Different Stages of Quiescence,Senescence, and Cell Stress Identified by Molecular Algorithm Based on the Expression of Ki67, RPS6, and Beta-Galactosidase Activity

During their life span, cells have two possible states: a non-cycling, quiescent state (G0) and a cycling, activated state. Cells may enter a reversible G0 state of quiescence or, alternatively, they may undergo an irreversible G0 state. The latter may be a physiological differentiation or, following a stress event, a senescent status. Discrimination among the several G0 states represents a significant investigation, since quiescence, differentiation, and senescence are progressive phenomena with intermediate transitional stages. We used the expression of Ki67, RPS6, and beta-galactosidase to identify healthy cells that progressively enter and leave quiescence through G0-entry, G0 and G0-alert states. We then evaluated how cells may enter senescence following a genotoxic stressful event. We identified an initial stress stage with the expression of beta-galactosidase and Ki67 proliferation marker. Cells may recover from stress events or become senescent passing through early and late senescence states. Discrimination between quiescence and senescence was based on the expression of RPS6, a marker of active protein synthesis that is present in senescent cells but absent in quiescent cells. Even taking into account that fixed G0 states do not exist, our molecular algorithm may represent a method for identifying turning points of G0 transitional states that continuously change.     

Digital PCR Panel for Sensitive Hematopoietic Chimerism Quantification after Allogeneic Stem Cell Transplantation

Accurate and sensitive determination of hematopoietic chimerism is a crucial diagnostic measure after allogeneic stem cell transplantation to monitor engraftment and potentially residual disease. Short tandem repeat (STR) amplification, the current “gold standard” for chimerism assessment facilitates reliable accuracy, but is hampered by its limited sensitivity (≥1%). Digital PCR (dPCR) has been shown to combine exact quantification and high reproducibility over a very wide measurement range with excellent sensitivity (routinely ≤0.1%) and thus represents a promising alternative to STR analysis. We here aimed at developing a whole panel of digital-PCR based assays for routine diagnostic. To this end, we tested suitability of 52 deletion/insertion polymorphisms (DIPs) for duplex analysis in combination with either a reference gene or a Y-chromosome specific PCR. Twenty-nine DIPs with high power of discrimination and good performance were identified, optimized and technically validated. We tested the newly established assays on retrospective patient samples that were in parallel also measured by STR amplification and found excellent correlation. Finally, a screening plate for initial genotyping with DIP-specific duplex dPCR assays was designed for convenient assay selection. In conclusion, we have established a comprehensive dPCR system for precise and high-sensitivity measurement of hematopoietic chimerism, which should be highly useful for clinical routine diagnostics.     

Identification of Proteins Modulated in the Date Palm Stem Infested with Red Palm Weevil (Rhynchophorus ferrugineus Oliv.) Using Two Dimensional Differential Gel Electrophoresis and Mass Spectrometry

A state of the art proteomic methodology using Matrix Assisted Laser Desorption/Ionization-Time of Flight (MALDI TOF) has been employed to characterize peptides modulated in the date palm stem subsequent to infestation with red palm weevil (RPW). Our analyses revealed 32 differentially expressed peptides associated with RPW infestation in date palm stem. To identify RPW infestation associated peptides (I), artificially wounded plants (W) were used as additional control beside uninfested plants, a conventional control (C). A constant unique pattern of differential expression in infested (I), wounded (W) stem samples compared to control (C) was observed. The upregulated proteins showed relative fold intensity in order of I > W and downregulated spots trend as W > I, a quite interesting pattern. This study also reveals that artificially wounding of date palm stem affects almost the same proteins as infestation; however, relative intensity is quite lower than in infested samples both in up and downregulated spots. All 32 differentially expressed spots were subjected to MALDI-TOF analysis for their identification and we were able to match 21 proteins in the already existing databases. Relatively significant modulated expression pattern of a number of peptides in infested plants predicts the possibility of developing a quick and reliable molecular methodology for detecting plants infested with date palm.     

Three-dimensional imaging of human stem cells using soft X-ray tomography

Three-dimensional imaging of human stem cells using transmission soft X-ray tomography (SXT) is presented for the first time. Major organelle types—nuclei, nucleoli, mitochondria, lysosomes and vesicles—were discriminated at approximately 50 nm spatial resolution without the use of contrast agents, on the basis of measured linear X-ray absorption coefficients and comparison of the size and shape of structures to transmission electron microscopy (TEM) images. In addition, SXT was used to visualize the distribution of a cell surface protein using gold-labelled antibody staining. We present the strengths of SXT, which include excellent spatial resolution (intermediate between that of TEM and light microscopy), the lack of the requirement for fixative or contrast agent that might perturb cellular morphology or produce imaging artefacts, and the ability to produce three-dimensional images of cells without microtome sectioning. Possible applications to studying the differentiation of human stem cells are discussed.     

The Bulk Osteosarcoma and Osteosarcoma Stem Cell Activity of a Necroptosis-Inducing Nickel(II)–Phenanthroline Complex

We report the anti-osteosarcoma and anti-osteosarcoma stem cell (OSC) properties of a nickel(II) complex, 1 . Complex 1 displays similar potency towards bulk osteosarcoma cells and OSCs, in the micromolar range. Notably, 1 displays similar or better OSC potency than the clinically approved platinum(II) anticancer drugs cisplatin and carboplatin in two- and three-dimensional osteosarcoma cell cultures. Mechanistic studies revealed that 1 induces osteosarcoma cell death by necroptosis, an ordered form of necrosis. The nickel(II) complex, 1 triggers necrosome-dependent mitrochondrial membrane depolarisation and propidium iodide uptake. Interestingly, 1 does not evoke necroptosis by elevating intracellular reactive oxygen species (ROS) or hyperactivation of poly ADP ribose polymerase (PARP-1). ROS elevation and PARP-1 activity are traits that have been observed for established necroptosis inducers such as shikonin, TRAIL and glutamate. Thus the necroptosis pathway evoked by 1 is distinct. To the best of our knowledge, this is the first report into the anti-osteosarcoma and anti-OSC properties of a nickel complex.     

Cell Sheet Comprised of Mesenchymal Stromal Cells Overexpressing Stem Cell Factor Promotes Epicardium Activation and Heart Function Improvement in a Rat Model of Myocardium Infarction

Cell therapy of the post-infarcted myocardium is still far from clinical use. Poor survival of transplanted cells, insufficient regeneration, and replacement of the damaged tissue limit the potential of currently available cell-based techniques. In this study, we generated a multilayered construct from adipose-derived mesenchymal stromal cells (MSCs) modified to secrete stem cell factor, SCF. In a rat model of myocardium infarction, we show that transplantation of SCF producing cell sheet induced activation of the epicardium and promoted the accumulation of c-kit positive cells in ischemic muscle. Morphometry showed the reduction of infarct size (16%) and a left ventricle expansion index (0.12) in the treatment group compared to controls (24–28%; 0.17–0.32). The ratio of viable myocardium was more than 1.5-fold higher, reaching 49% compared to the control (28%) or unmodified cell sheet group (30%). Finally, by day 30 after myocardium infarction, SCF-producing cell sheet transplantation increased left ventricle ejection fraction from 37% in the control sham-operated group to 53%. Our results suggest that, combining the genetic modification of MSCs and their assembly into a multilayered construct, we can provide prolonged pleiotropic effects to the damaged heart, induce endogenous regenerative processes, and improve cardiac function.     

Gestational Exposure to Bisphenol A Affects Testicular Morphology,Germ Cell Associations,and Functions of Spermatogonial Stem Cells in Male Offspring

Exposure to bisphenol A (BPA) in the gestational period damages the reproductive health of offspring; detailed evidence regarding BPA-induced damage in testicular germ cells of offspring is still limited. In this study, pregnant mice (F0) were gavaged with three BPA doses (50 μg, 5 mg, and 50 mg/kg body weight (bw)/day; tolerable daily intake (TDI), no-observed-adverse-effect-level (NOAEL), and lowest-observed-adverse-effect level (LOAEL), respectively) on embryonic days 7 to 14, followed by investigation of the transgenerational effects of such exposure in male offspring. We observed that the NOAEL- and LOAEL-exposed F1 offspring had abnormalities in anogenital distance, nipple retention, and pubertal onset (days), together with differences in seminiferous epithelial stages and testis morphology. These effects were eradicated in the next F2 and F3 generations. Moreover, there was an alteration in the ratio of germ cell population and the apoptosis rate in germ cells increased in F1 offspring at the LOAEL dose. However, the total number of spermatogonia remained unchanged. Finally, a reduction in the stemness properties of spermatogonial stem cells in F1 offspring was observed upon LOAEL exposure. Therefore, we provide evidence of BPA-induced disruption of physiology and functions in male germ cells during the gestational period. This may lead to several reproductive health issues and infertility in offspring.     

Immunogenic Cell Death of Breast Cancer Stem Cells Induced by an Endoplasmic Reticulum-Targeting Copper(II) Complex

Immunogenic cell death (ICD) offers a method of stimulating the immune system to attack and remove cancer cells. We report a copper(II) complex containing a Schiff base ligand and a polypyridyl ligand, 4 , capable of inducing ICD in breast cancer stem cells (CSCs). Complex 4 kills both bulk breast cancer cells and breast CSCs at sub-micromolar concentrations. Notably, 4 exhibits greater potency (one order of magnitude) towards breast CSCs than salinomycin (an established breast CSC-potent agent) and cisplatin (a clinically approved anticancer drug). Epithelial spheroid studies show that 4 is able to selectively inhibit breast CSC-enriched HMLER-shEcad spheroid formation and viability over non-tumorigenic breast MCF10 A spheroids. Mechanistic studies show that 4 operates as a Type II ICD inducer. Specifically, 4 readily enters the endoplasmic reticulum (ER) of breast CSCs, elevates intracellular reactive oxygen species (ROS) levels, induces ER stress, evokes damage-associated molecular patterns (DAMPs), and promotes breast CSC phagocytosis by macrophages. As far as we are aware, 4 is the first metal complex to induce ICD in breast CSCs and promote their engulfment by immune cells.     

异嗪皮啶对人乳腺癌干细胞凋亡相关基因Bcl-2与Caspase家族表达的影响

目的: 研究草珊瑚中异嗪皮啶成分对乳腺癌干细胞中凋亡相关基因通路Bcl-2、Caspase-3、Caspase-8基因表达影响，进而明确其抑制增殖、迁移和促凋亡的机制。方法: 无血清培养法诱导MDA-MB-231细胞株富集乳腺癌干细胞，流式细胞仪分选CD44⁺ /CD24 ^-/low干细胞群。各组分别给予0、17、50、150、450 μmol/L异嗪皮啶，CCK-8法观察给药后24、48、72 h的细胞活力；细胞划痕实验法检测给药后细胞痕道宽度变化值并判断其对细胞迁移能力的影响；Annexin V-FITC/PI染色法检测给药后细胞总凋亡率变化；实时荧光定量PCR测定各组Bcl-2、Caspase-3、Caspase-8基因的mRNA水平；Western blot检测上述基因的蛋白水平。结果: 与对照组比较，50、150和450 μmol/L异嗪皮啶组24、48、72 h的细胞活力降低，细胞迁移能力下降。与对照组比较，50、150和450 μmol/L异嗪皮啶组细胞总凋亡率高于对照组，Bcl-2基因mRNA和蛋白表达水平降低，Caspase-3和Caspase-8基因mRNA和蛋白表达水平升高。以上结果均呈明显剂量效应关系。结论: 异嗪皮啶能通过下调Bcl-2基因表达与激活Caspase基因家族诱导乳腺癌干细胞的凋亡，同时能抑制其增殖与迁移。