Use of Surface Properties to Control the Growth and Differentiation of Mouse Fetal Liver Stem/Progenitor Cell Colonies

Multilayers of poly-l-lysine/poly-l-glutamic acid (PLL/PLGA) were constructed by layer-by-layer deposition on an end-tethered cationic PLL brush film serving as an initial layer. Increasing the number of coupling layers increased the thickness and the hydration of the films, and decreased the films’ shear modulus and serum adsorption. These films were used to culture primary mouse fetal liver cells. Fetal liver stem/progenitor cells (FLSPCs) were isolated and maintained on the PLGA-terminal PLL/PLGA surfaces, forming colonies with clear boundaries that were partially attached to the surface, with cross-sectional areas of ∼500 to ∼2500 μm² after 2 days culture. Long-term studies showed that the cluster size of colonies slowly expanded and was correlated with the surface properties. For example, on the thicker films with shear modulus, <i>G</i>, less than 5 kPa, FLSPCs cluster size was constrained within a small distribution with less than 4000 μm² of projected area, whereas on the thinner films with <i>G</i> > 30 kPa, clusters were expanded and widely distributed, with projected areas over 4000 um². Immunostaining studies suggested that clusters with a small size maintained the self-renewal characteristics of stem cells, while the expanded clusters were clearly the results of spontaneous differentiation, exhibiting hepatocyte-like properties. On PLL-terminal t-(PLL/PLGA) films, which are less favorable for stem cell cultures than PLGA-terminal t-(PLL/PLGA) films, the cluster size distribution was also correlated with the film thickness, with more clusters of small size preserved on the thicker films. We observed that a soft, hydrated, serum-free surface could restrict the FLSPC expansion, resulting in self-maintenance of FLSPC colonies

Additional file 2 of Effects of the media conditioned by various macrophage subtypes derived from THP-1 cells on tunneling nanotube formation in pancreatic cancer cells

Additional file 2: Fig. S1. The mRNA levels of markers for M1 macrophage (a) IL-1β and (b) TLR-2, and (c) the marker for M2 macrophage CCL22, measured in the macrophages after the differentiation according to the protocols described in the Materials and Methods section. The data are from three independent experiments. ***, P < 0.005; *, P < 0.05 in comparison with those in THP-1 cells (post hoc Tukey’s test). Fig. S2. The ELISA results of EGF in (a) the conditioned media (CMs) of the THP-1 cells and macrophages, and (b) the CM of PANC-1 cells cultured in the macrophage CMs for 48 hours. The data are from three independent experiments. (c) The calibration curve of the optical density (OD) vs. the EGF concentrations. From this calibration curve, we learned that the EGF concentrations in panels (a) and (b) are all below the detection limit of ELISA. The ELISA kit was DY 236, DuoSet ELISA (R&D Systems, Minneapolis, MN, USA). The absorbance of the analytes were measured with a plate reader (Synergy 2, BioTek Instruments). Fig. S3. Formation of TNTs between two PANC-1 cells originally in contact (indicated by an arrow in the image at 0 min) in the M0 CM. This process is consistent with the “cell dislodgement” TNT formation mechanism. Fig. S4. Co-localization of kinesin (red) with the mitochondria (green) within a TNT. Most of the bright mitochondria were co-localized with the kinesin signal. The mitochondria were fused with green fluorescence protein in a stable cloned PANC-1 cell line. The kinesin was labeled with rabbit antibody (ab5629, abcam) then probed with DyLight 650-conjugated secondary antibody (ab96886, abcam)

Promoting the Selection and Maintenance of Fetal Liver Stem/Progenitor Cell Colonies by Layer-by-Layer Polypeptide Tethered Supported Lipid Bilayer

In this study, we designed and constructed a series of layer-by-layer polypeptide adsorbed supported lipid bilayer (SLB) films as a novel and label-free platform for the isolation and maintenance of rare populated stem cells. In particular, four alternative layers of anionic poly-l-glutamic acid and cationic poly-l-lysine were sequentially deposited on an anionic SLB. We found that the fetal liver stem/progenitor cells from the primary culture were selected and formed colonies on all layer-by-layer polypeptide adsorbed SLB surfaces, regardless of the number of alternative layers and the net charges on those layers. Interestingly, these isolated stem/progenitor cells formed colonies which were maintained for an 8 day observation period. Quartz crystal microbalance with dissipation measurements showed that all SLB-polypeptide films were protein resistant with serum levels significantly lower than those on the polypeptide multilayer films without an underlying SLB. We suggest the fluidic SLB promotes selective binding while minimizing the cell–surface interaction due to its nonfouling nature, thus limiting stem cell colonies from spreading

Inhibition of ABCG2 resulted in increases in levels of γH2AX, p53, phosphorylated p53 and phosphylated ATM.

<p>(A). RT-PCR data for genes involved in oxidative stress defense: SOD2, SOD4, TRX, and GPX2, with GAPDH as internal control for untreated and 10 µM FTC-treated mES-R1 cells 72 hours after addition of FTC. (B) Western blot of 10 µM FTC-treated mES-J1 cells stained for Nanog, γH2AX, p53 and Phospho-p53 Ser18, 23 and 389 as well as ATM and Phospho-ATM ser1981. Equal amounts of protein were used (40 µg per lane). (C–F) Immunofluorescence staining for γH2AX (C, D) and p53 (E, F) in untreated and 10 µM FTC treated mES-R1 cells taken at 72 hours after addition of FTC.</p

DataSheet_1_Inhibiting TLR7 Expression in the Retinal Pigment Epithelium Suppresses Experimental Autoimmune Uveitis.pdf

Experimental autoimmune uveitis (EAU), a model of human uveitis, is an organ-specific, T cell-mediated autoimmune disease. Autoreactive T cells can penetrate the blood-retinal barrier, which is a physical defense composed of tight junction-linked retinal pigment epithelial (RPE) cells. RPE cells serve as antigen-presenting cells (APCs) in the eye since they express MHC class I and II and Toll-like receptors (TLRs). Although previous studies have shown that supplementation with TLR agonists exacerbates uveitis, little is known about how TLR signaling in the RPE contributes to the development of uveitis. In this study, we isolated the RPE from EAU mice, which were induced by active immunization (aEAU) or adoptive transfer of antigen-specific T cells (tEAU). The expression of TLRs on RPE was determined, and both aEAU and tEAU mice exhibited induced tlr7 expression. The TLR7 agonist R848 was shown to induce aggressive disease progression, along with significantly elevated levels of the uveopathogenic cytokine IL-17. Furthermore, not only IL-17 but also R848 appeared to enhance the inflammatory response and to impair the barrier function of the RPE, indicating that TLR7 signaling is involved in the pathogenesis of EAU by affecting the behaviors of the RPE and consequently allowing the infiltration of autoreactive T cells intraocularly. Finally, local application of shRNA against TLR7 delivered by recombinant AAV effectively inhibited disease severity and reduced IFN-γ and IL-17. Our findings highlight an immunomodulatory role of RPE TLR7 in EAU development and provide a potential therapeutic strategy for autoimmune uveitis.</p

Inhibition of ABCG2 resulted in increases in PPIX and ROS levels.

<p>(A–D) PPIX fluorescence measured by flow cytometry for control vector and CMS-ABCG2 (A), scramble and shABCG2 transfected ES cells (B) as well as untreated and 10 µM FTC-treated mES-R1 cells 96 hours after addition of FTC (C). For positive control, mES-R1 cells were treated with 1 mM 5-aminovaleric acid (ALA) for 1 hour to induce production of PPIX (D). Diagram shown represents at least three independent experiments. (E–G) Cytosolic PPIX level measurements after induction with 1 mM ALA for 30 minutes and efflux for 1 hour for Untreated and 10 µM FTC (E) treated mES-R1 control vector and CMS-ABCG2 transfected mES-R1 (F) and scramble and shABCG2 transfected mES-R1(G). (H, I) Immunofluorescence staining showing downregulation of Nanog in untreated (H) as well as 2-hour ALA treated (I) mES cells. (J–L) ROS level measurements by flow cytometry using the ROS sensitive dye DCFH-DA for untreated and 1 µM (J) and 10 µM (K) FTC-treated mES-R1 cells as well as 2-hour treatment with ALA (L). Diagram shown represents at least three independent experiments.</p

Inhibition of ABCG2 led to downregulation of Nanog in ES cells.

<p>(A) RT-PCR data of ABCG2 in J1, R1, D3 and B6 mouse ES cell lines as well as in MEFs, with GAPDH as internal control. (B, C) Real-time PCR data showing relative mRNA expression level of ABCG2 (B) and Nanog (C) in mES-R1 cells treated with or without 10 µM of the ABCG2 inhibitor FTC, normalized to GAPDH. Data was calculated from at least three independent experiments. A t-test was performed to confirm statistical significance and * indicates p<0.05 (D, E) Immunofluorescence staining showing Nestin expression was observed in FTC treated mES-R1 cells. (F–T) Immunofluorescence staining for ABCG2, Oct-4 and Nanog in untreated mES-R1 and mES-J1 cells, in mES-R1 cells treated with 1 µM or 10 µM of the ABCG2 inhibitor FTC, and in mES-J1 cells treated with 10 µM FTC for 72 hours.</p

Knock-down of p53 prevented downregulation of Nanog in ABCG2-inhibited ES cells.

<p>(A–D) Immunofluorescence staining for Nanog on untreated (A, B) and 10 µM FTC-treated (C, D) J1_pSico-p53 cells with (B, D) or without (A, C) the addition of HTNCre to activate inhibition of p53. White arrows indicate GFP negative cells with high Nanog where p53 inhibition has rescued Nanog expression. Arrowheads indicate GFP positive cells with low or no Nanog.</p

Selection, Enrichment, and Maintenance of Self-Renewal Liver Stem/Progenitor Cells Utilizing Polypeptide Polyelectrolyte Multilayer Films

Recent progress has led to the identification of liver stem/progenitor cells as suitable sources for generating transplantable liver cells. However, the great variability in methods utilized to isolate liver stem/progenitor cells is a considerable challenge for clinical applications. The polyelectrolyte-multilayer technique can constitute a useful method for selective cell adhesion. Whether enrichment of liver stem/progenitor cells can be achieved utilizing polypeptide polyelectrolyte-multilayer films was investigated in current work. Fetal liver cells isolated from E13.5 mouse embryos were seeded on the poly-l-glutamic acid/poly-l-lysine alternating films, and we revealed that fetal liver stem/progenitor cells were selected and formed colonies. These undifferentiated colonies were maintained on the films composed of four alternating layers, with the topmost poly-l-glutamic acid layer judged by the constitutive expression of stem-cell markers such as Dlk-1, CD49f, and CD133 and self-renew marker-β-catenin. Our work has demonstrated that highly tunable polyelectrolyte-multilayer films were suitable for selective enrichment of liver stem/progenitor cells in vitro

shABCG2 and scramble control sequence.

<p>shABCG2 and scramble control sequence.</p