Changes in keratins and alpha-smooth muscle actin during three-dimensional reconstitution of eccrine sweat glands

We have examined the changes of keratins and alpha-SMA at various time points in order to investigate the development and differentiation of eccrine sweat gland cells during the course of three-dimensional (3D) reconstitution. Mixtures of eccrine sweat gland cells and Matrigel were injected subcutaneously into the inguinal regions of nude mice. At 1, 2, 4, 6, 8, 14, 21, 28, 35, and 42 days post-implantation, Matrigel plugs were removed and immunostained. We found that during 3D reconstitution, keratin and alpha-SMA expression changed in a time-dependent manner. At day 1, all cells stained positively for keratin isoforms K5, K14, and K15, with the staining intensity of K15 being weak and K5 and K14 being strong, but none of the cells displayed K7, K8, or alpha-SMA. As time progressed, spheroid-like structures formed with the inner layer acquiring K7 and K8, but losing K5 and K14 expression, and the outer layer acquiring alpha-SMA expression, but losing K15 expression. K8 expression was first noted at day 14, and K7 and alpha-SMA at day 21. The loss of K15 expression was first noted at day 14, K14 at day 21, and K5 at day 28. At 28, 35, and 42 days, the spheroid-like structures could be distinguished, by immunohistochemistry, as having secretory coil-like and coiled duct-like structures. We conclude that the changes in expression of keratins and alpha-SMA in 3D-reconstituted eccrine sweat glands are similar to those of native eccrine sweat glands, indicating that the 3D reconstitution of sweat glands provides an excellent model for studying the development, cytodifferentiation, and regulation of eccrine sweat glands.