing canine or porcine models for their translation into humans.</p

A glue for heterochromatin maintenance: stable SUV39H1 binding to heterochromatin is reinforced by the SET domain

Trimethylation of histone H3 lysine 9 and the subsequent binding of heterochromatin protein 1 (HP1) mediate the formation and maintenance of pericentromeric heterochromatin. Trimethylation of H3K9 is governed by the histone methyltransferase SUV39H1. Recent studies of HP1 dynamics revealed that HP1 is not a stable component of heterochromatin but is highly mobile (Cheutin, T., A.J. McNairn, T. Jenuwein, D.M. Gilbert, P.B. Singh, and T. Misteli. 2003. Science. 299:721–725; Festenstein, R., S.N. Pagakis, K. Hiragami, D. Lyon, A. Verreault, B. Sekkali, and D. Kioussis. 2003. Science. 299:719–721). Because the mechanism by which SUV39H1 is recruited to and interacts with heterochromatin is unknown, we studied the dynamic properties of SUV39H1 in living cells by using fluorescence recovery after photobleaching and fluorescence resonance energy transfer. Our results show that a substantial population of SUV39H1 is immobile at pericentromeric heterochromatin, suggesting that, in addition to its catalytic activity, SUV39H1 may also play a structural role at pericentromeric regions. Analysis of SUV39H1 deletion mutants indicated that the SET domain mediates this stable binding. Furthermore, our data suggest that the recruitment of SUV39H1 to heterochromatin is at least partly independent from that of HP1 and that HP1 transiently interacts with SUV39H1 at heterochromatin

Randomised controlled trial of first-line tyrosine-kinase inhibitor (TKI) versus intercalated TKI with chemotherapy for EGFR-mutated nonsmall cell lung cancer

Introduction Previous studies have shown interference between epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors and chemotherapy in the cell cycle, thus reducing efficacy. In this randomised controlled trial we investigated whether intercalated erlotinib with chemotherapy was superior compared to erlotinib alone in untreated advanced EGFR-mutated nonsmall cell lung cancer (NSCLC). Materials and methods Treatment-naïve patients with an activating EGFR mutation, ECOG performance score of 0–3 and adequate organ function were randomly assigned 1:1 to either four cycles of cisplatin-pemetrexed with intercalated erlotinib (day 2–16 out of 21 days per cycle) followed by pemetrexed and erlotinib maintenance (CPE) or erlotinib monotherapy. The primary end-point was progression-free survival (PFS). Secondary end-points were overall survival, objective response rate (ORR) and toxicity. Results Between April 2014 and September 2016, 22 patients were randomised equally into both arms; the study was stopped due to slow accrual. Median follow-up was 64 months. Median PFS was 13.7 months (95% CI 5.2–18.8) for CPE and 10.3 months (95% CI 7.1–15.5; hazard ratio (HR) 0.62, 95% CI 0.25–1.57) for erlotinib monotherapy; when compensating for number of days receiving erlotinib, PFS of the CPE arm was superior (HR 0.24, 95% CI 0.07–0.83; p=0.02). ORR was 64% for CPE versus 55% for erlotinib monotherapy. Median overall survival was 31.7 months (95% CI 21.8–61.9 months) for CPE compared to 17.2 months (95% CI 11.5–45.5 months) for erlotinib monotherapy (HR 0.58, 95% CI 0.22–1.41 months). Patients treated with CPE had higher rates of treatment-related fatigue, anorexia, weight loss and renal toxicity. Conclusion Intercalating erlotinib with cisplatin-pemetrexed provides a longer PFS compared to erlotinib alone in EGFR-mutated NSCLC at the expense of more toxicity

Dynamic assembly of chromatin complexes during cellular senescence: implications for the growth arrest of human melanocytic nevi

The retinoblastoma (RB)/p16INK4a pathway regulates senescence of human melanocytes in culture and oncogene-induced senescence of melanocytic nevi in vivo. This senescence response is likely due to chromatin modifications because RB complexes from senescent melanocytes contain increased levels of histone deacetylase (HDAC) activity and tethered HDAC1. Here we show that HDAC1 is prominently detected in p16INK4a-positive, senescent intradermal melanocytic nevi but not in proliferating, recurrent nevus cells that localize to the epidermal/dermal junction. To assess the role of HDAC1 in the senescence of melanocytes and nevi, we used tetracycline-based inducible expression systems in cultured melanocytic cells. We found that HDAC1 drives a sequential and cooperative activity of chromatin remodeling effectors, including transient recruitment of Brahma (Brm1) into RB/HDAC1 mega-complexes, formation of heterochromatin protein 1β (HP1β)/SUV39H1 foci, methylation of H3-K9, stable association of RB with chromatin and significant global heterochromatinization. These chromatin changes coincide with expression of typical markers of senescence, including the senescent-associated β-galactosidase marker. Notably, formation of RB/HP1β foci and early tethering of RB to chromatin depends on intact Brm1 ATPase activity. As cells reached senescence, ejection of Brm1 from chromatin coincided with its dissociation from HP1β/RB and relocalization to protein complexes of lower molecular weight. These results provide new insights into the role of the RB pathway in regulating cellular senescence and implicate HDAC1 as a likely mediator of early chromatin remodeling events

Leaping the hurdles in developing regenerative treatments for the intervertebral disc from preclinical to clinical

Chronic back and neck pain is a prevalent disability, often caused by degeneration of the intervertebral disc. Because current treatments for this condition are less than satisfactory, a great deal of effort is being applied to develop new solutions, including regenerative strategies. However, the path from initial promising idea to clinical use is frought with many hurdles to overcome. Many of the keys to success are not necessarily linked to science or innovation. Successful translation to clinic will also rely on planning and awareness of the hurdles. It will be essential to plan your entire path to clinic from the outset and to do this with a multidisciplinary team. Take advise early on regulatory aspects and focus on generating the proof required to satisfy regulatory approval. Scientific demonstration and societal benefits are important, but translation cannot occur without involving commercial parties, which are instrumental to support expensive clinical trials. This will only be possible when intellectual property can be protected sufficiently to support a business model. In this manner, commercial, societal, medical, and scientific partners can work together to ultimately improve patient health. Based on literature surveys and experiences of the co‐authors, this opinion paper presents this pathway, highlights the most prominent issues and hopefully will aid in your own transational endeavors

Heterochromatin and the molecular mechanisms of 'parent-of-origin' effects in animals.

Twenty five years ago it was proposed that conserved components of constitutive heterochromatin assemble heterochromatinlike complexes in euchromatin and this could provide a general mechanism for regulating heritable (cell-to-cell) changes in gene expressibility. As a special case, differences in the assembly of heterochromatin-like complexes on homologous chromosomes might also regulate the parent-of-origin-dependent gene expression observed in placental mammals. Here, the progress made in the intervening period with emphasis on the role of heterochromatin and heterochromatin-like complexes in parent-of-origin effects in animals is reviewed

PREditOR: A synthetic biology approach to removing heterochromatin from cells

It is widely accepted that heterochromatin is necessary to maintain genomic stability. However, direct experimental evidence supporting this is slim. Previous studies using either enzyme inhibitors, gene knockout or knockdown studies all are subject to the caveat that drugs may have off-target effects and enzymes that modify chromatin proteins to support heterochromatin formation may also have numerous other cellular targets as well. Here, we describe PREditOR (protein reading and editing of residues), a synthetic biology approach that allows us to directly remove heterochromatin from cells without either drugs or global interference with gene function. We find that removal of heterochromatin perturbs mitotic progression and causes a dramatic increase in chromosome segregation defects, possibly as a result of interfering with the normal centromeric localization of the chromosomal passenger complex. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s10577-016-9539-3) contains supplementary material, which is available to authorized users