Limited Replicative Lifespan Research Articles

Establishment of fishing cat cell biobanking for sustainable conservation

The fishing cat (Prionailurus viverrinus) is a vulnerable wild felid that is currently under threat from habitat destruction and other human activities. The zoo provides insurance to ensure the survival of the fishing cat population. Creating a biobank of fishing cats is a critical component of recent zoo strategies for securely stocking cell samples for long-term survival. Here, our goal was to compare cell biobanking techniques (tissue collection, primary culture, and reprogramming) and tissue sources (ear skin, abdominal skin, testis) from captive (n = 6)/natural (n = 6) vs. living (n = 8)/postmortem (n = 4) fishing cats. First, we show that dermal fibroblasts from the medial border of the helix of the ear pinna and abdominal tissues of living fishing cats can be obtained, whereas postmortem animals provided far fewer fibroblasts from the ears than from the testes. Furthermore, we can extract putative adult spermatogonial stem cells from the postmortem fishing cat's testes. The main barrier to expanding adult fibroblasts was early senescence, which can be overcome by overexpressing reprogramming factors through felid-specific transfection programs, though we demonstrated that reaching iPSC state from adult fibroblasts of fishing cats was ineffective with current virus-free mammal-based induction approaches. Taken together, the success of isolating and expanding primary cells is dependent on a number of factors, including tissue sources, tissue handling, and nature of limited replicative lifespan of the adult fibroblasts. This study provides recommendations for tissue collection and culture procedures for zoological research to facilitate the preservation of cells from both postmortem and living felids.

Development and Characterisation of a Human Chronic Skin Wound Cell Line-Towards an Alternative for Animal Experimentation.

Background: Chronic skin wounds are a growing financial burden for healthcare providers, causing discomfort/immobility to patients. Whilst animal chronic wound models have been developed to allow for mechanistic studies and to develop/test potential therapies, such systems are not good representations of the human chronic wound state. As an alternative, human chronic wound fibroblasts (CWFs) have permitted an insight into the dysfunctional cellular mechanisms that are associated with these wounds. However, such cells strains have a limited replicative lifespan and therefore a limited reproducibility/usefulness. Objectives: To develop/characterise immortalised cell lines of CWF and patient-matched normal fibroblasts (NFs). Methods and Results: Immortalisation with human telomerase resulted in both CWF and NF proliferating well beyond their replicative senescence end-point (respective cell strains senesced as normal). Gene expression analysis demonstrated that, whilst proliferation-associated genes were up-regulated in the cell lines (as would be expected), the immortalisation process did not significantly affect the disease-specific genotype. Immortalised CWF (as compared to NF) also retained a distinct impairment in their wound repopulation potential (in line with CWF cell strains). Conclusions: These novel CWF cell lines are a credible animal alternative and could be a valuable research tool for understanding both the aetiology of chronic skin wounds and for therapeutic pre-screening.

Stem cell-based strategies in vascular surgery

Critical chronic ischemia in patients with underlying arterial occlusive disease requires vascular reconstructive surgery. The limited supply of suitable small-diameter autologous vascular grafts in many patients and obvious disadvantages of synthetic bypass material demand the development of clinically usable tissue-engineered blood vessel substitutes. Despite substantial progress in the field over the last two decades, their implementation into the clinical routine has been challenging. The limited replicative life span of human adult vascular cells and their slow rate of collagenous matrix production in vitro have posed important problems in the development of mechanically robust and biologically functional engineered grafts. With recent advances in stem cell research, new cell sources for vascular tissue engineering have become available. In particular, the discovery of human induced pluripotent stem (iPS) cells derived from adult differentiated cells, as well as of human multipotent adult mesenchymal stem cells without gene modification requirements and related safety concerns, may advance the development of novel autologous cell-based tissue engineering approaches. Here we discuss recent developments in the field of vascular progenitor cells and opportunities and challenges for the clinical translation of stem cell-engineered vascular tissue substitutes.

Enhancement of the in vivo persistence and antitumor efficacy of CD19 chimeric antigen receptor T cells through the delivery of modified TERT mRNA.

Chimeric antigen receptor T cell immunotherapy is a promising therapeutic strategy for treating tumors, demonstrating its efficiency in eliminating several hematological malignancies in recent years. However, a major obstacle associated with current chimeric antigen receptor T cell immunotherapy is that the limited replicative lifespan of chimeric antigen receptor T cells prohibits the long-term persistence and expansion of these cells in vivo, potentially hindering the long-term therapeutic effects of chimeric antigen receptor T cell immunotherapy. Here we showed that the transient delivery of modified mRNA encoding telomerase reverse transcriptase to human chimeric antigen receptor T cells targeting the CD19 antigen (CD19 chimeric antigen receptor T cells) would transiently elevate the telomerase activity in these cells, leading to increased proliferation and delayed replicative senescence without risk of insertion mutagenesis or immortalization. Importantly, compared to conventional CD19 chimeric antigen receptor T cells, after the transient delivery of telomerase reverse transcriptase mRNA, these CD19 chimeric antigen receptor T cells showed improved persistence and proliferation in mouse xenograft tumor models of human B-cell malignancies. Furthermore, the transfer of CD19 chimeric antigen receptor T cells after the transient delivery of telomerase reverse transcriptase mRNA enhanced long-term antitumor effects in mouse xenograft tumor models compared with conventional CD19 chimeric antigen receptor T cell transfer. The results of the present study provide an effective and safe method to improve the therapeutic potential of chimeric antigen receptor T cells, which might be beneficial for treating other types of cancer, particularly solid tumors.

In This Issue

HomeCirculation ResearchVol. 117, No. 2In This Issue Free AccessResearch ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessResearch ArticlePDF/EPUBIn This Issue Ruth Williams Ruth WilliamsRuth Williams Search for more papers by this author Originally published3 Jul 2015https://doi.org/10.1161/RES.0000000000000066Circulation Research. 2015;117:106CRISPR/Cas9-Modification of Endothelial Cells (p 121)Download figureDownload PowerPointAbrahimi et al perform CRISPR/Cas9 gene editing in primary human endothelial cells for the first time.Endothelial cells play a critical role in numerous physiological and pathological processes, such as inflammation, wound healing, vascularization, and atherosclerosis. Cultured human umbilical vein endothelial cells (HUVECs) are a valuable resource for studying endothelial cell biology. However, these cells have a limited replicative lifespan and are difficult to genetically manipulate. Recently, endothelial colony forming cells (ECFCs), also derived from umbilical cords, have been identified as having far longer replicative lives. Furthermore, gene editing using CRISPR/Cas9 technology has revolutionized genetic manipulations. Abrahimi and colleagues have therefore now performed CRISPR/Cas9-mediated gene disruption in ECFCs. They targeted the gene encoding CIITA—the master regulator of expression of the immune protein MHC Class II. The approach successfully depleted MHC Class II from more than 40 percent of ECFCs. And aside from a lack of MHC class II expression, the cells behaved similar to normal ECFCs. The results serve as proof-of-principle evidence that such gene editing can be applied to ECFCs. But specifically, successful suppression of MHC class II immunogenicity in ECFCs suggests that these cells might be used for therapeutic applications without the issue of immune rejection.MMP17 in Aortic Pathology (p e13)Download figureDownload PowerPointMartín-Alonso et al report that deficiency of matrix metalloproteinase 17 (MMP17) can increase one’s risk of developing aortic aneurysm.Aortic aneurysms caused by weakening of the aortic wall can lead to dissection or rupturing of the aorta and are the underlying cause of 1 to 2 percent of deaths in the developed world. Genetic mutations affecting either the extracellular matrix (ECM) or vascular smooth muscle cells (VSMCs) have been associated with predisposition for aortic aneurysm, but up to 75 percent of patients with family histories of aneurysms have no known genetic cause. Martin-Alonso and colleagues have now performed whole exome sequencing of a number of individuals with such inherited predisposition and have identified a new mutation in the ECM enzyme MMP17. When expressed in mice, the mutant version of the protein caused the animals’ aortas to become dilated with altered VSMC morphology and aberrant ECM structure. Moreover, the mice had an increased susceptibility to aortic aneurysm. The team went on to show that an MMP17 target—osteopontin—failed to be cleaved in the mutant mice. And that expression of the correctly cleaved osteopontin protein could rescue the animals’ defective aortic structure. Screening for MMP17 mutations in patients with a family history of aneurysms may lead to early identification of the defect and ultimately to tailored therapies for these patients, the authors suggest.Altered BM Function in Hypertension (p 178)Download figureDownload PowerPointInflammatory bone marrow cells increase blood pressure, report Santisteban et al.Although in most patients, hypertension can be controlled with current anti-hypertensive drugs, up to 30 percent of patients remain resistant to current therapy. Reasons for such resistance are not known, but may relate to difference in drug metabolism, genetic predisposition and severity of disease. In addition, the multi-factorial nature of hypertension makes it difficult to control. Hypertension has both neurogenic and inflammatory components and has been linked to increased sympathetic nervous stimulation of bone marrow, which increases inflammatory cell activation. Moreover hypertension is exacerbated by both peripheral and neurological inflammation. To examine the complex relationship between hypertension, inflammation and the brain, Santisteban and colleagues transferred bone marrow from hypertensive rats into those with normal blood pressure and found that the recipient animals’ blood pressure increased. Conversely, hypertensive rats whose bone marrow was replaced with that of normotensive animals exhibited a fall in blood pressure. Peripheral and brain inflammation was also reduced in these animals. Using an anti-inflammatory drug called minocycline that crosses the blood brain barrier, the team was able to inhibit activation of microglia—the brain’s immune cells—and reduce blood pressure in hypertensive rats. These results suggest that reducing peripheral and brain inflammation might be an effective treatment for hypertensive patients resistant to current medications. Previous Back to top Next FiguresReferencesRelatedDetailsCited By Hou X, Chen G, Bracamonte‐Baran W, Choi H, Diny N, Sung J, Talor M, Hackam D, Klingel K, Davogustto G, Taegtmeyer H, Coppens I, Barin J and Cihakova D The Cardiac Microenvironment Instructs Divergent Monocyte Fates and Functions in Myocarditis, SSRN Electronic Journal, 10.2139/ssrn.3272232 July 3, 2015Vol 117, Issue 2 Advertisement Article InformationMetrics © 2015 American Heart Association, Inc.https://doi.org/10.1161/RES.0000000000000066 Originally publishedJuly 3, 2015 PDF download Advertisement

Efficient gene disruption in cultured primary human endothelial cells by CRISPR/Cas9.

The participation of endothelial cells (EC) in many physiological and pathological processes is widely modeled using human EC cultures, but genetic manipulation of these untransformed cells has been technically challenging. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) technology offers a promising new approach. However, mutagenized cultured cells require cloning to yield homogeneous populations, and the limited replicative lifespan of well-differentiated human EC presents a barrier for doing so. To create a simple but highly efficient method using CRISPR/Cas9 to generate biallelic gene disruption in untransformed human EC. To demonstrate proof-of-principle, we used CRISPR/Cas9 to disrupt the gene for the class II transactivator. We used endothelial colony forming cell-derived EC and lentiviral vectors to deliver CRISPR/Cas9 elements to ablate EC expression of class II major histocompatibility complex molecules and with it, the capacity to activate allogeneic CD4(+) T cells. We show the observed loss-of-function arises from biallelic gene disruption in class II transactivator that leaves other essential properties of the cells intact, including self-assembly into blood vessels in vivo, and that the altered phenotype can be rescued by reintroduction of class II transactivator expression. CRISPR/Cas9-modified human EC provides a powerful platform for vascular research and for regenerative medicine/tissue engineering.

Differential effects of the extracellular microenvironment on human embryonic stem cell differentiation into keratinocytes and their subsequent replicative life span.

Culture microenvironment plays a critical role in the propagation and differentiation of human embryonic stem cells (hESCs) and their differentiated progenies. Although high efficiency of hESC differentiation to keratinocytes (hESC-Kert) has been achieved, little is known regarding the effects of early culture microenvironment and pertinent extracellular matrix (ECM) interactions during epidermal commitment on subsequent proliferative capacity of hESC-Kert. The aim of this study is to evaluate the effects of the different ECM microenvironments during hESC differentiation on subsequent replicative life span of hESC-Kert. In doing so, H1-hESCs were differentiated to keratinocytes (H1-Kert) in two differentiation systems. The first system employed autologous fibroblast feeder support, in which keratinocytes (H1-Kert(ACC)) were derived by coculture of hESCs with hESC-derived fibroblasts (H1-ebFs). The second system employed a novel decellularized matrix from H1-ebFs to create a dermoepidermal junction-like (DEJ) matrix. H1-Kert(AFF) were derived by differentiation of hESCs on the feeder-free system employing the DEJ matrix. Our study indicated that the feeder-free system with the use of DEJ matrix was more efficient in differentiation of hESCs toward epidermal progenitors. However, the feeder-free system was not sufficient to support the subsequent replicative capacity of differentiated keratinocytes. Of note, H1-Kert(AFF) showed limited replicative capacity with reduced telomere length and early cellular senescence. We further showed that the lack of cell-cell interactions during epidermal commitment led to heightened production of TGF-β1 by hESC-Kert during extended culture, which in turn was responsible for resulting in the limited replicative life span with cellular senescence of hESC-Kert derived under the feeder-free culture system. This study highlights for the first time the importance of the culture microenvironment and cell-ECM interactions during differentiation of hESCs on subsequent replicative life span and cellular senescence of the differentiated keratinocytes, with implications for use of these cells for applications in tissue engineering and regenerative medicine.

Sublethal heat shock induces premature senescence rather than apoptosis in human mesenchymal stem cells

Stem cells in adult organism are responsible for cell turnover and tissue regeneration. The study of stem cell stress response contributes to our knowledge on the mechanisms of damaged tissue repair. Previously, we demonstrated that sublethal heat shock (HS) induced apoptosis in human embryonic stem cells. This study aimed to investigate HS response of human adult stem cells. Human mesenchymal stem cells (MSCs) cultivated in vitro were challenged with sublethal HS. It was found that sublethal HS did not affect the cell viability assessed by annexin V/propidium staining. However, MSCs subjected to severe HS exhibited features of stress-induced premature senescence (SIPS): irreversible cell cycle arrest, altered morphology, increased expression of senescence-associated β-galactosidase (SA-β-gal) activity, and induction of cyclin-dependent kinase inhibitor p21 protein. High level of Hsp70 accumulation induced by sublethal HS did not return to the basal level, at least, after 72 h of the cell recovery when most cells exhibited SIPS hallmarks. MSCs survived sublethal HS, and resumed proliferation sustained the properties of parental MSCs: diploid karyotype, replicative senescence, expression of the cell surface markers, and capacity for multilineage differentiation. Our results showed for the first time that in human MSCs, sublethal HS induced premature senescence rather than apoptosis or necrosis. MSC progeny that survived sublethal HS manifested stem cell properties of the parental cells: limited replicative life span and multilineage capacity.

Human telomerase reverse transcriptase and glucose-regulated protein 78 increase the life span of articular chondrocytes and their repair potential

BackgroundLike all mammalian cells, normal adult chondrocytes have a limited replicative life span, which decreases with age. To facilitate the therapeutic use of chondrocytes from older donors, a method is needed to prolong their life span.MethodsWe transfected chondrocytes with hTERT or GRP78 and cultured them in a 3-dimensional atelocollagen honeycomb-shaped scaffold with a membrane seal. Then, we measured the amount of nuclear DNA and glycosaminoglycans (GAGs) and the expression level of type II collagen as markers of cell proliferation and extracellular matrix formation, respectively, in these cultures. In addition, we allografted this tissue-engineered cartilage into osteochondral defects in old rabbits to assess their repair activity in vivo.ResultsOur results showed different degrees of differentiation in terms of GAG content between chondrocytes from old and young rabbits. Chondrocytes that were cotransfected with hTERT and GRP78 showed higher cellular proliferation and expression of type II collagen than those of nontransfected chondrocytes, regardless of the age of the cartilage donor. In addition, the in vitro growth rates of hTERT- or GRP78-transfected chondrocytes were higher than those of nontransfected chondrocytes, regardless of donor age. In vivo, the tissue-engineered cartilage implants exhibited strong repairing activity, maintained a chondrocyte-specific phenotype, and produced extracellular matrix components.ConclusionsFocal gene delivery to aged articular chondrocytes exhibited strong repairing activity and may be therapeutically useful for articular cartilage regeneration.

If you can’t beat me, join me: collaborating oncogenes circumventing senescence and causing cancer

death. Signaling routes commonly altered in cancer include the RAS-MAP Kinase and PI3 Kinase (PI3K) pathways. Activation of the first can take place at many levels, most frequently through mutations in members of the RAS and RAF families, while also further upstream receptor tyrosine kinases can be activated by mutation or amplification. PI3K pathway activation occurs largely through mutations in its catalytic subunit PIK3CA or amplification of AKT family members. Additionally, the Phosphatase and Tensin Homolog (PTEN), a phosphatase negatively regulating the PI3K pathway by dephosphorylating the lipid phosphatidylinositol-3,4,5-trisphosphate (PIP3), is inactivated in several tumor types by different mechanisms including mutation, deletion, and epigenetic silencing. Perhaps somewhat counter intuitively, although PI3K is an established downstream effector of RAS in oncogenic transformation, simultaneous mutations in RAS and in the PIK3CA ⁄ PTEN ⁄AKT pathway do occur in human cancer. Consistently, in several mouse models, they have been shown to cooperate in accelerating tumorigenesis. While it is reasonably well understood why mutant RAS and deficiencies in the pRB or p53 pathways collaborate in cancer, it is less clear what the mechanistic basis is for activating mutations at two points in a presumed single signaling pathway, like RAS and PI3K. Kennedy et al. recently reported on a molecular interplay model in which the second oncogene, acting downstream, counteracts the senescence-inducing effect of the first (Kennedy et al.,

Extending the Activities of Human Nucleus Pulposus Cells with Recombinant Adeno-Associated Virus Vector-Mediated Human Telomerase Reverse Transcriptase Gene Transfer

Even though human nucleus pulposus (HNP) cells have a limited replicative lifespan in vitro culture, the lifespan of ovine nucleus pulposus cells transferred by lipofectamine with human telomerase reverse transcriptase (hTERT) gene could be prolonged. However, the lipofectamine is a transient expression system with an expressed duration of hTERT for less than 73 days. Here we present a viral vector system to investigate whether recombinant adenoassociated virus vector-mediated hTERT gene (rAAV-hTERT) could safely prolong the activities of HNP cells. Our hypothesis was that the activities of HNP cells could be extended with rAAV-hTERT gene transfer. Therefore, we designed an in vitro and in vivo evaluation of a HNP cells gene transfectant. Second-generation HNP cells were transfected with rAAV-hTERT and the expression of hTERT determined by reverse transcription-polymerase chain reaction (RT-PCR) and western blot. Cumulative population doubling, senescence assay, real-time RT-PCR, and enzyme-linked immunosorbent assay were used to determine cellular activities. Genetic phenotype was validated by karyotypic analysis and ontogenicity by nude mice injected in vivo. The continuing expression of hTERT gene and production of extra cellular matrix for 120 days were found in the transfected HNP cells, and karyotypic instability was detected, but there was no evidence of polyploidy or oncogenicity in nude mouse. The results showed that this transfection model might provide a resource cells necessary for the biological treatment of degenerative disc disease.

Artificial tethering to nuclear pores promotes partitioning of extrachromosomal DNA during yeast asymmetric cell division

Asymmetric cell division in unicellular organisms enables sequestration of senescence factors to specific subpopulations. Accumulation of autonomously replicating sequence (ARS) plasmids, which frequently emerge from recombination within the highly repetitive ribosomal DNA locus, is linked to limited replicative life span of Saccharomyces cerevisiae cells [1]. During budding yeast cell division, ARS plasmids are retained in the ageing mother cell, such that only 1 out of 10 plasmids enters the rejuvenated bud [2]. Binding of ARS plasmids to nuclear structures retained in the mother cell was speculated to explain asymmetric plasmid segregation [2]. Association with nuclear pore complexes (NPCs) was proposed to underlie retention of ARS plasmids in the mother cell [3]. However, the role of NPCs in segregation of ARS plasmids is unclear, as NPCs are partitioned between mother and bud nuclei during mitosis [4,5]. Here we analyzed how segregation of ARS plasmids is influenced by their interaction with NPCs. We found that artificial tethering to NPCs promotes transport of ARS plasmids into the bud. Moreover, our experiments provide support for the notion that interaction with ARS plasmids does not affect movement of NPCs into the bud. We conclude that binding to NPCs cannot by itself contribute to asymmetric segregation of ARS plasmids.

Analysis of the effect of inoculum characteristics on the first stages of a growing yeast population in beer fermentations by means of an individual-based model

The yeast Saccharomyces cerevisiae has a limited replicative lifespan. The cell mass at division is partitioned unequally between a larger, old parent cell and a smaller, new daughter cell. Industrial beer fermentations maintain and reuse yeast. At the end of fermentation a portion of the yeast is 'cropped' from the vessel for 'serial repitching'. Harvesting yeast may select a population with an imbalance of young and aged individuals, but the output of any bioprocess is dependent on the physiology of each single cell in the population. Unlike continuous models, individual-based modelling is an approach that considers each microbe as an individual, a unique and discrete entity, with characteristics that change throughout its life. The aim of this contribution is to explore, by means of individual-based simulations, the effects of inoculum size and cell genealogical age on the dynamics of virtual yeast fermentation, focussing on: (1) the first stages of population growth, (2) the mean biomass evolution of the population, (3) the rate of glucose uptake and ethanol production, and (4) the biomass and genealogical age distributions. The ultimate goal is to integrate these results in order to make progress in the understanding of the composition of yeast populations and their temporal evolution in beer fermentations. Simulation results show that there is a clear influence of these initial features of the inocula on the subsequent growth dynamics. By contrasting both the individual and global properties of yeast cells and populations, we gain insight into the interrelation between these two types of data, which helps us to deal with the macroscopic behaviour observed in experimental research.

Abstract 4267: Eltrombopag, a non-peptide thrombopoietin receptor agonist, enhances the ex vivo expansion of human hematopoietic stem cells

Abstract Hematopoietic stem cells (HSCs) are characterized by their capacity for self-renewal and pluripotency, allowing them to replenish all mature, differentiated cells of the hematopoietic lineage. HSCs are not immortal but have a limited replicative potential and finite lifespan, properties which may be further negatively influenced by increased age or malignant disorders of the bone marrow. To overcome this problem, various combinations of cytokines, including recombinant human thrombopoietin (rhTPO), have been developed to support in vitro culture as well as ex vivo expansion of HSCs. Although rhTPO appears to enhance the mobilization of hematopoietic progenitors, its use in humans has been limited due to the generation of neutralizing antibodies. Eltrombopag is an orally-active, non-peptide small molecule TPO receptor agonist that has been shown to promote megakaryocyte proliferation and differentiation in a manner similar to that seen with rhTPO. We hypothesized that eltrombopag may enhance the ex vivo expansion of human HSCs to a degree equivalent to that of rhTPO. To test this hypothesis, human bone marrow derived CD34+ cells isolated from 5 healthy individuals were incubated in serum-free expansion medium containing SCF (50 ng/ml), IL-3 (10 ng/ml), IL-6 (10 ng/ml), LDL (40 μg/ml) and various concentrations of eltrombopag (0 - 100 μM) or rhTPO (0 - 100 ng/ml) and CD34+ cell numbers determined using FACS analysis over the course of a 14-day ex vivo culture. Both eltrombopag and rhTPO exhibited dose-dependent increases in CD34+ cell numbers, with 10 μM eltrombopag and 100 ng/ml rhTPO providing the most robust CD34+ cell expansion. Under these conditions, both 10 µM eltrombopag and 100 ng/ml rhTPO resulted in a five-fold greater CD34+ cell expansion compared with control (p < 0.05), an effect seen most prominently at day 11 of liquid culture. To confirm that expanded CD34+ cells maintained their pluripotency, hematopoietic colony-forming assays were performed in methylcellulose cultures containing SCF (50 ng/ml), IL-3 (10 ng/ml), G-CSF (10 ng/ml) and erythropoietin (1 U/ml). CD34+ cells expanded with eltrombopag or rhTPO increased the total number of granulocyte, monocyte, and erythroid colony forming units (CFUs) by 81% and 95%, respectively, compared to control (p < 0.01). The difference in total CFU formation observed between eltrombopag- and rhTPO-treated CD34+ cells was not statistically significant (p = 0.59). Taken together, these results serve as proof-of-principle showing eltrombopag to be an effective alternative to rhTPO for promoting the ex vivo expansion of human HSCs. Furthermore, these pre-clinical studies support the design of future clinical trials examining the use of eltrombopag in combination with G-CSF for the enhanced mobilization of human CD34+ hematopoietic progenitors in patients undergoing peripheral blood stem cell transplantation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4267.

Perturbation of wild‐type lamin A metabolism results in a progeroid phenotype

Mutations in the lamin A/C gene cause the rare genetic disorder Hutchinson-Gilford progeria syndrome (HGPS). The prevalent mutation results in the production of a mutant lamin A protein with an internal 50 amino acid deletion which causes a cellular aging phenotype characterized by growth defects, limited replicative lifespan, and nuclear membrane abnormalities. However, the relevance of these findings to normal human aging is unclear. In this study, we demonstrate that increased levels of wild-type lamin A in normal human cells result in decreased replicative lifespan and nuclear membrane abnormalities that lead to apoptotic cell death and senescence in a manner that is strongly reminiscent of the phenotype shown by HGPS cells. In contrast to the accelerated aging defects observed in HGPS cells, the progeroid phenotype resulting from increased expression of wild-type lamin A can be rescued by overexpression of ZMPSTE24, the metalloproteinase responsible for the removal of the farnesylated carboxyl terminal region of lamin A. Furthermore, farnesyltransferase inhibitors also serve to reverse the progeroid phenotype resulting from increased lamin A expression. Significantly, cells expressing elevated levels of lamin A display abnormal lamin A localization and similar alterations in the nuclear distribution of lamin A are also observed in cells from old-age individuals. These data demonstrate that the metabolism of wild-type lamin A is delicately poised and even in the absence of disease-linked mutations small perturbations in this system are sufficient to cause prominent nuclear defects and result in a progeroid phenotype.

Modulation of Replicative Senescence of Diploid Human Cells by Nuclear ERK Signaling

Normal somatic cells have a limited replicative lifespan, and serial subcultivation ultimately results in senescence. Senescent cells are irreversibly growth-arrested and show impaired responses to mitogens. Activation of the ERK signaling pathway, an absolute requirement for cell proliferation, results in nuclear relocalization of active ERKs, an event impaired in senescent fibroblasts. This impairment coincides with increased activity of the nuclear ERK phosphatase MKP2. Here we show that replicative lifespan can be altered by changes in nuclear ERK activity. Ectopic expression of MKP2 results in premature senescence. In contrast, knock-down of MKP2 expression, through transduction of MKP2 sequence-specific short hairpin RNA, or expression of the phosphatase resistant ERK2(D319N) mutant, abrogates the effects of increased endogenous MKP2 levels and senescence is postponed. Nuclear targeting of ERK2(D319N) significantly augments its effects and the transduced cultures show higher than 60% increase in replicative lifespan compared with cultures transduced with wt ERK2. Long-lived cultures senesce with altered molecular characteristics and retain the ability to express c-fos, and Rb is maintained in its inactive form. Our results support that MKP2-mediated inactivation of nuclear ERK2 represents a key event in the establishment of replicative senescence. Although it is evident that senescence can be imposed through multiple mechanisms, restoration of nuclear ERK activity can bypass a critical senescence checkpoint and, thus, extend replicative lifespan.

Accelerated immune senescence and HIV-1 infection

A recent consensus has emerged regarding the association between chronic immune activation and poor outcome in HIV-1 infection. However, its basis remains unclear. Accumulating evidence suggests that the cells of the immune system may have a limited replicative lifespan in vivo. In this context, persistent activation during chronic HIV infection may lead to an exhaustion of immune resources. This may occur at two levels: Clonal and Global. Some HIV-1-specific CD8+ T-cells start expressing the senescence marker CD57 soon after primary infection. Persistently activated HIV-1-specific T-cell clones may eventually reach stages of replicative senescence and disappear, resulting in the specific loss of CD8+ T-cell populations important to control viral replication. In addition, HIV-1 infected individuals are characterized by the accumulation of highly differentiated CD8+ and CD4+ T-cells overtime. Together with the decline of T-cell renewal capacities, this may reflect a general ageing of the lymphocyte population. Similar observations have been done in HIV non-infected elderly individuals, which suggests that premature immunosenescence occurs in HIV-1 infection, as a result of persistent immune activation.

Relevance and safety of telomerase for human tissue engineering

Tissue engineering holds the promise of replacing damaged or diseased tissues and organs. The use of autologous donor cells is often not feasible because of the limited replicative lifespan of cells, particularly those derived from elderly patients. Proliferative arrest can be overcome by the ectopic expression of telomerase via human telomerase reverse transcriptase (hTERT) gene transfection. To study the efficacy and safety of this potentially valuable technology, we used differentiated vascular smooth muscle cells (SMC) and vascular tissue engineering as a model system. Although we previously demonstrated that vessels engineered with telomerase-expressing SMC had improved mechanics over those grown with control cells, it is critical to assess the phenotypic impact of telomerase expression in donor cells, because telomerase up-regulation is observed in >95% of human malignancies. To study the impact of telomerase in tissue engineering, expression of hTERT was retrovirally induced in SMC from eight elderly patients and one young donor. In hTERT SMC, significant lifespan extension beyond that of control was achieved without population doubling time acceleration. Karyotype changes were seen in both control and hTERT SMC but were not clonal nor representative of cancerous change. hTERT cells also failed to show evidence of neoplastic transformation in functional assays of tumorigenicity. In addition, the impact of donor age on cellular behavior, particularly the synthetic capability of SMC, was not affected by hTERT expression. Hence, this tissue engineering model system highlights the impact of donor age on cellular synthetic function that appears to be independent of lifespan extension by hTERT.

An In Vivo Model To Investigate the Identity of the Cell-of-Origin of Multiple Myeloma.

Multiple myeloma (MM) is characterized by monoclonal expansion of bone marrow plasma cells. However, long-lived plasma cells resident in the marrow are terminally differentiated and possess a limited replicative lifespan; it is puzzling how they could be the source of aggressive and relapsing neoplasms. We postulate that the myeloma clonogenic progenitor may reside in a more immature compartment with greater self-renewal capacity, most probably a cell participating in, or having shortly exited the germinal center reaction. However, it is unclear whether critical mutations occur in the target cell prior to, or following commitment to the plasma cell fate. To investigate the nature of the MM cell-of-origin, we have created a novel flexible mouse model system that enables the delivery of stochastic, sequential, somatic mutations to precisely defined compartments of the germinal center in secondary lymphoid tissues. To this end, we have used BAC transgenic technology to express distinct types of avian leukosis virus (ALV) receptors, TVA and TVB, in the expanding centroblast of the dark zone and the committed plasmablast of the light zone, respectively. Mammalian tissues are refractory to transduction by retroviruses of the ALV family unless they ectopically express the cognate avian-derived receptors. Thus, the coding sequences for the TVA receptor, fused to a fluorescent protein tag were placed under the control of transcription factor A-myb, expressed in centroblasts of the dark zone. Similarly, sequences encoding a fluorescent-tagged TVB receptor were placed under the control of transcription factor Blimp1, expressed in the earliest committed plasmablasts as well as mature plasma cells. Analysis of the Blimp1: TVB mice showed that expression of the avian retroviral receptor in the hematopoietic system is limited to the light zone of germinal centers, extrafollicular collections of CD138+ cells in the spleen and lymph nodes as well as long-lived bone marrow plasma cells. Analysis of A-myb: TVA transgenic mice is currently underway. The system permits the introduction of a variety of molecular lesions to specific plasma cell precursors via retroviral transduction of oncogenes, shRNAs against tumor suppressor genes or inducible regulators of gene expression in an attempt to re-create the sequence of molecular lesions leading to MM in the relevant cellular context.

Analysis of telomerase activity and telomere function in cancer.

Telomeres are the structures at the ends of chromosomes, composed of repetitive sequences and associated proteins, which cap chromosome ends to maintain genomic stability. These structures are maintained by the enzyme complex telomerase in germ cells and some stem cells, but are absent in the majority of somatic cells. The consequence of this lack of telomerase in normal somatic cells is the shortening of the telomeric repeat, which results in a limited replicative life span. However, in cancer cells, which grow indefinitely, telomerase activity has been detected in a large number of different cancer cell types. This has lead to a great deal of interest in establishing techniques to measure telomerase activity, telomere length, and telomere function in both normal and cancer cells/tissue. Here we describe the TRAP (telomeric repeat amplification protocol) assay, a technique to measure telomerase activity in cells, TRF (terminal restriction fragment length) analysis to estimate telomere length, and both the anaphase bridge index and the frequency of dicentric chromosomes as indicators of telomere dysfunction.