Fig. 1: Prospective isolation of thymic epithelial (TEC) and interstitial cells (TIC).
a Left panel: Rhodamine-B staining of TEC: 500 cultured TEC were seeded in a dish for colony-forming efficiency (CFE); the dish was stained after 12 days of culture. TEC gave rise to colonies of variable sizes that stained differently with Rhodamine. Scale bar, 2 cm. Right panel: growth curve over serial weekly passaging of TEC (X axis, number of weekly passages; Y axis, cumulative cell number); significance, mean ± SD (n = 3; donor age from 2 months to 4 years). b Left panel: immunofluorescence labelling of thymic epithelial cells in human post-natal thymus where anti-CD205 antibody (green) marked cortical TEC (cTEC) and anti-CK5-14 (red) marked medullary TEC (mTEC). Scale bar, 50 μm. Right panel: FACS plot visualising cTEC (EpCAMlowCD205+, 4.87%), mTEC (EpCAMhighCD205−, 11.1%) and TIC (EpCAM−CD205−, 61.5%) in the CD45-negative population enriched for sorting (n = 18; age from 3 days to 6 years). Representative analysis, thymus donor age 4 months (live cells = 51600). c FACS plots showing CD90 expression in enriched EpCAMhigh mTEC (red), EpCAMlow cTEC (green) and EpCAM− TIC (blue) respectively. Gating was decided on negative unstained control value for each channel. Same thymus as in 1b. d Representative FACS analysis for CD49f and CD90 demonstrating CD49f+ (Type1) and CD49f− (Type2) populations. Same thymus as in 1b and 1c. e Rhodamine-stained indicator dishes of sorted cTEC and mTEC subtypes (4000 events/dish) cultivated for 12 days, colony-forming efficiency (CFE). mTECType1 is the most clonogenic population (2–4%) and cTECType1 (1–2%) is the clonogenic population of the cortex (n = 4). f Growth curve over serial weekly passaging of sorted mTECType1 and cTECType1 (X axis, number of weekly passages; Y axis, cumulative cell number; n = 4 independent cultures; data are presented as mean value ± SE; donor age from 2 months to 4 years old). g Gene expression signature of cultivated clonogenic TEC (unsorted, black), medullary (Type1 mTEC, red) and cortical TEC (Type1 cTEC, green). Heatmap and hierarchical clustering of samples according to expression of thymic identity genes (SIX1, EYA1, PAX1 and PAX9), markers of thymic cortex (CTSV, PRSS16 and LY75) and medulla (CD24 and CDH1).
