The reliability of colorimetry is precise(ly) as expected.

Figure S6. Gene ontology (GO) analysis of the differentially expressed genes between C1 and C2. (JPG 403 kb

Phenotypes of fES-4N and iPS-4N full-term pups after C-section at E18.5.

<p>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.</p

Improved Derivation Efficiency and Pluripotency of Stem Cells from the Refractory Inbred C57BL/6 Mouse Strain by Small Molecules

<div><p>The ability of small molecules to maintain self-renewal and to inhibit differentiation of pluripotent stem cells has been well-demonstrated. Two widely used molecules are PD 98059 (PD), an inhibitor of extracellular-signal-regulated kinase 1 (ERK), and SC1 (Pluripotin), which inhibits the RasGAP and ERK pathways. However, no studies have been conducted to compare their effects on the pluripotency and derivation of embryonic stem (ES) cells from inbred mice C57BL/6, an important mouse strain frequently used to model behavior, cognitive functions, immune system, and metabolic disorders in humans and also the first mouse strain chosen to be sequenced for its entire genome. We found significantly increased derivation efficiency of ES cells from in vivo fertilized embryos (fES) of C57BL/6 with the use of PD (71.4% over the control of 35.3%). Because fES and ES from cloned embryos (ntES) are not distinguishable in transcription or translation profiles, we used ntES cells to compare the effect of small molecules on their <i>in vitro</i> characteristics, <i>in vitro</i> differentiation ability, and the ability to generate full-term ntES-4N pups by tetraploid complementation. NtES cells exhibited typical ES characteristics and up-regulated Sox2 expression in media with either small-molecule. Higher rates of full term ntES-4N pup were generated by the supplementation of PD or SC1. We obtained the highest efficiency of ntES-4N pup generation ever reported from this strain by supplementing ES medium with SC1. Lastly, we compared the pluripotency of fES, ntES and induced pluripotent stem (iPS) cells of C57BL/6 background using the tetraploid complementation assay. A significant increase in implantation sites and the number of full-term pups were obtained when fES, ntES, and iPS cells were cultured with SC1 compared to the control ES medium. In conclusion, supplementing ES cell culture medium with PD and SC1 increases the derivation efficiency and pluripotency, respectively, of stem cells derived from the refractory inbred C57BL/6 strain.</p></div

Embryoid body (EB) formation and differentiation of C57BL/6 ntES cells after withdrawal of LIF and SC1 from SC1-supplemented medium.

<p>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.</p

Characterization of C57BL/6 ntES cells cultured in different media.

<p>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, <i>P</i><0.05.</p

Induction and <i>in vitro</i> characterization of iPS cells from C57BL/6 strain.

<p>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.</p

Effects of SC1 on the <i>in vivo</i> developmental potential of fES, ntES and iPS cells of the C57BL/6 strain by the tetraploid complementation assay.

a,b<p>Comparision of SC1 supplementation within each cell type; data with different superscripts are significantly different (p<0.05).</p>c,d,e<p>Comparisions among three cell types in control media only; data with different superscripts between cell types are significantly different (p<0.05).</p>f,g<p>Comparision among three cell types in SC1 supplemented media; data with different superscripts are significantly different (p<0.05).</p>1<p>Two of the iPS-4N mice showed umbilical hernia phenotype.</p>2<p>One of the iPS-4N mouse showed umbilical hernia phenotype.</p><p>Effects of SC1 on the <i>in vivo</i> developmental potential of fES, ntES and iPS cells of the C57BL/6 strain by the tetraploid complementation assay.</p

Derivation of ES cell lines in different ES culture media from fertilized embryos of the C57BL/6 strain.

<p>*the other 6 outgrowths appeared differentiated after trypsinization.</p>a,b<p>Values within columns with different superscripts differ, p<0.05.</p><p>Derivation of ES cell lines in different ES culture media from fertilized embryos of the C57BL/6 strain.</p

Morphology of newly derived C57BL/6 fES cells cultured in different ES media.

<p>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.</p

Alteration in morphology and cell cycle distribution of C57BL/6 nuclear transfer ES (ntES) cells cultured in different media.

<p>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).</p