Skin cell cultures express autoantigens and transcription factors important for thymic and T cell development. (A and B) Skin cell/HPC cultures express AIRE. Equal amounts of mRNA derived from skin cell/HPC cultures and from normal human thymus were analyzed by real-time RT-PCR for AIRE expression in the presence (+) or absence (–) of RT. RNA from skin cell–colonized matrices was isolated on days 0, 7, 14, 21, and 28 after the addition of HPCs. Signal for each sample was first normalized to cyclophilin A expression and then normalized to thymus expression. mw, molecular weight markers (100-kb ladder). (C) Fibroblasts in skin cell cultures express AIRE. AIRE expression was assayed by real-time RT-PCR in keratinocyte (Ker) and fibroblast (Fib) cocultures without HPC and in cultures of keratinocytes or fibroblasts alone. Combined cultures of skin cells were assayed after treatment with control medium (Con), IFN-γ, or IFN-γ followed by LTα stimulation. Signal for each sample was normalized to cyclophilin A and thymus expression. (D and E) Skin cell cocultures with and without HPC express the autoantigens MBP, PLP, S100β and thyroglobulin by quantitative real-time RT-PCR. RNA from skin cell/HPC cocultures harvested on day 14, matrices colonized with keratinocytes and fibroblasts alone, and thymus was analyzed in the presence (+) or absence (–) of RT. Signal for all samples was normalized to cyclophilin A and thymus expression. No product was detected in samples without RT. (F) Skin cell/HPC cultures express Foxn1 and Hoxa3, but do not express Pax1. Day-28 skin cell/HPC cultures were analyzed by real-time RT-PCR. Samples without RT had no signal in all samples. Expression was normalized to cyclophilin A, and expression of each factor was then normalized to thymus expression.