Paracetamol in vitro toxicity in stem-cell derived hepatocytes (hESC-heps) and primary human hepatocytes (PHH). hESC-heps (at day 17) and PHH (24 hours post- replating) were induced with different concentrations of paracetamol (0-50 mM) for 24 hours. The CellTitre viability assay (Promega) was used to measure the ATP levels. The IC50 was calculated from the function f(x)= ax + b.
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Mesenchymal stem cells (MSCs) increase secretion of exosomes upon exposure to PAD-like conditions. (A): Quantification of total protein content of vesicles derived from MSC under EX, IC, and PAD culture conditions using DC assay. (B): Scanning electron micrograph of MSCs cultured in EX culture conditions indicating microvesicle release (blue arrows) from the cell surface (scale bar = 5 μm, × 5k). (C): Scanning electron micrograph of MSCs cultured under PAD conditions (scale bar 2 μm, × 10k) indicating exosome adhesion to cell surface (red arrows). (D): Transmission electron micrograph of MSC derived exosomes with 2% uranyl acetate negative staining (scale bar 200 nm, × 25k). Abbreviations: EX, expansion condition; Exo, exosomes; IC, intermediate condition; MV, microvesicle; PAD, peripheral arterial disease.
Set1a is essential for embryonic stem (ES) maintenance. (A): Alkaline phosphatase (AP) staining of embryonic stem cell (ESC) colonies 4 days after treatment with shOct4 and shSet1a-4. (B): Immunofluorescent staining showing that knockdown of Set1a in CGR8 ESCs by shSet1a-4 resulted in reduced levels of ES core transcriptional factors Oct4, Sox2, and Nanog. (C): The effect of knockdown of Set1a in CGR8 ESCs by control shRNA (shCtrl) and shSet1a-4 on teratoma formation in nude mice. For each group 10 mice were injected. (D): Western blot analyses showing knockdown of Set1a by two different siRNAs led to reduced levels of Oct4, Sox2, and Nanog. (E): AP staining of ESC colonies 4 days after treatment with control or two siRNAs against Set1a. (F): Quantitation of the AP-positive colonies in (E). (G): Cell proliferation assay showing knockdown of Set1a by siRNAs reduced CGR8 ES proliferation. Abbreviations: AP, alkaline phosphatase; NC, negative control; WT, wild type.
Stem cell pool composition in homeostasis. The probability of either a neutral mutant (grey) or a mutant with increased proliferation rate rm=1.5 (blue) to constitute a certain frequency of the stem cell pool in homeostasis, if the mutant occurred early (initial frequency of 0.5, dots), at an intermediate (initial frequency 0.2, triangles), or late (initial frequency 0.005, squares) during the expansion of the stem cell pool. An early neutral mutation (black dots) undergoes a random walk and consequently constitutes any fraction of the stem cell pool with the same probability. An increased proliferation rate is a significant fitness advantage early (dark blue dots), but vanishes for mutants in late stages of development (light blue squares).
Characterization of skeletal muscle induction by SMI treatment. (A): Kinetics of Pax7 luciferase activity in Pax7 luciferase knock-in embryonic stem (ES) cells after treatment with SMI1 or DMSO. The bars indicate SD from independent EBs from a representative experiment. (B): The relative transcript levels at day 4+4 of MyoD, Myf5, and Myogenin were determined by real-time reverse transcription polymerase chain reaction analysis and plotted in relation to the GAPDH endogenous levels. Both compounds were added at 10 μM at day 1 and day 4. The bars indicate SEM for three independent pools of 10 EBs from a representative experiment. (C): Immunofluorescence staining for Myogenin (red) of a mouse EB at day 4+8 (upper panel, scale bar = 20 μm) and at day 4+20 for MHC (lower panel, scale bar = 160 μm). DAPI was used to stain the nucleus. The white square indicates the region with higher magnification demonstrating the striation of the muscle cells. (D): Immunofluorescence staining for MHC (red) of a whole single EB in a 24-well plate at day 4+20 either untreated or SMI2 treated. The image is a composition of many individual pictures in order to visualize the entire EB. DAPI was used to stain the nucleus. Scale bar = 1 mm. (E): The population of MHC positive cells from EB at day 4+20 was quantified as a percentage of MHC positive nuclei (left) and MHC positive area (right) to the total number of cells or the total area. The bars indicate SEM for three independent EBs from a representative experiment. (*, p < .05 by Student's t test, significance vs. control). Abbreviations: EB, embryoid body; SMI1,2, smooth muscle inducers 1, 2.
ErbB4 expressed in hPSC-CMs but not in undifferentiated hPSCs. (A): Immunostaining of undifferentiated hPSCs for ErbB receptor tyrosine kinases (RTKs) (green) and Oct4 (red), counterstained with DAPI. Note the absence of ErbB4 in hPSCs. Scale bar = 50 µm. (B): Western blot analysis of ErbB RTKs and Oct4 across five hPSC lines. Please see Materials and Methods for details of five hPSC lines. (C): Western blots showing time-kinetic evaluation of ErbB RTKs along with Oct4 and cardiac markers, NKx2.5, MEF2c, and cTnT (left). Densitometric data were normalized to housekeeping gene, GAPDH (right). Data presented as mean ± SEM of five independent experiments.*, p < .05, significantly different from day 0. (D): hPSC-CMs (top panel, i, ii) and mouse heart (bottom panel, iii, iv) stained with ErbB RTKs (green) and α-actinin (red), counterstained with DAPI, showing sarcolemmal localization of ErbB2 and ErbB4 (yellow arrows) and nuclear localization of ErbB4 (white arrows). Scale bar = 50 µm. Abbreviations: cTnT, cardiac troponin T; DAPI, 4',6-diamidino-2-phenylindole; EGFR, epidermal growth factor receptor; hPSC-CMs, human pluripotent stem cell-derived cardiomyocytes.
Cone-rod homeobox (CRX)/green fluorescent protein (GFP) expression is found in photoreceptor precursors at day 60 of human embryonic stem cell (hESC) differentiation. Immunocytochemistry with antibodies raised against GFP and Recoverin (A, B), OPN1SW (C), OPN1MW/LW (D), Calbindin (E), HuC/D (F), and Ki-67 (G). Scale bars = 20 µm (A, F), 5 µm (B), and 10 µm (C, D, E). Abbreviation: GFP, green fluorescent protein
Mesenchymal stem cells (MSCs) exposed to peripheral arterial disease-like (PAD) conditions increase expression of a diverse profile of angiogenic proteins which are subsequently packaged into exosome for secretion and induce angiogenesis in endothelial cells.
Growth arrest and DNA-damage inducible 45 alpha (Gadd45a) expression is induced by γ-irradiation in murine hematopoietic stem cells (HSC). GADD45A upregulation neither causes cell cycle arrest nor cell death in HSCs but essentially affects the balance between their self-renewal (SR) and differentiation (Diff). Elevated GADD45A levels robustly induce and accelerate the differentiation in HSCs via the GADD45A-mediated activation of MAP kinase.
The data presented in this manuscript suggest that in the Gata1low model, myelofibrotic stem cells are sustained in the spleen by a niche formed by the interaction of at least three distinctive cell populations. First neutrophils establish a P-selectin dependent interaction with megakaryocytes that leads to release of TGF-β. TGF-β induces the transition of a resident spleen cell population to activated fibrocytes which in turn interact with their protrusions with the megakaryocytes creating a super-cellular structure that hosts, and possibly supports, the proliferation of myelofibrotic stem cells in the spleen.