Morphological analysis of astrocytes cultured on glass with the additional of soluble factors.

<p>(A) Percentage of cells with protrusions. Control (N = 79), PDGF (N = 81), laminin (N = 68), bFGF (N = 71), LIF (N = 74). Immunofluorescence images of (B) control and (C) astrocytes treated with LIF stained for GFAP (green) and DAPI (blue).</p

Tortuosity of astrocytes.

<p>(A) Immunofluorescence image of an astrocyte on HBMEC-derived ECM. Endothelial cells were grown to confluence on glass-bottom dishes, and removed from the dish with a lysis buffer containing 0.5% Triton X-100 and 20 mM NH₄OH in PBS. (B) Immunofluorescence image of an astrocyte on 50 μm inner diameter fibronectin rings. Astrocytes tended to trace the rings and have smaller cell bodies like those seen in co-culture. Fibronectin (red), GFAP (green). (C) Tortuosity of astrocytes on surface coatings and in co-culture. The tortuosity (τ) is given by τ = <i>l</i>/c where <i>l</i> is the arc length of the processes and c is the shortest end-to-end distance. For a straight line τ = 1, whereas for a circle τ = ∞. While all of the surface coatings result in very small increases over the control, the astrocytes in co-culture have significantly higher tortuosities. Statistical significance was determined using a student’s t-test. ***P≤0.01, **P≤0.05, *P≤0.1.</p

Influence of surface coatings on astrocyte morphology.

<p>(A) The cell area defined by the area of the cell body. (B) the cell diameter is overall size defined by the diameter of the smallest circle that can enclose the cell and all of its processes. (C) The protrusion length is the total length of all protrusions. (D) The degree of branching is the number of branch points divided by the number of primary protrusions. (E) The number of primary protrusions represents the number of protrusions emanating from the cell body. (F) The number of secondary protrusions represents protrusions emanating from primary protrusions. (G) The number of tertiary protrusions represents protrusions emanating from secondary protrusions. (H) The number of branch points represents the sum of secondary and other higher order protrusions (equivalent to the number of bifurcations). Data represent mean ± SE. Statistical significance was determined using a student’s t-test test. ***P≤0.01, **P≤0.05, *P≤0.1. Only cells with astrocyte-like morphology were analyzed (the total number of cells and the fraction of cells with astrocyte-like morphology are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092165#pone-0092165-g001" target="_blank">Figure 1</a>).</p

Influence of ECM coating on astrocyte morphology after 24 hours.

<p>Fluorescence images of astrocytes stained for GFAP (green) and DAPI (blue) on (A) glass, (B) collagen I, (C) collagen IV, (D) fibronectin, (E) matrigel, and (F) co-culture on a confluent monolayer of HBMECs. (G) Astrocyte (from panel (F)) seeded on a confluent monolayer of HBMECs, stained for GFAP (green), DAPI (blue), and ZO-1 (red). (H) The percentage of cells with protrusions. Total number of cells analyzed: uncoated (N = 103), collagen I (N = 85), collagen IV (N = 61), fibronectin (N = 63), matrigel (N = 54), co-culture (N = 58).</p

Influence of soluble factors on astrocyte morphology.

<p>Data represent mean ± SE. Statistical significance was determined using a student’s t-test. ***P≤0.01, **P≤0.05, *P≤0.1. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092165#pone-0092165-g004" target="_blank">Figure 4</a> for the number of cells analyzed.</p

Stemness of primary AMSC lines demonstrated with differentiation along three mesenchymal lineages, Adipocyte (a, d [48], g), Osteocyte (b [48], e, h), and Chondrocyte (c [48], f, i), documented via lineage specific staining with Oil Red O, Alizarin Red, and Collagen II, respectively.

<p>The details of the staining protocols are provided in the methods section. Scale bar, 50 µm.</p

Growth curve documenting relative proliferation rates of commercial (Lonza) BMSC, commercial (Invitrogen) AMSC, and the two primary AMSC cell lines (671, 654).

<p>The values shown are the mean of three replicates per cell line per time point, and the S.E.M (*:p<0.05).</p

Primary and commercial AMSCs and commercial BMSCs have similar tropism to glioma conditioned media <i>in vitro</i>, although there is notable variability in the tropism of the primary AMSC lines.

<p>Analysis of individual cell line tumor tropism in a matrigel-coated insert Boyden chamber migration assay; migration for each line was normalized to the serum free media condition. Results are reported as mean ± S.E.M., n = 9. (* represents the statistically significant difference within groups and # represents the statistically significant difference between groups).</p

Stemness of the primary adipose-derived mesenchymal stem cell lines (AMSC), and the Invitrogen supplied commercial AMSC line, confirmed with FACS documenting the absence of hematopoetic surface antigens CD31/CD45 and the presence of cell adhesion markers CD73/CD90/CD105.

<p>Percentage of each antigen was analyzed using Kaluza software and labeled in the responsive graph.</p

(A) Schematic diagram of the pneumatic device and modified 96 well plate attached to a microscope stage

<p>(<b>B</b>) Pressure profile measurements of the simulated open field blast shockwave compared to a classical Friedlander curve of the same peak pressure and positive phase duration. Average of 6 measurements is shown with standard error.</p