Late Passage Cells Research Articles

Metformin restores autophagic flux and mitochondrial function in late passage myoblast to impede age-related muscle loss

Sarcopenia, which refers to age-related muscle loss, presents a significant challenge for the aging population. Age-related changes that contribute to sarcopenia include cellular senescence, decreased muscle stem cell number and regenerative capacity, impaired autophagy, and mitochondrial dysfunction. Metformin, an anti-diabetic agent, activates AMP-activated protein kinase (AMPK) and affects various cellular processes in addition to reducing hepatic gluconeogenesis, lowering blood glucose levels, and improving insulin resistance. However, its effect on skeletal muscle cells remains unclear. This study aimed to investigate the effects of metformin on age-related muscle loss using a late passage C2C12 cell model. The results demonstrated that metformin alleviated hallmarks of cellular senescence, including SA-β-gal activity and p21 overexpression. Moreover, treatment with pharmacological concentrations of metformin restored the reduced differentiation capacity in late passage cells, evident through increased myotube formation ability and enhanced expression of myogenic differentiation markers such as MyoD, MyoG, and MHC. These effects of metformin were attributed to enhanced autophagic activity, normalization of mitochondrial membrane potential, and improved mitochondrial respiratory capacity. These results suggest that pharmacological concentrations of metformin alleviate the hallmarks of cellular senescence, restore differentiation capacity, and improve autophagic flux and mitochondrial function. These findings support the potential use of metformin for the treatment of sarcopenia.

Effects of Replicative Senescence of Human Chorionic MSCs on their EV-miRNA Profile.

Chorionic mesenchymal stromal cells (CHO-MSCs) and their extracellular vesicles (EVs) are becoming increasingly popular, since chorion is ethically harmless and an easily accessible source of MSCs. However, until now there is only a limited number of studies with a thorough characterization of CHO-MSCs derived EVs and their miRNA profile. In this study, we monitored changes in the EV-miRNA profile between early and late passage of human CHO-MSCs. First, senescence of CHO-MSCs was induced by serial passaging and confirmed by morphological changes, shortened telomeres and changes in the expression of selected genes. The expression of MSCs-specific surface markers CD73, CD90, CD105 did not change with increasing passages. Next, EVs and their miRNA profiles were compared between early vs late passage cells. Number of EVs and their size were not significantly changed. Seven of the top 10 most expressed EV-miRNAs were common to both early and late passages. A differential expression study between early and late passages identified 37 significantly differentially expressed EV-miRNAs, out of which 23 were found to be associated with pathways of cellular senescence based on KEGG pathway analysis. A set of 9 miRNAs were identified as the most frequently associated with senescence and/or with the most altered expression between early and late passages, out of which miR-145-5p, miR-335-5p and miR-199b-3p were the most significant downregulated miRNAs in late passages. The most upregulated EV-miRNAs were miR-1307-3p, miR-3615 and miR320b. Targeting these miRNAs in future experiments may prolong the therapeutic potential of CHO-MSCs and their EVs.

Leveraging Cell Migration Dynamics to Discriminate Between Senescent and Presenescent Human Mesenchymal Stem Cells

PurposeThe suboptimal clinical performance of human mesenchymal stem cells (hMSCs) has raised concerns about their therapeutic potential. One major contributing factor to this issue is the heterogeneous nature of hMSCs. Senescent cell accumulation during stem cell expansion is a key driver of MSC heterogeneity. Current methodologies to eradicate senescent hMSCs have either shown limited success or lack clinical relevance. This study leverages the inherent capacity of hMSCs to migrate toward damaged tissues as a means to discern senescent from presenescent stem cells. Given the established deficiency of senescent cells to migrate through physiologically relevant environments, we hypothesized that a microfluidic device, designed to emulate key facets of in vivo cell motility, could serve as a platform for identifying presenescent cells.MethodsWe employed a Y-shaped microchannel assay, which allows fine-tuning of fluid flow rates and the degree of confinement.ResultsHighly migratory hMSCs detected by the device not only demonstrate increased speed, smaller size, and higher proliferative capacity but also manifest reduced DNA damage and senescence compared to non-migratory cells. Additionally, this assay detects presenescent cells in experiments with mixed early and late passage cells. The introduction of fluid flow through the device can further increase the fraction of highly motile stem cells, improving the assay's effectiveness to remove senescent hMSCs.ConclusionsCollectively, this assay facilitates the detection and isolation of a highly potent stem cell subpopulation. Given the positive correlation between the migratory potential of administered MSCs and the long-term clinical outcome, delivering homogeneous, highly motile presenescent hMSCs may benefit patient outcomes.

O16 Understanding the pleiotropic effects of the Rho-kinase inhibitor Y-27632 on human keratinocytes

Abstract Introduction and aims Successful epidermal transplant generation depends on sufficiently high numbers of self-renewing stem cells that give rise to clonal colonies termed ‘holoclones’. Treatment with Y-27632, a nonspecific Rho-kinase 1/2 inhibitor, conditionally immortalizes epidermal keratinocyte cultures. However, the pleiotropic signalling mechanisms downstream of Y-27632 remain largely unexplored. We have therefore characterized the effects of continuous long-term Y-27632 treatment on chromatin regulation, and investigated whether Y-27632 reprogrammes a holoclone-like state. Methods Neonatal foreskin-derived human epidermal keratinocytes were cultured from passage p5 to p30, with or without Y treatment. Samples were collected for RNA sequencing analysis from progenitor-enriched middle passage cells (p5, MPK), progenitor-depleted late-passage cells (p8, LPK) and Y-treated cells at p8 (LPK-Y). Samples were also collected for assay for transposase-accessible chromatin (ATAC)-Seq analysis to assess open and closed chromatin regions. Results Untreated cultures became depleted of proliferating cells by p11, whereas Y maintained a highly proliferative culture up to p30 and beyond. Y withdrawal at p11 resulted in rapid loss of proliferative capacity within a single passage. ATAC-Seq identified 196 differentially regulated chromatin sites in MPK vs. LPK. Y treatment maintained accessibility in approximately 40% of these MPK-enriched chromatin regions. Approximately 3000 differentially expressed genes (DEGs) were identified in MPK vs. LPK, while Y treatment induced approximately 11 000 DEGs. Activated genes in LPK-Y were enriched for a holoclone signature, with no evidence for activation of a precancerous transcriptome. Y treatment altered the expression of various genes associated with amino acid metabolism, enabling cells to overcome serine/glycine autotrophy. Conclusions Y acts by preserving accessibility of progenitor-enriched chromatin sites and further reprogrammes the keratinocyte transcriptome towards a more holoclone-like state. However, keratinocytes retain a ‘memory’ of their doubling number, which is conditionally suppressed by Y. The observed global transcriptomic changes are indicative of the highly pleiotropic nature of the Y-induced signalling changes, warranting further deconvolution.

Hyperoxia impairs induced pluripotent stem cell-derived endothelial cells and drives an atherosclerosis-like transcriptional phenotype

BackgroundInduced pluripotent stem cells (iPSCs) directed to endothelial identity (iPSC-ECs) are emerging as a potent tool for regenerative medicine in vascular disease. However, iPSC-ECs lose expression of key identity markers under standard in vitro conditions, limiting their clinical applications. MethodsTo model physiological in vivo conditions, we examined the bioenergetics, presence of key cell markers, and proliferative and angiogenic capacity in iPSC-ECs at late and early passage under hyperoxic (21%) and physiological (4%) oxygen concentrations. ResultsPhysoxia resulted in relative preservation of mitochondrial bioenergetic activity, as well as CD144 expression in late passage iPSC-ECs, but not proliferative capacity or tube formation. Single cell RNA sequencing (scRNA-seq) revealed that late passage hyperoxic iPSC-ECs develop an endothelial-to-mesenchymal phenotype. Comparing scRNA-seq data from iPSC-ECs and from atherosclerotic ECs revealed overlap of their transcriptional phenotypes. ConclusionsTaken together, our studies demonstrate that physiological 4% oxygen culture conditions were sufficient to improve mitochondrial function in high passage cells, but alone was insufficient to preserve angiogenic capacity. Furthermore, late passage cells under typical conditions take on an endothelial-to-mesenchymal phenotype with similarities to ECs found in atherosclerosis.

Biological Effects of "Inflammageing" on Human Oral Cells: Insights into a Potential Confounder of Age-Related Diseases.

The term "inflammageing" describes the process of inflammation-induced aging that leads living cells to a state of permanent cell cycle arrest due to chronic antigenic irritation. This in vitro study aimed to shed light on the mechanisms of "inflammageing" on human oral cells. Primary cultures of human gingival fibroblasts (hGFs) were exposed to variable pro-inflammatory stimuli, including lipopolysaccharide (LPS), Tumor Necrosis Factor-alpha (TNFa), and gingival crevicular fluid (GCF) collected from active periodontal pockets of systemically healthy patients. Inflammageing was studied through two experimental models, employing either late-passage ("aged") cells (p. 10) that were exposed to the pro-inflammatory stimuli or early-passage ("young") cells (p. 1) continuously exposed during a period of several passages (up to p. 10) to the above-mentioned stimuli. Cells were evaluated for the expression of beta-galactosidase activity (histochemical staining), senescence-associated genes (qPCR analysis), and biomarkers related to a Senescence-Associated Secretory Phenotype (SASP), through proteome profile analysis and bioinformatics. A significant increase (p < 0.05) in beta-galactosidase-positive cells was observed after exposure to each pro-inflammatory stimulus. The senescence-associated gene expression included upregulation for CCND1 and downregulation for SUSD6, and STAG1, a profile typical for cellular senescence. Overall, pro-inflammatory priming of late-passage cells caused more pronounced effects in terms of senescence than long-term exposure of early-passage cells to these stimuli. Proteomic analysis showed induction of SASP, evidenced by upregulation of several pro-inflammatory proteins (IL-6, IL-10, IL-16, IP-10, MCP-1, MCP-2, M-CSF, MIP-1a, MIP-1b, TNFb, sTNF-RI, sTNF-RII, TIMP-2) implicated in cellular aging and immune responses. The least potent impact on the induction of SASP was provoked by LPS and the most pronounced by GCF. This study demonstrates that long-term exposure of hGFs to various pro-inflammatory signals induced or accelerated cellular senescence with the most pronounced impact noted for the late-passage cells. The outcome of these analyses provides insights into oral chronic inflammation as a potential confounder of age-related diseases.

Abstract 2577: Pre-cancerous ovarian cells harboring a familial SMARCA4 mutation acquire in vitro phenotypes associated with transformation in SSCOHT

Abstract Introduction: Small Cell Carcinoma of the Ovary, Hypercalcemic Type (SCCOHT) is an extremely rare but deadly ovarian cancer associated with mutations in the SMARCA4 gene. Using a unique population of ovarian cells derived from a familial carrier of an SCCOHT-associated SMARCA4 mutation, we sought to determine if non-cancerous cells would spontaneously transform during in vitro culture, thereby providing a model system for studying formation of SCCOHT. Methods: Ovarian cells derived from a familial carrier of a SMARCA4 mutation (P590, SMARCA4. c.3081+1G&amp;gt;T) were serially passaged in vitro and interrogated for acquisition of phenotypes consistent with cellular transformation. Loss of contact-dependent inhibition was interrogated with focus-forming assays in confluent monolayers. The ability of cells to escape anoikis and grow independently as spheroids was tested in attachment-free growth conditions using both single and aggregate spheroid growth assays. Senescence-associated β-galactosidase (SA-β-gal), cellular necrosis, and cellular apoptosis was also measured. All experiments were conducted in parallel with an age-matched cell population derived from a benign ovarian surgery (P583). In experiments to determine if these differences could be attributed to loss of functional SMARCA4, non-mutant P583 cells were cultured in the presence of the SWI/SNF inhibitor BRM014 and interrogated in focus-forming and attachment-free growth assays. Results: P590 cells passaged over 12 twelve times (approx. 40 cell divisions) continued to proliferate in culture. In contrast, non-mutant P583 cells failed to grow beyond 11-12 passages. With prolonged culture, P590 cells lost expression of SMARCA4 protein, while P583 cells retained SMARCA4 expression. P590 cells grown to confluency spontaneously formed foci of multicellular aggregates, while P583 cells did not. In attachment-free culture conditions, P590 cells readily formed spheroids from both early-passage and late-passage cells. In contrast, P583 formed small spheroids that failed to expand in culture. Surprisingly, both P590 and P583 cells displayed elevated SA-β-gal staining, with neither cell populations showing elevated necrosis or apoptosis markers. Treating P583 cells with BRM014 increased focus formation and spheroid growth similar to that of mutant cells. Conclusions: SCCOHT is extremely rare, with very few representative samples useful for developing early-stage markers or improved therapeutics. Long-term culture of ovarian cells harboring inactivated forms of SMARCA4 result in the acquisition of several phenotypes consistent with early stages of cellular transformation. Our data suggest that mutating or inactivating SMARCA4 in non-transformed ovarian cells may be a way to create novel research tools for studying formation of SCCOHT. This work was supported by DoD award W81XWH-21-1-0512 Citation Format: Sonali Joshi, Shawn Campbell, Yukie Bean, Tanja Pejovic, Adam Krieg. Pre-cancerous ovarian cells harboring a familial SMARCA4 mutation acquire in vitro phenotypes associated with transformation in SSCOHT [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2577.

14 In vitro transformation of ovarian surface epithelium links a mutation in SWI/SNF Related, Matrix Associated, Actin Dependent Regulator Of Chromatin, Subfamily A, Member 4 to familial small carcinoma of the ovary hypercalcemic type

Using a unique population of cells derived from a familial carrier of an SCCOHT-associated SMARCA4 mutation (SMARCA4. c.3081+1G>T), we sought to determine if non-cancerous cells would spontaneously transform during in vitro culture, thereby providing a model system for studying the formation of SCCOHT. Ovarian surface epithelium (OSE) derived from a familial carrier of a SCCOHT-associated mutation in the SMARCA4 gene (P590, SMARCA4. c.3081+1G>T, prophylactic oophorectomy) were serially passaged in vitro, and interrogated for their ability to acquire characteristics of cellular transformation. Cells were grown for multiple passages to determine if cells would spontaneously immortalize. Loss of contact-dependent inhibition was interrogated with focus-forming assays of confluent monolayers. The ability of cells to escape anoikis and grow independently as spheroids was interrogated in attachment-free growth conditions. Senescenceassociated beta-galactosidase (SA-beta-gal), cellular necrosis, and cellular apoptosis was also measured. All experiments were conducted in parallel with an age-matched OSE population derived from a benign ovarian surgery, P590 cells that were passaged over 12 twelve times, representing at least 40 cellular doublings, continued to proliferate in culture. In contrast, non-mutant P583 cells failed to grow beyond 11–12 passages. P590 cells grown to confluency spontaneously formed foci of multicellular aggregates, while P583 cells did not. In attachment-free culture conditions, P590 cells readily formed spheroids from both early-passage and late-passage cells. In contrast, P583 formed small spheroids that failed to expand in culture. Surprisingly, both P590 and P583 cells displayed elevated SA-betagal staining, with neither cell populations showing elevated necrosis or apoptosis markers. OSE harboring a SMARCA4 mutation associated with familial SCCOHT display several phenotypes consistent with early stages of cellular transformation. In contrast, non-mutant cells failed to display signs of transformation. Our data suggest that OSE may represent a unique cell population for studying the early stages of SCCOHT formation, providing a useful tool for developing improved diagnostic and therapeutic approaches.

Transcriptomics and Proteomics Analyses Reveal JAK Signaling and Inflammatory Phenotypes during Cellular Senescence in Blind Mole Rats: The Reflections of Superior Biology.

Simple SummaryBlind mole rats (BMR) (Spalax, Nannospalax sp.) are extraordinary organisms with cancer resistance and a long lifespan for their size. Cellular senescence is a condition in which cells cease dividing irreversibly and secrete proinflammatory cytokines. To understand the mechanisms behind their superior traits, we utilized transcriptomics and proteomics tools in senescent BMR cells to compare them to similarly sized mice. The results revealed the alterations in Janus kinase (JAK) signaling and the cytokine-mediated pathway during the cellular senescence process in BMRs. These findings might reveal the novel mechanisms behind the unique biology of BMRs through cytokine-mediated adaptations.The blind mole rat (BMR), a long-living subterranean rodent, is an exceptional model for both aging and cancer research since they do not display age-related phenotypes or tumor formation. The Janus kinase–signal transducer and activator of transcription (JAK–STAT) signaling is a cytokine-stimulated pathway that has a crucial role in immune regulation, proliferation, and cytokine production. Therefore, the pathway has recently attracted interest in cellular senescence studies. Here, by using publicly available data, we report that JAK–STAT signaling was suppressed in the BMR in comparison to the mouse. Interestingly, our experimental results showed upregulated Jak1/2 expressions in BMR fibroblasts during the replicative senescence process. The transcriptomic analysis using publicly available data also demonstrated that various cytokines related to JAK–STAT signaling were upregulated in the late passage cells, while some other cytokines such as MMPs and SERPINs were downregulated, representing a possible balance of senescence-associated secretory phenotypes (SASPs) in the BMR. Finally, our proteomics data also confirmed cytokine-mediated signaling activation in senescent BMR fibroblasts. Together, our findings suggest the critical role of JAK–STAT and cytokine-mediated signaling pathways during cellular senescence, pointing to the possible contribution of divergent inflammatory factors to the superior resistance of aging and cancer in BMRs.

Role of H4K16 acetylation in 53BP1 recruitment to double-strand break sites in in vitro aged cells

Increased frequency of DNA double strand breaks (DSBs) with aging suggests an age-associated decline in DSB repair efficiency, which is also influenced by the epigenetic landscape. H4 acetylation at lysine 16 (H4K16Ac) has been related to DSB repair since deacetylation of this mark is required for efficient 53BP1 recruitment to DSBs. Although age-associated changes in H4K16Ac levels have been studied, their contribution to age-related DSB accumulation remains unknown. In vitro aged Human Dermal Fibroblasts (HDFs) display lower levels of H4K16A that correlate with reduced recruitment of 53BP1 to basal DSBs. Following DNA damage induction, early passage (EP) cells suffered from a transient H4K16 deacetylation that allowed proper 53BP1 recruitment to DSBs. In contrast, to reach this specific and optimum level, aged cells responded by increasing their overall lower H4K16Ac levels. Induced hyperacetylation of late passage (LP) cells using trichostatin A increased H4K16Ac levels but did not ameliorate 53BP1 recruitment. Instead, deacetylation induced by MOF silencing reduced H4K16Ac levels and compromised 53BP1 recruitment in both EP and LP cells. Age-associated decrease of H4K16Ac levels contributes to the repair defect displayed by in vitro aged cells. H4K16Ac responds to DNA damage in order to reach a specific, optimum level that allows proper 53BP1 recruitment. This response may be compromised with age, as LP cells depart from lower H4K16Ac levels. Variations in H4K16Ac following the activation of the DNA damage response and aging point at this histone mark as a key mediator between DNA repair and age-associated chromatin alterations.

The Long-Term Culture of Human Fibroblasts Reveals a Spectroscopic Signature of Senescence.

Aging is a complex process which leads to progressive loss of fitness/capability/ability, increasing susceptibility to disease and, ultimately, death. Regardless of the organism, there are some features common to aging, namely, the loss of proteostasis and cell senescence. Mammalian cell lines have been used as models to study the aging process, in particular, cell senescence. Thus, the aim of this study was to characterize the senescence-associated metabolic profile of a long-term culture of human fibroblasts using Fourier Transform Infrared and Nuclear Magnetic Resonance spectroscopy. We sub-cultivated fibroblasts from a newborn donor from passage 4 to passage 17 and the results showed deep changes in the spectroscopic profile of cells over time. Late passage cells were characterized by a decrease in the length of fatty acid chains, triglycerides and cholesterol and an increase in lipid unsaturation. We also found an increase in the content of intermolecular β-sheets, possibly indicating an increase in protein aggregation levels in cells of later passages. Metabolic profiling by NMR showed increased levels of extracellular lactate, phosphocholine and glycine in cells at later passages. This study suggests that spectroscopy approaches can be successfully used to study changes concomitant with cell senescence and validate the use of human fibroblasts as a model to monitor the aging process.

Osteosarcoma cells in early and late stages as cancer in vitro progression model for assessing the responsiveness of cells to silver nanoparticles.

Understanding of biology of osteosarcoma malignant progression is indispensable for enhancement of conventional chemotherapy by the use of silver nanoparticles (AgNPs). We presented an in vitro model of cancer progression closely resembling processes occurring in vivo in terms of protein profile. A comparison of cytotoxic and genotoxic potential of AgNPs in Saos-2 cells in early stages of cancerous progression (early passages) with the cells in advanced stages (late passages) demonstrated significantly reduced responsiveness of the late passage cells to nanoparticles toxicity. It was also confirmed by proteome analysis as we identified considerably higher number of differentially expressed proteins in Saos-2 cells in early passages compared to the late passage cells. Our studies showed that the ability of AgNPs as potential drug carriers to deliver a medication and/or to evoke toxic effects might be significantly diminished in advanced stages of cancer progression.

Characterization of Odontoblasts in Supernumerary Tooth-derived Dental Pulp Stem Cells between Passages by Real-Time PCR

The aim of this study is to compare the properties of odontoblast gene of early passage cells and late passage cells derived from impacted maxillary supernumerary teeth. Impacted supernumerary teeth with maxilla were extracted from 12 patients (8 males, 4 females) between 6 - 9 years old without medical history. Real-time polymerase chain reaction (PCR) was conducted to compare characterization of odontoblast cell in the 3rd and 10th passage, and between with bone inducing additive group and without additive group. Genes for odontoblasts characteristics are osteonectin (ONT), alkaline phosphatase (ALP), osteocalcin (OCN), dentin matrix protein 1 (DMP-1) and dentin sialophosphoprotein (DSPP). The level of gene expression was in a decreasing order of ONT, ALP, OCN, DMP-1 and DSPP in the 3rd passage, and in decreasing order of ONT, DMP-1, OCN, ALP, and DSPP in the 10th passage in the undifferentiation and differentiation group. The order of ONT, DMP-1, and OCN did not changed. ALP and DMP-1 were switched in order. ALP and DMP-1 may be used as important markers for differentiating between the 3rd passage and 10th passage cells. Considering that supernumerary tooth was extracted young age and the time required to cultured 10th passage was short, supernumerary tooth can be considered a useful donor site of dental pulp stem cells.

Protective effects of carotenoid fucoxanthin in fibroblasts cellular senescence

Fucoxanthin, as a main marine carotenoid, exhibit a wide variety of bioactivities, including antioxidant activity. Previously, we have shown the geroprotective activity of fucoxanthin on Drosophila and C. elegans. Our new study aimed to compare the antioxidant activity of fucoxanthin in early and late passage normal human cells LECh4(81) in physiological conditions and under oxidative stress. In addition, using the RNA-seq we have analyzed the transcriptomic changes during the replicative senescence of fibroblasts treated with fucoxanthin. Results showed that fucoxanthin at a concentration of 5 μM caused the most pronounced antioxidant effect in the late passage cells. Moreover, transcriptomic data showed the increased expression levels of genes related to the Nrf2/ARE pathway. According to the analysis of enriched KEGG pathways, fucoxanthin altered cellular processes like ribosome biogenesis, lipid metabolism, and cell cycle regulation including some age-related pathways such as Wnt, JAK-STAT, and FoxO signaling pathways. We suggest that fucoxanthin may have therapeutic potential for treating age-related diseases.

Endothelial cell aging detection by means of atomic force spectroscopy.

Endothelial cell aging is related to changes not only in cell phenotype, such as luminal changes, intimal and medial thickening, and increased vascular stiffness, but encompasses different cell responses to various substances including drugs or nanomaterials. In the present work, time- and dose-dependent elasticity changes evoked by silver nanoparticles in endothelial cells in early (below 15) passages were analyzed. Silver nanoparticle concentrations of 3, 3.6, and 16 μg/mL were selected for elasticity measurements for long incubation (24 hours) and of 1 and 3 μg/mL for monitoring dynamic elasticity changes of 1-, 3-, and 6-hour incubations. Surprisingly, a significant reduction in the cells elasticity modulus at lower number of passages exposed to silver nanoparticles used at 3 μg/mL for 24 hours was demonstrated. These results are in contrast to those obtained for endothelial cells in late (33-43) passages that may result from cellular aging in response to nanosilver. Furthermore, for short incubation times (1 and 3 hours), SNP-induced significant increase in the cell elasticity modulus was detected. In current work, we also attempted to answer the question whether the changes in cell elasticity were induced by the silver nanoparticles stabilized with polyvinyl pyrrolidone or by stabilizer itself. Elasticity measurements were supplemented by observations made with transmission electron microscopy and scanning electron microscopy, which confirmed the presence of silver nanoparticles inside the cells and on the cell membrane. Additionally, activation of reactive oxygen species was detected for cells exposed to SNPs for 1 and 3 hours, which was accompanied by increased cell elasticity modulus suggesting a possible mechanism of observed phenomenon.

Gene expression profile of immunoregulatory cytokines secreted from bone marrow and adipose derived human mesenchymal stem cells in early and late passages.

Mesenchymal Stem Cells (MSCs) have therapeutic potential in a variety of diseases; however, the safety and efficacy of their use remain ambiguous. Clinical applications of MSCs are under intensive investigation due to their immunomodulatory features and lack of immune reactivity. Therefore, having a clear perspective on the exact mechanisms underlying the regulation of cytokine secretion in different microenvironments seems crucially important.In the current study, samples from human bone marrow and adipose were collected, and peripheral blood mononuclear cells (PBMCs) were isolated and cultured in conventional medium. After MSC expansion, the cells from passage 3 (P3) and passage 9 (P9) were utilized to identify MSC cell surface markers and their differentiation capacity. The P3, P5, P7, and P9 cells were used for RNA extraction to qualify the expression of the main immunomodulatory cytokines IDO, VCAM-1, TGF-β, IL-6, IL-10, and PGE2 at mRNA levels. The results indicate that VCAM-1 expression in the subcultures was reduced in bone marrow-derived MSCs. After an increase in P5, P7, and P9, IL-6 expression was reduced. In adipose-derived MSCs, the mRNA levels of IL-10 in higher passages were decreased compared with P3; other studied cytokines had no significant changes in their expression levels in either bone marrow or adipose-derived MSCs. Based on these results, it can be concluded that a suitable source for MSCs in cell therapy with stable expression of main cytokines, even in higher subcultures, appears to be adipose-derived MSCs with the exception of IL-10 secretion.

Uncoupled inflammatory, proliferative, and cytoskeletal responses in senescent human gingival fibroblasts.

Aging is characterized by a decline in tissue structure and function that may be explained by the development of cellular senescence. However, the acquisition of specific phenotypic responses in senescent gingival fibroblasts is still poorly understood. Here, we have analyzed whether proliferation of primary cultures of human gingival fibroblasts may affect different cell functions relevant to cellular senescence and tissue deterioration. Human gingival fibroblasts from five young donors were expanded until cellular senescence was achieved. Cellular senescence was evaluated by determining modifications in cell size, cell proliferation, p16 and p21 mRNA levels, H2Ax phosphorylation, cell viability, and senescence-associated beta-galactosidase staining. Inflammation was evaluated by analyzing the secretion of cytokines and nuclear translocation of NF-κB. Collagen remodeling was evaluated using a collagen gel contraction assay. Immunofluorescence and confocal microscopy were used to determine changes in the localization of the cytoskeletal proteins. Data analysis was performed to identify changes between cultures of the same donor at early and late passages using the paired sample t test or the Wilcoxon matched-pairs signed-rank test. Late passage cells showed changes compatible with cellular senescence that included increased cell size, reduced cell proliferation, staining for SA-beta gal, phosphorylated H2Ax, and increased p16 and p21 mRNA levels. Late passage cells showed a decrease in collagen contraction and reduced co-localization between the cytoskeletal proteins actin and vinculin. Importantly, late passage cells neither demonstrated changes in the secretion of inflammatory cytokines nor NF-κB activation. Our results imply that cytoskeletal changes and inhibition of cell proliferation represent early modifications in the structure and function of senescent gingival fibroblasts that are not coupled with the acquisition of an inflammatory phenotype. Further studies are needed to clarify the impact of different senescence stages during aging of the periodontium.

Proliferation of adult human bronchial epithelial cells without a telomere maintenance mechanism for over 200 population doublings.

To date, there is no direct evidence of telomerase activity in adult lung epithelial cells, but typical culture conditions only support cell proliferation for 30-40 population doublings (PD), a point at which telomeres remain relatively long. Here we report that in in vitro low stress culture conditions consisting of a fibroblast feeder layer, rho-associated coiled coil protein kinase inhibitor (ROCKi), and low oxygen (2%), normal human bronchial epithelial basal progenitor cells (HBECs) divide for over 200 PD without engaging a telomere maintenance mechanism (almost four times the "Hayflick limit"). HBECs exhibit critically short telomeres at 200 PD and the population of cells start to undergo replicative senescence. Subcloning these late passage cells to clonal density, to mimic lung injury in vivo, selects for rare subsets of HBECs that activate low levels of telomerase activity to maintain short telomeres. CRISPR/Cas9 knockout of human telomerase reverse transcriptase or treatment with the telomerase-mediated telomere targeting agent 6-thio-2'deoxyguanosine abrogates colony growth in these late passage cultures (>200 PD) but not in early passage cultures (<200 PD). To our knowledge, this is the first study to report such long-term growth of HBECs without a telomere maintenance mechanism. This report also provides direct evidence of telomerase activation in HBECs near senescence when telomeres are critically short. This novel cell culture system provides an experimental model to understand how telomerase is regulated in normal adult tissues.

Safety evaluation of conditionally immortalized cells for renal replacement therapy.

End-stage kidney disease represents irreversible kidney failure. Dialysis and transplantation, two main treatment options currently available, present various drawbacks and complications. Innovative cell-based therapies, such as a bioartificial kidney, have not reached the clinic yet, mostly due to safety and/or functional issues. Here, we assessed the safety of conditionally immortalized proximal tubule epithelial cells (ciPTECs) for bioartificial kidney application, by using in vitro assays and athymic nude rats. We demonstrate that these cells do not possess key properties of oncogenically transformed cells, including anchorage-independent growth, lack of contact inhibition and apoptosis-resistance. In late-passage cells we did observe complex chromosomal abnormalities favoring near-tetraploidy, indicating chromosomal instability. However, time-lapse imaging of ciPTEC-OAT1, confined to a 3D extracellular matrix (ECM)-based environment, revealed that the cells were largely non-invasive. Furthermore, we determined the viral integration sites of SV40 Large T antigen (SV40T), human telomerase (hTERT) and OAT1 (SLC22A6), the transgenes used for immortalization and cell function enhancement. All integrations sites were found to be located in the intronic regions of endogenous genes. Among these genes, early endosome antigen 1 (EEA1) involved in endocytosis, and BCL2 Like 1 (BCL2L1) known for its role in regulating apoptosis, were identified. Nevertheless, both gene products appeared to be functionally intact. Finally, after subcutaneous injection in athymic nude rats we show that ciPTEC-OAT1 lack tumorigenic and oncogenic effects in vivo, confirming the in vitro findings. Taken together, this study lays an important foundation towards bioartificial kidney (BAK) development by confirming the safety of the cell line intended for incorporation.

Qualifying mscs for clinical applications -differential gene expression analyses of pluripotent, differentiation and surface marker genes of early and late passage mscs, in hypoxic and normal oxygen level in culture conditions.

Background & Aim With the increasing focus on the large scale expansion of MSCs required for the clinical application, the optimisation of in-vitro expansion of MSCs requires careful consideration to maintain the native MSCs characteristics. Physiological parameters such as oxygen concentration, media constituents and passage numbers have much influence on the properties of MSCs and may have major impact on the therapeutic potential of MSCs. Cells grown under hypoxic (low oxygen) conditions have been widely documented in clinical use however culturing MSCS in large scale requires bioreactor and it is challenging to maintain the low oxygen and other physiological parameters over several passages in big vessels of bioreactors. Similarly, the culture conditions in the bioreactor including pH, dissolved oxygen and the sheer force exerted on cells may change the cellular characteristics including gene expression Methods, Results & Conclusion These culture conditions may have different potency attributes clinically. For these reasons, our study investigated differential expression of 11 key genes of MSCs for pluripotent genes POU5F1, NANOG and KLF4, differentiation genes RUNX2, COL1A1, SOX9, COL2A1, PPARG and CD marker genes THY1, ENG and NT5E from adipose derived MSCs, grown under both the normal oxygen or hypoxic conditions were analysed. Two different cells age early (P5) and late passage (P9) cells. The result demonstrated marked differences in gene expression pattern of the early and late passage cells grown under normoxic (normal oxygen) when compared with the hypoxic conditions. Pluripotent markers KLF4 in early passage cells was expressed more than two-fold in the normoxic cells, and PPARG was expressed more in the late passage cells under normoxic condition. Differentiation marker for RUNX2 and NT5E were highly expressed in early passage cells. These differences in gene expression may have critical impact on the potency of the cells grown in large scale in bioreactor. Additionally, the effects of prolonged culture of MSCs in-vitro will need be qualified prior to use when manufactured in large scale bioreactor for the clinical dose to ensure the therapeutic efficacy.