alization view of Waddington’s epigenetic landscape, fates of somatic cells are progressively determined as cellular differentiation proceeds, like going downhill. It is generally believed that reversal of differentiated status requires artificial physical or genetic manipulation of nuclear function such as nuclear transfer, or the introduction of multiple transcription factors. Here we investigated the question of whether somatic cells can undergo nuclear reprogramming simply in response to external triggers without direct nuclear manipulation. This type of situation is known to occur in plants—drastic environmental changes can convert mature somatic cells (for example, dissociated carrot cells) into immature blastema cells, from which a whole plant structure, including stalks and roots, develops in the presence of auxins.
A challenging question is whether animal somatic cells have a similar potential that emerges under special conditions. Over the past decade, the presence of pluripotent cells (or closely relevant cell types) in adult tissues has been a matter of debate, for which conflicting conclusions have been reported by various groups. However, no study so far has proven that such pluripotent cells can arise from differentiated somatic cells.
Haematopoietic cells positive for CD45 (leukocyte common antigen) are typical lineage-committed somatic cells that never express pluripotency-related markers such as Oct4 unless they are reprogrammed. We therefore addressed the question of whether splenic CD45+ cells could acquire pluripotency by drastic changes in their external environment such as those caused by simple chemical perturbations.
CD45+ cells were sorted by fluorescence-activated cell sorting (FACS) from the lymphocyte fraction of postnatal spleens (1-week old) of C57BL/6 mice carrying an Oct4-gfp transgene, and were exposed to various types of strong, transient, physical and chemical stimuli (described below). We examined these cells for activation of the Oct4 promoter after culture for several days in suspension using DMEM/F12 medium supplemented with leukaemia inhibitory factor (LIF) and B27 (hereafter called LIF+B27 medium).
Spontaneous neural conversion from salamander animal caps by soaking the tissues in citrate-based acidic medium below pH 6.0 has been demonstrated previously.
