Initial characterization of dECM prepared from DMSC23 and CMSC29 cell lines.

<p>Immunofluorescence labelling of collagen type 1 (FITC) in dECM-DMSC23 and dECM-CMSC29 (A and B, respectively). Immunofluorescence labelling of fibronectin (Texas Red) in dECM-DMSC23 and dECM-CMSC29 (C and D, respectively). dECM-DMSC23 and dECM-CMSC29 proteoglycan visualized by Alcian blue (E and F, respectively). Scalebars are 100 μm. (G) SDS-PAGE protein profile of dECM-DMSC23 (lane 1), dECM-CMSC29 (lane 2), collagen I (lane 3), fibronectin (lane 4) and protein standards (lane 5).</p

Primary DMSC and CMSC proliferation on dECM substrates.

<p>(A) numbers of primary DMSC and (B) numbers of population doublings after 14 days of culture, and (C) representative photomicrographs showing primary DMSC cultured on dECM substrates and control surfaces on day 7. (D) Numbers of primary CMSC and (E) the numbers of population doublings after 14 days of culture, and (F) representative photomicrographs showing primary CMSC cultured on various dECM substrates and control surfaces on day 7. Significant increases in cell proliferation and population doubling levels were observed using one-way ANOVA with Tukey’s multiple comparison test. All values are mean ± SD; n = 3; *p < 0.05, **p < 0.01. Scalebar is 100 μm.</p

Decellularized extracellular matrices produced from immortal cell lines derived from different parts of the placenta support primary mesenchymal stem cell expansion

<div><p>Mesenchymal stem/stromal cells (MSCs) exhibit undesired phenotypic changes during <i>ex vivo</i> expansion, limiting production of the large quantities of high quality primary MSCs needed for both basic research and cell therapies. Primary MSCs retain many desired MSC properties including proliferative capacity and differentiation potential when expanded on decellularized extracellular matrix (dECM) prepared from primary MSCs. However, the need to use low passage number primary MSCs (passage 3 or lower) to produce the dECM drastically limits the utility and impact of this technology. Here, we report that primary MSCs expanded on dECM prepared from high passage number (passage 25) human telomerase reverse transcriptase (hTERT) transduced immortal MSC cell lines also exhibit increased proliferation and osteogenic differentiation. Two hTERT-transduced placenta-derived MSC cell lines, CMSC29 and DMSC23 [derived from placental chorionic villi (CMSCs) and <i>decidua basalis</i> (DMSCs), respectively], were used to prepare dECM-coated substrates. These dECM substrates showed structural and biochemical differences. Primary DMSCs cultured on dECM-DMSC23 showed a three-fold increase in cell number after 14 days expansion in culture and increased osteogenic differentiation compared with controls. Primary CMSCs cultured on the dECM-DMSC23 exhibited a two-fold increase in cell number and increased osteogenic differentiation. We conclude that immortal MSC cell lines derived from different parts of the placenta produce dECM with varying abilities for supporting increased primary MSC expansion while maintaining important primary MSC properties. Additionally, this is the first demonstration of using high passage number cells to produce dECM that can promote primary MSC expansion, and this advancement greatly increases the feasibility and applicability of dECM-based technologies.</p></div

Representative images showing decellularization of DMSC23 cultures.

<p>phase contrast (A) before and (D) after decellularization, fluorescence micrographs of DAPI staining (B) before and (E) after decellularization, and scanning electron micrographs (C) before and (F) after decellularization. Scale bar on phase contrast and fluorescence micrographs is 100 μm. Scale bar on SEM images is 5 μm.</p

Osteogenic differentiation of DMSC and CMSC on dECM substrates.

<p>Representative images of DMSC stained with Alizarin Red S dye after 14 days of osteogenic induction: (A) dECM-DMSC23, (B) dECM-CMSC29, (C) Fibronectin, and (D) TCP. Inset shows control uninduced DMSC. Scalebar is 100 μm. Osteoimage staining in (E) primary DMSC and (F) primary CMSC cultured on dECM and control surfaces after 14 days of osteogenic differentiation. All values are mean ± SD; n = 3; *p < 0.05, **p < 0.01, ***p < 0.001.</p

Fluorescence micrographs of DMSC seeded on dECM and control surfaces after 72 h of incubation.

<p>(A) dECM-DMSC23, (B) dECM-CMSC29, (C) fibronectin, and (D) tissue culture plastic. Scale bar is 100 μm for all images. (E) Average DMSC cell spread area. (F) Average CMSC cell spread area. All values are mean ± SD, *p < 0.05.</p

Histology of CMSCs and DMSCs transplants.

<p>Cross sections are representative of CMSCs transplants (A-B) and DMSCs transplants (E-F) after 8 weeks stained with Haematoxylin and Eosin (H&E). In the transplant, the HA/TCP carrier surfaces (dashed lines) are lined with new bone formation (b), areas of immature bone (ob) together with the surrounding fibrous and hematopoietic tissue (a) and blood vessel (bv). Representative BrdU staining for localization of implanted CMSCs (C-D) and DMSCs (G-H). BrdU-stained implanted cells were found lining the mineralized matrix (black arrows) and surrounding fibrous tissue. Brown nuclear staining is indicative of DAB reactivity. There was no immunoreactivity present in sections stained with isotype-matched antibodies. HA/TCP: hydroxyapatite/tricalcium phosphate particles. Magnification is 100X and scalebar is 500 μm.</p

EGb761 decreased BBB permeability in Aβ₁_–42 oligomer-induced bEnd.3 cells.

<p>BBB permeability, evaluated by the Na-F leakage test, was assessed after incubation with 10 µM Aβ_1–42 oligomer. Cells were incubated with or without various concentrations of EGb761 for 2 h, followed by incubation with Aβ_1–42 oligomer for 24 h. Then, the absorbance of Na-F was determined by fluorescence spectrophotometry. Results are shown as the Mean±S.E.M. (*<i>p</i><0.01, Aβ versus Control; #<i>p</i><0.01, EGb761+Aβ versus Aβ).</p

Antibodies used for characterizing CMSCs and DMSCs by flow cytometry.

<p>^a anti-human antibodies raised in mice.</p><p>Antibodies used for characterizing CMSCs and DMSCs by flow cytometry.</p

EGb761 attenuated the Aβ₁_–42 oligomer-induced increase of ROS.

<p>Panel A: ROS generation in bEnd.3 cells was evaluated by the oxidation of H₂DCF-DA to DCF (Fig. 3A, Control) and assessed by inverted fluorescent microscopy (100×). Following treatment for 24 h with 10 µM Aβ_1–42 oligomer, an increase in fluorescence was detected (Fig. 3A, Aβ). Cells treated with 100 µg/mL EGb761 for 2 h prior Aβ_1–42 oligomer treatment for 24 h, showed a decrease in fluorescence (Fig. 3A, EGb761+Aβ). Panel B shows the relative levels of intracellular ROS quantified by a microplate reader (488 nm excitation and 525 nm emission), with the results normalized to the control (set at 100). Results are shown as the Mean±S.E.M. (*<i>p</i><0.01, Aβ versus Control; #<i>p</i><0.01, EGb761+Aβ versus Aβ).</p