Fig. 5

Differentiation of engrafted eye-wall c-kit⁺/SSEA1⁻ progenitor cells (green) in the retinas of rd1 mice. (A–D) Cells were labeled using quantum nanocrystal dots (QDs) before transplantation. Cells cultured in a dish and incubated with QDs labeled with green fluorescence (A) and merged with phase contrast (A′). Under digestion conditions, a high rate of labeling was observed (B). After fixation, QDs in the cells could still be seen clearly (C). Representative western blot bands of recoverin (Rec) (D) and rhodopsin (Rho) (E) versus β-actin at 4 weeks after transplantation. The blots were then quantitated for expression of Rec (F) and Rho (G), corrected for β-actin, and plotted as the mean expression ± SD (n = 3 for each time point, *P < 0.05). Representative images of rd1 mice injected with c-kit⁺/SSEA1⁻ cells at 4 weeks (I, K, N) and 8 weeks (P, R, U) after transplantation and age-matched control mice including wild-type (WT) mice (H, L, O, S) and rd1 mice injected with phosphate-buffered saline (PBS; J, M, Q, T). The outer nuclear layer (ONL) of WT mice expressed Rec generally (H, O) and formed the structure of inner segment (IS) and outer segment (OS; L, S); there were few Rec-expressing photoreceptors remaining in the PBS control group and IS/OS was hardly observed (J, M, 4 weeks; Q, T, 8 weeks). Immunohistochemical detection of c-kit⁺/SSEA1⁻ cell-derived photoreceptors expressing Rec (I) and Rho (K) with rod a-transducin (Gnat1)-positive OS-like structure (N) at 4 weeks and 8 weeks (P, R, U) post transplantation. Area in the white boxes is shown at higher magnification. White arrow, engrafted cells could form IS/OS-like structures; red arrow, engrafted cells had condensed nuclei, similar to normal rod photoreceptors; white arrowhead, engrafted cells could develop synaptic button-like structures. DAPI 4′,6-diamidino-2-phenylindole, INL inner nuclear layer, GCL ganglion cell layer. Scale bars represent 5 μm (C), 10 μm (H1–H4, I1–I4, J1–J4, K1–K4, L, M, N, N1–N2, O1–O4, P1–P4, Q1–Q4, R1–R4, S, T, U, U1–U2), and 20 μm (A, B, H–K, O–R) (Color figure online)