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. 2015 Jun 16;23(7):1262–1277. doi: 10.1038/mt.2015.77

Figure 2.

Figure 2

Reconstruction of thymus organoids from decellularized thymus scaffolds. (a) Light microscopic images of a decellularized thymus scaffold (left panel) and a reconstructed thymus organoid (with CD45– thymic stromal cells and bone marrow cells of Lin– population at 1 : 1 ratio) cultured overnight in vitro (right panel). (b) Fluorescent microscopic images of thymic stromal cells (TSCs) cultured either as “hanging drop” overnight (left panel) or in the 3-D scaffold for 7 days (right panel). Live cells were discriminated from the dead cells by their intracellular esterase activities to generate green fluorescent calcein-AM (green) and their capabilities to exclude the red-fluorescent ethidium homodimer-1 (EthD-1, red) from entering the nucleus. (c,d) Representative immunohistochemical images of reconstructed thymus organoids cultured in vitro for 7 (d) or 21 (c) days. (c) Cryosections were stained with antibodies against Epcam (green), counterstained with either anti-CD45 (red, left panel) or anti-Ki67 (red, right panel) antibodies. In the left panel, white arrows show the presence of close interactions between the CD45+ thymocytes and the Epcam+ thymic epithelial cells (TECs). In the right panel, the yellow arrows show the presence of multicellular thymus nurse cell complex, whereas the red arrow shows a Ki67+Epcam+ TEC. (d) Cryosections were stained with endothelial cell-specific anti-CD31 antibodies (red, left panel) and fibroblast-specific antibodies (red, fibro, right panel). Both sections are counterstained with the anti-Epcam antibodies (green) and the Hoechst 33342 dye (blue) for TECs and nuclei, respectively. (e) Semiquantitative RT-PCR analyses of CD45– thymic stroma specific gene expression in TSCs, reconstructed thymus organoids cultured in vitro for 0 and 7 days (day 0 and day 7, respectively). Sample dilutions: undiluted, 1/4, 1/16, and 1/64. (f) RT-PCR analyses of tissue-specific antigen transcription in reconstructed thymus organoids cultured in vitro for 0, 7, 28, and 56 days. All the experiments were repeated at least once with similar results.