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. 2021 Jun 17;35(10):2964–2977. doi: 10.1038/s41375-021-01325-y

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© The Author(s) 2021

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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Fig. 2 — CD34⁺ cell population highly enriched in HSPCs was purified from CB units and cultured in a dedicated expansion medium containing hiPSC-EVs. A Visualization of hiPSC-EVs internalization into CB-derived CD34⁺ cells. Cells were incubated with copGFP+ hiPSC-EVs for 2 h. After the removal of unbound hiPSC-EVs, cell nuclei were stained with Hoechst 33342 dye (Hoe). Cell images were captured by Leica DMI6000B microscope with 100× NA-1.47 oil immersion objective. A representative merged image for differential interference contrast module and fluorescence channels for copGFP and Hoe is presented. White arrows indicate copGFP+ hiPSC-EVs accumulated inside the CB-HSPCs. White asterisks indicate aggregated copGFP+ hiPSC-EVs attached to the cells. Scale bar indicates 5 µm. B Kinetics of CB-HSPCs ex vivo expansion. Data are expressed as fold expansion compared to the number of isolated CB-HSPCs. Each dot represents data obtained from individual experimental repetition (N = 4) for cells expanded in the control medium (Ctrl) or hiPSC-EVs (+hiPSC-EVs) medium. C The effect of hiPSC-EVs on the metabolic activity of CB-derived HSPCs. Expanding CB-HSPCs were treated with hiPSC-EVs for 2, 24, or 48 h. Subsequently, a luminescence assay was performed to measure the concentration of ATP produced by the cells. Data on the graph present the ATP level in hiPSC-EVs-treated cells expressed as the percentage of the control (cells untreated with hiPSC-EVs) in individual experimental repetitions (N = 5). Black lines represent the mean value, whereas the red line indicates the level of the control (100%). *p < 0.05 for the control vs. hiPSC-EVs-treated cells, two-tailed Student’s t-test. D Kinetics of phenotypic changes in CB-HSPCs during ex vivo expansion. Cells were expanded for 14 days in control medium (Ctrl) or medium containing hiPSC-EVs (+hiPSC-EVs). On the indicated day of the expansion, cells were harvested and stained with fluorescent-conjugated antibodies against CD34 and hematopoietic lineage markers. The analysis of antigen expression was performed with the BD LSRFortessa flow cytometer. Data are presented as mean ± SD (N = 3). *p < 0.05 for control vs. hiPSC-EVs-treated cells, two-tailed Student’s t-test.