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The aim of this study was to induce organized layered tissues with characteristics of the urinary tract from embryonic stem (ES) cells alone. We seeded embryoid bodies (EBs) originating from mouse ES cells onto mono-layered collagen membranes and cultured them in four different media. Group 1 was grown in a mixed medium of keratinocyte serum-free medium (KSFM) and Medium 199, Group 2 in a mixed medium of KSFM and conditioned medium collected from 3T3 fibroblasts, Group 3 in an EB formation medium (control group), and Group 4 in KSFM only (control group). After 28 days, cultured tissues were transplanted into nude mice. Cultured tissues from Groups 1 and 2 formed four-layered structures comprising a stratified epithelium, a submucosal loose connective tissue layer, a smooth muscle cell layer identified immunohistochemically by alpha-smooth muscle actin, and a deep loose connective tissue layer identical to the adventitia. Immunohistochemistry showed that the epithelia were positive for cytokeratins. Tissues also expressed uroplakin as detected by reverse transcription/polymerase chain reaction. In contrast, specimens from Groups 3 and 4 demonstrated necrotic features. Uroplakin-positive (i.e., urothelium-like) cells developed only in Group 2 in the transplanted culture tissues in nude mice. In addition to inducing organized layered tissues from mouse ES cells directly in vitro, these findings demonstrate that tissues cultured in KSFM plus conditioned medium from 3T3 fibroblasts differentiate into luminal walls similar to those of urinary tract in vivo. These findings suggest a new approach to urinary tract regeneration.

Direct induction of layered tissues from mouse embryonic stem cells: potential for differentiation into urinary tract tissue