Population Doubling Research Articles

Biotic potential of Transeius montdorensis (Acari: Phytoseiidae) under technocenosis conditions

The subtropical mite species Transeius montdorensis is widely used to control thrips and other sucking pests in European countries. To develop a domestic technology for the mass rearing of T. montdorensis, its biotic potential has been evaluated under large scale production. The growth rate of T. montdorensis is 0.19±0.017, the doubling time is 4.7±0.29 days at 26–27 °C. In the tested range of mite densities (5–80 ind./ml), the growth rate tends to decrease as the density increases. This trend can be approximated by a logarithmic curve. The density 60–80 ind./ml causes not only a significant drop in the population growth rate but also the decrease in range of its fluctuations. Higher fluctuations of growth rate at lower predator densities (5–15 ind./ml) are probably due to the fact that, in some containers, the predator is unable to hold back outbreaks of its prey (the flour mite Tyrophagus entomophagus). As a result, the prey overpopulates the substrate, which causes its contamination by waste products stimulating microflora development. The growth rate in the density range of 5–15 ind./ml is lower than expected and does not differ significantly from that in the range of 20–30 ind./ml. This indicates that the prey uncontrolled growth inhibit the reproduction of T. montdorensis. The population doubling time is a reliable indicator for screening the best stock culture of T. montdorensis for its successful rearing up to density 60 ind./ml within 6–8 days, i.e., before mass growth of fungi in the substrate. The range of 20–30 ind./ml is the optimal starting density for mass rearing, and when the predator reaches a density of more than 60 ind./ml, it should be resettled or offered for sale.

New characterization and safety evaluation of human limbal stem cells used in clinical application: fidelity of mitotic process and mitotic spindle morphologies

BackgroundLimbal stem cells (LSCs) are crucial for the regeneration of the corneal epithelium in patients with limbal stem cell deficiency (LSCD). Thus, LSCs during cultivation in vitro should be in highly homogeneous amounts, while potency and expression of stemness without tumorigenesis would be desirable. Therefore, further characterization and safety evaluation of engineered limbal grafts is required to provide safe and high-quality therapeutic applications.MethodsAfter in vitro expansion, LSCs undergo laboratory characterization in a single-cell suspension, cell culture, and in limbal grafts before transplantation. Using a clinically applicable protocol, the data collected on LSCs at passage 1 were summarized, including: identity (cell size, morphology); potency (yield, viability, population doubling time, colony-forming efficiency); expression of putative stem cell markers through flow cytometry, immunofluorescence, and immunohistochemistry. Then, mitotic chromosome stability and normal mitotic outcomes were explored by using live-cell imaging. Finally, impurities, bacterial endotoxins and sterility were determined.ResultsExpression of the stemness marker p63 in single-cell suspension and in cell culture showed high values by different methods. Limbal grafts showed p63-positive cells (78.7 ± 9.4%), Ki67 proliferation (41.7 ± 15.9%), while CK3 was negative. Impurity with 3T3 feeder cells and endotoxins was minimized. We presented mitotic spindles with a length of 11.40 ± 0.54 m and a spindle width of 8.05 ± 0.55 m as new characterization in LSC culture. Additionally, live-cell imaging of LSCs (n = 873) was performed, and only a small fraction < 2.5% of aberrant interphase cells was observed; 2.12 ± 2.10% of mitotic spindles exhibited a multipolar phenotype during metaphase, and 3.84 ± 3.77% of anaphase cells had a DNA signal present within the spindle midzone, indicating a chromosome bridge or lagging chromosome phenotype.ConclusionThis manuscript provides, for the first time, detailed characterization of the parameters of fidelity of the mitotic process and mitotic spindle morphologies of LSCs used in a direct clinical application. Our data show that p63-positive CK3-negative LSCs grown in vitro for clinical purposes undergo mitotic processes with extremely high fidelity, suggesting high karyotype stability. This finding confirms LSCs as a high-quality and safe therapy for eye regeneration in humans.

Prediction of CHO cell line stability using expression of DNA repair genes.

Chinese hamster ovary (CHO) cells are essential to biopharmaceutical manufacturing and production instability, the loss of productivity over time, is a long-standing challenge in the industry. Accurate prediction of cell line stability could enable efficient screening to identify clones suitable for manufacturing saving significant time and costs. DNA repair genes may offer biomarkers to address this need. In this study, over 40 cell lines representing various host lineages from three companies/organizations were evaluated for expression of five DNA repair genes (Fam35a, Lig4, Palb2, Pari, and Xrcc6). Expression measured in cells with less than 30 population doubling levels (PDLs) was correlated to stability profiles at 60+ PDL. Principal component analysis identified markers which separate stable and unstable CHO-DG44 cell lines. Notably, two genes, Lig4 and Xrcc6, showed higher expression in unstable CHO-DG44 cell lines with copy number loss identified as the mechanism of production instability. Expression levels across all cell ages showed lower DNA repair gene expression was associated with increased cell age. Collectively, DNA repair genes provide critical insight into long-term behavior of CHO cells and their expression levels have potential to predict cell line stability in certain cases.

Extracellular Matrix Deposition Defines the Duration of Cell Sheet Assembly from Human Adipose-Derived MSC

Cell sheet (CS) engineering using mesenchymal stromal cells (MSC) draws significant interest for regenerative medicine and this approach translates to clinical use for numerous indications. However, little is known of factors that define the timing of CS assembly from primary cultures. This aspect is important for planning CS delivery in autologous and allogeneic modes of use. We used a comparative in vitro approach with primary donors’ (n = 14) adipose-derived MSCs and evaluated the impact of healthy subject’s sex, MSC culture features (population doubling time and lag-phase), and extracellular matrix (ECM) composition along with factors related to connective tissue formations (α-SMA and FAP-α) on CS assembly duration. Using qualitative and quantitative analysis methods, we found that, in seeded MSCs, high contents of collagen I and collagen IV had a direct correlation with longer CS assembly duration. We found that short lag-phase cultures faster turned to a ready-to-use CS, while age, sex, fibronectin, laminin, α-SMA, and FAP-α failed to provide a significant correlation with the timing of assembly. In detachable CSs, FAP-α was negatively correlated with the duration of assembly, suggesting that its concentration rose over time and contributed to MSC activation, transitioning to α-SMA-positive myofibroblasts and ECM turnover. Preliminary data on cell density and collagen I deposition suggested that the TGF-β1 signaling axis is of pivotal importance for ECM composition and construct maturation.

Chronic Alcohol Exposure Causes Myeloid-Biased Hematopoiesis Via Double-Strand RNA Sensors and Type 1 Interferon Response

Excessive alcohol use is well-known to cause cytopenias and bone marrow (BM) suppression. However, the mechanisms by which alcohol suppresses BM are poorly understood. Moreover, even though the prevalence of moderate drinking is high worldwide, the effects of chronic moderate alcohol consumption on the human hematopoietic system remain unknown. To fill this knowledge gap, we sought to investigate the effects of chronic moderate alcohol consumption on human hematopoiesis using cell-based and animal models. To examine the effects of alcohol on hematopoietic cells in vitro, we tested varying doses of alcohol (0-100 mM) on four leukemia cell lines (Jurkat, MOLM13, MV-4-11, and THP1) and measured population doubling over 6 days. Population doubling levels significantly decreased in a dose-dependent manner in all cell lines tested. We also measured mitochondrial (mt) ROS by MitoSOX-Red flow cytometry in Jurkat, which showed a dose-dependent increase of mtROS (trends only at 10, 25 mM; significantly increased at 50, 100 mM alcohol) compared to untreated. These findings confirm that alcohol negatively affects cell growth and increases mtROS in a physiologic range of doses. To determine the effects of alcohol on human hematopoietic stem progenitor cells (HSPCs) in vitro, we treated human BM CD34+ cells with 0 vs. 50 mM alcohol for 48 hours and performed bulk RNA-sequencing in triplicate. Gene Set Enrichment Analysis (GSEA) revealed that multiple pathways related to platelet activation and coagulation were enriched in alcohol-treated HSPCs, suggesting the induction of inflammation and/or platelet-biased differentiation by alcohol. To assess the effects of chronic moderate alcohol consumption on HSPCs in vivo, we employed a xenotransplant model. In brief, human BM CD34+ cells were transplanted to 5-to-6-week-old female NBSGW mice. We confirmed their engraftment by bone marrow aspiration at six weeks post-transplant, and then they were randomly assigned to the alcohol vs. control groups. After 2 weeks of adjustment, mice were fed a liquid diet (their only source of water and food) containing alcohol 5% vol/vol vs. iso-caloric control diet for 8 weeks. At 4 and 8 weeks, the alcohol-fed group showed significant myeloid bias, reaching greater than 3 times higher myeloid-to-lymphoid ratio than the control group at 8 weeks, while overall engraftment levels were not different. We will report the results of the secondary transplantation of this experiment at the annual meeting. After 8-week alcohol feeding, sorted human CD45+CD34+ cells from mouse BM were subject to 10x Genomics Chromium X 3' HT single-cell RNA-sequencing (scRNA-seq). Complementing the flow data, our transcriptomic data portrayed stark differences between the two groups. Over 50% of cells in the alcohol group comprised granulocyte progenitors, with B cell progenitors representing less than 25%. Inversely, control group data revealed approximately 30% granulocyte progenitors and 55% B cell progenitors, confirming the myeloid bias of HSPC caused by alcohol. Examining differentially expressed genes and GSEA highlighted a significant upregulation of genes associated with the response to virus and type 1 interferon (IFN-1) signature across all HSPC types. A closer look into the gene set showed increased expression of genes tied to cytosolic double-strand (ds) RNA sensing pathways and subsequent interferon type 1 response. Specifically, dsRNA sensors like MDA5, RIG1, PKR, and OAS1-3, as well as interferon regulatory or responsive genes, such as IRF7, ISG15, IFI6, IFI44L, MX1, and STAT1, were notably increased in HSCs and most progenitor populations. Unexpectedly, alcohol-exposed HSCs tended to be in the G0/G1 phase rather than the S or M phase compared to their counterpart upon cell cycle analysis using scRNA-seq data. This may be due to the activation of the PKR pathway, a known cell cycle inhibitor in mouse HSCs. In summary, our findings demonstrate that chronic moderate alcohol consumption can significantly reshape the hematopoietic system through myeloid-biased differentiation and potentiate inflammation by activating the cytosolic dsRNA sensing pathways and IFN-1 response.

IPSC-Derived KitD816V/Jak2V817F Double-Mutant Kitjak Cell Lines Allow Inexpensive Large-Scale Production of Enucleated Red Blood Cells in the Absence of SCF and Erythropoietin

Cultured Red Blood Cells (cRBCs) have the potential to supplement transfusion needs and to be used as drug carriers or immune tolerization agents. Several methods have been devised to amplify primary Hematopoietic Progenitor cells (HPCs) into cRBCs. Although these protocols have the potential to relieve local blood shortages, they are not ideal for large scale cRBC production because the primary HPCs must constantly be replenished. Immortalized human erythroid cell lines have been developed by overexpressing the E6/E7 proteins of human papilloma virus 16, c-Myc, Bcl-XL, Spi1 or Bmi-1. Many of these lines can be cultured for months and in some cases indefinitely, and some retain their ability to differentiate into cRBCs with rates of enucleation varying between 0.1-30%. These lines are useful as research tools and are a potential source of cells for some translational applications. Nevertheless, karyotypic instability, the cost of the media and cytokines, and cumbersome culture procedure to maintain some of these lines remain major obstacles for their large-scale use. We previously developed the Pluripotent Stem Cell Robust Erythroid Differentiation (PSC-RED) protocol to differentiate induced-pluripotent stem cells (iPSCs) into cRBCs. The PSC-RED protocol yields a large number of enucleated RBCs, does not require any albumin, and allows transferrin recycling. Despite these advantages, the PSC-RED protocol is not well-adapted for large-scale cRBC production because differentiation of an iPSC into a RBC takes up to 45 days and because of cytokine cost. To eliminate the requirement for Stem Cell Factor (SCF) and Erythropoietin (Epo) which accounts for more than 95% of total cytokine cost in the PSC-RED protocol, we have used CRSPR/cas9 technology to introduce in iPSCs the kitD816V mutation that allows the SCF receptor (the kit gene) to constitutive signal in the absence of SCF, and the Jak2V617F mutation that allows constitutive signaling through the JAK/STAT pathway in the absence of Epo. We report here that kitD816V, Jak2V617F double mutants iPSCs readily differentiate into self-renewing erythroid progenitors cells that can grow in the absence of any cytokines for &amp;gt;60 population doubling ( &amp;gt;10 19 fold expansion for &amp;gt;5 months) in an inexpensive culture medium containing little more than two small molecules and low amounts of recombinant transferrin. Importantly, KitJak cell lines can be reproducibly generated from iPSCs by a two-week differentiation procedure without any time lag, suggesting that no other mutations are necessary to generate these self-renewing erythroid progenitors. Indeed, we have shown using a next-generation sequencing approach that KitJak cells appear to be remarkably karyotypically stable since multiple experiments fail to detect any chromosomal aneuploidy after long-term culture. KitJak cells have an immuno-phenotype of late CFU-E/pro-Erythroblast, can be cultured at high density and can be induced to differentiate into cRBCs using a 10- to 12-day differentiation protocol that leads to a 20 to 40-fold cell amplification. The rate of enucleation is 30-40%, providing an overall yield of 7-14 RBCs per self-renewing KitJak cell. The cRBCs produced express mostly fetal hemoglobin, are slightly larger that adult cells, and have normal hemoglobin content. The development of these KitJak cell lines greatly facilitates the production of cRBCs and will pave the way to large-scale erythroid cell production for transfusion and other translational applications. As a first application, we show that the RBCs produced are fully compatible with the Immucor red blood cell typing system and can be used as reagent red blood cells.

Silicon application enhances wheat defence against Sitobion avenae F. by regulating plant physiological-biochemical responses

Sitobion avenae F. is a highly prevalent and devastating pest in wheat crops, leading to significant yield losses. Silicon (Si) has been widely recognized as an effective inducer of plant resistance against aphids. Nevertheless, the underlying mechanisms governing the physiological and biochemical responses of plants induced by Si defense against S. avenae F. remain incompletely understood. In this study, we conducted experiments by treating wheat leaves with varying concentrations of Tetraethyl orthosilicate (TEOS) spray under aphid infestation. We meticulously observed and recorded the life cycle of S. avenae F. and measured the content of plant hormones, secondary metabolites, and the activity of defense enzymes in wheat leaves. Furthermore, we utilized structural equation modeling to discern the causal correlation between aphid performance and the physiological-biochemical responses of wheat under TEOS sprays. Our findings revealed that a concentration of 3 mmol/L TEOS significantly shortened the net reproductive rate, intrinsic rate and finite rate of increase, and mean generation time of S. avenae F., while simultaneously prolonging the population doubling time. Additionally, the content of lignin, total phenolics, flavonoids, alkaloids, tannin, jasmonic acid (JA), and salicylic acid in wheat leaves exhibited a substantial increase. Furthermore, the activity of phenylalanine ammonia-lyase (PAL), polyphenol oxidase (PPO), catalase, and lipoxygenase in wheat leaves was significantly enhanced. Our results suggest that TEOS spray reduced the survival and population growth of S. avenae F. while enhancing the defense response of wheat against aphids by activating the activity of PAL and PPO in wheat, and increasing the content of total phenolic and JA. This work provides valuable insights for the development of appropriate Si fertilizers for effective pest management and offers robust theoretical support for wheat aphid control through agricultural fertilization strategies.

THE EFFECT OF CHRONOLOGICAL AGE AND GENDER AND DIFFERENT SOURCE OF TISSUE ON THE IN VITRO PROPERTIES OF MESENCHYMAL STROMAL CELLS

AbstractObjectivesOsteoarthritis is a common articular cartilage disorder and causes a significant global disease burden. Articular cartilage has a limited capacity of repair and there is increasing interest in the use of cell-based therapies to facilitate repair including the use of Mesenchymal Stromal Cells (MSCs). There is some evidence in the literature that suggests that advancing age and gender is associated with declining MSC function, including reduced proliferation and differentiation potential, and greater cellular apoptosis. In our study, we first performed a systematic review of the literature to determine the effects of chronological age and gender on the in vitro properties of MSCs, and then performed a laboratory study to investigate these properties.Methods and ResultsWe initially conducted a PRISMA systematic review of the literature to review the evidence base for the effects of chronological age and gender on the in vitro properties of MSCs including cell numbers, expansion, cell surface characterization and differentiation potential. This was followed by laboratory-based experiments to assess these properties. Compare the extent of the effect of age on MSC cell marker expression, proliferation and pathways. Tissue from patients undergoing total knee replacement surgery was used to isolate MSCs from the synovium, fat pad and bone fragments using a method developed in our laboratory. The growth kinetics was determined by calculating the population doublings per day. Following expansion in culture, MSCs at P2 were characterised for a panel of cell surface markers using flow cytometry. The cells were positive for CD73, CD90 and CD105, and negative for antibody cocktail (eg included CD34, CD45). The differentiation potential of the MSCs was assessed through tri-lineage differentiation assays. At P2 after extracting RNA, we investigate the gene analysis using Bulk seq. Clear differences between the younger and older patients and gender were indicated.ConclusionsChronological age and gender-related changes in MSC function have important implications on the use of these cells in clinical applications for an ageing population. The results from this study will be used to plan further work looking at the effects of chronological age and gender on cellular senescence and identify pathways that could be targeted to potentially reverse any age and gender-related changes.Declaration of Interest(b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project.

Effect of standard and physiological cell culture temperatures on in vitro proliferation and differentiation of primary broiler chicken pectoralis major muscle satellite cells.

Culture temperatures for broiler chicken cells are largely based on those optimized for mammalian species, although normal broiler body temperature is typically more than 3°C higher. The objective was to evaluate the effects of simulating broiler peripheral muscle temperature, 41°C, compared with standard temperature, 38°C, on the in vitro proliferation and differentiation of primary muscle-specific stem cells (satellite cells; SC) from the pectoralis major (PM) of broiler chickens. Primary SC cultures were isolated from the PM of 18-day-old Ross 708 × Yield Plus male broilers. SC were plated in triplicate, 1.8-cm2, gelatin-coated wells at 40,000 cells per well. Parallel plates were cultured at either 38°C or 41°C in separate incubators. At 48, 72, and 96h post-plating, the culture wells were fixed and immunofluorescence-stained to determine the expression of the myogenic regulatory factors Pax7 and MyoD as well as evaluated for apoptosis using a TUNEL assay. After 168h in culture, plates were immunofluorescence-stained to visualize myosin heavy chain and Pax7 expression and determine myotube characteristics and SC fusion. Population doubling times were not impacted by temperature (p ≥ 0.1148), but culturing broiler SC at 41°C for 96h promoted a more rapid progression through myogenesis, while 38°C maintained primitive populations (p ≤ 0.0029). The proportion of apoptotic cells increased in primary SC cultured at 41°C (p ≤ 0.0273). Culturing at 41°C appeared to negatively impact fusion percentage (p < 0.0001) and tended to result in the formation of thinner myotubes (p = 0.061) without impacting the density of differentiated cells (p = 0.7551). These results indicate that culture temperature alters primary broiler PM SC myogenic kinetics and has important implications for future in vitro work as well as improving our understanding of how thermal manipulation can alter myogenesis patterns during broiler embryonic and post-hatch muscle growth.

Development and reproduction of Tetranychus cinnabarinus (Acari: Tetranychidae) on transgenic insect-resistant cotton cultivars

Effects of transgenic insect-resistant Bt cotton Zhongkangza 5 and Lumianyan 23 and Bt + CpTI cotton sGK958 and Zhongmiansuo 45 on Tetranychus cinnabarinus growth and reproduction were compared with those of nontransgenic cotton (K836) under laboratory conditions. Developmental times of different T. cinnabarinus stages fed on transgenic Bt and Bt + CpTI cotton differed significantly from those fed on K836. Pre-oviposition (1.44 d) and oviposition (12.71 d) periods and female life span (14.11 d) were longer on K836 than on transgenic cotton. Maximum fecundity was 75.61 eggs per female on K836, while minimum fecundity was 54.68 eggs per female on sGK958. Net reproductive rate was highest with 55.00 female offspring on K836, whereas the lowest was 37.219 female offspring on Zhongkangza 5. The highest intrinsic rate of increase was 0.454 day−1 on K836, whereas the lowest was 0.246 day−1 on Zhongkangza 5. The highest finite rate of increase was 1.575 day−1 on K836, whereas the lowest was 1.278 day−1 on Zhongkangza 5. The shortest population doubling time was 1.526 d on K836, while the longest was 2.823 d on Zhongkangza 5. Thus, transgenic cotton inhibited T. cinnabarinus development and reproduction, indicating transgenic cotton could prevent damage by T. cinnabarinus or other pests.

Fratricide-Resistant Anti-CD2 Chimeric Antigen Receptor T-Cells with Endogenous CD2 Knockout Are Highly Effective Against T-Cell Neoplasms

T cell neoplasms, such as peripheral T cell lymphomas (PTCL) and acute T cell lymphoblastic leukemia (T-ALL), have some of the worst prognoses of all hematological malignancies. Poor outcomes are driven by a limited number of effective treatments, particularly in relapsed and refractory settings. While chimeric antigen receptor T cell (CAR-T) immunotherapy has been successful in other hematological diseases, its clinical use against T cell neoplasms is limited due to a lack of unique tumor-specific antigens. Additionally, CAR specificity against pan-T cell antigens causes CAR-T fratricide and insufficient manufacturability for therapeutic dosing. Early studies of CAR-T against CD5- and CD7-expressing T cell neoplasms have shown responses, however, 1) most patients ultimately relapse, and 2) in some patients, antigen negative relapse has occurred. To circumvent these obstacles, we hypothesized that targeting CD2, a pan-T cell antigen, using anti-CD2 CAR-T cells that are engineered to lack CD2, would permit effective CAR-T manufacturing against T cell neoplasms. In a retrospective analysis of clinical flow cytometry diagnostic samples, we found CD2 expression in 25 of 33 (75.8%) and 37 of 51 (72.5%) pediatric PTCL and T-ALL cases, respectively. Similarly, we found 82 of 89 (92.1%) adult PTCL cases with CD2 immunohistochemical expression, which exceeded CD7 expression (38 of 90, 42.2%) that is currently being evaluated in early phase clinical trials. We optimized early CD2 knockout (KO) during CAR-T cell manufacturing from human donor T cells (KO efficiency: 81.2% ± 2.4, n=9 donors). We next engineered a best-in-class, 4-1BB-costimulated, second-generation CD2KO anti-CD2 CAR-T product (CART2) via lentiviral transduction ( PANEL A) using seven distinct single-chain variable fragments (transduction efficiency: 60.2% ± 5.5, n=7 donors) based on cellular expansion and stressed in vivo efficacy against CD2+ T-ALL cells (Jurkat). There were no significant differences in population doublings during CART2 expansion as compared to both CD2 wild-type (CD2 WT) untransduced T cells (Mock UTD) and CD2KO untransduced T cells (CD2KO UTD) (6.20 ± 0.42 vs. 6.12 ± 0.62 vs. 6.67 ± 0.68, respectively, Day 15, n=3 donors), while CD2 WT CART2 cells failed to expand due to fratricide. CART2 cells had no significant differences in T cell memory phenotypes or T cell exhaustion markers, including PD1 and LAG3 expression, at the end of the expansion. Expanded CART2 cells eradicated primary patient-derived CD2+ Sezary cells and CD2+ T-ALL leukemic blasts as measured by in vitro cytotoxicity assays. Cytotoxicity correlated with CD8+ CART2 cells expressing IL-2 (20.4% vs. 0.2% vs. 0.35%), TNFα (28.5% vs. 0.27% vs. 0.2%), and IFNγ (11.27% vs. 0.18% vs. 0.14%) relative to Mock UTD and CD2KO UTD controls stimulated with CD2+ Jurkat cells (n=2 donors). Importantly, we also found both CD2KO UTD and CART2 cells were capable of TNFα and IFNγ release upon interaction with cytomegalovirus and influenza peptides, suggesting that T cells with CD2 perturbations are still able to mediate effector T cell responses. We further demonstrated that CART2 cells were highly effective in controlling systemic tumor burden of T-ALL patient-derived xenograft mouse (PDX) models and significantly improved median overall survival as compared to Mock UTD and CD2KO UTD controls (138 days vs. 30 days vs. 30 days, p=0.0007, Mantel-Cox) ( PANEL B). Long-term tumor control was proportional to CART2 peripheral blood persistence at Day 28 (CD2KO UTD: 186.6 ± 83.7, n=5 vs. CART2: 936.3 ± 241.7, n=5) and Day 100 (CD2KO UTD: N/A vs. CART2: 514.0 ± 441, n=3). We also found that rescue infusion of CART2 could eliminate relapsed T-ALL in PDX mice previously in a remission after initial treatment with anti-CD5 CAR-T cells, with long-term persistence of CD2-negative T cells thereafter. In conclusion, our data demonstrate that CART2 are manufacturable and highly efficacious against T-cell neoplasms. To our knowledge, this is the first report of prolonged efficacy using a CD2-targeted cellular monotherapy in human leukemia preclinical models. This product could be delivered alone or in combination with additional CARs for dual targeting, such as anti-CD5 or anti-CD7 CARs, to reduce the potential of antigen-negative escape. Further research is ongoing to study the effect of CD2 deletion in anti-CD19 CAR T cells and the possible implication for clinical translation.

Melphalan-Induced Multiple Myeloma Cells Exhibit a Senescent-like Dormant Phenotype

Multiple myeloma (MM) is an incurable plasma cell malignancy. Despite therapeutic advances that have vastly improved survival, all patients are expected to relapse. One mechanism that may contribute to MM relapse is tumor dormancy. Dormant MM cells may persist in the bone marrow, where they exhibit reduced proliferative capacity and resistance to treatment. These cells may then exit dormancy following treatment to drive disease relapse. Dormancy shares many features with cellular senescence, a state of stable growth arrest induced by cell stress. Chemotherapies that produce genotoxic stress have been associated with therapy-induced senescence (TIS). One such genotoxic agent, melphalan (Mel), is routinely administered to MM patients for myeloablation prior to autologous bone marrow transplant. We hypothesized that High Dose Mel (HDM) induces a senescent-like dormant phenotype in surviving MM cells. To test our hypothesis, we developed an in vitro model for HDM. We identified 10µM Mel as a concentration to induce &amp;gt;90% cell toxicity (HDM) in 5TGM1 mouse MM cells over 10 days. 5TGM1-GFP MM cells were cultured with HDM or vehicle (Veh) for 6 hours; cells were then cultured in normal growth medium in the presence of primary mouse (C57BL/6) bone marrow stromal cells (BMSCs). Co-cultures were imaged by fluorescent microscopy every other day to quantify cumulative population doublings (CPD) and cell morphology over the following 10 days. The effect of HDM on CPDs was also evaluated in the human RPMI-8226 MM cell line. 5TGM1-GFP cells were isolated by FACS on day 10 for evaluation of senescence histological characteristics and gene expression. All studies represent n=3 independent experiments unless otherwise indicated. HDM-5TGM1 cells exhibited a significant reduction in mean CPDs over 10 days compared to Veh (HDM 0.32±0.31, Veh 6.72±0.69, p&amp;lt;0.0001), indicating stable growth arrest in HDM treated cells consistent with senescence. HDM-5TGM1 cells were also significantly larger versus Veh (median size in pixels units: HDM 413.2±99.98, Veh 207±8.88, p&amp;lt;0.05), consistent with classical enlarged morphology in senescent cells. Preliminary assessment (n=1) of HDM-RPMI-8226 cells showed similar growth arrest (mean cumulative doublings: HDM 0.058±0.101, Veh 4.185±0.358) and enlargement (mean size in pixel units: HDM 804.1±6.048, Veh 526.9±2.115), suggesting a translatable mechanism in humans. FACS-sorted 5TGM1-GFP cells were stained for telomere associated foci (TAF), a marker of persistent DNA damage characteristic of senescent cells. HDM-5TGM1 cells showed a higher percentage of TAF+ cells (Percent TAF+ cells: HDM 39.7%±9.1%, Veh 10.6%±0.8%, n=2). Sorted HDM-5TGM1 cells also exhibited significant increases over Veh (p&amp;lt;0.001) in senescence markers ( Cdkn1a, Cdkn1c, Glb1), senescence cell anti-apoptosis pathway genes (SCAPs, Bcl2l1), and senescence associated secretory phenotype (SASP) genes ( Ccl5, Icam1, Mmp13). In addition, HDM-5TGM1 cells showed significant increases in myeloid markers that have previously been shown to be increased in dormant 5TGM1 ( Axl, Fcer1g, Mpeg1). Single cell RNA-sequencing was used to further evaluate senescence gene expression changes in growth arrested HDM-5TGM1 cells. Gene set enrichment analysis (GSEA) of genes differentially expressed in HDM-5TGM1 versus Veh cells was performed for custom senescence phenotyping gene sets (Senescence Up, SenUp; Senescence Growth Arrest, SenGA; Inflammatory SASP, iSASP; SCAPs; and Plasma Cell Senescence; PCSen) as well as mouse ortholog versions of a published senescence and biological aging gene set (SenMayo) and KEGG curated gene sets. GSEA of HDM-5TGM1 versus Veh cells confirmed significant (FDR&amp;lt;0.25) upregulation of iSASP, MayoSen, PCSen, SenUp, SCAPs, and SenGA gene sets. Concurrently, GSEA revealed significant downregulation of KEGG DNA replication, cell cycle, and nucleotide excision repair gene sets, supporting our observations of proliferative arrest and persistent DNA damage TAF. Our findings demonstrate that HDM-MM cells exhibit multiple features of senescence, suggesting a potential mechanistic role for TIS in MM dormancy and highlight a novel therapeutic target that may be present in patients post-HDM. Thus, targeting senescence may be a novel approach to eliminate dormant MM cells and prevent relapse.

Evaluation of Telenomusremus (Hymenoptera: Platygastridae) as a biocontrol agent of Spodopteralitura (Lepidoptera: Noctuidae) based on two-sex life table and functional response analyses

BackgroundTelenomusremus Nixon is an important egg parasitoid of Spodoptera spp. pests and, as such, has potential as a biological control agent. Spodopteralitura (Fabricius) is a significant pest of many economically important crops worldwide. This study was conducted to evaluate the demographic parameters and functional response of T.remus on the S.litura eggs.ResultsT.remus can lay 186.90 eggs/female in the lifetime, adult preoviposition period was 0 days, total preoviposition period was 10.03 days, and the ratio of female and male offspring was 0.495 and 0.421, respectively. In addition, most females emerged from 24 h-old eggs, whereas most males emerged from 48 h-old eggs. The intrinsic rate of increase, finite rate of increase, net reproductive rate, mean generation time, and population doubling time were 0.3506 d–1 1.4199 d–1 92.45 offspring/individua, 12.91 days and 1.98 days, respectively. The net killing rate of T.remus on S.litura was 101.49 eggs/female, indicating the high capacity of T.remus to parasitize S.litura eggs. Moreover, the higher the egg density, the higher the parasitism rate by female T.remus, although there was a trend of parasitism stabilization at an egg density of 100, indicating a type II functional response curve for this parasitoid.ConclusionOverall, these findings suggest that T.remus can be efficiently reared on S.litura eggs and shows potential as biocontrol agent for this economically important pest species.

Xanthium strumarium seed extract boosts osteogenesis in human dental pulp stem cell model

BackgroundIn traditional medicine, Xanthium strumarium is used as an anti-inflammatory and anti-arthritic plant-based medicine. Human Dental Pulp Stem Cells (hDPSCs) are an ideal in vitro model for drug and bioactive compound screening. This study assessed the potential of X. strumarium aqueous extract on hDPSCs differentiation towards the osteogenic lineage. Materials and methodsHDPSCs were isolated and cultured by explant method and characterized by surface marker expression, Colony Forming units fibroblasts (CFU-F), Population Doubling time (PDT), and tri-lineage differentiation. X. strumarium aqueous seed extract (XSE) was prepared and its cytotoxic effect on hDPSCs was examined by MTT assay. The effect of XSE on hDPSC differentiation into osteocytes was investigated by biochemical staining and gene expression. ResultsThe hDPSCs were positive for CD73, CD90, and CD105 and negative for CD34, CD45, and HLA-DR surface markers. The cells had a colony-forming ability with a PDT of 44.91 h. The hDPSCs differentiated into osteocytes, chondrocytes, and adipocytes. The XSE concentration of 15 μg/ml had a significant increase in hDPSC viability. Alizarin Red S staining revealed that XSE treatment enhanced calcium accumulation and matrix mineralization in hDPSCs. XSE treatment also increased osteonectin and IL-6 transcript expression in osteogenesis-induced hDPSCs. ConclusionX. strumarium aqueous extract is a suitable candidate for bone repair because it promotes osteogenic differentiation in hDPSCs. Therefore this could be explored further in the treatment of bone disorders.

Long-term cryopreservation of whole gingival tissue.

Stem cells obtained from the body tissue, such as adipose tissue, dental pulp and gingival tissue. Fresh tissue is often used to isolate and culture for regenerative medicine. However, availability of tissue as and when required is one of the measure issue in regenerative medicine. Cryopreservation of tissue provides benefit over tissue availability, storage for significant amount of period and helps preserve the original cell structures. The effects of cryopreservation of gingival tissue for mesenchymal stem cell (MSC) are not well documented; however this process is of increasing importance for regenerative therapies. This study examined the effect of cryopreservation on the long term survival the whole gingival biopsy tissue. We studied cell outgrowth, cell morphology, MSC surface-markers and differentiation of mesenchymal stem cells derived from cryopreserved gingiva. In this study, gingival tissue was cryopreserved for 3, 6, 9months. Cryopreserved tissue has been thawed and cells were isolated by using explant culture method. The fresh and cryopreserved gingival tissue cells were cultured and characterized for surface marker analysis, CFU-f, population doubling time, and osteogenic, chondrogenic and adipogenic differentiation. The fresh and cryopreserved tissue has similar stem cell properties. Results indicate that cryopreservation of the entire gingival tissue does not affect the properties of stem cells. This opens door for gingival tissue banking for future use in periodontology and regenerative medicine.

Clonal and Scalable Endothelial Progenitor Cell Lines from Human Pluripotent Stem Cells.

Human pluripotent stem cells (hPSCs) can be used as a renewable source of endothelial cells for treating cardiovascular disease and other ischemic conditions. Here, we present the derivation and characterization of a panel of distinct clonal embryonic endothelial progenitor cells (eEPCs) lines that were differentiated from human embryonic stem cells (hESCs). The hESC line, ESI-017, was first partially differentiated to produce candidate cultures from which eEPCs were cloned. Endothelial cell identity was assessed by transcriptomic analysis, cell surface marker expression, immunocytochemical marker analysis, and functional analysis of cells and exosomes using vascular network forming assays. The transcriptome of the eEPC lines was compared to various adult endothelial lines as well as various non-endothelial cells including both adult and embryonic origins. This resulted in a variety of distinct cell lines with functional properties of endothelial cells and strong transcriptomic similarity to adult endothelial primary cell lines. The eEPC lines, however, were distinguished from adult endothelium by their novel pattern of embryonic gene expression. We demonstrated eEPC line scalability of up to 80 population doublings (pd) and stable long-term expansion of over 50 pd with stable angiogenic properties at late passage. Taken together, these data support the finding that hESC-derived clonal eEPC lines are a potential source of scalable therapeutic cells and cell products for treating cardiovascular disease. These eEPC lines offer a highly promising resource for the development of further preclinical studies aimed at therapeutic interventions.

In-vitro Augmentation of Mesenchymal Stem Cells by Using Adult Bovine Serum.

Umbilical cord mesenchymal stem cells (UC-MSCs) are increasingly being utilized for immune-related disease therapies due to their low immunogenicity. However, the primary culture of UC-MSCs requires the supplementation of serum in the growth medium, which has posed a challenge due to ethical issues related to the collection method of the fetal bovine serum (FBS) that is routinely used in cell culture. In order to address this, the purpose of this research was to assess the effectiveness of adult bovine serum (ABS) as a different and more affordable source of serum for the in-vitro cultivation of UC-MSCs. UC-MSCs were isolated from the umbilical cord of Wharton's jelly of cow immediately after birth, by digestion with Collagenase type I. ABS was collected from fresh bovine sources and heat-inactivated. The morphology of UC-MSCs was observed under an inverted microscope, and growth patterns, proliferative index, and doubling time were calculated every two days to compare the efficacy of ABS with FBS. Immunocytochemistry for specific markers was also conducted on the MSCs. The results showed a notable difference in morphology, growth rate, population doubling, and proliferative index between ABS and FBS. Intriguingly, ABS proved to be an effective supplement in the growth medium for expanding UC-MSCs in vitro, providing a viable alternative to FBS.

One Hundred Priority Questions for the Development of Sustainable Food Systems in Sub-Saharan Africa

Sub-Saharan Africa is facing an expected doubling of human population and tripling of food demand over the next quarter century, posing a range of severe environmental, political, and socio-economic challenges. In some cases, key Sustainable Development Goals (SDGs) are in direct conflict, raising difficult policy and funding decisions, particularly in relation to trade-offs between food production, social inequality, and ecosystem health. In this study, we used a horizon-scanning approach to identify 100 practical or research-focused questions that, if answered, would have the greatest positive impact on addressing these trade-offs and ensuring future productivity and resilience of food-production systems across sub-Saharan Africa. Through direct canvassing of opinions, we obtained 1339 questions from 331 experts based in 55 countries. We then used online voting and participatory workshops to produce a final list of 100 questions divided into 12 thematic sections spanning topics from gender inequality to technological adoption and climate change. Using data on the background of respondents, we show that perspectives and priorities can vary, but they are largely consistent across different professional and geographical contexts. We hope these questions provide a template for establishing new research directions and prioritising funding decisions in sub-Saharan Africa.

Establishment and characterization of a skeletal myoblast cell line of grass carp (Ctenopharyngodon idellus).

Skeletal muscle myoblastic cell lines can provide a valuable new in vitro model for the exploration of the mechanisms that control skeletal muscle development and its associated molecular regulation. In this study, the skeletal muscle tissues of grass carp were digested with trypsin and collagenase I to obtain the primary myoblast cell culture. Myoblast cells were obtained by differential adherence purification and further analyzed by cryopreservation and resuscitation, chromosome analysis, immunohistochemistry, and immunofluorescence. A continuous grass carp myoblast cell line (named CIM) was established from grass carp (Ctenopharyngodon idellus) muscle and has been subcultured > 100 passages in a year and more. The CIM cells revived at 79.78-95.06% viability after 1-6 months of cryopreservation, and shared a population doubling time of 27.24 h. The number of modal chromosomes of CIM cells was 48, and the mitochondrial 12S rRNA sequence of the CIM cell line shared 99% identity with those of grass carp registered in GenBank. No microorganisms (bacteria, fungi, or mycoplasma) were detected during the whole study. The cell type of CIM cells was proven to be myoblast by immunohistochemistry of specific myogenic protein markers, including CD34, desmin, MyoD, and MyHC, as well as relative expression of key genes. And the myogenic rate and fusion index of this cell line after 10 days of induced differentiation were 8.96 ~ 9.42% and 3-24%, respectively. The telomerase activity and transfection efficiency of CIM cell line were 0.027 IU/mgprot and 23 ~ 24%, respectively. These results suggest that a myoblast cell line named CIM with normal biological function has been successfully established, which may provide a valuable tool for related in vitro studies.

Urban scale, production efficiency, and dynamic development paths

The rise and fall of a city is the inevitable result of its response to sustainable development over time. Using the scaling analysis framework of urban total factor productivity (TFP), we find that it can reveal how the rise and fall of a city occurs and changes on a time scale. We estimate the scaling of TFP for 299 prefecture-level cities in China based on urban wage and labor force data from 1999 to 2019. We show that a doubling of urban population increases productivity by about 18 %. The estimation of the scaling helps us identify which cities are more advantaged. Surprisingly, resource-based cities are typical examples of high-productivity cities, with an average efficiency advantage of 6.8 % higher. However, there are significant differences within resource-based cities, which seem to show advantages only in growth and maturity cities. We also describe the trajectory of urban development based on the local dynamics of urban wages and labor and discuss the general trend and potential mechanisms behind this process. In conclusion, our analysis of urban economic efficiency is conducive to understanding the rise-and-fall of urban development, and provides new methods, ideas and research perspectives for describing this dynamic. Most importantly, we find the difference between developing and developed countries is whether local dynamics can be maintained for a long time. It is of great significance to enrich the research of urban scaling theory in developing countries.