Analysis of pancreatic cancer stem cell markers.

<p>Expression patterns of stem cell markers such as CD44, CD24, and ESA were compared between 2D (A) and 3D cultures (B). Percentages of the cell population expressing CD44, CD24, and ESA in Panc-1 cells cultured under 2D and 3D conditions are summarized in the table.</p

The miRNA expression profiles were compared between 2D and 3D cultures.

<p>A heatmap of differentially expressed miRNAs generated by unsupervised hierarchical cluster analysis (A). Differential expression levels of miRNAs between 2D and 3D cultures were confirmed using qRT-PCR for several genes selected from microarray data. Among the differentially expressed miRNAs, up-regulated miRNAs including miR-34b-5p, miR-578, miR-1304, and miR-324-5p and down-regulated miRNAs including miR-7-5p, and miR-34b-3p were analyzed using primers for mature mRNAs (B). Data are expressed as the mean ± SE of triplicates. * p<0.05, n = 3.</p

Schematic overview of pancreatic tumor spheroids (TS) generated in concave microwells.

<p>Pancreatic cancer cells cultured in agarose-coated 96 well plates produced only loose aggregates (A). The TS formation process in a BSA-coated concave microwell plate (B). The TS structure, compared with an <i>in vivo</i> tumor, showed a close resemblance to the <i>in vivo</i> condition. Cells in the TS retained the characteristics of <i>in vivo</i> tumors under 3D culture conditions.</p

Concave PDMS microwell plate for culture and growth of pancreatic tumor spheroids (TS).

<p>Shape and dimension of concave microwell plate 600 (A). Changes in size of three different pancreatic spheroids cultured in microwell 600 (B). Size distribution of Panc-1 spheroids cultured in three different sized-microwell plate during 13 days of culture (C). Data are expressed as mean ± SE of a minimum of 10 spheroids cultured in one microwell plate.</p

Expression of proteins associated with tumor growth and drug resistance.

<p>Spheroids were cultured for 5-β1, CTGF, and MT1-MMP (A) and for ECM proteins such as collagen type I, fibronectin, and laminin (B). (Scale bar  = 20 μm).</p

Differentially expressed miRNAs in Panc-1 cell cultured under 2D and 3D conditions. Fold change indicates 3D intensity/2D intensity.

<p>Differentially expressed miRNAs in Panc-1 cell cultured under 2D and 3D conditions. Fold change indicates 3D intensity/2D intensity.</p

Morphology and histological examination of tumor spheroids (TS) cultured in concave microwell 600 or in 96 well plates.

<p>Representative images of H&E stained paraffin sections or toluidine blue stained semi-thin sections, SEM and TEM images of HT-29 (A), Panc-1 (B), Aspc-1 (C), and Capan-2 (D) spheroids cultured in concave microwell 600 plates for 5 and 13 days. Aggregates of Panc-1, Aspc-1, and Capan-2 cells formed in agarose-coated 96 well plates shown as an H&E stained paraffin section or bright field images (E). Arrow: desmosome; dotted lines: gap junctions; arrow head: tight junction; cross: lipid droplet; I: invagination structure; N: necrotic regions. The scale bars indicate 100 μm, 50 μm, 2 μm and 500 μm, in H&E or toluidine blue stained, SEM, TEM and bright-field images, respectively.</p

Heterogeneous Niche Activity of <i>Ex-Vivo</i> Expanded MSCs as Factor for Variable Outcomes in Hematopoietic Recovery

<div><p><i>Ex-vivo</i> expanded mesenchymal stromal cells (MSCs) are increasingly used for paracrine support of hematopoietic stem cell (HSC) regeneration, but inconsistent outcomes have hindered ongoing clinical trials. Here, we show that significant heterogeneity in the niche activity of MSCs is created during their culture in various serum-supplemented media. The MSCs cultured under stimulatory or non-stimulatory culture conditions exhibited differences in colony forming unit-fibroblast contents, expression levels of cross-talk molecules (Jagged-1 and CXCL-12) and their support for HSC self-renewal. Accordingly, the enhancing effects of MSCs on hematopoietic engraftment were only visible when HSCs were co-transplanted with MSCs under stimulatory conditions. Of note, these differences in MSCs and their effects on HSCs were readily reversed by switching the cultures, indicating that the difference in niche activity can be caused by distinct functional state, rather than by clonal heterogeneity. Supporting the findings, transcriptomic analysis showed distinct upstream signaling pathways such as inhibition of P53 and activation of ER-stress response gene ATF4 for MSCs under stimulatory conditions. Taken together, our study shows that the niche activity of MSCs can vary rapidly by the extrinsic cues during culture causing variable outcomes in hematopoietic recoveries, and point to the possibility that MSCs can be pre-screened for more predictable efficacy in various cell therapy trials.</p></div

Effect of MSC culture conditions on the hematopoietic supporting activity during in-vitro co-culture.

<p>(A) Schematic illustration of the experimental scheme. CD34⁺ cells from UCB were co-cultured with hMSCs passage cultured under each serum condition (SS1,2, NSS1,2) and analyzed for their phenotype, CFC and long-term culture-initiating cells. (B) Effect of co-culture on the colony forming cells (CFC). Shown are the mean numbers ± SEM of CFCs in semi-solid medium derived from 300 input CD34⁺ cells co-cultured with each MSCs for 5 days (n = 3; ***, p < .0001). (C) CD34⁺ cells that had been co-cultured on each MSCs were analyzed for expansion of primitive hematopoietic repopulating cells (CD34⁺/CD90⁺). Total numbers of CD34⁺/90⁺ cells after co-culture were shown with SEM (n = 8; ***, p < .0001). (D) CD34⁺ cells co-cultured with each MSCs were subjected to 6 weeks’ long-term culture and analyzed for CFC content (n = 3; **, p < .001).</p

Penetration of DOX into Panc-1 spheroids.

<p>Penetration was evaluated by imaging DOX autofluorescence in sections of spheroids. Spheroids were cultured in a concave microwell 600 plate for 5 μM for 12 h. Fluorescence intensity across sections was measured and expressed as the mean ± SE of 5 replicates. (Scale bar  = 100 μm) * and **, p<0.01 and p<0.001, respectively.</p