Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2020 Dec 11;11:6372. doi: 10.1038/s41467-020-20082-7

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© 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/.

PMC Copyright notice

Fig. 6 — a H&E staining of histological sections of thymic scaffolds harvested at different time points post-transplant (8, 11, 18 and 22 wpt). Asterisks indicates Hassall’s Bodies (HB); n = 18 scaffolds in 3 independent experiments. Scale bar, 100 μm. b H&E of histological section of a thymic scaffold not seeded with CD34⁺ HSC at grafting and harvested at 18 wpt; Asterisks indicates Hassall’s Bodies (HB). Scale bar, 100 μm. c Immunofluorescence analysis for CD3, E-Cadherin, CK5, Vimentin (VIM) and mouse Endomucin (Emcn) at 11 wpt for thymic scaffolds repopulated with stroma and CD34⁺ HSC; n = 4 scaffolds in two independent experiments. Scale bar, 50 μm. d Immunohistochemistry for AIRE-1 of thymus scaffolds harvested at 11, 18 and 22 wpt demonstrate progressive increase of AIRE-1⁺ cells; n = 8 scaffolds in three independent experiments. Scale bar, 50 μm. e HLA-DR was detected in cytokeratin-positive medullary (CK5) and cortical (CK8) cells in repopulated scaffolds by immunofluorescence; n = 18 scaffolds in three independent experiments. Scale bar, 25 μm. f Representative FACS analysis of a dissociated thymic scaffold at 11 wpt showing presence of CD3⁺ cells (80% of total human (h) CD45⁺ population; n = 9 scaffolds). g FACS analysis of dissociated thymic scaffolds demonstrates both double positive (DP) and single positive (SP) CD4 and CD8 cells. SP CD4 and CD8 cells also expressed CD3 and TCRαβ, demonstrating the presence of immature single positive (ISP) as well as fully mature thymocytes at both 11 wpt and 18 wpt time points (n = 9 scaffolds, live cells = 1100–3800). h Representative FACS analysis of sorted and in vitro expanded CD3⁺ cells. CD3⁺ cells were sorted either from repopulated thymus scaffolds or from the endogenous NSG thymus 18 wpt, expanded in vitro prior to FACS analysis that shows presence of CD4⁺ and CD8⁺ SP cells. Expanded CD4⁺ and CD8⁺ cells were stimulated by PMA-ionomycin and intracellular cytokine staining performed: CD4⁺ and CD8⁺ isolated from endogenous mouse thymus were able to produce IL2, TNFα and IFNγ, while SP CD4 and CD8 cells developed within thymus scaffolds were able to produce only limited amount of IL2. CD8 SP cells, though in minor number in each scaffold compared to CD4⁺ were able to produce the highest level of IFNγ and TNFα; n = 2 independent experiments (live cells from scaffolds = 7000 and from endogenous thymus = 52000).