scholarly journals Comparing the cytotoxicity of electronic cigarette fluids, aerosols and solvents

2017 ◽  
Vol 27 (3) ◽  
pp. 325-333 ◽  
Author(s):  
Rachel Z Behar ◽  
Yuhuan Wang ◽  
Prue Talbot
Keyword(s):  
Stem Cells ◽  
Cultured Cells ◽  
Low Voltage ◽  
Embryonic Stem ◽  
Electronic Cigarette ◽  
Lung Epithelial Cells ◽  
Potential Health ◽  
Differentiated Cells ◽  
Do It Yourself

BackgroundAs thousands of electronic cigarette (e-cigarette) refill fluids continue to be formulated and distributed, there is a growing need to understand the cytotoxicity of the flavouring chemicals and solvents used in these products to ensure they are safe. The purpose of this study was to compare the cytotoxicity of e-cigarette refill fluids/solvents and their corresponding aerosols using in vitro cultured cells.MethodsE-cigarette refill fluids and do-it-yourself products were screened in liquid and aerosol form for cytotoxicity using the MTT (3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide) assay. The sensitivity of human pulmonary fibroblasts, lung epithelial cells (A549) and human embryonic stem cells to liquids and aerosols was compared. Aerosols were produced using Johnson Creek’s Vea cartomizer style e-cigarette.ResultsA hierarchy of potency was established for the aerosolised products. Our data show that (1) e-cigarette aerosols can produce cytotoxic effects in cultured cells, (2) four patterns of cytotoxicity were found when comparing refill fluids and their corresponding aerosols, (3) fluids accurately predicted aerosol cytotoxicity 74% of the time, (4) stem cells were often more sensitive to aerosols than differentiated cells and (5) 91% of the aerosols made from refill fluids containing only glycerin were cytotoxic, even when produced at a low voltage.ConclusionsOur data show that various flavours/brands of e-cigarette refill fluids and their aerosols are cytotoxic and demonstrate the need for further evaluation of e-cigarette products to better understand their potential health effects.

scholarly journals Defining totipotency using criteria of increasing stringency

2020 ◽  
Author(s):  
Eszter Posfai ◽  
John Paul Schell ◽  
Adrian Janiszewski ◽  
Isidora Rovic ◽  
Alexander Murray ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Single Cell ◽  
Pluripotent Stem Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Differentiated Cells ◽  
Totipotent Cell

AbstractTotipotency is the ability of a single cell to give rise to all the differentiated cells that build the conceptus, yet how to capture this property in vitro remains incompletely understood. Defining totipotency relies upon a variety of assays of variable stringency. Here we describe criteria to define totipotency. We illustrate how distinct criteria of increasing stringency can be used to judge totipotency by evaluating candidate totipotent cell types in the mouse, including early blastomeres and expanded or extended pluripotent stem cells. Our data challenge the notion that expanded or extended pluripotent states harbor increased totipotent potential relative to conventional embryonic stem cells under in vivo conditions.

scholarly journals Human Hematopoietic Stem Cells Can Survive In Vitro for Several Months

2009 ◽  
Vol 2009 ◽  
pp. 1-7 ◽  
Author(s):  
Taro Ishigaki ◽  
Kazuhiro Sudo ◽  
Takashi Hiroyama ◽  
Kenichi Miharada ◽  
Haruhiko Ninomiya ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Cultured Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
Hematopoietic Stem ◽  
Immunodeficient Mice ◽  
Cell Sample

We previously reported that long-lasting in vitro hematopoiesis could be achieved using the cells differentiated from primate embryonic stem (ES) cells. Thus, we speculated that hematopoietic stem cells differentiated from ES cells could sustain long-lasting in vitro hematopoiesis. To test this hypothesis, we investigated whether human hematopoietic stem cells could similarly sustain long-lasting in vitro hematopoiesis in the same culture system. Although the results varied between experiments, presumably due to differences in the quality of each hematopoietic stem cell sample, long-lasting in vitro hematopoiesis was observed to last up to nine months. Furthermore, an in vivo analysis in which cultured cells were transplanted into immunodeficient mice indicated that even after several months of culture, hematopoietic stem cells were still present in the cultured cells. To the best of our knowledge, this is the first report to show that human hematopoietic stem cells can survive in vitro for several months.

scholarly journals Differentiation of trophoblast cells from human embryonic stem cells: to be or not to be?

Reproduction ◽  
2014 ◽  
Vol 147 (5) ◽  
pp. D1-D12 ◽  
Author(s):  
R Michael Roberts ◽  
Kyle M Loh ◽  
Mitsuyoshi Amita ◽  
Andreia S Bernardo ◽  
Katsuyuki Adachi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Full Range ◽  
Embryonic Stem ◽  
Cell Types ◽  
Embryonic Cells ◽  
Cell Type ◽  
Differentiated Cells ◽  
Developmental Hierarchy

It is imperative to unveil the full range of differentiated cell types into which human pluripotent stem cells (hPSCs) can develop. The need is twofold: it will delimit the therapeutic utility of these stem cells and is necessary to place their position accurately in the developmental hierarchy of lineage potential. Accumulated evidence suggested that hPSC could develop in vitro into an extraembryonic lineage (trophoblast (TB)) that is typically inaccessible to pluripotent embryonic cells during embryogenesis. However, whether these differentiated cells are truly authentic TB has been challenged. In this debate, we present a case for and a case against TB differentiation from hPSCs. By analogy to other differentiation systems, our debate is broadly applicable, as it articulates higher and more challenging standards for judging whether a given cell type has been genuinely produced from hPSC differentiation.

Expression of Imprinted Genes Kcnq1 and Cdkn1c During the Course of Differentiation from Mouse Embryonic Stem Cells into Islet-like Cells in vitro

2017 ◽  
Vol 126 (04) ◽  
pp. 249-254
Author(s):  
Feng Liu ◽  
Peng yu-huan ◽  
Li Qiang ◽  
Liu Chanchan
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Islet Cell ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells ◽  
Imprinted Genes ◽  
Rt Pcr ◽  
Differentiated Cells ◽  
Pcr Rflp

AbstractTo study the effects of inducement on the expression of mouse embryonic stem cells SF1-G imprinted genes, Kcnq1 and Cdkn1c during the course of differentiation into islet-like cells in vitro. Mouse embryonic fibroblasts (MEFs) were isolated from pregnant mice embryos and fibroblast feeder cells were prepared by treating 3–5th generations MEFs with Mitomycin C. Moreover, mouse embryonic stem cells were induced to differentiate into islet-like cells directly. RT-PCR and Immunofluorescence staining were used to test the expression of islet cell-specific markers. Cells were collected at various stages throughout the differentiation process and the imprinted genes Kcnq1 and Cdkn1c were tested by reverse transcription-polymerase chain reaction fragment length polymorphism (RT-PCR/RFLP). In the present study, we found that cells appear islet cell-specific gene expression. Furthermore, immunofluorescence shows us that the islet cell-specific hormone protein can be measured at stage, which confirms that the embryonic stem cells can be successfully induced into islet-like cells in vitro. RT-PCR/RFLP analysis showsthat imprinted genes Kcnq1 and Cdkn1c are biallelic expression in the differentiated cells, suggestive of loss of imprinting (LOI), while these genes demonstrate maternal monoallelic expression in the undifferentiated cells’ continued subculture; this marks the maintenance of imprinting (MOI). Our data indicate that mouse embryonic stem cells are induced into islet-like cells in vitro. The gene imprinting status of Kcnq1 and Cdkn1c may be changed in differentiated cells during the induction in vitro.

Generating gastruloids with somite-like structures from mouse embryonic stem cells

2020 ◽  
Author(s):  
Vincent van Batenburg ◽  
Susanne Carina van den Brink ◽  
Marloes Blotenburg ◽  
Anna Alemany ◽  
Naomi Moris ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Cultured Cells ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells ◽  
Hands On ◽  
Somite Formation ◽  
Key Aspects ◽  
Post Implantation

Abstract Gastruloids are aggregates of mouse embryonic stem cells that can be used to study key aspects of mammalian post-implantation development in vitro1–4. Gastruloids generated with previously published protocols do not generate somite-like structures4–6. Here, we describe a modified version of the gastruloids culture protocol5,6 that results in gastruloids that do generate somite-like structures in vitro (van den Brink et al., Nature, 2020)7. Under these conditions, about 50% of the gastruloids generated form structures with features that are characteristic of somites7.This protocol takes 6 days, with relatively little hands-on time. The protocol starts with the aggregation of the cultured cells. Then, the Wnt-agonist Chiron is added 2 days (48h) later. The medium of the aggregates is replaced 3 days (72h) after aggregation. To induce somite-formation, gastruloids are embedded in Matrigel 4 days (96h) after aggregation. After 5 days (120h) of culture, gastruloids resemble E8.5 mouse embryos. At this timepoint they can be fixed (fixative is added on day 5 and washed away on day 6 after overnight incubation in PFA) to prepare them for staining or microscopy experiments.

scholarly journals Prunellae Spica Extract Suppresses Teratoma Formation of Pluripotent Stem Cells through p53-Mediated Apoptosis

Nutrients ◽  
2020 ◽  
Vol 12 (3) ◽  
pp. 721 ◽  
Author(s):  
Aeyung Kim ◽  
Seo-Young Lee ◽  
Chang-Seob Seo ◽  
Sun-Ku Chung
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Apoptotic Cell ◽  
Embryonic Stem ◽  
M Cell ◽  
In Ovo ◽  
Differentiated Cells ◽  
Teratoma Formation

Induced pluripotent stem cells (iPSCs) have similar properties to embryonic stem cells in terms of indefinite self-renewal and differentiation capacity. After in vitro differentiation of iPSCs, undifferentiated iPSCs (USCs) may exist in cell therapy material and can form teratomas after in vivo transplantation. Selective elimination of residual USCs is, therefore, very important. Prunellae Spica (PS) is a traditional medicinal plant that has been shown to exert anti-cancer, antioxidant, and anti-inflammatory activities; however, its effects on iPSCs have not been previously characterized. In this study, we find that ethanol extract of PS (EPS) effectively induces apoptotic cell death of USCs through G2/M cell cycle arrest, generation of intracellular reactive oxygen species, alteration of mitochondrial membrane potentials, and caspase activation of USCs. In addition, EPS increases p53 accumulation and expression of its downstream targets. In p53 knockout (KO) iPSCs, the EPS did not induce apoptosis, indicating that EPS-mediated apoptosis of USCs was p53-dependent. In addition, EPS was not genotoxic towards iPSCs-derived differentiated cells. EPS treatment before injection efficiently prevented in ovo teratoma formation of p53 wild-type (WT) iPSCs but not p53KO iPSCs. Collectively, these results indicate that EPS has potent anti-teratoma activity and no genotoxicity to differentiated cells. It can, therefore, be used in the development of safe and efficient iPSC-based cell therapies.

scholarly journals BMAL1 coordinates energy metabolism and differentiation of pluripotent stem cells

2020 ◽  
Vol 3 (5) ◽  
pp. e201900534 ◽  
Author(s):  
Cristina Ameneiro ◽  
Tiago Moreira ◽  
Alejandro Fuentes-Iglesias ◽  
Alba Coego ◽  
Vera Garcia-Outeiral ◽  
...  
Keyword(s):  
Stem Cells ◽  
Energy Metabolism ◽  
Cell Fate ◽  
Adult Stem Cells ◽  
Embryonic Stem ◽  
Loss Of Function ◽  
Differentiation Potential ◽  
Differentiated Cells ◽  
Permanent Loss

BMAL1 is essential for the regulation of circadian rhythms in differentiated cells and adult stem cells, but the molecular underpinnings of its function in pluripotent cells, which hold a great potential in regenerative medicine, remain to be addressed. Here, using transient and permanent loss-of-function approaches in mouse embryonic stem cells (ESCs), we reveal that although BMAL1 is dispensable for the maintenance of the pluripotent state, its depletion leads to deregulation of transcriptional programs linked to cell differentiation commitment. We further confirm that depletion of Bmal1 alters the differentiation potential of ESCs in vitro. Mechanistically, we demonstrate that BMAL1 participates in the regulation of energy metabolism maintaining a low mitochondrial function which is associated with pluripotency. Loss-of-function of Bmal1 leads to the deregulation of metabolic gene expression associated with a shift from glycolytic to oxidative metabolism. Our results highlight the important role that BMAL1 plays at the exit of pluripotency in vitro and provide evidence implicating a non-canonical circadian function of BMAL1 in the metabolic control for cell fate determination.

scholarly journals 168 ESTABLISHMENT AND MOLECULAR CHARACTERIZATION OF PIG PARTHENOGENETIC EMBRYONIC STEM CELLS

2005 ◽  
Vol 17 (2) ◽  
pp. 235 ◽  
Author(s):  
T.A.L. Brevini ◽  
F. Cillo ◽  
F. Gandolfi
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Therapeutic Potential ◽  
Embryonic Stem ◽  
Culture Conditions ◽  
Embryoid Bodies ◽  
Morphological Examination ◽  
Differentiation Markers ◽  
Differentiated Cells

Parthenogenetic embryonic stem cells have been obtained in mouse and in primates. However, it would be desirable to have an alternative experimental model that could be used to investigate the therapeutic potential of these cells. For this purpose, we generated parthenogenetic pig blastocysts from in vitro-matured oocytes activated by sequential exposure to 10 μM ionomycin for 5 min and 2 mM 6-DMAP for 3 h. Inner cell masses were isolated by immunosurgery and plated on mitotically inactivated STO fibroblast feeder layers in 4-well dishes. Cells were incubated in 5% CO2 at 37°C in low glucose DMEM/F10 medium supplemented with 1000 IU/mL of mouse recombinant LIF, 10% Knockout serum replacer (Gibco, Italy), and 5% FBS. Within 3 days, circular colonies with distinct margins of small round cells were observed on both substrates. When a colony enlarged enough to cover half or more of the well surface, cells were trypsinized in clumps never reaching single-cell suspension and passaged to a newly prepared well. The expression of a gene panel was examined by RT-PCR on a portion of the cells at each passage. Oct-4 and nanog were used as markers of pluripotency. Interferon-τ, α-Amilase, Bone Morphogenetic Protein-4, and Neurofilament were used as markers of trophectoderm, endoderm, mesoderm, and ectoderm differentiation respectively. After 4 passages, three colonies expressed Oct-4 and nanog and were negative for all four differentiation markers. Two colonies at the 5th and 7th passages maintained nanog but not Oct-4 expression, while remaining negative to all of the other genes. To induce the formation of embryoid bodies (EBs), cells were cultured in 50-μL droplets of medium without LIF. Initiation of differentiation of EBs was confirmed through both morphological examination and molecular analysis; mesodermal, ectodermal, and endodermal markers were all expressed by Day 9 of culture and Oct-4 and nanog expression was completely down-regulated. Interestingly, when EBs were returned to adherent culture conditions patches of differentiated cells tended to form, spontaneously differentiating into mesodermal, endodermal, or neuroectodermal cell monolayers. The present data suggest that it is possible to establish putative embryonic stem cells from pig parthenotes. Further studies are in progress to determine their ability to stably maintain the undifferentiated state. This work was supported by MIUR COFIN 20022074357 and Fondazione CARIPLO.

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