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. 2020 Oct 2;11:4964. doi: 10.1038/s41467-020-18802-0

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© The Author(s) 2020

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 — a Confocal microscopy imaging demonstrated the internalization of isolated PMPs (green) into CD34⁺-HSPCs (cytoplasm, red; nuclei, blue) (top: monochrome, bottom: merge. Incubation time: 72 h. Scale bars: 5 μm). b FCM analysis of CD34⁺-HSPCs showed that the majority of the cells internalized the supplemented PMPs stained with PKH67. PMPs did not apparently alter CD34⁺-HSPC surface marker (CD107a) expression during a 72 h culture in MK induction medium (n = 3 donors, two-way repeated-measures ANOVA with Bonferroni’s multiple comparison tests, p < 0.0001). c PMPs promoted the proportion of CD61⁺ and CD41⁺/CD61⁺ cells on day 15. Representative FCM plots are shown in Supplementary Fig. 4a (mean ± S.E.M. of n = 7 donors). d Cycloheximide (CHX) inhibited the PMP-mediated promotion of the proportion of CD61⁺ and CD41⁺/CD61⁺ cells on day 10. (mean ± S.E.M of three donors, two-way repeated-measures ANOVA with Bonferroni’s multiple comparison tests). e PMPs expanded CFU-MK colonies from 72 h-treated CD34⁺-HSPCs. Colonies were stained with human CD41 antibody, and the size was calculated (Scale bars: 100 μm). (n = 3 donors) f Twenty days after PMP internalization, megakaryocytes derived from the CD34⁺-HSPCs showed enlarged cell dimensions. Cytospin cells were stained with Wright-Giemsa solutions and measured from five random views. The error bars represent the standard deviation. (Two-tailed unpaired t-tests with Welch’s correction, p < 0.0001). g The percentage of cultured CD61⁺-promegakaryocytes five days after PMP internalization in each ploidy is shown. Representative FCM plots are shown in Supplementary Fig. 4e. (n = 4 donors) h The number of platelets released in the culture media normalized to each CD61⁺-promegakaryocyte is shown. Representative FCM plots are shown in Supplementary Fig. 4f. (n = 4 donors) i FCM analysis of the megakaryocytic lines K562, Meg-01 and UT-7 showed that PMP supplementation demonstrated similar or even better megakaryocytic promotion effects than PMA treatment. Megakaryocyte differentiation was determined by the CD41⁺/CD61⁺ proportions. Representative FCM plots are shown in Supplementary Fig. 3c (n = 3 independent experiments, one-way ANOVA with Bonferroni’s multiple comparison tests, p < 0.0001). All data are expressed as mean ± S.D from two-tailed paired-samples t-tests, P-values unless otherwise specified: Source data are provided as a Source Data file *P < 0.05, **P < 0.01, ***P < 0.001.