DOCSLIB.ORG

Understanding the Molecular Interplay Between Senescence, Rejuvenation, and Healthy Ageing. Eleanor Tyler

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Understanding the Molecular Interplay Between Senescence, Rejuvenation, and Healthy Ageing. Eleanor Tyler Understanding the molecular interplay between senescence, rejuvenation, and healthy ageing. Eleanor Tyler A thesis presented for the degree of Doctor of Philosophy 2016 Supervisors: Dr Cleo L. Bishop Prof. Mike P. Philpott Centre for Cell Biology and Cutaneous Research The Blizard Institute Barts and the London School of Medicine and Dentistry Queen Mary University of London Statement of originality I, Eleanor Tyler, confirm that the research included within this thesis is my own work or that where it has been carried out in collaboration with, or supported by others, that this is duly acknowledged below and my contribution indicated. I attest that I have exercised reasonable care to ensure that the work is original, and does not to the best of my knowledge break any UK law, infringe any third party’s copyright or other Intellectual Property Right, or contain any confidential material. I accept that the College has the right to use plagiarism detection software to check the electronic version of the thesis. I confirm that this thesis has not been previously submitted for the award of a degree by this or any other university. The copyright of this thesis rests with the author and no quotation from it or information derived from it may be published without the prior written consent of the author. Signature: Eleanor Tyler Date: 19.12.16 1 Abstract Senescence is classically defined as an irreversible cell cycle arrest. There is now convincing evidence that senescent cells accumulate during human ageing, potentially driving age-related dysfunction through depletion of mitotically active cells and stimulation of chronic inflammation. Recently, a landmark paper demonstrated that removal of p16INK4A (p16)-positive senescent cells in mice prolonged healthy lifespan, suggesting a direct link between senescence and age-related dysfunction. As such, restraining the senescent pool or slowing their rate of accumulation presents an attractive therapeutic strategy for extending healthspan. Previously, our laboratory has demonstrated that siRNA transfection can reverse deep senescence in p16-positive primary adult human mammary epithelial cells using a panel of senescence markers. Subsequent siRNA screening revealed 28 hits which strongly induced reversal in the deeply senescent HMECs. In this project, siRNA knockdown of p16 combined with p21WAF1/CIP1 (p21) was found to reverse deep replicative senescence in primary adult human mammary fibroblasts, as defined by a panel of senescence markers. This discovery provided the opportunity to screen for novel siRNAs which induce reversal in both cell types. Screening in the deeply senescent HMFs of the 28 shortlisted candidates and 33 protein interactors identified using bioinformatics revealed 45 siRNAs which significantly increased cell number compared to the negative siRNA control. Subsequent immunofluorescence staining and high content analysis of the top 14 candidates identified 10 hit siRNAs which induced senescence reversal as defined by a panel of markers. Interestingly, these 10 hits were enriched for cytoskeletal and cell adhesion processes, suggesting an interplay between external forces and senescence induction. The top siRNA hit, early growth response 2 or EGR2, a transcription factor, was validated and selected for further exploration as a novel driver of senescence. Bioinformatics analysis revealed an enrichment for EGR2 binding sites within genes dynamically expressed in HMEC senescence, including p16, p21, and nine hits identified to reverse senescence in HMFs and HMECs. Furthermore, deeply senescent HMFs and HMECs were found to have a significantly increased nuclear EGR2 foci number compared to proliferating cells, and this was significantly decreased in reversed HMFs. In conclusion, it is proposed that EGR2 may represent a novel driver of both HMF and HMEC senescence. 2 Acknowledgements This work was supported by a Medical Research Council PhD Studentship (Grant number: MR/K501372/1). First and foremost, I would like to thank my supervisor, Dr Cleo Bishop, for her invaluable, unwavering support and guidance throughout my PhD. Your relentless optimism has been an inspiration. Thank you. I am also grateful for the support from my second supervisor, Prof. Mike Philpott, who has been there for me throughout all the PhD milestones. To Deb, the members of the Bishop lab., and everyone in the Blizard (Anke, Heather, Hannah, to name but a few), thank you for making our institute such a fun place to work. In particular, I would like to thank Dr Maddi Moore. We grew up together in the lab. and I will always look to you as my academic big sister. Special thanks also to Mat for sharing his seemingly endless wealth of knowledge. A big thank you goes to my support at home, Mike, who made sure I was always fed and watered. You are my rock. Also, to my friends and family, Kim, Sylvie, Alex, Gemma, Mum and Dad, who were always there for me and always okay when I couldn’t be there. Thank you all. 3 Contents Chapter 1 Introduction .................................................................................................... 20 1.1 The cell cycle .......................................................................................................... 21 1.1.1 Cell cycle regulation ........................................................................................ 21 1.1.2 The INK4/ARF locus ......................................................................................... 24 1.1.3 p21 .................................................................................................................. 26 1.2 Fibroblast replicative senescence ......................................................................... 28 1.3 Other types of senescence .................................................................................... 31 1.3.1 Epithelial cellular senescence ......................................................................... 31 1.3.2 Premature senescence ................................................................................... 32 1.3.3 Senescence in other cell types ........................................................................ 33 1.4 Markers of senescence .......................................................................................... 34 1.4.1 Commonly used markers of senescence ........................................................ 34 1.4.2 Senescence-associated secretory phenotype (SASP) ..................................... 35 1.5 Senescence in vivo ................................................................................................. 38 1.5.1 Senescence, tissue dysfunction, and ageing .................................................. 38 1.5.2 Other roles of senescence in vivo ................................................................... 42 1.6 Senescence as a therapeutic target ...................................................................... 44 1.7 Reversal of senescence.......................................................................................... 47 1.8 Early growth response 2 (EGR2) ............................................................................ 49 1.8.1 EGR2 gene locus, isoforms, and family ........................................................... 49 1.8.2 EGR2 and development .................................................................................. 50 1.8.3 EGR2 and the peripheral nervous system ...................................................... 51 1.8.4 EGR2 and the immune system ........................................................................ 52 1.8.5 EGR2, cancer, and senescence ....................................................................... 54 1.9 Small interfering RNA (siRNA) technology ............................................................ 57 1.10 Project aims ......................................................................................................... 59 Chapter 2 Materials and Methods .................................................................................. 60 4 2.1 Mammalian cell culture .................................................................................... 61 2.1.1 Culture of normal human mammary fibroblasts ...................................... 61 2.1.2 Culture of wildtype and p21 double knockout human fibroblasts ........... 62 2.1.3 Culture of normal human mammary epithelial cells ................................ 62 2.2 UV irradiation-induced senescence ................................................................. 62 2.3 Paracrine senescence ....................................................................................... 63 2.4 Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western Blotting ......................................................................................................... 63 2.4.1 Protein lysate preparation ........................................................................ 63 2.4.2 SDS-PAGE .................................................................................................. 64 2.4.3 Immunoblot analysis ................................................................................. 64 2.4.4 Densitometry............................................................................................. 66 2.4.5 Membrane stripping ................................................................................. 67 2.5 siRNA forward-transfection optimisation ........................................................ 67
Load more

Recommended publications
  • Disruptive Chemicals, Senescence and Immortality
    Disruptive chemicals, senescence and immortality Carnero, A., Blanco-Aparicio, C., Kondoh, H., Lleonart, M. E., Martinez-Leal, J. F., Mondello, C., ... & Yasaei, H. (2015). Disruptive chemicals, senescence and immortality. Carcinogenesis, 36(Suppl 1), S19-S37. doi:10.1093/carcin/bgv029 10.1093/carcin/bgv029 Oxford University Press Version of Record http://cdss.library.oregonstate.edu/sa-termsofuse Carcinogenesis, 2015, Vol. 36, Supplement 1, S19–S37 doi:10.1093/carcin/bgv029 Review review Disruptive chemicals, senescence and immortality Amancio Carnero*, Carmen Blanco-Aparicio1, Hiroshi Kondoh2, Matilde E. Lleonart3, Juan Fernando Martinez-Leal4, Chiara Mondello5, A.Ivana Scovassi5, William H.Bisson6, Amedeo Amedei7, Rabindra Roy8, Jordan Woodrick8, Annamaria Colacci9, Monica Vaccari9, Jayadev Raju10, Fahd Al-Mulla11, Rabeah Al- Downloaded from Temaimi11, Hosni K. Salem12, Lorenzo Memeo13, Stefano Forte13, Neetu Singh14, Roslida A. Hamid15, Elizabeth P. Ryan16, Dustin G. Brown16, John Pierce Wise Sr17, Sandra S.Wise17 and Hemad Yasaei18 http://carcin.oxfordjournals.org/ Instituto de Biomedicina de Sevilla (IBIS/CSIC/HUVR/Univ. Sevilla), Oncohematology and Genetics Department, Avda Manuel siurot sn, 41013 Sevilla, Spain, 1Spanish National Cancer Research Center, Experimental Therapuetics Department, Melchor Fernandez Almagro, 3, 28029 Madrid, Spain, 2Department of Geriatric Medicine, Kyoto University Hospital, 54 Kawaharacho, Shogoin, Sakyo-ku Kyoto 606-8507, Japan, 3Institut De Recerca Hospital Vall D’Hebron, Passeig Vall d’Hebron, 119–129,
    [Show full text]
  • The Emergence of Senescent Surface Biomarkers As Senotherapeutic Targets
    cells Review The Emergence of Senescent Surface Biomarkers as Senotherapeutic Targets Martina Rossi and Kotb Abdelmohsen * Laboratory of Genetics and Genomics, RNA Regulation Section, National Institute on Aging Intramural Research Program, National Institutes of Health, 251 Bayview Blvd., Baltimore, MD 21224, USA; [email protected] * Correspondence: [email protected] Abstract: Senescence is linked to a wide range of age-associated diseases and physiological declines. Thus, senotherapeutics are emerging to suppress the detrimental effects of senescence either by senomorphics or senolytics. Senomorphics suppress the traits associated with senescence phenotypes, while senolytics aim to clear senescent cells by suppressing their survival and enhancing the apoptotic pathways. The main goal of these approaches is to suppress the proinflammatory senescence- associated secretory phenotype (SASP) and to promote the immune recognition and elimination of senescent cells. One increasingly attractive approach is the targeting of molecules or proteins specifically present on the surface of senescent cells. These proteins may play roles in the maintenance and survival of senescent cells and hence can be targeted for senolysis. In this review, we summarize the recent knowledge regarding senolysis with a focus on novel surface biomarkers of cellular senescence and discuss their emergence as senotherapeutic targets. Keywords: senescence; surface proteins; surfaceome; senolytics; senolysis; senostatic; senescent cell clearance; senotherapeutics; senotherapy Citation: Rossi, M.; Abdelmohsen, K. The Emergence of Senescent Surface Biomarkers as Senotherapeutic Targets. Cells 2021, 10, 1740. 1. Introduction https://doi.org/10.3390/cells Cellular senescence is a phenotype associated with limited replicative capacity and 10071740 irreversible growth arrest of primary cells first described by Leonard Hayflick in the early 1960s [1].
    [Show full text]
  • Does Senescence Promote Fitness in Caenorhabditis Elegans by Causing Death?
    Institute of Healthy Ageing Preprint 1st Nov 2018 Essay Does senescence promote fitness in Caenorhabditis elegans by causing death? Jennifer N. Lohr1, Evgeniy R. Galimov1 and David Gems* Institute of Healthy Ageing, and Research Department of Genetics, Evolution and Environment, University College London, London WC1E 6BT, UK. 1These authors contributed equally. ∗ Corresponding author. E-mail address: [email protected] (D. Gems). 1 Institute of Healthy Ageing Preprint 1st Nov 2018 Abstract A widely appreciated conclusion from evolutionary theory is that senescence (aging) is of no adaptive value to the individual that it afflicts. Yet studies of C. elegans and S. cerevisiae are increasingly revealing the presence of processes which actively cause senescence and death, leading some biogerontologists to wonder about the established theory. Here we argue that programmed death that increases fitness could occur in C. elegans and S. cerevisiae, and that this is consistent with the classic evolutionary theory of aging. This is because of the special conditions under which these organisms have evolved, particularly the existence of clonal populations with limited dispersal and, in the case of C. elegans, the brevity of the reproductive period caused by protandry. Under these conditions, death-promoting mechanisms could promote worm fitness by enhancing inclusive fitness, or worm colony fitness through group selection. Such altruistic, adaptive death is not expected to evolve in organisms with outbred, dispersed populations. The plausibility of adaptive death in C. elegans is supported by computer modelling studies, and new knowledge about the ecology of this species. To support these arguments we also review the biology of adaptive death, and distinguish three forms: consumer sacrifice, biomass sacrifice and defensive sacrifice.
    [Show full text]
  • Dissecting Aging and Senescence—Current Concepts and Open Lessons
    cells Review Dissecting Aging and Senescence—Current Concepts and Open Lessons 1,2, , 1,2, 1 1,2 Christian Schmeer * y , Alexandra Kretz y, Diane Wengerodt , Milan Stojiljkovic and Otto W. Witte 1,2 1 Hans-Berger Department of Neurology, Jena University Hospital, 07747 Jena, Thuringia, Germany; [email protected] (A.K.); [email protected] (D.W.); [email protected] (M.S.); [email protected] (O.W.W.) 2 Jena Center for Healthy Ageing, Jena University Hospital, 07747 Jena, Thuringia, Germany * Correspondence: [email protected] These authors have contributed equally. y Received: 2 October 2019; Accepted: 13 November 2019; Published: 15 November 2019 Abstract: In contrast to the programmed nature of development, it is still a matter of debate whether aging is an adaptive and regulated process, or merely a consequence arising from a stochastic accumulation of harmful events that culminate in a global state of reduced fitness, risk for disease acquisition, and death. Similarly unanswered are the questions of whether aging is reversible and can be turned into rejuvenation as well as how aging is distinguishable from and influenced by cellular senescence. With the discovery of beneficial aspects of cellular senescence and evidence of senescence being not limited to replicative cellular states, a redefinition of our comprehension of aging and senescence appears scientifically overdue. Here, we provide a factor-based comparison of current knowledge on aging and senescence, which we converge on four suggested concepts, thereby implementing the newly emerging cellular and molecular aspects of geroconversion and amitosenescence, and the signatures of a genetic state termed genosenium.
    [Show full text]
  • Aging, Life Span, and Senescence
    Proc. Natl. Acad. Sci. USA Vol. 95, pp. 11034–11036, September 1998 From the Academy This paper is summary of a session presented at the Ninth Annual Frontiers of Science Symposium, held November 7–9, 1997, at the Arnold and Mabel Beckman Center of the National Academies of Sciences and Engineering in Irvine, CA. Aging, life span, and senescence LEONARD GUARENTE*, GARY RUVKUN†, AND RICHARD AMASINO‡ *Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139; †Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114; and ‡Department of Biochemistry, University of Wisconsin, Madison, WI 53706 Research in the field of aging recently has entered a new era. in old sgs1 mother cells were enlarged and fragmented, Model systems have begun to provide insight into cellular, changes that are also found in very old wild type cells. molecular, and organismal changes that are related intimately What is the molecular basis of this fragmentation and does to aging. One important approach has been the identification it cause aging? Examination of the rDNA in old cells revealed of genes that determine the life span of an organism. The very an accumulation of extrachromosomal rDNA circles (ERCs) existence of genes that when mutated can extend life span of discrete sizes representing oligomers of the rDNA unit (7). suggests that one or a few processes may be critical in aging and ERCs arise from recombination within the rDNA of chromo- that a slowing of these processes may slow aging itself. some XII in the early part of the life span (Fig. 1). Each subsequent cell cycle, the ERCs replicate via the replication Yeast sequence in the rDNA repeats and then segregate asymmetri- cally into mother cells.
    [Show full text]
  • A Biomarker That Identifies Senescent Human Cells in Culture and in Aging Skin in Vivo (Replicative Senescence/Tumor Suppression/18-Galactosidase) GOBERDHAN P
    Proc. Natl. Acad. Sci. USA Vol. 92, pp. 9363-9367, September 1995 Cell Bioiogy A biomarker that identifies senescent human cells in culture and in aging skin in vivo (replicative senescence/tumor suppression/18-galactosidase) GOBERDHAN P. DIMRI*, XINHUA LEEt, GEORGE BASILE*, MEILEEN ACOSTA*, GLYNIS SCOrrt, CALVIN ROSKELLEY*, ESTELA E. MEDRANO§, MAARTEN LINSKENSI, IVICA RUBELJII, OLIVIA PEREIRA-SMITHII, MONICA PEACOCKEt, AND JUDITH CAMPISI* ** *Department of Cell and Molecular Biology, Lawrence Berkeley Laboratory, University of California, Berkeley, CA 94720; tDepartment of Dermatology, New England Medical Center, Tufts University Medical School, Boston, MA 02111; iDepartment of Dermatology and Pathology, University of Rochester School of Medicine, Rochester, NY 14642; §Department of Cell Biology and IDivision of Molecular Virology, Huffington Center on Aging, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030; and IGeron Corporation, 200 Constitution Drive, Menlo Park, CA 94025 Communicated by Arthur B. Pardee, Dana-Farber Cancer Institute, Boston, MA, June 12, 1995 (received for review March 28, 1995) ABSTRACT Normal somatic cells invariably enter a state The idea that cellular senescence is tumor suppressive stems of irreversibly arrested growth and altered function after a from molecular, cellular, and in vivo data. Immortality greatly finite number of divisions. This process, termed replicative increases the susceptibility to malignant transformation, in senescence, is thought to be a tumor-suppressive mechanism culture and in vivo (5, 6). Indeed, many tumors contain and an underlying cause ofaging. There is ample evidence that immortal cells or cells with an extended replicative lifespan (7). escape from senescence, or immortality, is important for In addition, some oncogenes act primarily to immortalize or malignant transformation.
    [Show full text]
  • Life Extension Pseudoscience and the SENS Plan
    Life Extension Pseudoscience and the SENS Plan Preston W. Estep III, Ph.D. President and CEO, Longenity Inc. Matt Kaeberlein, Ph.D. Department of Pathology University of Washington Pankaj Kapahi, Ph.D. Buck Institute for Age Research Brian K. Kennedy, Ph.D. Department of Biochemistry University of Washington Gordon J. Lithgow Ph.D. Buck Institute for Age Research George M. Martin, M.D. Department of Pathology University of Washington Simon Melov, Ph.D. Buck Institute for Age Research R. Wilson Powers III Department of Genome Sciences University of Washington Heidi A. Tissenbaum, Ph.D. Program in Gene Function and Expression Program in Molecular Medicine University of Massachusetts Medical School Abstract Recent scientific advances have taken gerontological research to challenging and exciting new frontiers, and have given many scientists increased confidence that human aging is to some degree controllable. We have been on the front lines of some of these developments and the speculative discussions they have engendered, and we are proud to be part of the increasingly productive biomedical effort to reduce the pathologies of aging, and age-associated diseases, to the greatest degree possible—and to extend healthy human life span to the greatest degree possible. In contrast to clearly justifiable speculations regarding future advances in human longevity a few have made claims that biological immortality is within reach. One, Aubrey de Grey, claims to have developed a “detailed plan to cure human aging” called Strategies for Engineered Negligible Senescence (SENS) [1, 2]. This is an extraordinary claim, and we believe that extraordinary claims require extraordinary evidentiary support. In supplementary material posted on the Technology Review web site we evaluate SENS in detail.
    [Show full text]
  • Cellular Senescence
    infoaging guides BIOLOGY OF AGING CELLULAR SENESCENCE An introduction to aging science brought to you by the American Federation for Aging Research WHAT IS CELLULAR no longer divide become what is that activate oncogenes—normal SENESCENCE? called “senescent.” Cells taken cellular genes that, when mutated, from older humans generally have contribute to the development Cellular senescence is a process shorter telomeres than cells from of cancer. These diverse triggers associated with aging that occurs young humans, so cells from older support the idea that cellular at the level of our cells. Cellular humans generally divide fewer senescence evolved primarily to senescence is sometimes called times before becoming replica- protect organisms from cancer. replicative senescence. Forty years tively senescent. ago, Dr. Leonard Hayflick and his Scientists have also noted that colleague, Dr. Paul Moorhead, Critically short telomeres, which senescent cells are different from discovered that many human resemble broken chromosomes, their younger counterparts. Like cells—particularly fibroblast cells, are not the only causes of ”senes- older people themselves, cells ap- which secrete substances that cence,” and so scientists often proaching senescence incur many provide structure to tissues—had use the more general term ”cellular biological losses or take on new a limited capacity to reproduce senescence.” Cellular senescence functions. Where younger cells themselves in culture by dividing. produce structural or functional They found that these and many proteins that maintain tissues in other normal human cells derived a healthy state, cells approaching from fetal, embryonic, or newborn senescence release enzymes that tissue can undergo between 40 break down these proteins. and 60 cell divisions, but then can divide no more.
    [Show full text]
  • A Gene Involved in Control of Human Cellular Senescence on Human Chromosome Lq PATRICK J
    MOLECULAR AND CELLULAR BIOLOGY, Apr. 1994, p. 2291-2297 Vol. 14, No. 4 0270-7306/94/$04.00+0 Copyright © 1994, American Society for Microbiology A Gene Involved in Control of Human Cellular Senescence on Human Chromosome lq PATRICK J. HENSLER,'* LOIS A. ANNAB,2 J. CARL BARRETT,2 AND OLIVIA M. PEREIRA-SMITH 3'4 Roy M. and Phyllis Gough Huffington Center on Aging,3 Division of Molecular Virology,' and Department of Medicine,4 Baylor College of Medicine, Houston, Texas 77030, and Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 277092 Received 8 October 1993/Returned for modification 6 December 1993/Accepted 3 January 1994 Normal cells in culture exhibit limited division potential and have been used as a model for cellular senescence. In contrast, tumor-derived or carcinogen- or virus-transformed cells are capable of indefinite division. Fusion of normal human diploid fibroblasts with immortal human cells yielded hybrids having limited life spans, indicating that cellular senescence was dominant. Fusions of various immortal human cell lines with each other led to the identification of four complementation groups for indefinite division. The purpose of this study was to determine whether human chromosome 1 could complement the recessive immortal defect of human cell lines assigned to one of the four complementation groups. Using microcell fusion, we introduced a single normal human chromosome 1 into immortal human cell lines representing the complementation groups and determined that it caused loss of proliferative potential of an osteosarcoma-derived cell line (TE85), a cytomegalovirus-transformed lung fibroblast cell line (CMV-Mj-HEL-1), and a Ki-ras+-transformed derivative of TE85 (143B TK-), all of which were assigned to complementation group C.
    [Show full text]
  • Cellular Senescence in Cultured Human Brainstem Astrocytes: Effect of Oxidative Stress Authors: Zane Leibhart and Dr
    Cellular Senescence in Cultured Human Brainstem Astrocytes: Effect of Oxidative Stress Authors: Zane Leibhart and Dr. Madhan Subramanian* Abstract: Cardiovascular diseases are one of the leading causes of mortality in the elderly. A central role in the contraction of cardiovascular diseases by means of aging could be played by cellular senescence. Senescent cells accumulate with age in various tissues of the body including the brain. Recent studies from our lab have shown that there is increased senescent cell accumulation in the brainstem in aged mice. Neurons in the brainstem is responsible for the control of sympathetic nerve activity to several vascular beds and overactivity of the sympathetic neurons would lead to increased risk for cardiovascular diseases. It is well documented that neurons are post mitotic and do not undergo senescence, however glial cells could undergo senescence and have implications in neuronal function. In this study we developed a cell culture model to study senescence in vitro. We have used cultured human astrocytes and subjected them to oxidative stress by means of hydrogen peroxide treatment. We believe that increasing levels of hydrogen peroxide will lead to an increase in the number of senescent cells present within the cultured human brain stem astrocytes. Our results showed that there are increased levels of senescence marker p16 and senescence associated secretory phenotype IL6 upregulated after hydrogen peroxide treatment along with decreased levels of laminB1. These results suggest that we were able to induce senescence in human brainstem astrocytes and it could be used as a suitable model to study senescence in vitro. We were able to establish a model using human cells, replicating the cellular senescence in the mice and our future goal would involve understanding the mechanism by which senescence cells in the brainstem would affect the sympathetic regulation.
    [Show full text]
  • Multi-Pronged Approach to Human Mesenchymal Stromal Cells
    www.nature.com/scientificreports OPEN Multi‑pronged approach to human mesenchymal stromal cells senescence quantifcation with a focus on label‑free methods Weichao Zhai1, Jerome Tan1, Tobias Russell2, Sixun Chen1, Dennis McGonagle2, May Win Naing1,3, Derrick Yong3* & Elena Jones2* Human mesenchymal stromal cells (hMSCs) have demonstrated, in various preclinical settings, consistent ability in promoting tissue healing and improving outcomes in animal disease models. However, translation from the preclinical model into clinical practice has proven to be considerably more difcult. One key challenge being the inability to perform in situ assessment of the hMSCs in continuous culture, where the accumulation of the senescent cells impairs the culture’s viability, diferentiation potential and ultimately leads to reduced therapeutic efcacies. Histochemical β ‑galactosidase staining is the current standard for measuring hMSC senescence, but this method is destructive and not label‑free. In this study, we have investigated alternatives in quantifcation of hMSCs senescence, which included fow cytometry methods that are based on a combination of cell size measurements and fuorescence detection of SA‑β‑galactosidase activity using the fuorogenic substrate, C 12FDG; and autofuorescence methods that measure fuorescence output from endogenous fuorophores including lipopigments. For identifcation of senescent cells in the hMSC batches produced, the non‑destructive and label‑free methods could be a better way forward as they involve minimum manipulations of the cells of interest, increasing the fnal output of the therapeutic‑grade hMSC cultures. In this work, we have grown hMSC cultures over a period of 7 months and compared early and senescent hMSC passages using the advanced fow cytometry and autofuorescence methods, which were benchmarked with the current standard in β‑galactosidase staining.
    [Show full text]
  • Age-Related Inflammation Triggers Skeletal Stem/Progenitor Cell Dysfunction
    Age-related inflammation triggers skeletal stem/ progenitor cell dysfunction Anne Marie Josephsona,b, Vivian Bradaschia-Correaa, Sooyeon Leea, Kevin Leclerca, Karan S. Patela, Emma Muinos Lopeza, Hannah P. Litwaa, Shane S. Neibarta, Manasa Kadiyalaa, Madeleine Z. Wonga, Matthew M. Mizrahia, Nury L. Yima, Austin J. Rammea, Kenneth A. Egola, and Philipp Leuchta,b,1 aDepartment of Orthopedic Surgery, New York University School of Medicine, New York, NY 10003; and bDepartment of Cell Biology, New York University School of Medicine, New York, NY 10016 Edited by Helen M. Blau, Stanford University, Stanford, CA, and approved February 25, 2019 (received for review June 21, 2018) Aging is associated with impaired tissue regeneration. Stem cell Results number and function have been identified as potential culprits. We Skeletal Stem Cell Frequency Decreases with Aging. To investigate a first demonstrate a direct correlation between stem cell number and clinically relevant age-associated effect on skeletal stem cell fre- time to bone fracture union in a human patient cohort. We then quency and function, we first examined iliac crest bone graft devised an animal model recapitulating this age-associated decline in (ICBG) samples from 36 patients (20 male, 16 female) with ages bone healing and identified increased cellular senescence caused by a ranging from 24 to 89 y who underwent operative fixation of an systemic and local proinflammatory environment as the major contrib- upper- or lower-extremity fracture. FACS with CD271 as a human utor to the decline in skeletal stem/progenitor cell (SSPC) number and skeletal stem cell marker (8–11) revealed that SSPC frequency function.
    [Show full text]