Stepwise renal lineage differentiation from mouse embryonic stem cells

This protocol describes induction of renal lineages from mESCs, namely, the induction of mesoderm (Stage I), intermediate mesoderm (Stage II) and renal lineages (Stage III). For mesoderm induction, sequential addition of 3 factors: Activin-A, BMP-4 and lithium proved most effective to induce the mesodermal marker, Brachyury. For intermediate mesoderm induction, the addition of retinoic acid in the continuous presence of previous 3 factors is most effective in inducing intermediate mesodermal markers, such as Pax2 and Lim1. For renal lineages induction, in resemblance to the reciprocal induction between ureteric bud (UB) and metanephric mesenchyme (MM) during kidney development, the exposure to conditioned media derived from UB and MM cells is effective in inducing MM and UB markers, respectively.