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. 2019 Oct 10;9:14490. doi: 10.1038/s41598-019-50899-2

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Optimization of mBax_499 construct for enrichment of hESC-CMs. (a) General scheme of possible outcome after treatment with mBax_499 mRNA. The scheme includes basic scientific illustrations downloaded from somersault1824.com. (b) Quantitative PCR analysis of miR-499 and its corresponding host gene MYH7B expression levels during directed cardiac differentiation. (c) hESC-CMs purified by FACS AriaII sorter after staining with SIRPa antibody were treated with various concentrations of mBax_499 mRNA construct. Presto Blue viability assay was performed for undifferentiated hESCs (red) and purified hESC-CMs (blue) after 2 h (plane bars) and 24 h (stripe bars) of treatment (n = 2). (d,e) Undifferentiated hESCs and cardiac differentiated hESCs were transfected with eGFP mRNA labelled with Cy5 using the Stemfect reagent. After 4 h of incubation, the cells were analyzed by Imagestream^X. (d) Selected images of single cells presenting cellular uptake and localization of eGFP mRNA (red) and protein (green) captured by ImageStream^X. (e) Analysis for Cy5 and GFP intensity distribution. Populations: blue - untreated cells, red - cells treated with eGFP mRNA labelled with Cy5. All bar graphs indicate mean (SD). p values were generated using two-tailed unpaired student’s t-test. *p < 0.05.