Tunable Structural and Mechanical Properties of Cellulose Nanofiber Substrates in Aqueous Conditions for Stem Cell Culture

Thin cellulose nanofiber (CNF) nanostructured substrates with varying roughness, stiffness (Young’s modulus), porosity, and swelling properties were produced by varying the conditions used during fabrication. It was shown that with increased heat exposure, CNF substrate porosity in an aqueous state decreased while Young’s modulus in a water submerged state increased. In this study, the adhesion and viability of mesenchymal stem cells (MSCs) cultured on this CNF substrate will be presented. Viability of D1/BALBc MSCs were assessed for 24 and 48 h, and it was shown that depending on the CNF substrate the viability varied significantly. The adhesion of MSCs after 6 and 24 h was conditional on material mechanical properties and porosity of the CNF in cell culture conditions. These results suggest that material properties of CNF nanostructured substrate within the aqueous state can be easily tuned with curing step without any chemical modification to the CNF and that these changes can affect MSC viability in cell culture

Schedule of drug-escalating selection of GNA, CV-N, ConA and GRFT resistant HCV strains as a function of time.

<p>HuH-7-RFP-NLS-IPS cells were infected in the presence of GNA (<b>A</b>), CV-N (<b>B</b>), ConA (<b>C</b>) or GRFT (<b>D</b>). Infected cells were subcultured every three to four days in the presence of the lectins. When 100% cells were infected (indicated by arrows), supernatants were recovered and used to infect naive cells in the presence of the lectins. The concentrations of each lectin were increased in a stepwise manner as indicated. Stars indicate the time points when the virus isolates were recovered and sequenced.</p

Conservation of the mutated amino acids.

<p>Conservation of the mutated amino acids.</p

Effect of the E1E2p7 mutations on sensitivity to inhibition by GNA, CV-N, ConA and GRFT.

<p>Inhibition assays were performed by incubating WT or mutant HCVcc with various concentrations of GNA (<b>A</b>), CV-N (<b>B</b>), ConA (<b>C</b>) or GRFT (<b>D</b>). After a 1 h incubation at 37°C, mixes were put into contact with target cells for 4 h. Luciferase assays were performed on infected cells at 72 h post-infection. Results are expressed as percentages of infectivity compared to infection in absence of inhibitory protein and are reported as the means ± S.D. of at least three independent experiments.</p

Effect of the E1E2p7 mutations on E1E2 glycoprotein expression.

<p>HuH-7-RFP-NLS-IPS cells were transfected with WT or mutated HCV genomes. An assembly-deficient virus (ΔE1E2) was used as control. Expression of E1E2 viral glycoproteins and actin was analyzed in cell lysates 72h post-electroporation by western blotting with specific MAbs (A4 [anti-E1], 3/11 [anti-E2] and C4 [anti-actin]).</p

Effect of the E1E2p7 mutations on sensitivity to inhibition by MAb 3/11 and CD81-LEL.

<p>Inhibition assays were performed by incubating WT or mutant HCVcc with various concentrations of MAb 3/11 (<b>A</b>) or CD81-LEL (<b>B</b>). After a 1 h incubation at 37°C, mixes were put into contact with target cells for 4 h. Luciferase assays were performed on infected cells at 72 h post-infection. Results are expressed as percentages of infectivity compared to infection in absence of inhibitory protein and are reported as the means ± S.D. of at least three independent experiments.</p

DataSheet1_Interplay between integrins and cadherins to control bone differentiation upon BMP-2 stimulation.PDF

Introduction: Upon BMP-2 stimulation, the osteoblastic lineage commitment in C2C12 myoblasts is associated with a microenvironmental change that occurs over several days. How does BMP-2 operate a switch in adhesive machinery to adapt to the new microenvironment and to drive bone cell fate is not well understood. Here, we addressed this question for BMP-2 delivered either in solution or physically bound of a biomimetic film, to mimic its presentation to cells via the extracellular matrix (ECM).Methods: Biommetics films were prepared using a recently developed automated method that enable high content studies of cellular processes. Comparative gene expressions were done using RNA sequencing from the encyclopedia of the regulatory elements (ENCODE). Gene expressions of transcription factors, beta chain (1, 3, 5) integrins and cadherins (M, N, and Cad11) were studied using quantitative PCR. ECM proteins and adhesion receptor expressions were also quantified by Western blots and dot blots. Their spatial organization in and around cells was studied using immuno-stainings. The individual effect of each receptor on osteogenic transcription factors and alkaline phosphatase expression were studied using silencing RNA of each integrin and cadherin receptor. The organization of fibronectin was studied using immuno-staining and quantitative microscopic analysis.Results: Our findings highlight a switch of integrin and cadherin expression during muscle to bone transdifferentiation upon BMP-2 stimulation. This switch occurs no matter the presentation mode, for BMP-2 presented in solution or via the biomimetic film. While C2C12 muscle cells express M-cadherin and Laminin-specific integrins, the BMP-2-induced transdifferentiation into bone cells is associated with an increase in the expression of cadherin-11 and collagen-specific integrins. Biomimetic films presenting matrix-bound BMP-2 enable the revelation of specific roles of the adhesive receptors depending on the transcription factor.Discussion: While β3 integrin and cadherin-11 work in concert to control early pSMAD1,5,9 signaling, β1 integrin and Cadherin-11 control RunX2, ALP activity and fibronectin organization around the cells. In contrast, while β1 integrin is also important for osterix transcriptional activity, Cadherin-11 and β5 integrin act as negative osterix regulators. In addition, β5 integrin negatively regulates RunX2. Our results show that biomimetic films can be used to delinate the specific events associated with BMP-2-mediated muscle to bone transdifferentiation. Our study reveals how integrins and cadherins work together, while exerting distinct functions to drive osteogenic programming. Different sets of integrins and cadherins have complementary mechanical roles during the time window of this transdifferentiation.</p

Increase of HCV titers after successive infections.

<p>HuH-7 cells were electroporated in the presence of JFH1-CS-A4 RNA. Ten days later, the supernatant of electroporated cells was recovered (denoted supernatant i0) and used to perform successive infections in HuH-7-RFP-NLS-IPS. Each time the cells were 100% infected, the supernatant was recovered (supernatants recovered after “n” infection, denoted i1 to i24) and used to infect naive HuH-7-RFP-NLS-IPS cells. (<b>A</b>, <b>B</b>) The amount of HCV RNA (<b>A</b>) and Core protein (<b>B</b>) were quantified in these supernatants by RT-qPCR and fully automated chemiluminescent microparticle immunoassay, respectively. Results are expressed as HCV RNA copies/mL and fmol/L of HCV Core protein, respectively, and are reported as the mean ± S.D. of duplicate and triplicate measurements, respectively. (<b>C</b>) Viral titers were determined by ffu assay for i0, i6, i9, i12 and i24. Results are expressed as ffu/mL and are reported as the mean ± S.D. of three independent experiments. (<b>D</b>) HuH-7-RFP-NLS-IPS cells were inoculated with the different supernatants at low MOI. Foci of infected cells, identified by translocation of the cleavage product RFP-NLS to the nucleus, were visualized at 24 and 48 h. Images are representative of three independent experiments.</p

Cytopathic effects induced by cell culture adapted HCV.

<p>HuH-7-RFP-NLS-IPS cells were infected with i24 at different MOIs. Non-diluted virus that had been inactivated at 60°C for 30 min was used as control (ctrl). Infected cell viability was evaluated 3 days after infection. The results are expressed as percentages of viability compared to non-infected cells and are reported as means ± S.D. of three independent experiments.</p

Infection of PHHs with cell culture adapted HCV.

<p>PHHs from one representative donor were inoculated for 6 h with non-adapted HCV (i0; MOI = 0.01 HuH-7 infectious units per cell) or i24 (MOI = 1000 HuH-7 infectious units per cell), in the presence or absence of 2′CMC (10 µM) or py6 (500 nM). After inoculation, cells were washed three times with PBS and new media containing the drugs were added and replaced every day. (<b>A</b>) Infection of PHHs that had previously been transduced with lentivirus expressing RFP-NLS-IPS, was visualized 48 h post-infection by translocation of the cleavage product RFP-NLS to the nucleus (“Infection” panel). The supernatants of inoculated cells were recovered 48 h post-infection, centrifuged and used to inoculate naive HuH-7-RFP-NLS-IPS in the absence or presence of py6 (“Re-infection” and “Re-infection with py6” panels, respectively) to check the production of progeny virus. Infected HuH-7 cells were visualized 48 h post-infection. (<b>B</b>) Intracellular HCV RNA was quantified by RT-qPCR, after inoculation of non-transduced PHHs. Results are expressed as means ± S.D. of duplicates. (<b>C</b>) Expression of the viral proteins E1 and E2 was analyzed 48 h post-infection in cell lysates by Western blotting using specific MAbs (A4 [anti-E1], 3/11 [anti-E2]<b>,</b> and C4 [anti-β-actin]). HuH-7 cells infected in the same conditions were used as control. (<b>D</b>, <b>E</b>, <b>F</b>) IFN-β, IL-28A/B and IL-29 expression in infected PHHs was determined in duplicate by RT-qPCR. The results are normalized to GAPDH endogenous control and presented as fold-increase over pre-infection levels, using the ΔΔCt method.</p