Hypoxic conditions promote a proliferative, poorly differentiated phenotype in COPD lung tissue progenitor cells in vitro.

PURPOSE: Chronic obstructive pulmonary disease (COPD) patients experience hypoxemia and lung tissue hypoxia, causing vasoconstriction, and at its most severe Cor pulmonale. However, minimal attention has been given to the effects of hypoxia at the cellular level. We hypothesize that a persistent progenitor cell population undergoes an aberrant differentiation process, influenced by changes in oxygen. METHODS: Distal lung progenitor cells from two emphysematous donors were cultured in 21% and 2% oxygen. Proliferation was determined on collagen-coated plastic and in 3T3-J2 co-culture. Epithelial (E-cadherin, pan-cytokeratin) and progenitor (TP63, cytokeratin 5) marker expressions were examined. Cells were differentiated at air-liquid interface, and ciliated, mucus-producing, and club cell populations identified by immunofluorescence. MUC5AC, MUC5B, CC10, and TP63 expression were determined using qRT-PCR, mucin5AC, and mucin5B protein levels by ELISA, and secreted mucin by periodic acid biotin hydrazide assay. RESULTS: Cells were positive for epithelial and progenitor markers at isolation and passage 5. Passage 5 cells in hypoxia increased the proportion of TP63 by 10% from 51.6 ± 1.2% to 62.6 ± 2.3% (p ≤ 0.01). Proliferative capacity was greater on 3T3J2 cells and in 2% oxygen, supporting the emergence of a proliferation unrestricted population with limited differentiation capacity. Differentiation resulted in ßIV tubulin positive-ciliated cells, mucin5AC, mucin5B, and CC10 positive secretory cells. Epithelial barrier formation was reduced (p ≤ 0.0001) in hypoxia-expanded cells. qRT-PCR showed higher mucin expression in 2% cells, significantly so with MUC5B (p ≤ 0.05). Although overall mucin5AC and mucin5B content was greater in 21% cells, normalization of secreted mucin to DNA showed a trend for increased mucin by low oxygen cells. CONCLUSIONS: These results demonstrate that hypoxia promotes a proliferative phenotype while affecting subsequent progenitor cell differentiation capacity. Furthermore, the retained differentiation potential becomes skewed to a more secretory phenotype, demonstrating that hypoxia may be contributing to disease symptoms and severity in COPD patients.

Endotenon-Derived Type II Tendon Stem Cells Have Enhanced Proliferative and Tenogenic Potential.

Tendon injuries caused by overuse or age-related deterioration are frequent. Incomplete knowledge of somatic tendon cell biology and their progenitors has hindered interventions for the effective repair of injured tendons. Here, we sought to compare and contrast distinct tendon-derived cell populations: type I and II tendon stem cells (TSCs) and tenocytes (TNCs). Porcine type I and II TSCs were isolated via the enzymatic digestion of distinct membranes (paratenon and endotenon, respectively), while tenocytes were isolated through an explant method. Resultant cell populations were characterized by morphology, differentiation, molecular, flow cytometry, and immunofluorescence analysis. Cells were isolated, cultured, and evaluated in two alternate oxygen concentrations (physiological (2%) and air (21%)) to determine the role of oxygen in cell biology determination within this relatively avascular tissue. The different cell populations demonstrated distinct proliferative potential, morphology, and transcript levels (both for tenogenic and stem cell markers). In contrast, all tendon-derived cell populations displayed multipotent differentiation potential and immunophenotypes (positive for CD90 and CD44). Type II TSCs emerged as the most promising tendon-derived cell population for expansion, given their enhanced proliferative potential, multipotency, and maintenance of a tenogenic profile at early and late passage. Moreover, in all cases, physoxia promoted the enhanced proliferation and maintenance of a tenogenic profile. These observations help shed light on the biological mechanisms of tendon cells, with the potential to aid in the development of novel therapeutic approaches for tendon disorders.

Physoxia Influences Global and Gene-Specific Methylation in Pluripotent Stem Cells.

Pluripotent stem cells (PSC) possess unlimited proliferation, self-renewal, and a differentiation capacity spanning all germ layers. Appropriate culture conditions are important for the maintenance of self-renewal, pluripotency, proliferation, differentiation, and epigenetic states. Oxygen concentrations vary across different human tissues depending on precise cell location and proximity to vascularisation. The bulk of PSC culture-based research is performed in a physiologically hyperoxic, air oxygen (21% O2) environment, with numerous reports now detailing the impact of a physiologic normoxia (physoxia), low oxygen culture in the maintenance of stemness, survival, morphology, proliferation, differentiation potential, and epigenetic profiles. Epigenetic mechanisms affect multiple cellular characteristics including gene expression during development and cell-fate determination in differentiated cells. We hypothesized that epigenetic marks are responsive to a reduced oxygen microenvironment in PSCs and their differentiation progeny. Here, we evaluated the role of physoxia in PSC culture, the regulation of DNA methylation (5mC (5-methylcytosine) and 5hmC (5-hydroxymethylcytosine)), and the expression of regulatory enzyme DNMTs and TETs. Physoxia enhanced the functional profile of PSC including proliferation, metabolic activity, and stemness attributes. PSCs cultured in physoxia revealed the significant downregulation of DNMT3B, DNMT3L, TET1, and TET3 vs. air oxygen, accompanied by significantly reduced 5mC and 5hmC levels. The downregulation of DNMT3B was associated with an increase in its promoter methylation. Coupled with the above, we also noted decreased HIF1A but increased HIF2A expression in physoxia-cultured PSCs versus air oxygen. In conclusion, PSCs display oxygen-sensitive methylation patterns that correlate with the transcriptional and translational regulation of the de novo methylase DNMT3B.

Human Mesenchymal Stem Cell Secretome Driven T Cell Immunomodulation Is IL-10 Dependent.

The Human Mesenchymal Stem Cell (hMSC) secretome has pleiotropic effects underpinning its therapeutic potential. hMSC serum-free conditioned media (SFCM) contains a variety of cytokines, with previous studies linking a changed secretome composition to physoxia. The Jurkat T cell model allowed the efficacy of SFCM vs. serum-free media (SFM) in the suppression of immunological aspects, including proliferation and polarisation, to be explored. Cell growth in SFM was higher [(21% O2 = 5.3 × 105 ± 1.8 × 104 cells/mL) and (2% O2 = 5.1 × 105 ± 3.0 × 104 cells/mL)], compared to SFCM [(21% O2 = 2.4 × 105 ± 2.5 × 104 cells/mL) and (2% O2 = 2.2 × 105 ± 5.8 × 103 cells/mL)]. SFM supported IL-2 release following activation [(21% O2 = 5305 ± 211 pg/mL) and (2% O2 = 5347 ± 327 pg/mL)] whereas SFCM suppressed IL-2 secretion [(21% O2 = 2461 ± 178 pg/mL) and (2% O2 = 1625 ± 159 pg/mL)]. Anti-inflammatory cytokines, namely IL-4, IL-10, and IL-13, which we previously confirmed as components of hMSC SFCM, were tested. IL-10 neutralisation in SFCM restored proliferation in both oxygen environments (SFM/SFCM+antiIL-10 ~1-fold increase). Conversely, IL-4/IL-13 neutralisation showed no proliferation restoration [(SFM/SFM+antiIL-4 ~2-fold decrease), and (SFM/SFCM+antiIL-13 ~2-fold decrease)]. Present findings indicate IL-10 played an immunosuppressive role by reducing IL-2 secretion. Identification of immunosuppressive components of the hMSC secretome and a mechanistic understanding of their action allow for the advancement and refinement of potential future cell-free therapies.

Therapeutic Effects of Hypoxic and Pro-Inflammatory Priming of Mesenchymal Stem Cell-Derived Extracellular Vesicles in Inflammatory Arthritis.

Mesenchymal stem cells (MSCs) immunomodulate inflammatory responses through paracrine signalling, including via secretion of extracellular vesicles (EVs) in the cell secretome. We evaluated the therapeutic potential of MSCs-derived small EVs in an antigen-induced model of arthritis (AIA). EVs isolated from MSCs cultured normoxically (21% O2, 5% CO2), hypoxically (2% O2, 5% CO2) or with a pro-inflammatory cytokine cocktail were applied into the AIA model. Disease pathology was assessed post-arthritis induction through swelling and histopathological analysis of synovial joint structure. Activated CD4+ T cells from healthy mice were cultured with EVs or MSCs to assess deactivation capabilities prior to application of standard EVs in vivo to assess T cell polarisation within the immune response to AIA. All EVs treatments reduced knee-joint swelling whilst only normoxic and pro-inflammatory primed EVs improved histopathological outcomes. In vitro culture with EVs did not achieve T cell deactivation. Polarisation towards CD4+ helper cells expressing IL17a (Th17) was reduced when normoxic and hypoxic EV treatments were applied in vitro. Normoxic EVs applied into the AIA model reduced Th17 polarisation and improved Regulatory T cell (Treg):Th17 homeostatic balance. Normoxic EVs present the optimal strategy for broad therapeutic benefit. EVs present an effective novel technology with the potential for cell-free therapeutic translation.

Clerodane Diterpenoids from an Edible Plant Justicia insularis: Discovery, Cytotoxicity, and Apoptosis Induction in Human Ovarian Cancer Cells.

OBJECTIVES: The toxicity of chemotherapeutic anticancer drugs is a serious issue in clinics. Drug discovery from edible and medicinal plants represents a promising approach towards finding safer anticancer therapeutics. Justicia insularis T. Anderson (Acanthaceae) is an edible and medicinal plant in Nigeria. This study aims to discover cytotoxic compounds from this rarely explored J. insularis and investigate their underlying mechanism of action. METHODS: The cytotoxicity of the plant extract was evaluated in human ovarian cancer cell lines and normal human ovarian surface epithelia (HOE) cells using a sulforhodamine B assay. Bioassay-guided isolation was carried out using column chromatography including HPLC, and the isolated natural products were characterized using GC-MS, LC-HRMS, and 1D/2D NMR techniques. Induction of apoptosis was evaluated using Caspase 3/7, 8, and 9, and Annexin V and PI based flow cytometry assays. SwissADME and SwissTargetPrediction web tools were used to predict the molecular properties and possible protein targets of identified active compounds. Key finding: The two cytotoxic compounds were identified as clerodane diterpenoids: 16(α/ß)-hydroxy-cleroda-3,13(14)Z-dien-15,16-olide (1) and 16-oxo-cleroda-3,13(14)E-dien-15-oic acid (2) from the Acanthaceous plant for the first time. Compound 1 was a very abundant compound (0.7% per dry weight of plant material) and was shown to be more potent than compound 2 with IC50 values in the micromolar range against OVCAR-4 and OVCAR-8 cancer cells. Compounds 1 and 2 were less cytotoxic to HOE cell line. Both compounds induced apoptosis by increasing caspase 3/7 activities in a concentration dependent manner. Compound 1 further increased caspase 8 and 9 activities and apoptosis cell populations. Compounds 1 and 2 are both drug like, and compound 1 may target various proteins including a kinase. CONCLUSIONS: Clerodane diterpenoids (1 and 2) in J. insularis were identified as cytotoxic to ovarian cancer cells via the induction of apoptosis, providing an abundant and valuable source of hit compounds for the treatment of ovarian cancer.

In Vitro Innovation of Tendon Tissue Engineering Strategies.

Tendinopathy is the term used to refer to tendon disorders. Spontaneous adult tendon healing results in scar tissue formation and fibrosis with suboptimal biomechanical properties, often resulting in poor and painful mobility. The biomechanical properties of the tissue are negatively affected. Adult tendons have a limited natural healing capacity, and often respond poorly to current treatments that frequently are focused on exercise, drug delivery, and surgical procedures. Therefore, it is of great importance to identify key molecular and cellular processes involved in the progression of tendinopathies to develop effective therapeutic strategies and drive the tissue toward regeneration. To treat tendon diseases and support tendon regeneration, cell-based therapy as well as tissue engineering approaches are considered options, though none can yet be considered conclusive in their reproduction of a safe and successful long-term solution for full microarchitecture and biomechanical tissue recovery. In vitro differentiation techniques are not yet fully validated. This review aims to compare different available tendon in vitro differentiation strategies to clarify the state of art regarding the differentiation process.

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.

Measurement of C-reactive protein, procalcitonin and neutrophil elastase in saliva of COPD patients and healthy controls: correlation to self-reported wellbeing parameters.

BACKGROUND: Saliva is increasingly promoted as an alternative diagnostic bio-sample to blood; however its role in respiratory disease requires elucidation. Our aim was to investigate whether C-reactive protein (CRP), procalcitonin (PCT) and neutrophil elastase (NE) could be measured in unstimulated whole saliva, and to explore differences between COPD patients and controls with normal lung function. We also determined the relationship between these salivary biomarkers and self-reported COPD-relevant metrics. METHODS: Salivary CRP, PCT and NE levels were measured at each of 3 visits over a 14-day period alongside spirometry and a daily self-assessment dairy in 143 subjects: 20 never-smokers and 25 smokers with normal spirometry; 98 COPD patients [GOLD Stage I, 16; Stage II, 32; Stage III, 39; Stage IV, 11]. Twenty-two randomly selected subjects provided simultaneous blood samples. RESULTS: Levels of each salivary biomarker could distinguish between the above cohorts. Significant differences remained for salivary CRP and NE (p < 0.05) following adjustment for age, gender, sampling time, gum disease and total co-morbidities; but not for BMI except for salivary NE, which remained higher in smokers compared to non-smokers and stable COPD subjects (p < 0.001). Patients with acute COPD exacerbations had a median increase in all 3 salivary biomarkers (p < 0.001); CRP: median 5.74 ng/ml, [interquartile range (IQR) 2.86-12.25], PCT 0.38 ng/ml, [IQR 0.22-0.94], and NE 539 ng/ml, [IQR 112.25-1264]. In COPD patients, only salivary CRP and PCT levels correlated with breathing scores (r = 0.14, p < 0.02; r = 0.13, p < 0.03 respectively) and sputum features but not with activities of daily living. Salivary CRP and PCT concentrations strongly correlated with serum counterparts [r = 0.82, (95% CI: 0.72-0.87), p < 0.001 by Spearman's; and r = 0.53, (95% CI: 0.33-0.69), p < 0.006 respectively]; salivary NE did not. CONCLUSIONS: CRP, PCT and NE were reliably and reproducibly measured in saliva, providing clinically-relevant information on health status in COPD; additionally NE distinguished smoking status. All 3 salivary biomarkers increased during COPD exacerbations, with CRP and PCT correlating well with patient-derived clinical metrics. These results provide the conceptual basis for further development of saliva as a viable bio-sample in COPD monitoring and exacerbation management.

TERT Promoter Methylation Is Oxygen-Sensitive and Regulates Telomerase Activity.

Telomere repeats protect linear chromosomes from degradation, and telomerase has a prominent role in their maintenance. Telomerase has telomere-independent effects on cell proliferation, DNA replication, differentiation, and tumorigenesis. TERT (telomerase reverse transcriptase enzyme), the catalytic subunit of telomerase, is required for enzyme activity. TERT promoter mutation and methylation are strongly associated with increased telomerase activation in cancer cells. TERT levels and telomerase activity are downregulated in stem cells during differentiation. The link between differentiation and telomerase can provide a valuable tool for the study of the epigenetic regulation of TERT. Oxygen levels can affect cellular behaviors including proliferation, metabolic activity, stemness, and differentiation. The role of oxygen in driving TERT promoter modifications in embryonic stem cells (ESCs) is poorly understood. We adopted a monolayer ESC differentiation model to explore the role of physiological oxygen (physoxia) in the epigenetic regulation of telomerase and TERT. We further hypothesized that DNMTs played a role in physoxia-driven epigenetic modification. ESCs were cultured in either air or a 2% O2 environment. Physoxia culture increased the proliferation rate and stemness of the ESCs and induced a slower onset of differentiation than in ambient air. As anticipated, downregulated TERT expression correlated with reduced telomerase activity during differentiation. Consistent with the slower onset of differentiation in physoxia, the TERT expression and telomerase activity were elevated in comparison to the air-oxygen-cultured ESCs. The TERT promoter methylation levels increased during differentiation in ambient air to a greater extent than in physoxia. The chemical inhibition of DNMT3B reduced TERT promoter methylation and was associated with increased TERT gene and telomerase activity during differentiation. DNMT3B ChIP (Chromatin immunoprecipitation) demonstrated that downregulated TERT expression and increased proximal promoter methylation were associated with DNMT3B promoter binding. In conclusion, we have demonstrated that DNMT3B directly associates with TERT promoter, is associated with differentiation-linked TERT downregulation, and displays oxygen sensitivity. Taken together, these findings help identify novel aspects of telomerase regulation that may play a role in better understanding developmental regulation and potential targets for therapeutic intervention.

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.

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

BACKGROUND: Mesenchymal 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. METHODS: To 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. RESULTS: We 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. CONCLUSION: These 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.

Injectable hydrogels: An emerging therapeutic strategy for cartilage regeneration.

The impairment of articular cartilage due to traumatic incidents or osteoarthritis has posed significant challenges for healthcare practitioners, researchers, and individuals suffering from these conditions. Due to the absence of an approved treatment strategy for the complete restoration of cartilage defects to their native state, the tissue condition often deteriorates over time, leading to osteoarthritic (OA). However, recent advancements in the field of regenerative medicine have unveiled promising prospects through the utilization of injectable hydrogels. This versatile class of biomaterials, characterized by their ability to emulate the characteristics of native articular cartilage, offers the distinct advantage of minimally invasive administration directly to the site of damage. These hydrogels can also serve as ideal delivery vehicles for a diverse range of bioactive agents, including growth factors, anti-inflammatory drugs, steroids, and cells. The controlled release of such biologically active molecules from hydrogel scaffolds can accelerate cartilage healing, stimulate chondrogenesis, and modulate the inflammatory microenvironment to halt osteoarthritic progression. The present review aims to describe the methods used to design injectable hydrogels, expound upon their applications as delivery vehicles of biologically active molecules, and provide an update on recent advances in leveraging these delivery systems to foster articular cartilage regeneration.

Tendon tissue engineering: An overview of biologics to promote tendon healing and repair.

Tendons are dense connective tissues with a hierarchical polarized structure that respond to and adapt to the transmission of muscle contraction forces to the skeleton, enabling motion and maintaining posture. Tendon injuries, also known as tendinopathies, are becoming more common as populations age and participation in sports/leisure activities increases. The tendon has a poor ability to self-heal and regenerate given its intrinsic, constrained vascular supply and exposure to frequent, severe loading. There is a lack of understanding of the underlying pathophysiology, and it is not surprising that disorder-targeted medicines have only been partially effective at best. Recent tissue engineering approaches have emerged as a potential tool to drive tendon regeneration and healing. In this review, we investigated the physiochemical factors involved in tendon ontogeny and discussed their potential application in vitro to reproduce functional and self-renewing tendon tissue. We sought to understand whether stem cells are capable of forming tendons, how they can be directed towards the tenogenic lineage, and how their growth is regulated and monitored during the entire differentiation path. Finally, we showed recent developments in tendon tissue engineering, specifically the use of mesenchymal stem cells (MSCs), which can differentiate into tendon cells, as well as the potential role of extracellular vesicles (EVs) in tendon regeneration and their potential for use in accelerating the healing response after injury.

Stem cell-based therapy for human diseases.

Recent advancements in stem cell technology open a new door for patients suffering from diseases and disorders that have yet to be treated. Stem cell-based therapy, including human pluripotent stem cells (hPSCs) and multipotent mesenchymal stem cells (MSCs), has recently emerged as a key player in regenerative medicine. hPSCs are defined as self-renewable cell types conferring the ability to differentiate into various cellular phenotypes of the human body, including three germ layers. MSCs are multipotent progenitor cells possessing self-renewal ability (limited in vitro) and differentiation potential into mesenchymal lineages, according to the International Society for Cell and Gene Therapy (ISCT). This review provides an update on recent clinical applications using either hPSCs or MSCs derived from bone marrow (BM), adipose tissue (AT), or the umbilical cord (UC) for the treatment of human diseases, including neurological disorders, pulmonary dysfunctions, metabolic/endocrine-related diseases, reproductive disorders, skin burns, and cardiovascular conditions. Moreover, we discuss our own clinical trial experiences on targeted therapies using MSCs in a clinical setting, and we propose and discuss the MSC tissue origin concept and how MSC origin may contribute to the role of MSCs in downstream applications, with the ultimate objective of facilitating translational research in regenerative medicine into clinical applications. The mechanisms discussed here support the proposed hypothesis that BM-MSCs are potentially good candidates for brain and spinal cord injury treatment, AT-MSCs are potentially good candidates for reproductive disorder treatment and skin regeneration, and UC-MSCs are potentially good candidates for pulmonary disease and acute respiratory distress syndrome treatment.

Telomerase Regulation: A Role for Epigenetics.

Telomerase was first described by Greider and Blackburn in 1984, a discovery ultimately recognized by the Nobel Prize committee in 2009. The three decades following on from its discovery have been accompanied by an increased understanding of the fundamental mechanisms of telomerase activity, and its role in telomere biology. Telomerase has a clearly defined role in telomere length maintenance and an established influence on DNA replication, differentiation, survival, development, apoptosis, tumorigenesis, and a further role in therapeutic resistance in human stem and cancer cells including those of breast and cervical origin. TERT encodes the catalytic subunit and rate-limiting factor for telomerase enzyme activity. The mechanisms of activation or silencing of TERT remain open to debate across somatic, cancer, and stem cells. Promoter mutations upstream of TERT may promote dysregulated telomerase activation in tumour cells but additional factors including epigenetic, transcriptional and posttranscriptional modifications also have a role to play. Previous systematic analysis indicated methylation and mutation of the TERT promoter in 53% and 31%, respectively, of TERT expressing cancer cell lines supporting the concept of a key role for epigenetic alteration associated with TERT dysregulation and cellular transformation. Epigenetic regulators including DNA methylation, histone modification, and non-coding RNAs are now emerging as drivers in the regulation of telomeres and telomerase activity. Epigenetic regulation may be responsible for reversible silencing of TERT in several biological processes including development and differentiation, and increased TERT expression in cancers. Understanding the epigenetic mechanisms behind telomerase regulation holds important prospects for cancer treatment, diagnosis and prognosis. This review will focus on the role of epigenetics in telomerase regulation.

Epigenetic features in regulation of telomeres and telomerase in stem cells.

The epigenetic nature of telomeres is still controversial and different human cell lines might show diverse histone marks at telomeres. Epigenetic modifications regulate telomere length and telomerase activity that influence telomere structure and maintenance. Telomerase is responsible for telomere elongation and maintenance and is minimally composed of the catalytic protein component, telomerase reverse transcriptase (TERT) and template forming RNA component, telomerase RNA (TERC). TERT promoter mutations may underpin some telomerase activation but regulation of the gene is not completely understood due to the complex interplay of epigenetic, transcriptional, and posttranscriptional modifications. Pluripotent stem cells (PSCs) can maintain an indefinite, immortal, proliferation potential through their endogenous telomerase activity, maintenance of telomere length, and a bypass of replicative senescence in vitro. Differentiation of PSCs results in silencing of the TERT gene and an overall reversion to a mortal, somatic cell phenotype. The precise mechanisms for this controlled transcriptional silencing are complex. Promoter methylation has been suggested to be associated with epigenetic control of telomerase regulation which presents an important prospect for understanding cancer and stem cell biology. Control of down-regulation of telomerase during differentiation of PSCs provides a convenient model for the study of its endogenous regulation. Telomerase reactivation has the potential to reverse tissue degeneration, drive repair, and form a component of future tissue engineering strategies. Taken together it becomes clear that PSCs provide a unique system to understand telomerase regulation fully and drive this knowledge forward into aging and therapeutic application.

Physoxia alters human mesenchymal stem cell secretome.

The human mesenchymal stem cell (hMSC) secretome has pleiotropic effects which underpin their therapeutic potential. hMSC serum-free conditioned media (SFCM) has been determined to contain a variety of cytokines with roles in regeneration and suppression of inflammation. Physiological oxygen (physoxia) has been demonstrated to impact upon a number of facets of hMSC biology and we hypothesized that the secretome would be similarly modified. We tested a range of oxygen conditions; 21% O2 (air oxygen (AO)), 2% O2 (intermittent hypoxia (IH)) and 2% O2 Workstation (physoxia (P)) to evaluate their effect on hMSC secretome profiles. Total protein content of secretome was upregulated in IH and P (>3 fold vs AO) and IH (>1 fold vs P). Focused cytokine profiling indicated global upregulation in IH of all 31 biomolecules tested in comparison to AO and P with basic-nerve growth factor (bNGF) and granulocyte colony-stimulating factor (GCSF) (>3 fold vs AO) and bNGF and Rantes (>3 fold vs P) of note. Similarly, upregulation of interferon gamma-induced protein 10 (IP10) was noted in P (>3 fold vs AO). Interleukin-2 (IL2) and Rantes (in AO and P) and adiponectin, IL17a, and epidermal growth factor (EGF) (in AO only) were entirely absent or below detection limits. Quantitative analysis validated the pattern of IH-induced upregulation in vascular endothelial growth factor (VEGF), placental growth factor-1 (PIGF1), Tumor necrosis factor alpha (TNFa), IL2, IL4, and IL10 when compared to AO and P. In summary, modulation of environmental oxygen alters both secretome concentration and composition. This consideration will likely impact on delivering improved mechanistic understanding and potency effects of hMSC-based therapeutics.

DNMT3B Is an Oxygen-Sensitive De Novo Methylase in Human Mesenchymal Stem Cells.

The application of physiological oxygen (physoxia) concentrations is becoming increasingly commonplace within a mammalian stem cell culture. Human mesenchymal stem cells (hMSCs) attract widespread interest for clinical application due to their unique immunomodulatory, multi-lineage potential, and regenerative capacities. Descriptions of the impact of physoxia on global DNA methylation patterns in hMSCs and the activity of enzymatic machinery responsible for its regulation remain limited. Human bone marrow-derived mesenchymal stem cells (BM-hMSCs, passage 1) isolated in reduced oxygen conditions displayed an upregulation of SOX2 in reduced oxygen conditions vs. air oxygen (21% O2, AO), while no change was noted for either OCT-4 or NANOG. DNA methylation marks 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) showed decreases in 2% O2 environment (workstation) (2% WKS). DNMT3B (DNA methyltransferase 3B) and TET1 (Ten-eleven translocation enzyme 1) displayed reduced transcription in physoxia. Consistent with transcriptional downregulation, we noted increased promoter methylation levels of DNMT3B in 2% WKS accompanied by reduced DNMT3B and TET1 protein expression. Finally, a decrease in HIF1A (Hypoxia-inducible factor 1A) gene expression in 2% WKS environment correlated with protein levels, while HIF2A was significantly higher in physoxia correlated with protein expression levels vs. AO. Together, these data have demonstrated, for the first time, that global 5mC, 5hmC, and DNMT3B are oxygen-sensitive in hMSCs. Further insights into the appropriate epigenetic regulation within hMSCs may enable increased safety and efficacy development within the therapeutic ambitions.

Mesenchymal Stromal Cell Differentiation for Generating Cartilage and Bone-Like Tissues In Vitro.

In the field of tissue engineering, progress has been made towards the development of new treatments for cartilage and bone defects. However, in vitro culture conditions for human bone marrow mesenchymal stromal cells (hBMSCs) have not yet been fully defined. To improve our understanding of cartilage and bone in vitro differentiation, we investigated the effect of culture conditions on hBMSC differentiation. We hypothesized that the use of two different culture media including specific growth factors, TGFß1 or BMP2, as well as low (2% O2) or high (20% O2) oxygen tension, would improve the chondrogenic and osteogenic potential, respectively. Chondrogenic and osteogenic differentiation of hBMSCs isolated from multiple donors and expanded under the same conditions were directly compared. Chondrogenic groups showed a notable upregulation of chondrogenic markers compared with osteogenic groups. Greater sGAG production and deposition, and collagen type II and I accumulation occurred for chondrogenic groups. Chondrogenesis at 2% O2 significantly reduced ALP gene expression and reduced type I collagen deposition, producing a more stable and less hypertrophic chondrogenic phenotype. An O2 tension of 2% did not inhibit osteogenic differentiation at the protein level but reduced ALP and OC gene expression. An upregulation of ALP and OC occurred during osteogenesis in BMP2 containing media under 20% O2; BMP2 free osteogenic media downregulated ALP and also led to higher sGAG release. A higher mineralization was observed in the presence of BMP2 during osteogenesis. This study demonstrates how the modulation of O2 tension, combined with tissue-specific growth factors and media composition can be tailored in vitro to promote chondral or endochondral differentiation while using the same donor cell population.