Cumulative Population Doublings Research Articles

GDF11 induces differentiation and apoptosis and inhibits migration of C17.2 neural stem cells via modulating MAPK signaling pathway.

GDF11, a member of TGF-β superfamily, has recently received widespread attention as a novel anti-ageing/rejuvenation factor to reverse age-related dysfunctions in heart and skeletal muscle, and to induce angiogenesis and neurogenesis. However, these positive effects of GDF11 were challenged by several other studies. Furthermore, the mechanism is still not well understood. In the present study, we evaluated the effects of GDF11 on C17.2 neural stem cells. GDF11 induced differentiation and apoptosis, and suppressed migration of C17.2 neural stem cells. In addition, GDF11 slightly increased cell viability after 24 h treatment, showed no effects on proliferation for about 10 days of cultivation, and slightly decreased cumulative population doubling for long-term treatment (p < 0.05). Phospho-proteome profiling array displayed that GDF11 significantly increased the phosphorylation of 13 serine/threonine kinases (p < 0.01), including p-p38, p-ERK and p-Akt, in C17.2 cells, which implied the activation of MAPK pathway. Western blot validated that the results of phospho-proteome profiling array were reliable. Based on functional analysis, we demonstrated that the differentially expressed proteins were mainly involved in signal transduction which was implicated in cellular behavior. Collectively, our findings suggest that, for neurogenesis, GDF11 might not be the desired rejuvenation factor, but a potential target for pharmacological blockade.

Proliferative and osteogenic differentiation capacity of mesenchymal stromal cells: Influence of harvesting site and donor age

Human mesenchymal stromal cells (hMSCs) are the cellular source of new bone formation and an essential component of autologous bone grafts. Autologous bone graft harvesting is routinely conducted at the iliac crest, although alternative donor sites with lower complication rates are available. Thus, the aim of this study was to compare hMSCs harvested from the iliac crest and the proximal tibia regarding their proliferative and osteogenic differentiation capacity. Furthermore, we investigated the influence of donor age on these biological properties.HMSCs were isolated from iliac crest or proximal tibia bone grafts of 46 patients. Proliferative capacity was assessed by cumulative population doublings, population doubling time, colony forming units and cell proliferation assays. Osteogenic capacity was assessed by quantification of extracellular calcium deposition and marker gene expression levels. The number of hMSCs per gram harvested tissue was determined. Furthermore, the adipogenic and chondrogenic differentiation capacity were quantified using BODIPY and Safranin Orange staining, respectively. Additional analyses were carried out after grouping young (18–49 years) and aged (≥50 years) donors.HMSCs derived from the proximal tibia featured a comparable proliferative and osteogenic differentiation capacity. No significant differences were found for any analysis conducted, when compared to hMSCs obtained from the iliac crest. Furthermore, no significant differences could be revealed when comparing young and aged donors. This was equally true for hMSCs from both donor sites after comparison within the same age group.Our study demonstrates comparable biological properties of hMSCs derived from both donor sites, the iliac crest and the proximal tibia. Furthermore, aging does not alter proliferative and osteogenic differentiation capacity. Consequently, the proximal tibia should be considered more closely as an alternative donor site in patients of all age groups.

Standardizing the Isolation, Culture‐Expansion, and Cryopreservation of Canine Umbilical‐Cord Derived Mesenchymal Stromal Cells

Mesenchymal stromal cells (MSCs) hold great promise in the field of regenerative medicine due to their ability to enable functional recovery in inflammatory injuries such as Crohn's disease and osteoarthritis. Currently, the MSC literature is focused on rodents for preclinical models of disease or clinical testing in human patients. We are interested in using the canine as a large animal model of human diseases. In this regard, the canine MSC literature lags behind human and rodent MSC, and most work on canine MSCs has focused upon bone marrow and adipose tissue‐derived with wide variation in isolation techniques. From surveying the literature and our pilot work, canine MSCs are more difficult to culture than human or rodent MSCs, have longer population doubling times, and earlier senesce. Here, a reproducible protocol for isolation and expansion of canine umbilical cord MSCs is provided. This method produces 10× greater yield per gram of tissue than other published methods. Gelatin‐coating of tissue culture plates greatly increases colony‐forming units‐fibroblast efficiency and decreases population doubling times. In addition, MSCs were able to be cultured longer before senescing, thereby increasing their cumulative population doublings. In summary, our understanding of the culture conditions needed for canine MSCs lags behind that of other species. Gelatin‐coating of tissue culture plates appears to be important for reproducible canine MSC culture. Currently, we are testing differentiation and characterization protocols to offer as standardized methods for the field.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

An Effect of Culture Media on Epithelial Differentiation Markers in Breast Cancer Cell Lines MCF7, MDA-MB-436 and SkBr3.

Background and objectives: Cell culture is one of the mainstays in the research of breast cancer biology, although the extent to which this approach allows to preserve the original characteristics of originating tumor and implications of cell culture findings to real life situations have been widely debated in the literature. The aim of this study was to determine the role of three cell culture media on transcriptional expression of breast cancer markers in three breast cancer reference cell lines (MCF7, SkBr3 and MDA-MB-436). Materials and methods: Cell lines were conditioned in three studied media (all containing 5% fetal bovine serum (FBS) + hormones/growth factors; different composition of basal media) for four passages. Population growth was characterized by cumulative population doubling levels, average generation time, cell yield and viability at the fourth passage. Transcriptional expression of breast cancer differentiation markers and regulatory transcriptional programs was measured by qPCR. Results: Differences in the composition of growth media significantly influenced the growth of studied cell lines and the expression of mammary lineage governing transcriptional programs and luminal/basal markers. Effects of media on transcriptional expression were more pronounced in luminal cell lines (MCF7, SkBr3), than in the basal cell line (MDA-MB-436). Changes in growth media in terms of supplementation and basal medium delayed growth of cells, but improved cell yields. Conclusions: The expression of breast cancer cell differentiation phenotypic markers depends on the composition of cell growth medium, therefore cell culture as a tool in phenotypic studies should be used considering this effect. The findings of such studies should always be interpreted with caution. The formulation of cell growth media has greater effect on the expression of phenotypic markers in luminal, rather than basal cell lines. Media containing mitogens and higher vitamin content improved efficacy of cell culture in terms of cell yields, although greatly increased growth times.

Reduced proliferation capacity of lung cells in chronic obstructive pulmonary disease.

The prevalence of chronic obstructive pulmonary disease (COPD) and lung emphysema increases with age and both lung diseases are again risk factors for lung cancer. Since areduced capacity of fibroblasts for proliferation is agood indicator of tissue aging, we studied the cell proliferation of lung fibroblasts from normal and tumor tissue of lung cancer patients depending on lung comorbidities. Fibroblasts were isolated from tumor and normal lung tissue of 40lung cancer patients. Cumulative population doubling (CPD) was determined to assess the proliferation capacity, and the PCR technique was used to measure telomere lengths. Since many patients had previously been exposed to severe air pollution, we also studied the effect of air pollution particles on the fibroblast CPD in vitro. Fibroblasts from tumor and normal lung tissue had comparable CPDs; however, the CPD of fibroblasts from both tumor and normal lung tissue was significantly reduced in patients also suffering from COPD. This CPD reduction was highest in COPD patients who had already developed emphysema or were smokers. Asignificant correlation between CPD and telomere length was identified only for fibroblasts of non-COPD patients. Further studies also showed an adverse effect of air pollution particles on the CPD of lung fibroblasts. Lung cells of COPD patients are characterized by accelerated senescence which must have been initiated prior to lung tumorigenesis and cannot depend on telomere shortening only. In addition to smoking as aknown risk factor for COPD and lung cancer, air pollution particles could be another reason for the accelerated senescence of lung cells.

Manufacture of endothelial colony‐forming progenitor cells from steady‐state peripheral blood leukapheresis using pooled human platelet lysate

Endothelial colony-forming progenitor cells (ECFCs) are promising candidates for cell therapies. However, ECFC translation to the clinic requires optimized isolation and manufacture technologies according to good manufacturing practice (GMP). ECFCs were manufactured from steady-state peripheral blood (PB) leukapheresis (11 donors), using GMP-compliant technologies including pooled human platelet (PLT) lysate, and compared to human umbilical cord endothelial cells, human aortic endothelial cells, and human cerebral microvascular endothelial cells. Specific variables assessed were growth kinetics, phenotype, trophic factors production, stimulation of tube formation, and Dil-AcLDL uptake. ECFCs could be isolated from PB leukapheresis units with mean processed volume of 5411 mL and mean white blood cell (WBC) concentration factor of 8.74. The mean frequency was 1.44×10-8 ECFCs per WBC, corresponding to a mean of 177.8 ECFCs per apheresis unit. Expandable for up to 12 cumulative population doublings, calculated projection showed that approximately 730×103 ECFCs could be manufactured from 1 apheresis unit. ECFCs produced epidermal growth factor, hepatocyte growth factor, vascular endothelial growth factor (VEGF)-A, PLT-derived growth factor-B, interleukin-8, and monocyte chemoattractant protein-1, featured high potential for capillary-like tubes formation, and showed no telomerase activity. They were characterized by CD29, CD31, CD44, CD105, CD117, CD133, CD144, CD146, and VEGF-R2 expression, with the most common subpopulation CD34+CD117-CD133-. Compared to controls, ECFCs featured greater Dil-AcLDL uptake and higher expression of CD29, CD31, CD34, CD44, CD144, and VEGF-R2. Here we show that isolation of ECFCs with proangiogenic profile from steady-state PB leukapheresis is feasible, marking a first step toward ECFC product manufacture according to GMP.

Derivation of Multipotent Mesenchymal Stromal Cells from Ovine Bone Marrow.

In the field of orthopedics, translational research of novel therapeutic approaches involves the use of large animal models (such as sheep, goat, pig, dog, and horse) due to the similarities with humans in weight, size, joint structure, and bone/cartilage healing mechanisms. Particularly in the development of cell-based therapies, the lack of manageable immunocompromised preclinical large animal models prevents the use of human cells, which makes it necessary to produce equivalent homologous cell types for the study of their pharmacodynamics, pharmacokinetics, and toxicology. The methods described herein allow for the isolation, expansion, manipulation, and characterization of fibroblastic-like ovine bone marrow-derived multipotent mesenchymal stromal cells (BM-MSC) that, similar to human BM-MSC, adhere to standard plastic surfaces; express specific surface markers such as CD44, CD90, CD140a, CD105, and CD166; and display trilineage differentiation potential in vitro. Homogeneous cell cultures result from a 3-week bioprocess yielding cell densities in the range of 2-4 × 104 MSC/cm2 at passage 2, which corresponds to ∼8 cumulative population doublings. Large quantities of BM-MSC resulting from following this methodology can be readily used in proof of efficacy and safety studies in the preclinical development stage. © 2018 by John Wiley & Sons, Inc.

Time-Dependent Effects of POT1 Knockdown on Proliferation, Tumorigenicity, and HDACi Response of SK-OV3 Ovarian Cancer Cells.

The roles of protection of telomeres 1 (POT1) in human ovarian cancer have not been fully elucidated. Here, we investigated the impact of POT1 knockdown (POT1-KD) on in vitro cell proliferation, tumorigenesis, and histone deacetylase inhibitor (HDACi) response in human ovarian cancer-derived SK-OV3 cells. The POT1 gene was knocked down by infection with POT1 lenti-shRNA. POT1, c-Myc, and hTERT mRNA levels and relative telomere length were determined by qRT-PCR; POT1 protein levels were determined by western blot. The relative telomerase activity levels were detected using qTRAP; cell proliferation was assessed using cumulative population doubling (cPD) experiments. Cell tumorigenicity was evaluated by anchorage-independent cell growth assays, and cell response to HDACi was determined by luminescence cell viability assays. Results indicate that lenti-shRNA-mediated POT1-KD significantly reduced POT1 mRNA and protein expression. POT1-KD immediately downregulated c-Myc expression, which led to the inhibition of cell proliferation, tumorigenesis, and HDACi response. However, after brief suppression, c-Myc expression increased in the medium term, which resulted in enhanced cell proliferation, tumorigenesis, and HDACi response in the POT1-KD cells. Furthermore, we discovered that c-Myc regulated cell proliferation and tumorigenesis via hTERT/telomerase/telomere pathway.

Abstract B34: Pyrimidobenzimidazoles as a potential new drug to treat human melanoma

Abstract Melanoma is the most aggressive skin tumor due to its high capacity to metastasize and resistance to therapy, leading to poor prognosis and high mortality rates. Thus, the aim of this study was to investigate a possible antitumoral action of pyrimidobenzimidazole compounds in human melanoma lineages, SK-Mel-103, SK-Mel-28, and SK-Mel-28 resistant to vemurafenib. Using acute treatment (72 hours) it was possible to select two, among the seven compounds tested, by their effectiveness in decrease the cell viability, by MTT assay, in low concentrations. Further, using chronic assay (120 hours) it was possible to find the IC50 of the best compounds, PBZs 1 and 4. For cell line SK-Mel-103 the IC50 value was 3.2 µM and 4.0 µM, for the two more potent compounds respectively, and for cell line SK-Mel-28 IC50 of 9.7 µM and 9.6 µM was obtained for PBZ01 and PBZ04, respectively. These compounds, alone or in combination with DTIC, were used to analyze the cumulative population doubling, in an attempt to simulate the treatment cycle used for patients. The results show that PBZs 1 and 4, especially when in combination with dacarbazine, were able to cause a significant decrease in the population growth rate and in the number of colonies in clonogenic assay, of the SK-Mel-28, better than dacarbazine alone and in lower concentration. Also, it is important to highlight that the effect of PBZs was selective to melanoma, considering that it did not cause relevant effect in melanocyte and in the proliferative nontumoral cell line, MRC5. The cell cycle analysis showed that the treatment alters the cell complexity, increasing the size (FSC) and granularity (SSC). Furthermore, when vemurafenib-resistant cells were treated with PBZ (cotreatment with vemurafenib), there were a population decrease in the population doubling assay more than vemurafenib alone. These results are important and encourage the study of the mechanism by which these compounds affect the melanomas' viability as well as the development of a preclinical model to confirm their effectiveness in vivo. Citation Format: Jéssica Gonçalves Azevedo, Iago Carvalho Schultz, Jacqueline Fraga de Souza Santos, Silvya Stuchi Maria-Engler, Guido Lenz, Marcia Rosângela Wink. Pyrimidobenzimidazoles as a potential new drug to treat human melanoma [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B34.

Mechanisms underlying the antiproliferative effects of a series of quinoxaline-derived chalcones

The present study aimed to characterize the effects of quinoxaline-derived chalcones, designed on the basis of the selective PI3Kγ inhibitor AS605240, in oral cancer cells. Three lead compounds, namely N9, N17 and N23, were selected from a series of 20 quinoxaline-derived chalcones, based on an initial screening using human and rat squamous cell carcinoma lineages, representing compounds with at least one methoxy radical at the A-ring. The selected chalcones, mainly N9 and N17, displayed marked antiproliferative effects, via apoptosis and autophagy induction, with an increase of sub-G1 population and Akt inhibition. The three chalcones displayed marked in vitro antitumor effects in different protocols with standard chemotherapy drugs, with acceptable toxicity on normal cells. There was no growth retrieval, after exposure to chalcone N9 alone, in a long-term assay to determine the cumulative population doubling (CPD) of human oral cancer cells. A PCR array evaluating 168 genes related to cancer and inflammation, demonstrated striking actions for N9, which altered the expression of 74 genes. Altogether, our results point out quinoxalinic chalcones, mainly N9, as potential strategies for oral cancer treatment.

Cytotoxic and cytostatic effects of digitoxigenin monodigitoxoside (DGX) in human lung cancer cells and its link to Na,K-ATPase

Cardiac glycosides (CGs) are natural compounds widely used to treat several cardiac conditions and more recently have been recognized as potential antitumor agents. They are known as Na,K-ATPases ligands, which is a promising drug target in cancer. In this study, the short and long-lasting cytotoxic effects of the natural cardenolide digitoxigenin monodigitoxoside (DGX) were evaluated against two non-small cell lung cancer lines (A549 and H460 cells). It was found that DGX induced cytotoxic effects in both cells and the apoptotic effects were more pronounced on H460 cells. In long-term analysis, using the clonogenic and the cumulative population doubling (CPD) assays, DGX showed a reduction of cell survival, after 15days without re-treatment. To better understand DGX effects in A549 cells, several assays were conducted. In cell cycle analysis, DGX caused an arrest in S and G2/M phases. This compound also increased the number of cells in subG1 phase in a concentration- and time-dependent manner. The presence of β-galactosidase positive cells, large nucleus and flattened cells indicated senescence. Additionally, DGX inhibited Na,K-ATPase activity in A549 cells, as well as in purified pig kidney and in human red blood cell membrane preparations, at nanomolar range. Moreover, results of molecular docking showed that DGX binds with high efficiency (−11.4Kcal/mol) to the Na,K-ATPase (PDB:4HYT). Taken together, our results highlight the potent effects of DGX both in A549 and H460 cells, and disclose its link with Na,K-ATPase inhibition.

Isolation and prolonged expansion of oral mesenchymal stem cells under clinical-grade, GMP-compliant conditions differentially affects \u201cstemness\u201d properties

BackgroundDevelopment of clinical-grade cell preparations is central to meeting the regulatory requirements for cellular therapies under good manufacturing practice-compliant (cGMP) conditions. Since addition of animal serum in culture media may compromise safe and efficient expansion of mesenchymal stem cells (MSCs) for clinical use, this study aimed to investigate the potential of two serum/xeno-free, cGMP culture systems to maintain long-term “stemness” of oral MSCs (dental pulp stem cells (DPSCs) and alveolar bone marrow MSCs (aBMMSCs)), compared to conventional serum-based expansion.MethodsDPSC and aBMMSC cultures (n = 6/cell type) were established from pulp and alveolar osseous biopsies respectively. Three culture systems were used: StemPro_MSC/SFM_XenoFree (Life Technologies); StemMacs_MSC/XF (Miltenyi Biotek); and α-MEM (Life Technologies) with 15% fetal bovine serum. Growth (population doublings (PDs)), immunophenotypic (flow cytometric analysis of MSC markers) and senescence (β-galactosidase (SA-β-gal) activity; telomere length) characteristics were determined during prolonged expansion. Gene expression patterns of osteogenic (ALP, BMP-2), adipogenic (LPL, PPAR-γ) and chondrogenic (ACAN, SOX-9) markers and maintenance of multilineage differentiation potential were determined by real-time PCR.ResultsSimilar isolation efficiency and stable growth dynamics up to passage 10 were observed for DPSCs under all expansion conditions. aBMMSCs showed lower cumulative PDs compared to DPSCs, and when StemMacs was used substantial delays in cell proliferation were noted after passages 6–7. Serum/xeno-free expansion produced cultures with homogeneous spindle-shaped phenotypes, while serum-based expansion preserved differential heterogeneous characteristics of each MSC population. Prolonged expansion of both MSC types but in particular the serum/xeno-free-expanded aBMMSCs was associated with downregulation of CD146, CD105, Stro-1, SSEA-1 and SSEA-4, but not CD90, CD73 and CD49f, in parallel with an increase of SA-gal-positive cells, cell size and granularity and a decrease in telomere length. Expansion under both serum-free systems resulted in “osteogenic pre-disposition”, evidenced by upregulation of osteogenic markers and elimination of chondrogenic and adipogenic markers, while serum-based expansion produced only minor changes. DPSCs retained a diminishing (CCM, StemPro) or increasing (StemMacs) mineralization potential with passaging, while aBMMSCs lost this potential after passages 6–7 under all expansion conditions.ConclusionsThese findings indicate there is still a vacant role for development of qualified protocols for clinical-grade expansion of oral MSCs; a key milestone achievement for translation of research from the bench to clinics.

Downregulation of Pin1 in human atherosclerosis and its association with vascular smooth muscle cell senescence

ObjectivePin1 is prevalently overexpressed in human cancers and implicated to regulate cell growth and apoptosis. Thus far, however, no role for Pin1 has been described in modulating vascular smooth muscle cell (VSMC) senescence. MethodsImmunohistochemistry and Western blotting were used to assess Pin1 protein level in human normal and atherosclerotic tissues. β-galactosidase staining, cumulative population doubling level, telomerase activity, and relative telomere length measurement were used to confirm VSMC senescence. The expressions of Pin1 and other genes involved in this research were analyzed by quantitative reverse-transcription polymerase chain reaction and Western blotting in VSMCs. Apolipoprotein E gene-deleted mice (ApoE−/−) fed a high-fat diet were treated with juglone or 10% ethanol, respectively, for 3 weeks. The extent of atherosclerosis was evaluated by Oil Red O, Masson trichrome staining, and immunohistology. ResultsPin1 protein level decreased in human atherosclerotic tissues and VSMCs, synchronously with increased VSMC senescence. Adenoviral-mediated Pin1 overexpression rescued cellular senescence in atherosclerotic VSMCs, with concurrent down-regulation of P53, p21, growth arrest and DNA-damage-inducible protein 45-alpha (Gadd45a), phosphorylated retinoblastoma (p-pRb), p65 and upregulation of cyclin subfamilies (cyclin B, D, and E), and cyclin-dependent kinase subfamilies (2, 4, and 6), whereas Pin1 knockdown resulted in the converse effects, indicating that VSMC senescence mediated by Pin1 is an integrated response to diverse signals. In vivo data from ApoE−/− mice showed that treatment of juglone led to accelerated atherosclerosis development. ConclusionsAltogether this work supports a role for Pin1 as a vital modulator of VSMC senescence, thereby providing a novel target for regulation and control of atherosclerosis.

Ionizing radiation response of primary normal human lens epithelial cells.

Whilst the cataractogenic potential of ionizing radiation has been known for over the past 120 years, little is known about radiation responses of lens cells. Our previous work was the first to evaluate the radiosensitivity of lens cells with the clonogenic assay, documenting that the survival of HLEC1 human lens epithelial cells is comparable to that of WI-38 human lung fibroblasts. Moreover, HLEC1 cells were found to contain subsets where irradiation stimulates proliferation or facilitates formation of abortive colonies with fewer cells than human fibroblasts. This study aims to gain insights into these mechanisms. Irradiation of HLEC1 cells with 10% survival dose caused a growth delay but did not reduce viability. HLEC1 cells at high cumulative population doubling level were more susceptible to radiogenic premature senescence than WI-38 cells. Concerning p53 binding protein 1 (53BP1) foci, HLEC1 cells harbored less spontaneous foci but more radiogenic foci than in WI-38 cells, and the focus number returned to spontaneous levels within 48 h postirradiation both in HLEC1 and WI-38. The chemical inhibition of DNA repair kinases ataxia telangiectasia mutated, DNA-dependent protein kinase or both delayed and attenuated the appearance and disappearance of radiogenic 53BP1 foci, increased radiogenic premature senescence and enhanced clonogenic inactivation. The DNA microarray analysis suggested both radiogenic stimulation and inhibition of cell proliferation. Treatment with conditioned medium from irradiated cells did not change growth and the plating efficiency of nonirradiated cells. These results partially explain mechanisms of our previous observations, such that unrepaired or incompletely repaired DNA damage causes a growth delay in a subset of HLEC1 cells without changing viability through induction of premature senescence, thereby leading to clonogenic inactivation, but that growth is stimulated in another subset via as yet unidentified mechanisms, warranting further studies.

Interference of ursolic acid treatment with glioma growth: An in vitro and in vivo study

Glioblastoma multiforme is the most devastating tumor in the brain. Ursolic acid (UA) is found in a variety of plants, and exhibits several pharmacological activities. In this study, we investigated the effects of UA in vitro, clarifying the mechanisms that mediate its toxicity and the long-lasting actions of UA in C6 glioma cells. We also evaluated the antitumor activity of UA in an in vivo orthotopic glioma model. Cell numbers were assessed using the Trypan blue exclusion test, and the cell cycle was characterized by flow cytometry using propidium iodide staining. Apoptosis was analyzed using an Annexin V kit and by examining caspase-3. Akt immunocontent was verified by Western blot and the long-lasting actions of UA were measured by cumulative population doubling (CPD). In vivo experiments were performed in rats to measure the effects on tumor size, malignant features and toxicological parameters. In vitro results showed that UA decreased glioma cell numbers, increased the sub-G1 fraction and induced apoptotic death, accompanied by increased active caspase-3 protein levels. Akt phosphorylation/activation in cells was also diminished by UA. With regard to CPD, cell proliferation was almost completely restored upon single UA treatments, but when the UA was added again, the majority of cells died, demonstrating the importance of re-treatment cycles with chemotherapeutic agents for abolishing tumor growth. In vivo, ursolic acid slightly reduced glioma tumor size but did not decrease malignant features. Ursolic acid may be a potential candidate as an adjuvant for glioblastoma therapy.

Adult venous endothelium is a niche for highly proliferative and vasculogenic endothelial colony-forming cells

ObjectivePostnatal resident endothelium of blood vessels has been proposed to represent terminally differentiated tissue that does not replicate. We previously isolated endothelial colony-forming cells (ECFCs) from human umbilical cord blood (CB) and term placenta by using colony-forming assays and immunocytochemistry. We showed that ECFCs are highly proliferative and form functioning vessels in vivo, the defining characteristics of a true endothelial progenitor cell. This exploratory investigation was conducted to determine whether the endothelium of healthy adult blood vessels contained resident ECFCs. MethodsThe endothelium of great saphenous vein (GSV) obtained from vein stripping procedures was collected with mechanical scraping, and ECFCs were isolated according to established protocols. ResultsGSV ECFCs incorporated acetylated low-density lipoprotein, formed tubules in Matrigel (BD Biosciences, San Jose, Calif) at 24 hours, and expressed endothelial antigens cluster of differentiation (CD) 144, CD31, CD105, and kinase insert domain receptor but not hematopoietic antigen CD45. Using cumulative population doublings and single-cell assays, we demonstrated that GSV ECFCs exhibited comparable proliferative capacities compared with CB ECFCs, including similar numbers of highly proliferative cells. When injected in collagen/fibronectin gels implanted in nonobese diabetic/severe combined immune deficiency mice, GSV ECFCs formed blood vessels with circulating murine red blood cells, demonstrating their vasculogenic potential. ConclusionsThe ECFCs of the GSV contain a hierarchy of progenitor cells with a comparable number of highly proliferative clones as ECFCs of CB. The results of this investigation demonstrate that the adult endothelium contains resident progenitor cells that may have a critical role in vascular homeostasis and repair and could potentially be used as a source of autologous cells for cell therapies focusing on vasculogenesis.

P 245 - Compromised proteasomal function in endothelial cell senescence

Senescence is thought to contribute to endothelial dysfunction, which ultimately leads to development of cardiovascular disease. The current project characterizes proteasomal protein degradation in endothelial cells undergoing replicative or stress-induced senescence in vitro or chronological ageing in vivo. Our results demonstrate that human endothelial cells become senescent in vitro within 25 cumulative population doublings. Senescence was verified by increased number of senescence-associated β-galactosidase (SA-β-gal)-positive cells and increased cell size and granularity. In addition, protein carbonylation and nitration as well as reduced GSH levels were observed. Studies on the expression of proteasomal subunits revealed a decline of the β5 and β2 subunits in senescent cells. In parallel, trypsin- and chymotrypsin-like activities were decreased. Similar observations were made in primary endothelial cells derived from old mice (24 months old) when compared to young mice (4 months old). Transient inhibition of proteasome in endothelial cells led to premature senescence. In parallel, persistent accumulation of carbonylated proteins was observed indicating a role of oxidative stress in the development of senescence. From these results we suggest a relationship between proteolytic insufficiency and senescence, which may contribute to vascular ageing.

Dynamic adhesive environment alters the differentiation potential of young and ageing mesenchymal stem cells

Engineering dynamic stem cell niche-like environment offers opportunity to obtain better control of the fate of stem cells. We identified, for the first time, that periodic changes in the adhesive environment of human adipose derived mesenchymal stem cells (ADSCs) alters dramatically their asymmetric division but not their ability for symmetric renewal. Hereby, we used smart thermo-responsive polymer (PNIPAM) to create a dynamic adhesive environment for ADSCs by applying periodic temperature cycles to perturb adsorbed adhesive proteins to substratum interaction. Cumulative population doubling time (CPDT) curves showed insignificant decline in the symmetric cell growth studied for up to 13th passages accompanied with small changes in the overall cell morphology and moderately declined fibronectin (FN) matrix deposition probably as a functional consequence of ADSCs ageing. However, a substantial alteration in the differentiation potential of ADSCs from both early and late passages (3rd and 14th, respectively) was found when the cells were switched to osteogenic differentiation conditions. This behavior was evidenced by the significantly altered alkaline phosphatase activity and Ca deposition (Alizarin red) assayed at 3, 14 and 21day in comparison to the control samples of regular TC polystyrene processed under same temperature settings.

Cytotoxic effects of the cardenolide convallatoxin and its Na,K-ATPase regulation.

Cardenolides are cardiac glycosides, mostly obtained from natural sources. They are well known for their inhibitory action on the Na,K-ATPase, an effect that regulates cardiovascular alterations such as congestive heart failure and atrial arrhythmias. In recent years, they have also sparked new interest in their anticancer potential. In the present study, the cytotoxic effects of the natural cardenolide convallatoxin (CON) were evaluated on non-small cell lung cancer (A549 cells). It was found that CON induced cytostatic and cytotoxic effects in A549 cells, showing essentially apoptotic cell death, as detected by annexin V-propidium iodide double-staining, as well as changes in cell form. In addition, it prompted cell cycle arrest in G2/M and reduced cyclin B1 expression. This compound also increased the number of cells in subG1 in a concentration- and time-dependent manner. At a long term, the reduction of cumulative population doubling was shown along with an increase of β-galactosidase positive cells and larger nucleus, indicative of senescence. Subsequently, CON inhibited the Na,K-ATPase in A549 cells at nM concentrations. Interestingly, at the same concentrations, CON was unable to directly inhibit the Na,K-ATPase, either in pig kidney or in red blood cells. Additionally, results of docking calculations showed that CON binds with high efficiency to the Na,K-ATPase. Taken together, our data highlight the potent anticancer effects of CON in A549 cells, and their possible link with non-classical inhibition of Na,K-ATPase.

Effect of Cryopreservation and cumulative population doublings on Senescence of Umbilical Cord Mesenchymal Stem Cells

Background: Multiple harvest explant method (MHEM) derived umbilical cord mesenchymal stem cells (UC-MSCs) was shown to undergo senescent beginning at passage-10 (P-10). However, for the same cells, there are no senescent data after cryopreservation and passage. Therefore, this study aimed to analyze the senescent profile of cryopreserved MHEM derived UC-MSCs after serial passages. Methods: MHEM derived UC-MSCs were isolated and cultured in platelet lysate (PL) containing medium as described previously. The cells were cryopreserved at passage-1 (P-1) in 10% dimethyl sulfoxide (DMSO) and 10% PL containing alpha minimal essential medium (αMEM). Cell density at cryopreservation was 500 000 cells/mL. After one month, the cells were thawed and recultured until P-8 in six 12-well plate. At 80-90% confluent, two wells were harvested and the cells were recultured into six wells, and the four remaining wells were subjected to senescent (β-galactosidase) staining, which was done for all passages. Random photographs were taken from all stained wells, and senescent percentage was recorded. Results: No senescent cells were observed at P-2. Senescent cells began to appear at P-3. Percentage (mean ± SD) of senescent cells from P-3 through P-8 were 0.04 ± 0.02, 1.18 ± 1.82, 0.14 ± 0.18, 0.49 ± 0.00, 0.58 ±0.91, and 0.07 ± 0.07, respectively. Results: No senescent cells were observed at P-2. Senescent cells began to appear at P-3. Percentage (mean ± SD) of senescent cells from P-3 through P-8 were 0.04 ± 0.02, 1.18 ± 1.82, 0.14 ± 0.18, 0.49 ± 0.00, 0.58 ±0.91, and 0.07 ± 0.07, respectively. Conclusion: No senescent cells were observed at P-2 (cumulative population doubling [CPD] > 9.38). Senescent cells began to appear at P-3 (CPD > 14.06), but in all passages until P-8 (CPD >34.34) the senescent percentage was below 5%.