Xiuchun (Cindy) Tian has been serving as an academic editor since 2011. However, this does not alter the authors’ adherence to PLOS ONE Editorial policies and criteria.
Conceived and designed the experiments: C-JL XT. Performed the experiments: C-JL YT. Analyzed the data: C-JL XT. Contributed reagents/materials/analysis tools: TA YT. Contributed to the writing of the manuscript: C-JL XT.
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
Small molecules have increasingly been applied to ES cell research to improve derivation efficiency and pluripotency maintenance. It has been postulated that the maintenance of ES cells at the ground state is not restricted to the LIF pathway
The mouse strain C57BL/6 is the most widely used inbred strain and the first stain chosen for genome sequencing. Although ES cell lines can be obtained using embryos from C57BL/6 mice
Unless otherwise indicated, chemicals were purchased from Sigma-Aldrich (St. Louis, MO).
Experimental mice were purchased from Charles River Laboratories (Wilmington, MA). Animal use and handling procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Connecticut. CD1 and C57BL/6 strains mice were used as embryo donors by superovulation
The control ES culture medium was prepared as follows: Knockout-DMEM medium (Invitrogen, Carlsbad, CA) supplemented with 15% Knockout Serum Replacement (KSR; Invitrogen), 100 mM nonessential amino acids, 100 mM 2-mercaptoethanol, 2 mM L-glutamine, 1000 U LIF (Millipore, Billerica, MA)
For derivation of C57BL/6 ES cell lines from fertilized embryos (fES), we harvested C57BL/6×C57BL/6
The ES cell line from embryos by somatic cell nuclear transfer (ntES)
NtES cells were trypsinized and dissociated into single cell suspensions, then plated onto gelatinized dishes for 30 min to remove feeder cells. For cell cycle analysis, unattached ntES cells were washed twice with DPBS+0.1% BSA, then centrifuged. Cell pellets were resuspended in 75% ethanol solution and stored at −20°C. For staining, cells were pelleted and resuspended in DPBS containing 10 µg/ml of propidium iodide (PI).
For the expression of specific-stage embryonic antigen-1 (SSEA-1), ntES cells were blocked in DPBS with 2% BSA for 15 min at room temperature. Cell lines were divided into two groups and stained with 10 µg/ml mouse IgM monoclonal SSEA-1 antibody (MAB4301, Millipore) or isotype control (IgM negative control, for outer membrane protein 1 of various strains of Neisseria gonorrhoeae, MABC008, Millipore) for 30 min on ice. After being washed in DPBS, cells were stained with the secondary antibody (Alexa Fluor 488 goat anti-mouse IgM, A21042, Invitrogen). Cells were washed and suspended in DPBS for flow cytometry analysis.
Flow cytometry was performed using a FACSCalibur or FACSAriaII flow cytometer (Becton Dickinson, Franklin Lakes, NJ). A total of 104 cells were collected from each batch. Cell debris, dead cells, and doublets were excluded by properly gating on the forward/side scatters. SSEA-1 isotype expression signals were acquired using the FL1-H or FL2-A channel. Cell cycle distributions were computed using the Dean/Jett/Fox module of the FlowJo software (Tree Star, Inc., Ashland, OR).
Detection of alkaline phosphatase (AP) activity was performed according to the manufacturer’s instruction (alkaline phosphatase substrate kit, Vector Laboratories, Burlingame, CA).
When typical ES cell colonies appeared, immunocytochemistry for pluripotency markers was performed. Briefly, cells were cultivated on glass cover slips with MEF feeder cells for 2–3 days, and fixed in 4% paraformaldehyde (PFA)/1% sucrose solution at 4°C overnight. Fixed samples were then washed in DPBS containing 0.25% Tween-20 (PBST) and treated with 0.5% Triton X-100 for permeabilization. Next, permeabilized samples were washed in PBST. Nonspecific signals were blocked by incubating cells in DPBS plus 2% BSA solution for 1 h at room temperature. ES specific markers, Oct 3/4, Nanog, Sox2 and SSEA-1, were detected with the following specific antibodies: rabbit polyclonal Oct 3/4 (sc-9081, Santa Cruz Biotechnology, Santa Cruz, CA), mouse monoconal Nanog (N3038), rabbit polyclonal Sox2 (ab-5603, Millipore), and SSEA-1 at concentration of 10 µg/ml at 37°C for 2 h. Species and isotype specific secondary antibodies (Alexa Fluor 488,546, Invitrogen) were chosen and incubated at 37°C for 1 h. After being washed in PBST, cell nuclei were counterstained with TO-PRO-3 iodide dye (T3605, Invitrogen) and mounted in ProLong Gold antifade reagent (P36930, Invitrogen). All immunostained images were created using a laser-scanning confocal microscope (Leica TCS SP2, Mannheim, Germany).
In addition to immunostains, RT-PCR and real-time quantitative RT-PCR were also performed as described in our previous report
NtES cells were trypsinized into single-cell suspensions and plated on gelatin-coated plates for 30 min to remove feeder cells. To create uniform-sized EBs, 3–5×105 unattached ES cells were added to AggreWell plates (Stemcell Technologies, Vancouver, BC, Canada) for 24 h. EBs and cystic EBs formed after 5 days of culture and were cultured for an additional 5 days. Total RNA was extracted and two representative markers from each of the three germ layers were selected for RT-PCR analysis:
Tetraploid (4N) embryos were generated as described in our previous report
ES cells were trypsinized and placed on ice for 30 min before 4N blastocyst microinjection. Fifteen to 20 cells were inserted into the blastocoel of the blastocysts using a piezo injection system (Prime Tech, Japan). The injected blastocysts were cultured for 1–2 h for recovery before being transferred into the uteri of E 2.5 recipient mice. C-section was performed at E 18.5; implantation sites, stillborn fetuses, or live pups were counted; and the weights of the bodies and placentas of 4N pups were recorded.
qRT-PCR results were analyzed with unpaired t-test. The efficiencies of ES derivation and full-term pup generation were analyzed with Pearson Chi-Square test using the Minitab software. Statistical significance was considered when p<0.05.
To evaluate the effect of small molecules on ES cell derivation, we used in vivo fertilized embryos because they are of superb quality in general and are therefore more suitable to derive ES cells in a refractory strain. Expanded blastocysts (
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.
Supplying either PD or SC1 increased the derivation efficiency of fES cell lines as shown in
Medium | No. of blastocysts | No. of attachedembryos | No. of outgrowthpicked | No. of welltrypsinization | No. of ES-like colonies(% of blastocysts) |
Control−LIF | 4 | 2 | 1 | 1 | 0a |
Control+LIF | 17 | 15 | 14 | 12 | 6 |
PD (5.6 µM)+LIF | 14 | 13 | 11 | 10 | 10 (71.4%)b |
SC1 (300 nM)+LIF | 23 | 19 | 16 | 13 | 13 (56.5%)a,b |
*the other 6 outgrowths appeared differentiated after trypsinization.
Values within columns with different superscripts differ, p<0.05.
To characterize the effects of the two molecules on ES cell maintenance, we compared the characteristics of ES cells cultured in three ES media: control ES medium and those supplemented with PD and SC1. In this series of experiments, we used an ES cell line generated from embryos cloned from C57BL/6 fibroblasts
First, we observed that after adapting the ntES cells in different media for 2–3 passages, the cells exhibited morphological changes (
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).
We then determined whether the colony morphology changes by small molecules were related to the expression of ES-specific markers by performing a series of ES cell characterizations. NtES cells expressed high activity of alkaline phosphates in all media studied (
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,
Typical ES cell pluripotent markers (Oct4, Nanog, and Sox2) were used to assess the in vitro pluripotency of ntES cells under the three culture conditions by both immunocytochemistry and real-time quantitative RT-PCR. NtES cells in all three groups were positively stained for Oct4, Nanog and Sox2 (
We also evaluated the
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.
The most stringent test for stem cell pluripotency is the tetraploid complementation assay. To investigate whether small molecules improve the
Cell type | Medium | Cell passageNo. | No. of embryostransferred | No. ofrecipients | No. of pregnantrecipients | No. of implantation sites(% of transferred embryos) | No. of stillbirths | No. of full-term pups(% of transferred embryos) |
fES | Control | P5 | 86 | 3 | 3 | 5 (5.8) | 1 | 1 (1.2)a,c |
SC1 (100 nM) | P3–P4 | 68 | 2 | 2 | 22 (32.4)b | 2 | 3 (4.4)a,f | |
ntES | Control | P6, P7 | 66 | 3 | 1 | 9 (13.6)a | 2 | 0a,d |
SC1 (100 nM) | P5 P6 | 68 | 3 | 2 | 42 (61.8)b | 0 | 7 (10.3)b,g | |
iPS | Control | P4 | 87 | 3 | 2 | 14 (16.1)a | 7 | 4 |
SC1 (100 nM) | P3 | 89 | 3 | 3 | 40 (44.9)b | 3 | 6 |
Comparision of SC1 supplementation within each cell type; data with different superscripts are significantly different (p<0.05).
Comparisions among three cell types in control media only; data with different superscripts between cell types are significantly different (p<0.05).
Comparision among three cell types in SC1 supplemented media; data with different superscripts are significantly different (p<0.05).
Two of the iPS-4N mice showed umbilical hernia phenotype.
One of the iPS-4N mouse showed umbilical hernia phenotype.
To compare the pluripotency of fES, ntES and iPS and to determine whether small molecule-supplementation enhances their pluripotency, we subjected all three stem cell types to the tetraploid complementation assay. We chose to use SC1 at 100 mM for maintenance culture, because this was the lowest effect dose of SC1 supplementation tested in our study. PD was not used here because it did not significantly improve the in vivo differentiation potential of ntES cells from the above experiment.
FES cells were selected from one of the C57BL/6 lines cultured in SC1-supplemented medium. The iPS cells were generated from C57BL/6 tail-tip fibroblasts that harbor and express the fibroblast marker fibrillin-2 (TTFs,
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.
We found that cells cultured in SC1-supplemented medium formed significantly more implantation sites in the 4N complementation tests (
Surprisingly, a high portion (3 out of 10) of the full-term pups derived from iPS cells showed a dilated umbilicus phenotype (
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.
Culture media and culture conditions for ES cells have been constantly modified and improved in the past three decades
Maintenance of the self-renewal property of ES cells has been proposed to involve not only the LIF/Jak/Stat3 pathway, but also other signaling molecules
The spontaneous in vitro differentiation capability of C57BL/6 ntES cells in regards to EBs formation, however, was not affected by the presence of small molecules. ntES cells cultured in all three media were capable of forming EBs and cystic EBs as well as expressing markers of the multiple germ layers after the withdrawal of LIF and small molecules. Small molecules, however, dramatically improved the pluripotency of ntES cells as determined by the tetraploid complementation assay: cells adapted in either the PD- or SC1-supplemented medium resulted in significantly more implantation sites and full-term ntES-4N pups from both supplemented groups (
Our data also support the hypothesis that modification of the ES culture medium with small molecules rescues the quality of fES, ntES, and iPS cell lines in this important mouse strain. Both PD and SC1 inhibit ERK kinase1 activity and prevent pluripotent cell lines from committing to a differentiation path. SC1 also inhibits the RasGAP pathway
During normal development, umbilical herniation ceases at 16.5 dpc, when the ventral body wall closes completely. Mice display umbilical hernia or omphalocele phenotypes when the ventral body wall fails to close
In summary, we demonstrated that all three types of pluripotent stem cells of the C57BL/6 background could generate full-term pups with high efficiency when cultured with SC1 (
Primer sequences and amplicon sizes of PCRs in this study.
(DOCX)
We thank the office of animal research services for taking care of experimental mice; thank Dr. Carol Norris for assistance in the confocal microscopy and flow cytometry; also appreciate veterinary medical diagnostic laboratory for the pathologic diagnosis of iPS-4N mouse.