Functional demonstration of a mortality phenotype associated with 4cen-q23

Normal human keratinocytes possess a finite replicative lifespan whereas most advanced squamous cells carcinomas are immortal. The mechanisms, whose abrogation's can be considered necessary to achieve immortality, include those involving the negative cell cycle regulators p53, p16INK4A, and the telomere repair reverse-transcriptase enzyme, telomerase. Other specific chromosomes have also been demonstrated to carry functions whose loss is necessary for the development of immortality, through immortal phenotype reversion upon reintroduction into an immortal cell line. We demonstrate here the phenotypic reversion of immortal HNSCC-derived keratinocyte cell lines to a mortal growth-arrest upon reintroduction of a resistance marker tagged wild-type human chromosome 4. We further demonstrate that this phenotypic reversion occurs only in cell lines, which display LOH on 4q (BICR6 and BICR31), and not in those with intact endogenous chromosome 4 copies (BICR3 and BICR19), and that it is chromosome 4-specific, chromosomes 6, 11, and 15 having no effect on proliferative lifespan following introduction into BICR6 by MMCT. Through XMMCT-based truncated chromosomal fragment generation the functional complementation was localised to 4cen-q23, whilst fine mapping in segregants arising from the MMCT experiments identified an approximate 1.5Mb locus containing a minimal number of candidate genes. Through biological assay we have further determined the growth-arrest to have characteristics of crisis. This was determined through low BrdU incorporation balanced with high levels of apoptosis to statistically significant levels (less than 0.05). We found no evidence for involvement of telomeric attrition in the observed phenotype, through insufficient phenotypic lag (3-10 MPD) and growth-arrest in the presence of ectopic hTERT expression, suggesting the operation of an alternative mechanism. This suggests the presence of gene(s) at 4cen-q23 whose loss is advantageous to the development of immortality in advanced tumours including HNSCC

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

Financial support was provided by the Ministry of Higher Education and Scientific Research, Iraq (S1443) and the Guy Hilton Asthma Trust.Peer reviewedPublisher PD

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

The work described in this paper was principally funded by the Iraqi Ministry of Higher Education and Scientific Research, University of Baghdad, RMKAJ (S1453), Baghdad, Iraq. We also wish to thank the Turkish Ministry of National Education for their support.Peer reviewedPublisher PD

Tuning the properties of all natural polymeric scaffolds for tendon repair with cellulose microfibers

Funding Information: This paper is a part of dissemination activities of the project P4FIT. This project has received funding from the European Union's Horizon 2020 Research and Innovation Programme under the Maria Skłodowska-Curie Grant Agreement N° 955685 .Peer reviewe

Injectable hydrogels : An emerging therapeutic strategy for cartilage regeneration

Funding Information: The authors acknowledge the funding support from the North Staffordshire Medical Institute (NSMI Research Awards 2021). Arjan Atwal gratefully thanks Faculty of Medicine and Health Sciences , Keele University , for funding his PhD studentship.Peer reviewedPublisher PD

The role of dissolved oxygen levels on human Mesenchymal Stem Cell culture success, regulatory compliance and therapeutic potential

Most cells in the human body, including human mesenchymal stem cells (hMSCs), have evolved to survive and function in a low, physiological, oxygen (O2) environment. Investigators have become increasingly aware of the effects of O2 levels on hMSC biology and culture and are mimicking the natural niche of these cells in vitro to improve cell culture yields. This presents many challenges in relation to hMSC identity and function and in the maintenance of a controlled O2 environment for cell culture. The aim of this review is to discuss a “hMSC checklist” as a guide to establishing which identity and potency assays to implement when studying hMSCs. The checklist includes markers, differentiation potential, proliferation & growth, attachment & migration, genomic stability and paracrine activity. Evidence drawn from the current literature demonstrates that low O2 environments could improve most “hMSC checklist” attributes. However, there are substantial inconsistencies around both the terminology and the equipment used in low O2 studies. Therefore, “hypoxia” as a term and as a culture condition are discussed. The biology of short (acute) vs long-term (chronic) hypoxia is considered and a nascent hypothesis to explain the behaviour of hMSCs in long-term hypoxia is presented. It is hoped that by establishing an ongoing discourse, and driving towards a regulatory recognisable “hMSC checklist”, we may be better able to provide the patient population with safe and efficacious regenerative treatments

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

Acknowledgements The authors would like to acknowledge the support of the UHNM theatre staff, Dr. Sana Iftikhar and Dr. Daniel Gey van Pittius for their assistance in acquiring participant lung tissue. Funding Information: This work was supported by funding from the North Staffordshire Medical Institute 50th Anniversary Award and The Royal Society-Newton Mobility Grant. The authors would like to acknowledge the support of the UHNM theatre staff, Dr. Sana Iftikhar and Dr. Daniel Gey van Pittius for their assistance in acquiring participant lung tissue.Peer reviewedPublisher PD

Investigating the relationship between surface properties of polymers and protein adsorption [Abstract]

Investigating the relationship between surface properties of polymers and protein adsorption [Abstract

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

Funding Information: The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The authors acknowledge operating grant support from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 955685, www.helsinki.fi/p4fit .Peer reviewedPublisher PD

Ectopic Telomerase Expression Fails to Maintain Chondrogenic Capacity in Three-Dimensional Cultures of Clinically Relevant Cell Types.

The poor healing capacity of cartilage and lack of effective treatment for associated disease and trauma makes it a strong candidate for a regenerative medicine approach. Potential therapies tested to date, although effective, have met with a number of intrinsic difficulties possibly related to limited autologous chondrocyte cell yield and quality of cartilage produced. A potential mechanism to bypass limited cell yields and improve quality of differentiation is to immortalize relevant cell types through the ectopic expression of telomerase. Pellet cultures of human chondrocytes (OK3), bone marrow mesenchymal stem cells (BMA13), and embryonic stem cell (H1 line)-derived cells (1C6) and their human telomerase reverse transcriptase (hTERT) transduced counterparts were maintained for 20 days in standard maintenance medium (MM) or transforming growth factor-ß3-supplemented prochondrogenic medium (PChM). Pellets were assessed for volume and density by microcomputed tomography. Quantitative gene expression (COL1A2, COL2A1, COL3A1, COL6A3, COL10A1, ACAN, COMP, SOX9); sulfated glycosaminoglycans (sGAGs), and DNA quantification were performed. Histology and immunohistochemistry were used to determine matrix constituent distribution. Pellet culture in PChM resulted in significantly larger pellets with an overall increased density when compared with MM culture. Gene expression analysis revealed similarities in expression patterns between telomerase-transduced and parental cells in both MM and PChM. Of the three parental cell types examined OK3 and BMA13 produced similar amounts of pellet-associated sGAG in PChM (4.62?±?1.20 and 4.91?±?1.37?µg, respectively) with lower amounts in 1C6 (2.89?±?0.52?µg), corresponding to 3.1, 2.3, and 1.6-fold increases from day 0. In comparison, telomerase-transduced cells all had much lower sGAG with OK3H at 2.74?±?0.11?µg, BMA13H 1.29?±?0.34?µg, and 1C6H 0.52?±?0.01?µg corresponding to 1.2, 0.87, and 0.34-fold changes compared with day 0. Histology of day 20 pellets displayed reduced staining overall for collagens and sGAG in telomerase-transduced cells, most notably with alterations in aggrecan and collagen VI; all cells stained positively for collagen II. We conclude that while telomerase transduction may be an effective technique to extend cellular proliferative capacity, it is not sufficient in isolation to sustain a naive chondrogenic phenotype across multiple cell types