PMC:4996362 / 8943-13818
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{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/4996362","sourcedb":"PMC","sourceid":"4996362","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4996362","text":"3. Results\n\n3.1. CMA Technology to Screen for Bonafide Pluripotent Stem Cells and Evaluation of Population Heterogeneity within the Lines\nCMA is highly informative to immunophenotypically characterize any cell line although it is easier for those cells that grow in suspension since embedding allows the preservation of the 3D structures and the multi-cellular organization. Here, we applied CMA technology to validate the differentiation of the human adherent and neuralized iPS cell model AF22 [13]. We incorporated the AF22 cells into an array to demonstrate the usefulness of this technique and quickly and efficiently assess their differentiation using antigen expression. Harvested cells were prepared and fixed in formalin, suspended in low-melting agarose and embedded in paraffin to produce a cell block. The semi‑automatic tissue microarrayer Galileo CK 4500 was used to remove in triplicate 1 mm-cores from each cell block and to transfer them into a recipient paraffin block at precise coordinates (x; y). The three-dimensional distribution of cells was evaluated by quantifying hematoxylin-stained (HE) 5‑μm sections. Figure 1 shows the distribution AF22 cells grown on coverslip (A), scraping-harvested cells embedded in the donor block (Hematoxylin-eosin, HE, stained, B) and from a selected CMA core slice (C) with subsequent HE staining (D). Image analysis results show that the mean number of cells/mm2 in donor block sections (B) is close to those cells cultured on coverslip (A). This is possible since scraping and immediate embedding keeps the cell density intact in addition to good 3D distribution within matrix. Not only the scraping techniques maintains the cells density (A), but also the morphology after CMA-coring (B).\nFigure 1 AF22 iPS-derived cells grown on cover slip (A); cell density and distribution of paraffin embedded cells in the donor block, using cell scraping and hematoxylin-eosin stain (B) and 1mm diameter cell core showing the distribution and architecture of the scraped cells within each core cell microarray (CMA) (C) and stained with hematoxylin-eosin (D). In order to try to preserve cell morphology we assessed two different methodological approaches in cell harvesting: trypsinization and mechanical scraping using a rubber policeman. Figure 2 shows that PFA fixation followed by mechanical scraping preserves both the cell morphology and architecture (B and D); on the other hand trypsin treatment disrupts the elongated cell morphology with consequent decrease in staining visibility (A and C).\nFigure 2 Morphologic differences between trypsinized (A and C) and scraped cells (B and D). Images A and C show the cells with a round morphology at different magnifications. The mechanical scraping instead displays a better preservation of cell morphologycompared to those grown on coverslip (Figure 1A), facilitating visualization of the nucleus and cytoplasm staining.\n\n3.2. Immunocharacterization of AF22 iPS-Derived Cells by CMA Technology versus Cover Slip\nAF22 iPS-derived cell line was induced to differentiate into neuronal lineage for 21 days in three different methodological conditions (coverslip, trypsinization and mechanical scraping as described in the material and method section). Agarose cell pellets were embedded in paraffin and CMA construction was assessed using the Galileo CK4500 platform. IF analysis was performed to investigate the expression and distribution of the stem cell markers Nestin, Sox 2, SEL1L and β3‑tubulin, as previously described (Figure 3) [17]. The mechanical harvesting (3D) of the cells facilitates the immunofluorescence analysis when compared to the enzymatic treatments (3C). Detachment of the cells using a rubber policeman gathers the majority of the cells from the flask maintaining the number of cells and preserving the elongated neuronal morphology. Moreover, this approach allows better marker localization (more similar to coverslip grown cells) if compared to trypsinized cells. These characteristics are very suitable for CMA applications.\nFigure 3 Immunocharacterization of the AF22 iPS-derived cell line by CMA technology versus coverslip. Undifferentiated (A) and differentiated AF22 cells (B) grown on coverslip were analyzed for Nestin, Sox2 and β3-tubulin expression by immunofluorescence. Undifferentiated cells were uniformly immunopositive for the neural precursor cell markers Nestin, Sox2 and SEL1L, a protein involved in neural lineage commitment but negative for the neuronal differentiation markers β3-tubulin. A clear decrease of Nestin, Sox2 and SEL1L is shown in the differentiated (B) cells with the concomitant increase of β3-tubulin expression. AF22 cells grown in stemness and differentiated conditions were simultaneously grown in flasks and collected either by trypsinization (C) or with a rubber policemen (D). Scale bars: 100 µm.\n\n4.","divisions":[{"label":"Title","span":{"begin":0,"end":10}},{"label":"Section","span":{"begin":12,"end":2930}},{"label":"Title","span":{"begin":12,"end":137}},{"label":"Figure caption","span":{"begin":1749,"end":2110}},{"label":"Figure caption","span":{"begin":2554,"end":2929}},{"label":"Section","span":{"begin":2928,"end":4874}},{"label":"Title","span":{"begin":2928,"end":3017}},{"label":"Figure caption","span":{"begin":4056,"end":4873}}],"tracks":[{"project":"2_test","denotations":[{"id":"27600341-22272239-69477248","span":{"begin":497,"end":499},"obj":"22272239"},{"id":"27600341-17308349-69477249","span":{"begin":3541,"end":3543},"obj":"17308349"}],"attributes":[{"subj":"27600341-22272239-69477248","pred":"source","obj":"2_test"},{"subj":"27600341-17308349-69477249","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#93ecc6","default":true}]}]}}