English: "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)."