Tanigawa S, Sharma N, Hall MD, Nishinakamura R, Perantoni AO. Preferential propagation of competent SIX2+ nephronic progenitors by LIF/ROCKi treatment of the metanephric mesenchyme. Stem Cell Reports 5(3):435-447, 2015.
Understanding the mechanisms responsible for nephrogenic stem cell preservation and commitment is fundamental to harnessing the potential of metanephric mesenchyme (MM) for nephron regeneration. Accordingly, we have established a culture model that preferentially expands the Six2+ progenitor pool using leukemia inhibitory factor (LIF), a Rho Kinase inhibitor (ROCKi), and extracellular matrix. Passaged MM cells express key stem cell regulators, Six2 and Pax2, and remain competent to respond to Wnt4 induction and to form mature tubular epithelia and glomeruli (Figure). Mechanistically, LIF activates Stat, which binds to a Stat consensus sequence in the Six2 proximal promoter and regulates Six2 levels. ROCKi attenuates the LIF-induced differentiation activity of Jnk and upregulatesSlug expression only when LIF is present. Concomitantly, the combination of LIF/ROCKi activates Yap, which regulates the expression of Six2, Pax2, and Sall1. Using this novel model, our study underscores the pivotal roles of Six2 and Yap in MM stem cell renewal.
Figure. Schematic of the rat MM cell culture method. Isolated rat MMs were explanted on extracellular matrices-coated culture dishes in the presence of LIF and Y27632 in media without serum. Expanded cells are retained competence to form renal tubules and glomeruli.
