Figure 4

Characterization of KSP+ cells derived from hESCs.

(a) A protocol to obtain KSP+ cells using flow cytometry from stochastically differentiated hESCs for 7 days after primitive streak induction with BIO for 3 days. (b,c) Characterization of KSP+ cells purified from hESCs (KSP+) (white bars) by comparison with unsorted differentiated hESCs on day 10 of differentiation (BIO+, unsorted) (black bars) and primary renal proximal tubular epithelial cells (RPTEC) (gray bars). Relative expression levels of the kidney tubular marker genes indicated were determined by real-time PCR for KSP+ cells, relative to the unsorted differentiated hESCs (b) and RPTEC (c). (d,e) Functional assay of KSP+ cells was determined by GGT activity. (d) Relative expression levels of GGT were determined by real-time PCR for RPTEC, relative to the KSP+ cells. (e) The concentration of p-nitroaniline (pNA) produced by KSP+ cells and RPTEC was measured and normalized to the cell number. (n = 3). (f–k) A protocol to form tubular organoids by KSP+ cells in 3D Matrigel. KSP+/TRA1-60 negative cells purified by flow cytometry after 10 d of differentiation were transferred onto Matrigel and cultured for 24–48 h. Light microscopy (g), electron microscopy of a cross section (h) and immunostaining for acetylated tubulin (i) of tubular organoids formed by KSP+ cells. (j–m) After 24 h, KSP+ cells co-cultured with NIH3T3-Wnt4 formed tubular organoids that contained MEGALIN (k), AQP1 (l), or AQP2 (m) positive cells. After 48 h, the relative expression levels of the kidney tubular marker genes indicated were determined by real-time PCR for KSP-positive cells before and after tubular formation (n). Scale bar; 500 μm (g), 2 μm (h,i), (j) 500 μm (left), 100 μm (right), 50 μm (k–m). Transcript expression levels were normalized to GAPDH (n = 2–6). Values shown are the means ± SEM. P-values were determined by a Student’s t-test. *P < 0.05; **P < 0.01.