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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. 3 — A Clonogenic potential of CB-HSPCs expanded with hiPSC-EVs. Cells were cultured for 14 days in the control medium (Ctrl) or hiPSC-EVs medium (+hiPSC-EVs). On selected days of expansion 5 × 10² cells were seeded in a dedicated methylcellulose-based medium for colony-forming cell (CFC) assays. Hematopoietic colonies were counted after 14 days. The total number of colonies generated by the cells seeded for CFC assays on the indicated day of expansion is presented as mean ± SD (N = 3). *p < 0.05 for control vs. hiPSC-EVs-treated cells, two-tailed Student’s t-test. B The effect of short-term treatment with hiPSC-EVs on the clonogenic potential of expanded cells. Prior experiment, cells were expanded for 7 days in the control medium without hiPSC-EVs and were subsequently treated with hiPSC-EVs for 2, 6, or 24 h. Data on the graph present the clonogenic potential of hiPSC-EVs-treated cells expressed as the percentage of the control (cells untreated with hiPSC-EVs) in individual experimental repetitions (N = 4). Black lines represent the mean value, whereas the red line indicates the level of the control (100%). *p < 0.05 for control vs. hiPSC-EVs-treated cells, two-tailed Student’s t-test. C The cytoprotective effect of hiPSC-EVs on CB-HSPCs. Prior experiment, cells were expanded for 7 days in the control medium without hiPSC-EVs and were then treated with hiPSC-EVs for 2 h. Subsequently, staurosporine (100 nM) was added to the culture medium for 24 h. Cell viability was assessed by Annexin V Apoptosis Detection Kit and BD LSRFortessa flow cytometer. In gating strategy, live (Anx V- 7-AAD-), early-apoptotic (Anx V+ 7-AAD-), late apoptotic (Anx V+ 7-AAD+), and necrotic cells (Anx V- 7-AAD+) cells were analyzed. Left panel: representative dot plots with the percentage of cells present in individual gates. Right panel: summarized data of cell viability presented as mean ± SD (N = 4). Data on the graph demonstrate the percentage of live cells. The table summarizes the percentage of cells in particular cell death phases. *p < 0.05 for control vs. hiPSC-EVs-treated cells, two-tailed Student’s t-test.