Journal: Advanced Healthcare Materials
Article Title: Droplet Microarray Based Screening Identifies Proteins for Maintaining Pluripotency of hiPSCs
doi: 10.1002/adhm.202200718
Figure Lengend Snippet: Validation of the screening protocol. A) Representative confocal laser scanning microscope (CLSM) images of hiPSCs cultivated in 200 nL droplets on DMA slides coated with Matrigel and stained for six pluripotency markers: SOX2, OCT‐4A, NANOG, TRA‐1‐60, SSEA4, and TRA‐1‐81. Three independent experiments ( n = 3) obtained comparable results. DAPI was used to counterstain nuclei. Scale bars: 50 µm. B) Bright‐field images of hiPSCs cultivated on positive (Matrigel, MG + ) and negative (no Matrigel, MG − ) controls. Scale bar: 100 µm. C) NANOG expression level was used as a read out for the primary screening. IF staining was carried out on DMA followed by automated microscopy. All colonies in one image were from the single droplet. Scale bar: 50 µm. D) Quantification of mean brightness (mean fluorescence intensity) of hiPSCs cultured on positive and negative controls and stained for the pluripotency marker NANOG. The mean fluorescence intensity was calculated as the total fluorescence intensity divided by the area (in pixels). *** p < 0.001, significant differences positive control and negative controls. ( Z ʹ value is between 0.5 and 1, corresponding to a high‐quality screening assay). Data were presented as mean ± 3SD. ( n = 3) E) Representative CLSM images of E‐cadherin expression in hiPSCs cultured on positive and negative control coatings. E‐cadherin mediates cell–cell interactions and contributes to stem cell colony formation and pluripotency. Thus, on the positive control coating, undifferentiated hiPSCs show high expression of E‐cadherin, while differentiated hiPSCs on the negative control coating show low‐to‐no E‐cadherin expression. DAPI was used to counterstain nuclei. Scale bar: 25 µm.
Article Snippet: DMA slides with 1 mm side length square spots (catalogue number: G‐np‐102) were purchased from Aquarray GmbH (Eggenstein‐Leopoldshafen, Germany).
Techniques: Biomarker Discovery, Laser-Scanning Microscopy, Staining, Expressing, Microscopy, Fluorescence, Cell Culture, Marker, Positive Control, Screening Assay, Negative Control