Early Passage Research Articles

Establishment and Validation of Patient-Derived Non-Small Cell Lung Cancer Organoids as In Vitro Lung Cancer Models

Background: Recent advances in the personalized treatment of non-small cell lung cancer (NSCLC) require representative in vitro model systems that reflect tumor heterogeneity and maintain the characteristic genetic aberrations. We therefore aimed to establish patient-derived NSCLC organoids that offer a reliable platform for further investigations. Methods: NSCLC organoids were cultured between May 2020 and February 2022 from surgically resected NSCLC tissue specimens. After histological and immunohistochemical validation, genetic validation was performed by targeted next-generation sequencing of tissue and organoid specimens using the Oncomine Focus Assay (ThermoFisher Scientific). Results: From 37 resected NSCLC samples, 18 primary organoid cultures were successfully established and expanded during early passages. Upon histomorphological validation, organoids showed complementary characteristics when compared to the resected parental tumor, including adenocarcinoma, squamous cell carcinoma, mucoepidermoid carcinoma, and lung carcinoid differentiation. Among nine parental tumors, traceable genetic alterations were detected, and three corresponding organoids lines retained this mutational profile, including a KRAS p.Gly12Val mutation, KRAS p.Gly12Cys mutation, and RET-fusion. Conclusions: The establishment of primary NSCLC organoids from surgically resected tissue is feasible. Histological, immunohistochemical, and genetic validation is essential to identify representative NSCLC organoids that maintain the characteristics of the parental tumor. Overall, low establishment rates remain a challenge for broad clinical applications.

Longitudinal analysis of genetic and epigenetic changes in human pluripotent stem cells in the landscape of culture-induced abnormality.

Human embryonic stem cells (hESCs) are naturally equipped to maintain genome integrity to minimize genetic mutations during early embryo development. However, genetic aberration risks and subsequent cellular changes in hESCs during in vitro culture pose a significant threat to stem cell therapy. While a few studies have reported specific somatic mutations and copy number variations (CNVs), the molecular mechanisms underlying the acquisition of 'culture-adapted phenotypes' by hESCs are largely unknown. Therefore, we conducted comprehensive genomic, single-cell transcriptomic, and single-cell ATAC-seq analyses of an isogenic hESC model displaying definitive 'culture-adapted phenotypes'. We found that hESCs lacking TP53, in which loss-of-function mutations were identified in human pluripotent stem cells (hPSCs), presented a surge in somatic mutations. Notably, hPSCs with a copy number gain of 20q11.21 during early passage did not present 'culture-adapted phenotypes' or BCL2L1 induction. Single-cell RNA-seq and ATAC-seq analyses revealed active transcriptional regulation at the 20q11.21 locus. Furthermore, the induction of BCL2L1 and TPX2 to trigger 'culture-adapted phenotypes' was associated with epigenetic changes facilitating TEA domain (TEAD) binding. These results suggest that 20q11.21 copy number gain and additional epigenetic changes are necessary for expressing 'culture-adapted phenotypes' by activating gene transcription at this specific locus.

Analysis of Legal Verbiage in State Legislation for Insurer Coverage of Early Eyedrop Refills in the United States

State laws on early eyedrop refills, implemented to help patients obtain their medications when they run out early, have many inconsistencies possibly impacting their efficacy and functionality. This study sought to examine different state laws and elucidate unique features and commonalities that may influence their effectiveness. A review of all state legislation to date regarding early eyedrop refills was performed. All 50 states were included, with an in-depth review of the 33 states that had early eyedrop refill legislation. The law database Nexis Uni (formerly LexisNexis) and each state's individual law code site were used to identify relevant laws (or proposed legislation) using keywords such as "eyedrop," "eye," "refill," and "early." Political data based on the year these laws were passed was obtained from the National Governor's Association and National Conference of State Legislatures. This data was aggregated and analyzed using descriptive statistics. State law wording was analyzed for percentage of dosage period passed, days passed, and other requirements. Of the 33 states with early eyedrop refill laws, 14 were only days based, 8 were only percentage based, 4 had both, 4 had neither percentage nor days criteria, and 3 had neither but cited CMS guidelines. These laws were passed between 2009-2023, with a notable increase from 2014-2018, rising from 10 to 29 states. Regionally, the Northeast had the highest adoption rate (88%), followed by the West (77%), the South (56%), and the Midwest (50%). Political climates varied: 14 states had Republican control, 2 had Democratic control, and 17 had mixed party control. Of the 17 states without an early eyedrop refill law 4 attempted passage but were not put into law for various reasons with the other 13 not appearing to have had any attempts at law passage. State laws providing coverage for early eyedrop refills vary in terms of verbiage and requirements necessary to obtain a covered early eyedrop refill. Region, political climate, and year appear to play minor roles in early eyedrop refill verbiage and passage.

The Senolytic Effect of Indole-3-Carbinol (I3C) on Mouse Embryonic (MEF) and Human Fibroblast Cell Lines.

Senescence and apoptosis are two fundamental cellular processes that play crucial roles in various physiological and pathological conditions. Senescence refers to the irreversible growth arrest that cells undergo in response to various stimuli, including telomeric alterations, stress, and oncogenic signaling. Pharmacological and/or genetic removal of senescent cells, also referred to as senolysis, triggers organ rejuvenation and tissue regeneration. Indole-3-carbinol (I3C) is a natural compound contained in Brassicaceae plants and identified in multiple in vitro and in vivo studies as a well-tolerated and effective compound in cancer prevention and therapy. Its anti-cancer properties have been attributed at least in part to its inhibitory activity of proto-oncogenic HECT E3-ubiquitin ligases such as NEDD4 and WWP1. While the tumor suppressive effects of I3C in cancer cell lines have been reported in multiple studies, little is known regarding the biological effects of I3C in primary normal cells, which attain spontaneous cellular senesce over serial passaging. To this end, we used two model systems: mouse embryonic fibroblasts (MEFs) and human primary dermal fibroblasts. Here, we surprisingly show that I3C does increase cellular senescence at early passages, while dramatically reducing the number of senescent cells through the induction of apoptosis in both mouse and human primary cells. Thus, our findings support the notion that I3C acts as a senolytic compound with important therapeutic implications for the prevention and treatment of aging manifestations. The notion can be readily tested in future clinical trials in humans also in view of the high tolerability and safety previously displayed by I3C in preclinical and clinical studies.

Genome Instability Precedes Viral Integration in Human Papilloma Virus Transformed Tonsillar Keratinocytes.

Approximately 70% of oropharyngeal squamous carcinomas (OPSCC) are associated with human papillomavirus (HPV). While patients with HPV-positive tumors generally have better outcomes than those with HPV-negative tumors, a subset of HPV-positive patients do have poor outcomes. Our previous work suggested that tumors with integrated virus exhibit significantly greater genome wide genomic instability than those with only episomal viral genomes and patients with HPV+ OPSCC with episomal viral genomes had better outcomes. To explore the causal relation between viral integration and genomic instability, we have examined the time course of viral integration and genetic instability in tonsillar keratinocytes transformed with HPV16. HPV-infected human tonsil keratinocyte cell lines were continuously passaged and every fifth passage some cells were retained for genomic analysis. Whole genome sequencing and optical genomic mapping confirmed that virus integrated in five of six cell lines while remaining episomal in the sixth. In all lines genome instability occurred during early passages, but essentially ceased following viral integration but continued to occur later passages in the episomal line. To test tumorigenicity of the cell lines, cells were injected subcutaneously into the flanks of nude mice. A cell line with the integrated virus induced tumors following injection in the nude mouse while that with the episomal virus did not. Implications: Genomic instability in HPV OPSCC tumors is not the result of viral integration but likely promotes integration. Moreover, transformants with episomal virus appear to be less tumorigenic than those with integrated virus.

Establishment of the first dermal fibroblast cell line derived from the Indo-Pacific Bottlenose Dolphin (Tursiops aduncus) and its response to pollutant exposure

The Indo-Pacific bottlenose dolphin (Tursiops aduncus), classified as a new species since 1998, is underexplored in ecotoxicology owing to ethical constraints and the lack of specific in vitro models. Herein, we established the first skin fibroblast cell line (TaSF) from an Indo-Pacific bottlenose dolphin stranded along the Pearl River Estuary, China. TaSF cells exhibited strong proliferation in early passages but ceased mitosis at passage 19, likely owing to reaching the Hayflick limit. Morphology and immunofluorescence tests confirmed the fibroblastic nature of these cells. Karyotyping revealed 21 pairs of autosomes and 1 pair of sex chromosomes (XY), consistent with many cetaceans. To facilitate long-term future studies, TaSF cells were immortalized with exogenous simian virus 40 T antigen, creating the TaSFT cell line. The cytotoxic effects of 27 contaminants, including 6 organotins (OTs), 10 per- and polyfluoroalkyl substances (PFASs), and 11 phthalates (PAEs), on TaSFT cells were evaluated after 24-h exposure. Among these chemicals, OTs exhibited the strongest cytotoxicity, and both OTs and PFASs showed structure-related toxicity. These findings confirm the feasibility of TaSFT cells as a novel tool for ecotoxicity research on the Indo-Pacific bottlenose dolphin and highlight the need for further investigation into the environmental contaminant pressures on this species.

TRACER Perspectives on Gulf-Breeze and Bay-Breeze Circulations and Coastal Convection

Abstract This study explores gulf-breeze circulations (GBCs) and bay-breeze circulations (BBCs) in Houston–Galveston, investigating their characteristics, large-scale weather influences, and impacts on surface properties, boundary layer updrafts, and convective clouds. The results are derived from a combination of datasets, including satellite observations, ground-based measurements, and reanalysis datasets, using machine learning, changepoint detection method, and Lagrangian cell tracking. We find that anticyclonic synoptic patterns during the summer months (June–September) favor GBC/BBC formation and the associated convective cloud development, representing 74% of cases. The main Tracking Aerosol Convection Interactions Experiment (TRACER) site located close to the Galveston Bay is influenced by both GBC and BBC, with nearly half of the cases showing evident BBC features. The site experiences early frontal passages ranging from 1040 to 1630 local time (LT), with 1300 LT being the most frequent. These fronts are stronger than those observed at the ancillary site which is located further inland from the Galveston Bay, including larger changes in surface temperature, moisture, and wind speed. Furthermore, these fronts trigger boundary layer updrafts, likely promoting isolated convective precipitating cores that are short lived (average convective lifetime of 63 min) and slow moving (average propagation speed of 5 m s−1), primarily within 20–40 km from the coast.

Creation and the Revelation of God: Interwoven and Interactive Themes across John 1:1–2:12

Abstract John 1:19–51 provides a bridge from the prologue (1:1–18) into the body of the narrative and the inauguration of Jesus’s public ministry at a wedding feast (2:1–12). The primary theme underlying these opening days has long been debated. Further, do the days articulated by the regular temporal markers tally to six or seven? The evangelist presents the prologue in a chiasm that turns on the pivot of v. 12b. The opening and closing elements correspond to one another (creation imagery in vv. 1–5; revelation imagery in vv. 16–18), but the latter has been fundamentally affected by the pivot: the incarnation of the Word of God and the human response to it. Moreover, as a prologue, its structure and content profoundly affect what follows. Based upon these foundations, this essay argues that themes of both creation and revelation are interwoven and interactive across these early passages.

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.

Abstract A058: Primary cancer-associated fibroblasts alter tumor organoid chemosensitivity through epigenetic regulation

Abstract Pancreatic adenocarcinoma (PDAC) is the 3rd leading cause of cancer-related deaths in Canada with a 5-year survival rate at ∼12%. A major cause of this poor survival rate is the inefficiency of current treatments. Only 20% of patients are eligible for surgical resection while the other 80% are treated with chemotherapies (nab-paclitaxel, Gemcitabine, FOLFIRINOX) having limited efficacy. This resistance is due to the PDAC tumour microenvironment (TME), including cancer-associated fibroblasts (CAFs). Numerous studies show CAFs and the TME affect tumour growth, chemoresistance and metastasis, but the mechanisms underlying the effects are unknown. Epigenetic mechanisms, including DNA methylation and histone modifications, link environmental factors to altered gene expression. Genes encoding epigenetic mediators are often mutated or over-expressed in PDAC, but a role in resistance has yet to be defined. We hypothesize that epigenetic reprogramming occurs in pancreatic cancer cells in response to secreted factors from CAFs leading to increased therapy resistance. Methods: Most work to date on PDAC resistance has been performed with immortalized cell lines and in two dimensions. Therefore, to test our hypothesis, we established patient-derived organoids (PDO) and CAFs obtained from endoscopic ultrasound (EUS). Clinical data was obtained through the DERIVE (Determination of Response to Therapy in Individual Patients) database. To examine the effects of CAFs on PDOs, we obtained conditioned media (CM) from four, early passage CAFs, grown 48 hours in culture. Organoid media was supplemented 1:1 with CAF-CM and six PDOs grown in this media for up to 14 days. PDO growth and the response to gemcitabine, FOLFIRINOX or radiation was monitored using an Incucyte system. To identify changes in the epigenome and how this reflects changes in gene expression, DNA methylation arrays and ATAC-Seq was performed before and after treatment with CAF-CM and aligned to RNA-seq analysis on RNA isolated at the same time point. Results: Exposure of PDOs to CAF-CM resulted in altered phenotypes and molecular profiles of PDOs in a patient-specific fashion. Increased resistance to chemotherapy (Gemcitabine) and radiotherapy was observed in multiple PDOs. Changes in DNA methylation and chromatin accessibility was aligned with transcriptomic data and identified known and novel pathways linked to chemoresistance. Metabolomic and cytokine analysis on CAFs-CM identified differential enrichment of metabolites linked to epigenetic mediators. We are currently determining if these factors secreted by the CAFs promote chemo and radio-resistance. Conclusions: This study shows the importance in integrating TME components when examining novel therapeutic options. In addition, we show (1) CAFs secrete factors that enhance therapeutic resistance in PDOs and (2) these changes likely involve altered epigenetic reprogramming. Future experiments will identify the epigenetic factors that can be targeted to counteract chemoresistance. Citation Format: Emilie Jaune-Pons, Zachary Klassen, Joana Ribeiro Pinto, Maria Nica, Nadeem Hussain, Michael Sey, Ken Leslie, Matthew Cecchini, Crystal Engelage, Danielle Porplycia, Stephen Welch, Brian Yan, Christopher Pin. Primary cancer-associated fibroblasts alter tumor organoid chemosensitivity through epigenetic regulation [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A058.

Abstract C043: Hyperinsulinemia promotes pancreatic cancer progression by altering tumor metabolism

Abstract Metabolic diseases, such as type 2 diabetes (T2D), insulin resistance, and obesity, often accompany pancreatic ductal adenocarcinoma (PDAC), and they are associated with reduced survival. Hyperinsulinemia is a common hallmark symptom shared by those disorders and is independently associated with reduced survival of PDAC patients. While it has been established that endogenous hyperinsulinemia accelerates PDAC initiation by promoting the formation of KRAS-driven pre-cancerous lesions, its role in the progression of established tumors remains poorly understood. This represents a major knowledge gap in our understanding of whether insulin needs to be monitored and controlled during PDAC treatment, as it is not typically considered in current treatment paradigms. We hypothesized that hyperinsulinemia promotes the progression of PDAC tumors. Using patient-derived PDAC organoids (PDOs) and a mouse model of metabolic disorders, we found that insulin may promote PDOs’ growth by reprogramming tumor metabolism. A High-fat diet (HFD) treatment that induces hyperinsulinemia, but not hyperglycemia in mice accelerated the growth of PDO-derived orthotopic xenografts. Importantly, the fasting insulin level showed a significant positive correlation with the endpoint tumor volume, suggesting that endogenous insulin may positively contribute to PDOs’ growth in vivo. To assess the direct effect of insulin on the growth and molecular profile of PDOs, we treated an early passage PDO line with different concentrations of insulin and glucose at physiologically relevant levels. We observed a ∼1.2-fold increase in cell number after 7 days of culture in high insulin (10 nM) conditions with high (15 mM) or physiological (6 mM) levels of glucose, relative to the low insulin control. Phosphoproteomic analysis demonstrated significantly greater PI3K/AKT/mTOR, but not RAF/MEK/ERK, activity under high insulin with high or physiological levels of glucose compared to the control, directing the investigation toward the metabolic effects of insulin. Consistently, unbiased proteomics profiling revealed that metabolic proteins were significantly enriched in both high insulin conditions, and interestingly insulin enriched different sets of proteins in various metabolic pathways depending on glucose levels. Together, our preliminary results suggest that hyperinsulinemia may enhance the growth and progression of PDAC by altering tumor metabolism to support cell growth. Further experiments are needed to functionally link the insulin-mediated metabolic changes with increased PDO growth, as well as testing the direct effect of endogenous insulin on PDAC cells in vivo. Citation Format: Jeffrey Lin, James D Johnson, David Schaeffer, Vincent Richard, Christoph Borchers, Janel L Kopp. Hyperinsulinemia promotes pancreatic cancer progression by altering tumor metabolism [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C043.

Analysis of infrasound array data from tornadic storms in the southeastern United States

Data recorded by several infrasound sensor arrays deployed in the southeastern United States during the spring of 2018 have been analyzed during a period when a storm front passed through and spawned several tornado touchdowns. The tornadoes ranged from EF-0 to EF-2. Accurate bearings have been obtained, corresponding to tornadoes at ranges up to more than 100 km. Earlier in the day, a convective storm cell passed and triggered a tornado warning but no verified tornado touchdowns. During this earlier passage, a weaker signal was detected with bearings that tracked this convective storm cell. The analyses reveal that on the systems deployed, the dominant band of coherent infrasound measured from the tornadic storms was between 2 and 6 Hz. Atmospheric conditions are known to have a significant influence on signal detection because of propagation effects and local wind noise conditions. Propagation modeling and wind noise analysis were undertaken and are observed to be consistent with signal detection from the tornadoes. Because of the static sources, as well as the multiple strong cells that were in the region simultaneously, it was necessary to use array signal processing methods that are capable of resolving multiple sources.

Leishmania major telomerase RNA knockout: From altered cell proliferation to decreased parasite infectivity

This study focuses on the biological impacts of deleting the telomerase RNA from Leishmania major (LeishTER), a parasite responsible for causing leishmaniases, for which no effective treatment or prevention is available. TER is a critical player in the telomerase ribonucleoprotein complex, containing the template sequence copied by the reverse transcriptase component during telomere elongation. The success of knocking out both LeishTER alleles was confirmed, and no off-targets were detected. LmTER−/− cells share similar characteristics with other TER-depleted eukaryotes, such as altered growth patterns and partial G0/G1 cell cycle arrest in early passages, telomere shortening, and elevated TERRA expression. They also exhibit increased γH2A phosphorylation, suggesting that the loss of LeishTER induces DNA damage signaling. Moreover, pro-survival autophagic signals and mitochondrion alterations were shown without any detectable plasma membrane modifications. LmTER−/− retained the ability to transform into metacyclics, but their infectivity capacity was compromised. Furthermore, the overexpression of LeishTER was also deleterious, inducing a dominant negative effect that led to telomere shortening and growth impairments. These findings highlight TER's vital role in parasite homeostasis, opening discussions about its potential as a drug target candidate against Leishmania.

Morphologic, Proliferative, and Cytogenetic Changes during In Vitro Propagation of Cat Adipose Tissue-Derived Mesenchymal Stromal/Stem Cells.

Stem cell therapy in cat patients needs a high quantity of mesenchymal stromal/stem cells (MSCs) requiring in vitro propagation under culture conditions which may potentially impact cellular characteristics and genetic stability. This study aimed to assess the in vitro characteristics and cytogenetic stability of cat adipose tissue-derived MSCs (cAT-MSCs). For this purpose, morphological features, clonogenic potential, and proliferative capacity of cAT-MSCs were assessed at passages 2 (P2), P4, and P6. Multipotency and immunophenotype were evaluated. Cytogenetic analyses were conducted up to P6. The cAT-MSCs exhibited a spindle-shaped morphology in early passages. The doubling time increased from 2.5 days at P2 to 9.4 at P4 and 10.5 at P6, accompanied by the observation of nuclear abnormalities such as cluster formation, karyorrhexis, karyolysis, and a decline in the mitotic index at P4. Cells demonstrated multipotency capacity and were CD45-, CD90+, and CD44+. Metaphase analysis at P2 and P4 revealed some indications of structural instability such as gaps, breaks, deletions, duplications, and early chromatid segregation, but these alterations did not show an increase across passages. In conclusion, cAT-MSCs decreased their proliferative capacity after P4, accompanied by morphological alterations and signs of structural instability.

Alu methylation level, morphological, and senescence changes during in vitro aging of human dental pulp stem cells

IntroductionHuman dental pulp stem cells (DPSCs) are pivotal in tissue engineering and cell-based therapies due to their significant differentiation potential and accessibility. A major challenge in in vitro cell expansion is their replicative senescence, which impacts their regeneration and differentiation capabilities. While genetic factors influence these processes, epigenetic regulations such as Alu methylation also play crucial roles. Changes in Alu methylation have been associated with human aging and age-related diseases, contributing to cellular dysfunction and stem cell senescence. Despite this, the implications of Alu methylation alterations in stem cell senescence remain underexplored. This study focuses on examining Alu methylation during the replicative senescence of DPSCs. MethodsThe methylation status of Alu elements in serially passaged, long-term cultured human DPSCs was assessed using combined bisulfite restriction analysis. Morphological changes and indicators of replicative senescence were also evaluated. DPSCs were divided into three passage groups for analysis: early, middle, and late. Methylation levels across these groups were compared to identify trends correlating with passage number. ResultsSignificant morphological changes and markers of replicative senescence were observed predominantly in the late-passage DPSCs. These cells exhibited notably lower levels of Alu methylation and higher proportions of hypomethylated Alu CpG sites compared to those in early passages. ConclusionThe study confirmed that alterations in Alu methylation are evident in the replicative senescence of human DPSCs, suggesting that epigenetic modifications could influence the aging process of these cells and potentially impact their therapeutic efficacy.

Stable isotope-resolved metabolomics analyses of metabolic phenotypes reveal variable glutamine metabolism in different patient-derived models of non-small cell lung cancer from a single patient.

Stable isotope tracers have been increasingly used in preclinical cancer model systems, including cell culture and mouse xenografts, to probe the altered metabolism of a variety of cancers, such as accelerated glycolysis and glutaminolysis and generation of oncometabolites. Comparatively little has been reported on the fidelity of the different preclinical model systems in recapitulating the aberrant metabolism of tumors. We have been developing several different experimental model systems for systems biochemistry analyses of non-small cell lung cancer (NSCLC1) using patient-derived tissues to evaluate appropriate models for metabolic and phenotypic analyses. To address the issue of fidelity, we have carried out a detailed Stable Isotope-Resolved Metabolomics study of freshly resected tissue slices, mouse patient derived xenografts (PDXs), and cells derived from a single patient using both 13C6-glucose and 13C5,15N2-glutamine tracers. Although we found similar glucose metabolism in the three models, glutamine utilization was markedly higher in the isolated cell culture and in cell culture-derived xenografts compared with the primary cancer tissue or direct tissue xenografts (PDX). This suggests that caution is needed in interpreting cancer biochemistry using patient-derived cancer cells in vitro or in xenografts, even at very early passage, and that direct analysis of patient derived tissue slices provides the optimal model for ex vivo metabolomics. Further research is needed to determine the generality of these observations.

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.

Changes in Stemness Properties of Human Adenoid-Derived Mesenchymal Stem Cells by in Vitro Aging

Mesenchymal stem cells (MSCs) have significant therapeutic potential in gene therapy. In vitro replicative senescence causes slow proliferation capacity of MSCs and changes in stem cell properties. In this study, the stem cell properties and proliferation potential of MSCs obtained from adenoid tissue, a new source of MSC, were investigated after long-term in vitro replicative senescence. Adenoid-derived MSCs (A-MSCs) were cultured up to passage 20 and were analyzed for cell morphology, proliferative capacity, differentiation potential and surface marker expression. In addition, the expression profile of cell cycle, apoptosis and senescence-related genes were evaluated. After in vitro replicative senescence, A-MSCs did not show significant morphological differences. The proliferation potential of A-MSCs was rapid up to passage 16, and a reduction in the proliferation potential of senescent cells in vitro was observed depending on the passage number. The differentiation potential of late-passage A-MSCs was also reduced compared to early-passage cells. A-MSCs also provided significant closure at the 8th hour in early passages in terms of closure of the scratch area, while late passage A-MSCs exhibited a similar closure profile at the 24th hour. In conclusion, A-MSCs have significant potential for clinical use due to the sustainability of MSC properties and their ability to proliferate and migrate with long-term culture.

Tablets (Du) and Passages (Zhang): the Material Conditions for the Formation of Early Short Passage Texts

Abstract Several studies have addressed the question of whether the people of early China wrote with thin bamboo strips held in their hands or whether they wrote with the support of flat desks. However, scholars have not addressed the distinction between jian (bamboo strips) and larger du (wooden tablets) in everyday writing. In early China, tablets would be held in the hand to write, while strips would be laid flat on a writing desk. Tablets were the main medium of everyday writing during the period spanning the pre-Qin and Western and Eastern Han dynasties. In the drafting of various literary texts, taking court records, and taking classroom notes, tablets were the primary writing medium. Among written materials from before the Western Han dynasty, duan zhang (short passages) were the most common style of writing, and most texts were composed of short passages. Among the early Chinese manuscripts that have been unearthed, short passages are also very common. However, almost no one has raised the question of why a documentary system dominated by short passages was formed in the pre-Qin and Western and Eastern Han dynasties period. The number of characters that a writing tablet can accommodate essentially coincides with the number of characters in short passages in early Chinese manuscripts. In view of its wide use, I propose that the formation of the short passage form was potentially influenced by the material writing medium of the tablet.

Human Rotaviruses of Multiple Genotypes Acquire Conserved VP4 Mutations during Serial Passage.

Human rotaviruses exhibit limited tropism and replicate poorly in most cell lines. Attachment protein VP4 is a key rotavirus tropism determinant. Previous studies in which human rotaviruses were adapted to cultured cells identified mutations in VP4. However, most such studies were conducted using only a single human rotavirus genotype. In the current study, we serially passaged 50 human rotavirus clinical specimens representing five of the genotypes most frequently associated with severe human disease, each in triplicate, three to five times in primary monkey kidney cells then ten times in the MA104 monkey kidney cell line. From 13 of the 50 specimens, we obtained 25 rotavirus antigen-positive lineages representing all five genotypes, which tended to replicate more efficiently in MA104 cells at late versus early passage. We used Illumina next-generation sequencing and analysis to identify variants that arose during passage. In VP4, variants encoded 28 mutations that were conserved for all P[8] rotaviruses and 12 mutations that were conserved for all five genotypes. These findings suggest there may be a conserved mechanism of human rotavirus adaptation to MA104 cells. In the future, such a conserved adaptation mechanism could be exploited to study human rotavirus biology or efficiently manufacture vaccines.