Nicholas R. Forsyth

Comparative biology of mammalian telomeres: hypotheses on ancestral states and the roles of telomeres in longevity determination

Progressive telomere shortening from cell division (replicative aging) provides a barrier for human tumor progression. This program is not conserved in laboratory mice, which have longer telomeres and constitutive telomerase. Wild species that do/do not use replicative aging have been reported, but the evolution of different phenotypes and a conceptual framework for understanding their uses of telomeres is lacking. We examined telomeres/telomerase in cultured cells from > 60 mammalian species to place different uses of telomeres in a broad mammalian context. Phylogeny‐based statistical analysis reconstructed ancestral states. Our analysis suggested that the ancestral mammalian phenotype included short telomeres (< 20 kb, as we now see in humans) and repressed telomerase. We argue that the repressed telomerase was a response to a higher mutation load brought on by the evolution of homeothermy. With telomerase repressed, we then see the evolution of replicative aging. Telomere length inversely correlated with lifespan, while telomerase expression co‐evolved with body size. Multiple independent times smaller, shorter‐lived species changed to having longer telomeres and expressing telomerase. Trade‐offs involving reducing the energetic/cellular costs of specific oxidative protection mechanisms (needed to protect < 20 kb telomeres in the absence of telomerase) could explain this abandonment of replicative aging. These observations provide a conceptual framework for understanding different uses of telomeres in mammals, support a role for human‐like telomeres in allowing longer lifespans to evolve, demonstrate the need to include telomere length in the analysis of comparative studies of oxidative protection in the biology of aging, and identify which mammals can be used as appropriate model organisms for the study of the role of telomeres in human cancer and aging.

Developmental differences in the immortalization of lung fibroblasts by telomerase

The role of ambient (21%) and physiological oxygen (2–5%) in the immortalization of fetal vs. adult human lung fibroblasts was examined. Growth in low oxygen and antioxidants extended the lifespan of both fetal and adult strains. As the ectopic expression of telomerase could immortalize adult lung fibroblasts cultured in ambient oxygen, the lifespan‐shortening effects of 21% oxygen must have been largely limited to telomeres. By contrast, fetal lung fibroblasts could not be immortalized in ambient oxygen in spite of telomere elongation by telomerase, suggesting more widespread oxidative damage. The long‐term culture requirements for the immortalization of WI‐38 fetal lung fibroblasts included supplementation with N‐(tert) butyl hydroxylamine, dexamethasone, zinc and vitamin B12, in addition to growth in physiological oxygen. The mechanisms regulating telomere shortening remain controversial. The present results suggest that both end‐replication and oxidative damage events contribute to telomere shortening in lung fibroblasts in vitro. These observations emphasize the need for better analytical techniques to distinguish whether the correlation of short telomeres with disease and mortality in humans reflects the consequences of increased proliferation, telomere shortening as a result of oxidative damage or some combination of these processes.

Mesenchymal stem cells promote alveolar epithelial cell wound repair in vitro through distinct migratory and paracrine mechanisms

BackgroundMesenchymal stem cells (MSC) are in clinical trials for widespread indications including musculoskeletal, neurological, cardiac and haematological disorders. Furthermore, MSC can ameliorate pulmonary fibrosis in animal models although mechanisms of action remain unclear. One emerging concept is that MSCs may have paracrine, rather than a functional, roles in lung injury repair and regeneration.MethodsTo investigate the paracrine role of human MSC (hMSC) on pulmonary epithelial repair, hMSC-conditioned media (CM) and a selected cohort of hMSC-secretory proteins (identified by LC-MS/MS mass spectrometry) were tested on human type II alveolar epithelial cell line A549 cells (AEC) and primary human small airway epithelial cells (SAEC) using an in vitro scratch wound repair model. A 3D direct-contact wound repair model was further developed to assess the migratory properties of hMSC.ResultsWe demonstrate that MSC-CM facilitates AEC and SAEC wound repair in serum-dependent and –independent manners respectively via stimulation of cell migration. We also show that the hMSC secretome contains an array of proteins including Fibronectin, Lumican, Periostin, and IGFBP-7; each capable of influencing AEC and SAEC migration and wound repair stimulation. In addition, hMSC also show a strong migratory response to AEC injury as, supported by the observation of rapid and effective AEC wound gap closure by hMSC in the 3D model.ConclusionThese findings support the notion for clinical application of hMSCs and/or their secretory factors as a pharmacoregenerative modality for the treatment of idiopathic pulmonary fibrosis (IPF) and other fibrotic lung disorders.

Tendon repair through stem cell intervention: Cellular and molecular approaches

Tendon injuries are common in either the workplace or sport activities, with some 3 to 5 million tendon and ligament injuries occurring annually worldwide. Management of tendon injury currently follows two routes: Conservative (rehabilitation and pain relief), or surgical. Irrespective of which of these primary treatment routes are followed, even if healing does occur, it may not result in a full gain of function. The inability of the tendon to self-repair and the relative inefficiency of current treatment regimens suggest that identifying alternative strategies is a priority. One such alternative is the use of stem cells to repair damage, either through direct application or in conjunction with scaffolding. We describe the current state of the art in terms of: (i) Molecular markers of tendon development, (ii) stem cell applicability to human tendon repair, (iii) scaffolding for in vitro tendon generation, and (iv) chemical/molecular approaches to both induce stem cell differentiation into tenocytes and maintain their proliferation in vitro.

Transcriptome alterations due to physiological normoxic (2% O2) culture of human embryonic stem cells

UNLABELLED Human embryonic stem cells (hESCs) hold great promise therapeutically. In order to deliver on this promise the correct defined conditions for long-term propagation must first be established. Researchers have now provided reports describing the benefits of culturing hESCs in physiologically approximate levels of oxygen. These physiological values fall in the range of 2 to 5% O2. Benefits include reduced spontaneous differentiation, enhanced chromosomal stability and increased clonality. AIMS The aim of our study was to examine the transcriptional consequences of culturing hESCs in physiological normoxia (2% O2) using microarray technology. METHODS Three karyoptically normal hESC lines (H1, H9 and RH1) were examined. At the initiation of this experiment, established hESC lines were redesignated as passage (p) 0 in 21% O2, then bifurcated into 21% O2 and 2% O2, and maintained for a further ten passages at which time samples were again collected. RNA was extracted from all sample points and subjected to microarray analysis using the Affymetrix U133 Plus 2.0 platform. Bioinformatic analysis was performed using dChip and GoStat. RESULTS We performed grouped analyses of gene expression of early (p0) versus late (p10) air-cultured cells. This revealed relative stability with six (air p0 baseline vs p10 experimental) and one (air p10 baseline vs p0 experimental) gene(s) displaying both greater than twofold and statistically significant upregulation. Conversely, we identified 302 gene upregulations and 56 downregulations when comparing 21% O(2) (p0p10) with 2% O2 (p10). These significantly upregulated changes clustered into 82 over-represented and 9 under-represented ontology terms. These terms were indicative of signaling pathways, developmental potential and metabolism. Hierarchical clustering indicated a trend for 2% O2 cultured cells to cluster collectively with reduced heterogeneity when compared with 21% O2 cultured cells. CONCLUSIONS The gene changes associated with 2% O2 culture may be predictive of novel cellular requirements for stable self-renewal, maintenance of pluripotency, and a reduction of hESC-line heterogeneity.

The application of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) scaffolds for tendon repair in the rat model

Tendon injuries and defects present a substantial burden to global healthcare economies. There are no synthetic/biosynthesised implants available which can restore full function or match the mechanical properties of native tendon. Therefore, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) was investigated for its utility as a scaffold in a rat Achilles tendon repair model. Porous PHBHHx tubes and fibres were prepared with particle leaching and extrusion methods, respectively. Collagen gels reinforced by polymer fibres were inserted into the lumen of scaffold tubes to create the operational scaffold unit. Mechanical testing demonstrated that PHBHHx scaffolds had comparable mechanical properties to rat tendon, with maximal loads of 23.73 ± 1.08 N, compared to 17.35 ± 1.76 N in undamaged rat Achilles tendon. Sprague-Dawley (SD) rats were split into four experimental groups: control, PHBHHx scaffold only, PHBHHx scaffold and collagen, PHBHHx scaffold, collagen and tenocyte compositions for implantation to repair an induced Achilles tendon defect. No secondary immune response to PHBHHx was observed over a 40 days period of implantation. Movement was restored in PHBHHx scaffold-collagen-tenocyte recipient rats at an earlier time point than in other experimental groups, with complete load-bearing and function returning 20 days post-surgery as determined by the Achilles Functional Index. In vitro testing of tendon constructs after 40 days demonstrated reductions in PHBHHx molecular weight and polydispersity index accompanied by an increase in mean chain length indicating degradation of smaller polymer chain subunits. Similarly a reduction in PHBHHx tube ultimate tensile strength and elastic modulus was observed. Histological analysis provided evidence of tissue remodelling and cell alignment. In summary, PHBHHx scaffolds have been successfully applied in an in vivo tendon repair model raising promise for future utility in tissue engineering applications.

Three-dimensional hypoxic culture of human mesenchymal stem cells encapsulated in a photocurable, biodegradable polymer hydrogel: a potential injectable cellular product for nucleus pulposus regeneration

Nucleus pulposus (NP) tissue damage can induce detrimental mechanical stresses and strains on the intervertebral disc, leading to disc degeneration. This study demonstrates the potential of a novel, photo-curable, injectable, synthetic polymer hydrogel (pHEMA-co-APMA grafted with polyamidoamine (PAA)) to encapsulate and differentiate human mesenchymal stem cells (hMSC) towards a NP phenotype under hypoxic conditions which could be used to restore NP tissue function and mechanical properties. Encapsulated hMSC cultured in media (hMSC and chondrogenic) displayed good cell viability up to day 14. The genotoxicity effects of ultraviolet (UV) on hMSC activity confirmed the acceptability of 2.5min of UV light exposure to cells. Cytotoxicity investigations revealed that hMSC cultured in media containing p(HEMA-co-APMA) grafted with PAA degradation product (10% and 20%v/v concentration) for 14days significantly decreased the initial hMSC adhesion ability and proliferation rate from 24hrs to day 14. Successful differentiation of encapsulated hMSC within hydrogels towards chondrogenesis was observed with elevated expression levels of aggrecan and collagen II when cultured in chondrogenic media under hypoxic conditions, in comparison with culture in hMSC media for 14days. Characterization of the mechanical properties revealed a significant decrease in stiffness and modulus values of cellular hydrogels in comparison with acellular hydrogels at both day 7 and day 14. These results demonstrate the potential use of an in vivo photo-curable injectable, synthetic hydrogel with encapsulated hMSC for application in the repair and regeneration of NP tissue.

Lagomorphs (rabbits, pikas and hares) do not use telomere-directed replicative aging in vitro

Telomere shortening is used for replicative aging in primates and ungulates but not rodents. We examined telomere biology in rabbits to expand the comparative biology of telomere-directed replicative senescence within mammals. The order Lagomorpha consists of two families; Leporidae and Ochotonidae. We examined telomere biology in species representing three leporid genera (European White Rabbit, Black-tailed Jack Rabbit, and Swamp Rabbit) and the monotypic ochotonid genus (North American Pika). Of the leporids one species was a laboratory strain and the others were wild caught. The leporids neither exhibited cellular senescence after sustained periods in culture nor displayed detectable telomerase activity. Continued culture was possible because of their extremely long telomeric arrays. Immunofluorescence showed robust telomere signals at chromosome ends and significant internal chromosomal staining in some instances. Pika was unique in displaying endogenous telomerase activity throughout time in culture. These results show that it is unlikely that lagomorphs use telomere shortening and replicative senescence as a tumor protective mechanism.

Lung Regeneration: Endogenous and Exogenous Stem Cell Mediated Therapeutic Approaches

The tissue turnover of unperturbed adult lung is remarkably slow. However, after injury or insult, a specialised group of facultative lung progenitors become activated to replenish damaged tissue through a reparative process called regeneration. Disruption in this process results in healing by fibrosis causing aberrant lung remodelling and organ dysfunction. Post-insult failure of regeneration leads to various incurable lung diseases including chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis. Therefore, identification of true endogenous lung progenitors/stem cells, and their regenerative pathway are crucial for next-generation therapeutic development. Recent studies provide exciting and novel insights into postnatal lung development and post-injury lung regeneration by native lung progenitors. Furthermore, exogenous application of bone marrow stem cells, embryonic stem cells and inducible pluripotent stem cells (iPSC) show evidences of their regenerative capacity in the repair of injured and diseased lungs. With the advent of modern tissue engineering techniques, whole lung regeneration in the lab using de-cellularised tissue scaffold and stem cells is now becoming reality. In this review, we will highlight the advancement of our understanding in lung regeneration and development of stem cell mediated therapeutic strategies in combating incurable lung diseases.

Club cells inhibit alveolar epithelial wound repair via TRAIL-dependent apoptosis

Club cells (Clara cells) participate in bronchiolar wound repair and regeneration. Located in the bronchioles, they become activated during alveolar injury in idiopathic pulmonary fibrosis (IPF) and migrate into the affected alveoli, a process called alveolar bronchiolisation. The purpose of this migration and the role of club cells in alveolar wound repair is controversial. This study was undertaken to investigate the role of club cells in alveolar epithelial wound repair and pulmonary fibrosis. A direct-contact co-culture in vitro model was used to evaluate the role of club cells (H441 cell line) on alveolar epithelial cell (A549 cell line) and small airway epithelial cell (SAEC) wound repair. Immunohistochemistry was conducted on lung tissue samples from patients with IPF to replicate the in vitro findings ex vivo. Our study demonstrated that club cells induce apoptosis in alveolar epithelial cells and SAECs through a tumour necrosis factor-related apoptosis-inducing ligand (TRAIL)-dependent mechanism resulting in significant inhibition of wound repair. Furthermore, in IPF lungs, TRAIL-expressing club cells were detected within the affected alveolar epithelia in areas of established fibrosis, together with widespread alveolar epithelial cell apoptosis. From these findings, we hypothesise that the extensive pro-fibrotic remodelling associated with IPF could be driven by TRAIL-expressing club cells inducing apoptosis in alveolar epithelial cells through a TRAIL-dependent mechanism.