Figure 1.
Morphology of newly derived C57BL/6 fES cells cultured in different ES media.
A. Expanded blastocysts of C57BL/6 mouse. B, C. Embryonic outgrowth from blastocyst after 2 (B) and 9 days (C) of culture on MEF feeder cells, respectively. D, E, F. ES-like colonies appeared after trypsinization in control ES medium (D), PD- (E) and SC1-supplemented media (F). G. Differentiated colony appeared after trypsinization in control ES medium. Scale bar = 50 µm.
Table 1.
Derivation of ES cell lines in different ES culture media from fertilized embryos of the C57BL/6 strain.
Figure 2.
Alteration in morphology and cell cycle distribution of C57BL/6 nuclear transfer ES (ntES) cells cultured in different media.
A, B, C. Slight differences of C57BL/6 ntES colonies cultured in control ES medium (A), PD- (B) and SC1-supplemented media (C). Scale bar = 50 µm. Arrow indicates the slight differentiation morphology in control ES medium. D, E, F. Histograms of cell cycle distribution of C57BL/6 ntES cells in the present in control ES medium (D), PD- (E) and SC1-supplemented media (F).
Figure 3.
Characterization of C57BL/6 ntES cells cultured in different media.
A. Alkaline phosphatase activity of ntES colonies cultured in different ES media. Scale bar = 50 µm. B. SSEA-1 expression profiles of ntES cells cultured in different ES media and characterized by flow cytometry. Pink lines refer to SSEA-1 profiles; green lines refer to negative isotype control. C. Immunocytochemistry detection of ES specific markers Oct4 (green) and Nanog (red) of C57BL/6 ntES cells cultured in different ES media. Scale bar = 25 µm. D. Immunocytochemistry detection of ES specific markers Sox2 (green) and Nanog (red) of C57BL/6 ntES cells cultured in different ES media. E. Effect of small molecules on ES specific marker expression level determined by real-time RT-PCR. a,b,cValues within grouped bar chart with different superscripts differ, P<0.05.
Figure 4.
Embryoid body (EB) formation and differentiation of C57BL/6 ntES cells after withdrawal of LIF and SC1 from SC1-supplemented medium.
A, B. EBs formation in microwells by C57BL/6 ntES cells 24 h after LIF and SC1 withdraw. C. Morphology of enlarged EBs and cystic EBs after 7 days of culture in suspension. D. RT-PCR analysis of day 10 EBs for markers of the three germ layers.
Table 2.
Effects of SC1 on the in vivo developmental potential of fES, ntES and iPS cells of the C57BL/6 strain by the tetraploid complementation assay.
Figure 5.
Induction and in vitro characterization of iPS cells from C57BL/6 strain.
A. Primary culture of fibroblasts from biopsy of tail-tip from C57BL/6 mouse. Scale bar = 200 µm. B, C. Morphology of C57BL/6 iPS colonies cultured in control ES medium (B) and SC1-supplemented medium (C), respectively. Scale bar = 50 µm. D. Alkaline phosphatase activity of C57BL/6 iPS cells. Arrows indicate the weakly stained colonies. Scale bar = 25 µm. E. RT-PCR detection of ES specific and fibroblast markers from C57BL/6 tail-tip fibroblast and iPS cells. F, G. Immunocytochemistry of C57BL/6 iPS cells by Oct4 (green) and Nanog (red; F) and Sox2 (green) and SSEA-1 (red; G) markers, DNA were counterstained by TO-PRO-3 (blue) Scale bar = 25 µm.
Figure 6.
Phenotypes of fES-4N and iPS-4N full-term pups after C-section at E18.5.
A. Normal phenotype of fES-4N pups. B. Phenotypes of normal and dilated umbilici found in iPS-4N pups. Arrow indicates region of the dilated umbilicus. Scale bar = 1 cm.