Cultured Mouse Embryonic Stem Cells Research Articles

Development of Apolipoprotein E–Deficient Mice

The first mouse carrying a deletion mutation in its apolipoprotein E (apoE) gene was born in our mouse colony in the summer of 1991. Our ongoing experiments to that point had indicated that this would happen, but the realization that the mouse in front of my eyes had inherited a mutation that we had designed and made in the laboratory filled me with awe. It did not occur to me immediately that this mouse would develop hypercholesterolemia and atherosclerosis and dictate the direction of my research for the next 20 years. I here briefly describe the background and my personal experience that led to the development of apoE-deficient mice as a model for human atherosclerosis. As a research scientist in the Department of Genetics at the University of Wisconsin, my main interest during the early 1980s was directed toward understanding how different types of DNA recombination contribute to the architecture and diversity of individual genomes within human populations and to DNA differences between humans and nonhuman primates. My studies clearly showed that new alleles and new genes are constantly being formed in the human genome by recombinational events, particularly between existing members of multigene families. There also appeared to be hot spots for recombination. By tracing a history of the human haptoglobin-gene cluster from the present-day structures in human populations and in other primates, I found that a single recombination event between related genes often produces much more drastic changes in the genome than those that result from simple point mutations.1 Whether this frequent yet natural occurrence of homologous recombination in the mammalian genome could be used to alter the genome of mammalian cells in culture in a preplanned fashion became a question of interest to several laboratories.2,3 An affirmative answer was first obtained in 1985, …

Ionizing radiation is a potent inducer of mitotic recombination in mouse embryonic stem cells

Maintenance of genomic integrity in embryonic cells is pivotal to proper embryogenesis, organogenesis and to the continuity of species. Cultured mouse embryonic stem cells (mESCs), a model for early embryonic cells, differ from cultured somatic cells in their capacity to remodel chromatin, in their repertoire of DNA repair enzymes, and in the regulation of cell cycle checkpoints. Using 129XC3HF1 mESCs heterozygous for Aprt, we characterized loss of Aprt heterozygosity after exposure to ionizing radiation. We report here that the frequency of loss of heterozygosity mutants in mESCs can be induced several hundred-fold by exposure to 5–10 Gy of X-rays. This induction is 50–100-fold higher than the induction reported for mouse adult or embryonic fibroblasts. The primary mechanism underlying the elevated loss of heterozygosity after irradiation is mitotic recombination, with lesser contributions from deletions and gene conversions that span Aprt. Aprt point mutations and epigenetic inactivation are very rare in mESCs compared to fibroblasts. Mouse ESCs, therefore, are distinctive in their response to ionizing radiation and studies of differentiated cells may underestimate the mutagenic effects of ionizing radiation on ESC or other stem cells. Our findings are important to understanding the biological effects of ionizing radiation on early development and carcinogenesis.

Exocyst Complex Component 3-like 2 (EXOC3L2) Associates with the Exocyst Complex and Mediates Directional Migration of Endothelial Cells

The exocyst is a protein complex that ensures spatial targeting of exocytotic vesicles to the plasma membrane. We present microarray data obtained from differentiating mouse embryonic stem cell cultures that identify an up-regulation of exocyst complex component 3-like 2 (exoc3l2) mRNA in sprouting blood vessels. Vascular expression of exoc3l2 is confirmed by qPCR analysis of different mouse tissues and immunofluorescence analyses of mouse brain sections. We detect an up-regulation of exoc3l2 mRNA synthesis in primary human endothelial cells in response to VEGFA, and this response is enhanced when the cells are grown on a three-dimensional collagen I matrix. Myc-tagged EXOC3L2 co-precipitates with the exocyst protein EXOC4, and immunofluorescence detection of EXOC3L2 shows partial subcellular colocalization with EXOC4 and EXOC7. Finally, we show that exoc3l2 silencing inhibits VEGF receptor 2 phosphorylation and VEGFA-directed migration of cultured endothelial cells.

Identification of a truncated form of methionine sulfoxide reductase a expressed in mouse embryonic stem cells

BackgroundMethionine Sulfoxide Reductase A (MsrA), an enzyme in the Msr gene family, is important in the cellular anti-oxidative stress defense mechanism. It acts by reducing the oxidized methionine sulfoxide in proteins back to sulfide and by reducing the cellular level of reactive oxygen species. MsrA, the only enzyme in the Msr gene family that can reduce the S-form epimers of methionine sulfoxide, has been located in different cellular compartments including mitochondria, cytosol and nuclei of various cell lines.MethodsIn the present study, we have isolated a truncated form of the MsrA transcript from cultured mouse embryonic stem cells and performed eGFP fusion protein expression, confocal microscopy and real time RT-PCR studies.ResultsResults show a different expression response of this truncated transcript to oxygen deprivation and reoxygenation treatments in stem cells, compared to the longer full length form. In addition, a different subcellular localization pattern was noted with most of the eGFP fusion protein detected in the cytosol.ConclusionOne possibility for the existence of a truncated form of the MsrA transcripts could be that with a smaller protein size, yet retaining a GCWFG action site, this protein might have easier access to oxidize methionine residues on proteins than the longer form of the MsrA protein, thus having an evolutionary selection advantage. This research opens the door for further study on the role and function of the truncated MsrA embryonic mouse stem cells.

Pituitary adenylyl cyclase‐activating peptide counteracts hedgehog‐dependent motor neuron production in mouse embryonic stem cell cultures

Pituitary adenylyl cyclase-activating peptide (PACAP; ADCYAP1) is a neuropeptide that regulates a wide array of functions within the brain and periphery. We and others have previously demonstrated that PACAP and its high-affinity receptor PAC1 are expressed in the embryonic mouse neural tube, suggesting that PACAP plays a role in early brain development. Moreover, we previously showed that PACAP antagonizes the mitotic action of Sonic hedgehog (Shh) in postnatal cerebellar granule precursors. In the present study, we demonstrate that PACAP completely blocked Shh-dependent motor neuron generation from embryonic stem cell cultures and reduced mRNA levels of the Shh target gene Gli-1 and several ventral spinal cord patterning genes. In vivo examination of motor neuron and other patterning markers in embryonic day 12.5 spinal cords of wild-type and PACAP-deficient mice by immunofluorescence, on the other hand, revealed no obvious alterations in expressions of Islet1/2, MNR2, Lim1/2, Nkx2.2, or Shh, although the Pax6-positive area was slightly expanded in PACAP-deficient spinal cord. Caspase-3 staining revealed low, and similar, numbers of cells undergoing apoptosis in embryonic wild-type vs. PACAP-deficient spinal cords, whereas a slight but significant increase in number of mitotic cells was observed in PACAP-deficient mice. Thus, although PACAP has a strong capacity to counteract Shh signaling and motor neuron production in vitro, corresponding patterning defects associated with PACAP loss may be obscured by compensatory mechanisms.

Recombinase-mediated cassette exchange (RMCE) and BAC engineering via VCre/VloxP and SCre/SloxP systems

Site-specific recombination is a powerful biotechnological tool for genome engineering. We previously reported two novel site-specific recombination systems, VCre/VloxP and SCre/SloxP, that do not cross-react with Cre/loxP and Flp/FRT in culture cells and mouse embryonic stem (ES) cells. In this study, a site-specific recombination assay in Escherichia coli was used to examine the activity of mutant VCre (H314L and Y349F) and mutant SCre (H317L and Y352F), in which both mutated residues lie within the active center of Cre recombination. The site-specific recombination activity of both mutants was significantly decreased. Recombinase-mediated cassette exchange (RMCE) using VloxP and the Vlox2272 mutant site was performed in E. coli by introducing a cassette bearing VloxP and Vlox2272 into a recipient plasmid bearing the same sites. RMCE using SloxP and Slox2272 was also performed by SCre recombinase. Moreover, BAC engineering via Red recombination and VCre/VloxP were demonstrated. First, the DNA cassette for modification was introduced into a BAC clone via Red recombination; second, the antibiotics resistance gene flanked by VloxP was removed from the BAC clone by induction of VCre recombinase. Such site-specific recombination systems may effectively be used in combination with other site-specific recombination systems or engineering tools (e.g., Red recombination).

Subregional Specification of Embryonic Stem Cell-Derived Ventral Telencephalic Tissues by Timed and Combinatory Treatment with Extrinsic Signals

During early telencephalic development, the major portion of the ventral telencephalic (subpallial) region becomes subdivided into three regions, the lateral (LGE), medial (MGE), and caudal (CGE) ganglionic eminences. In this study, we systematically recapitulated subpallial patterning in mouse embryonic stem cell (ESC) cultures and investigated temporal and combinatory actions of patterning signals. In serum-free floating culture, the dorsal-ventral specification of ESC-derived telencephalic neuroectoderm is dose-dependently directed by Sonic hedgehog (Shh) signaling. Early Shh treatment, even before the expression onset of Foxg1 (also Bf1; earliest marker of the telencephalic lineage), is critical for efficiently generating LGE progenitors, and continuous Shh signaling until day 9 is necessary to commit these cells to the LGE lineage. When induced under these conditions and purified by fluorescence-activated cell sorter, telencephalic cells efficiently differentiated into Nolz1(+)/Ctip2(+) LGE neuronal precursors and subsequently, both in culture and after in vivo grafting, into DARPP32(+) medium-sized spiny neurons. Purified telencephalic progenitors treated with high doses of the Hedgehog (Hh) agonist SAG (Smoothened agonist) differentiated into MGE- and CGE-like tissues. Interestingly, in addition to strong Hh signaling, the efficient specification of MGE cells requires Fgf8 signaling but is inhibited by treatment with Fgf15/19. In contrast, CGE differentiation is promoted by Fgf15/19 but suppressed by Fgf8, suggesting that specific Fgf signals play different, critical roles in the positional specification of ESC-derived ventral subpallial tissues. We discuss a model of the antagonistic Fgf8 and Fgf15/19 signaling in rostral-caudal subpallial patterning and compare it with the roles of these molecules in cortical patterning.

Differentiation of Embryonic Stem Cells Using Pancreatic Bud-Conditioned Medium Gives Rise to Neuroectoderm-Derived Insulin-Secreting Cells

Human embryonic stem cells can be differentiated into insulin-secreting cells by emulating in vitro the key processes that occur during embryonic development. However, the resulting cells are generally immature; thus, further research must be performed to identify the necessary factors to complete the differentiation. To this end, we cultured mouse embryonic stem cells with pancreatic bud-conditioned medium, based on a recent publication. Unlike in humans, mouse cells present two types of insulin, which can be used to identify different cell lineages. As a result, the cell product presented a neuroectodermal genetic expression pattern, with no expression of any definitive endodermal marker analyzed. Also, nonglucose-dependent insulin release was detected. Altogether, this previously published protocol results in neuroectoderm, and not definitive endoderm, derived insulin-positive cells. This further confirms the difficulty of obtaining true cell types of this germ layer. Finally, we identified a 16-kDa protein band that was present in pancreatic bud-conditioned medium. Sequencing this band revealed the presence of Reg proteins. The role of pancreatic bud-conditioned medium remains to be tested in definitive endoderm committed cells.

Anoxia, acidosis, and intergenic interactions selectively regulate methionine sulfoxide reductase transcriptions in mouse embryonic stem cells

Methionine sulfoxide reductases (Msr) belong to a gene family that contains one MsrA and three MsrBs (MsrB1, MsrB2, and MsrB3). We have identified all four of the genes that are expressed in mouse embryonic stem cell cultures. The vital cellular functions of the Msr family of genes are to protect cells from oxidative damage by enzymatically reducing the oxidized sulfide groups of methionine residues in proteins from the sulfoxide form (--SO) back to sulfide thus restoring normal protein functions as well as reducing intracellular reactive oxygen species (ROS). We have performed studies on the Msr family genes to examine the regulation of gene expression. Our studies using real-time RT-PCR and Western blotting have shown that expression levels of the four Msr family genes are under differential regulation by anoxia/reoxygenation treatment, acidic culture conditions and interactions between MsrA and MsrB. Results from these in vitro experiments suggest that although these genes function as a whole in oxidative stress protection, each one of the Msr genes could be responsive to environmental stimulants differently at the tissue level.

Soft Substrates Promote Homogeneous Self-Renewal of Embryonic Stem Cells via Downregulating Cell-Matrix Tractions

Maintaining undifferentiated mouse embryonic stem cell (mESC) culture has been a major challenge as mESCs cultured in Leukemia Inhibitory Factor (LIF) conditions exhibit spontaneous differentiation, fluctuating expression of pluripotency genes, and genes of specialized cells. Here we show that, in sharp contrast to the mESCs seeded on the conventional rigid substrates, the mESCs cultured on the soft substrates that match the intrinsic stiffness of the mESCs and in the absence of exogenous LIF for 5 days, surprisingly still generated homogeneous undifferentiated colonies, maintained high levels of Oct3/4, Nanog, and Alkaline Phosphatase (AP) activities, and formed embryoid bodies and teratomas efficiently. A different line of mESCs, cultured on the soft substrates without exogenous LIF, maintained the capacity of generating homogeneous undifferentiated colonies with relatively high levels of Oct3/4 and AP activities, up to at least 15 passages, suggesting that this soft substrate approach applies to long term culture of different mESC lines. mESC colonies on these soft substrates without LIF generated low cell-matrix tractions and low stiffness. Both tractions and stiffness of the colonies increased with substrate stiffness, accompanied by downregulation of Oct3/4 expression. Our findings demonstrate that mESC self-renewal and pluripotency can be maintained homogeneously on soft substrates via the biophysical mechanism of facilitating generation of low cell-matrix tractions.

Nonmitogenic survival-enhancing autocrine factors including cyclophilin A contribute to density-dependent mouse embryonic stem cell growth

An improved understanding of the role of extracellular factors in controlling the embryonic stem cell (ESC) phenotype will aid the development of cell-based therapies. While the role of extracellular factors in controlling the pluripotency and differentiation of embryonic stem cells (ESCs) has been the subject of much investigation, the identity and role of extrinsic factors in modulating ESC growth under conditions supporting self-renewal remain largely unknown. We demonstrate that mouse ESC (mESC) growth is density dependent and that one of the mechanisms underlying this phenomenon is the action of survival-enhancing autocrine factors. Proteomic analysis of proteins secreted by mouse ESCs demonstrates significant levels of cyclophilin A which increases the growth rate of mouse ESCs in a dose-dependent manner. Additionally, inhibition of the cyclophilin A receptor CD147 decreases the growth rate of mESCs. These findings identify cyclophilin A as a novel survival-enhancing autocrine factor in mouse ESC cultures.

Abrogation of E-Cadherin-Mediated Cellular Aggregation Allows Proliferation of Pluripotent Mouse Embryonic Stem Cells in Shake Flask Bioreactors

BackgroundA fundamental requirement for the exploitation of embryonic stem (ES) cells in regenerative medicine is the ability to reproducibly derive sufficient numbers of cells of a consistent quality in a cost-effective manner. However, undifferentiated ES cells are not ideally suited to suspension culture due to the formation of cellular aggregates, ultimately limiting scalability. Significant advances have been made in recent years in the culture of ES cells, including automated adherent culture and suspension microcarrier or embryoid body bioreactor culture. However, each of these methods exhibits specific disadvantages, such as high cost, additional downstream processes or reduced cell doubling times.Methodology/Principal FindingsHere we show that abrogation of the cell surface protein E-cadherin, using either gene knockout (Ecad-/-) or the neutralising antibody DECMA-1 (EcadAb), allows culture of mouse ES cells as a near-single cell suspension in scalable shake flask culture over prolonged periods without additional media supplements. Both Ecad-/- and EcadAb ES cells exhibited adaptation phases in suspension culture, with optimal doubling times of 7.3 h±0.9 and 15.6 h±4.7 respectively and mean-fold increase in viable cell number of 95.1±2.0 and 16±0.9-fold over 48 h. EcadAb ES cells propagated as a dispersed cell suspension for 15 d maintained expression of pluripotent markers, exhibited a normal karyotype and high viability. Subsequent differentiation of EcadAb ES cells resulted in expression of transcripts and proteins associated with the three primary germ layers.Conclusions/SignificanceThis is the first demonstration of the culture of pluripotent ES cells as a near-single cell suspension in a manual fed-batch shake flask bioreactor and represents a significant improvement on current ES cell culture techniques. Whilst this proof-of-principle method would be useful for the culture of human ES and iPS cells, further steps are necessary to increase cell viability of hES cells in suspension.

Methionine sulfoxide reductase A (MsrA) protects cultured mouse embryonic stem cells from H2O2‐mediated oxidative stress

Methionine sulfoxide reductase A (MsrA), a member of the Msr gene family, can reduce methionine sulfoxide residues in proteins formed by oxidation of methionine by reactive oxygen species (ROS). Msr is an important protein repair system which can also function to scavenge ROS. Our studies have confirmed the expression of MsrA in mouse embryonic stem cells (ESCs) in culture conditions. A cytosol-located and mitochondria-enriched expression pattern has been observed in these cells. To confirm the protective function of MsrA in ESCs against oxidative stress, a siRNA approach has been used to knockdown MsrA expression in ES cells which showed less resistance than control cells to hydrogen peroxide treatment. Overexpression of MsrA gene products in ES cells showed improved survivability of these cells to hydrogen peroxide treatment. Our results indicate that MsrA plays an important role in cellular defenses against oxidative stress in ESCs. Msr genes may provide a new target in stem cells to increase their survivability during the therapeutic applications.

Interactions of Wnt/ -Catenin Signaling and Sonic Hedgehog Regulate the Neurogenesis of Ventral Midbrain Dopamine Neurons

Signaling mechanisms involving Wnt/beta-catenin and sonic hedgehog (Shh) are known to regulate the development of ventral midbrain (vMB) dopamine neurons. However, the interactions between these two mechanisms and how such interactions can be targeted to promote a maximal production of dopamine neurons are not fully understood. Here we show that conditional mouse mutants with region-specific activation of beta-catenin signaling in vMB using the Shh-Cre mice show a marked expansion of Sox2-, Ngn2-, and Otx2-positive progenitors but perturbs their cell cycle exit and reduces the generation of dopamine neurons. Furthermore, activation of beta-catenin in vMB also results in a progressive loss of Shh expression and Shh target genes. Such antagonistic effects between the activation of Wnt/beta-catenin and Shh can be recapitulated in vMB progenitors and in mouse embryonic stem cell cultures. Notwithstanding these antagonistic interactions, cell-type-specific activation of beta-catenin in the midline progenitors using the tyrosine hydroxylase-internal ribosomal entry site-Cre (Th-IRES-Cre) mice leads to increased dopaminergic neurogenesis. Together, these results indicate the presence of a delicate balance between Wnt/beta-catenin and Shh signaling mechanisms in the progression from progenitors to dopamine neurons. Persistent activation of beta-catenin in early progenitors perturbs their cell cycle progression and antagonizes Shh expression, whereas activation of beta-catenin in midline progenitors promotes the generation of dopamine neurons.

The role of mechanical forces in stem cell differentiation to vascular lineage

The overall goal of our study is to evaluate the role of wall shear stress (WSS) and cyclic strain (CS) on the development of vascular cells from mouse embryonic stem cells (MESCs) differentiated in vitro. The aim is to generate an early marker of endothelial cells (ECs), platelet endothelial cell adhesion molecule‐1 (PECAM‐1+ cells), from ESCs differentiated in vitro, subjecting these cells to WSS and CS, and analyzing the functionality of these cells by testing their permeability to solutes of varying molecular weight and expression of key components of the inter‐endothelial tight junctions. If we are successful, the results will make an important contribution to vascular tissue regeneration.Results1) We have been able to sort PECAM‐1+ cells from the rest of the MESC culture; 2) The endothelial‐like cells display border staining for VE‐cadherin; 3) ES‐derived endothelial cells form multidimensional tubular structures on Matrigel; and 4) ES‐derived endothelial cells uptake acetylated low density lipoprotein (ac‐LDL).ConclusionOur sorting for PECAM‐1+ cells has been successful and these cells express hallmark of the endothelial phenotypes. We can use these cells for evaluating the role of WSS and CS on their development into mature endothelial cells.Supported by: NIH/NHLBI 1 RO1 HL086543‐01 and State of New York Department of Health (NYSTEM)

Characterization of Trophoblast and Extraembryonic Endoderm Cell Lineages Derived from Rat Preimplantation Embryos

BackgroundPrevious attempts to isolate pluripotent cell lines from rat preimplantation embryo in mouse embryonic stem (ES) cell culture conditions (serum and LIF) were unsuccessful, however the resulting cells exhibited the expression of such traditional pluripotency markers as SSEA-1 and alkaline phosphatase. We addressed the question, which kind of cell lineages are produced from rat preimplantation embryo under “classical” mouse ES conditions.ResultsWe characterized two cell lines (C5 and B10) which were obtained from rat blastocysts in medium with serum and LIF. In the B10 cell line we found the expression of genes known to be expressed in trophoblast, Cdx-2, cytokeratin-7, and Hand-1. Also, B10 cells invaded the trophectodermal layer upon injection into rat blastocysts. In contrast to mouse Trophoblast Stem (TS) cells proliferation of B10 cells occurred independently of FGF4. Cells of the C5 line expressed traditional markers of extraembryonic-endoderm (XEN) cells, in particular, GATA-4, but also the pluripotency markers SSEA-1 and Oct-4. C5 cell proliferation exhibited dependence on LIF, which is not known to be required by mouse XEN cells.ConclusionsOur results confirm and extend previous findings about differences between blastocyst-derived cell lines of rat and mice. Our data show, that the B10 cell line represents a population of FGF4-independent rat TS-like cells. C5 cells show features that have recently become known as characteristic of rat XEN cells. Early passages of C5 and B10 cells contained both, TS and XEN cells. We speculate, that mechanisms maintaining self-renewal of cell lineages in rat preimplantation embryo and their in vitro counterparts, including ES, TS and XEN cells are different than in respective mouse lineages.

Development of the circadian oscillator during differentiation of mouse embryonic stem cells in vitro

The molecular oscillations underlying the generation of circadian rhythmicity in mammals develop gradually during ontogenesis. However, the developmental process of mammalian cellular circadian oscillator formation remains unknown. In differentiated somatic cells, the transcriptional-translational feedback loops (TTFL) consisting of clock genes elicit the molecular circadian oscillation. Using a bioluminescence imaging system to monitor clock gene expression, we show here that the circadian bioluminescence rhythm is not detected in the mouse embryonic stem (ES) cells, and that the ES cells likely lack TTFL regulation for clock gene expression. The circadian clock oscillation was induced during the differentiation culture of mouse ES cells without maternal factors. In addition, reprogramming of the differentiated cells by expression of Sox2, Klf4, Oct3/4, and c-Myc genes, which were factors to generate induced pluripotent stem (iPS) cells, resulted in the re-disappearance of circadian oscillation. These results demonstrate that an intrinsic program controls the formation of the circadian oscillator during the differentiation process of ES cells in vitro. The cellular differentiation and reprogramming system using cultured ES cells allows us to observe the circadian clock formation process and may help design new strategies to understand the key mechanisms responsible for the organization of the molecular oscillator in mammals.

Interrogating cell signalling network sensitively monitors cell fate transition during early differentiation of mouse embryonic stem cells

The different cell types in an animal are often considered to be specified by combinations of transcription factors, and defined by marker gene expression. This paradigm is challenged, however, in stem cell research and application. Using a mouse embryonic stem cell (mESC) culture system, here we show that the expression level of many key stem cell marker genes/transcription factors such as Oct4, Sox2 and Nanog failed to monitor cell status transition during mESC differentiation. On the other hand, the response patterns of cell signalling network to external stimuli, as monitored by the dynamics of protein phosphorylation, changed dramatically. Our results also suggest that an irreversible alternation in cell signalling network precedes the adjustment of transcription factor levels. This is consistent with the notion that signal transduction events regulate cell fate specification. We propose that interrogating cell signalling network can assess the cell property more precisely, and provide a sensitive measurement for the early events in cell fate transition. We wish to bring up attention to the potential problem of cell identification using a few marker genes, and suggest a novel methodology to address this issue.

The use of SC1 (Pluripotin) to Support mESC Self-renewal in the Absence of LIF

Mouse embryonic stem (ES) cells are conventionally cultured with Leukemia Inhibitory Factor (LIF) to maintain self-renewal.1 However, LIF is expensive and activation of the LIF/JAK/STAT3 pathway is not absolutely required to maintain the self-renewal state.2 The SC1 small molecule may be an economical alternative to LIF. SC1 functions through dual inhibition of Ras-GAP and ERK1.3 Illustration of its mechanism of action makes it a useful tool to study the fundamental molecular mechanism of self-renewal. Here we demonstrate the procedure for culturing mouse ES cells in the presence of SC1 and show that they are able to maintain self-renewal in the absence of LIF. Cells cultured with SC1 showed similar morphology compared to cells maintained with LIF. Both exhibited typical mouse ES morphology after five passages. Expression of typical pluripotency markers (Oct4, Sox2, Nanog, and SSEA1) was observed after five passages in the presence of SC1. Furthermore, SC1 caused no overt toxicity on mouse ES cells.

The use of SC1 (Pluripotin) to Support mESC Self-renewal in the Absence of LIF

Mouse embryonic stem (ES) cells are conventionally cultured with Leukemia Inhibitory Factor (LIF) to maintain self-renewal.(1) However, LIF is expensive and activation of the LIF/JAK/STAT3 pathway is not absolutely required to maintain the self-renewal state.(2) The SC1 small molecule may be an economical alternative to LIF. SC1 functions through dual inhibition of Ras-GAP and ERK1.(3) Illustration of its mechanism of action makes it a useful tool to study the fundamental molecular mechanism of self-renewal. Here we demonstrate the procedure for culturing mouse ES cells in the presence of SC1 and show that they are able to maintain self-renewal in the absence of LIF. Cells cultured with SC1 showed similar morphology compared to cells maintained with LIF. Both exhibited typical mouse ES morphology after five passages. Expression of typical pluripotency markers (Oct4, Sox2, Nanog, and SSEA1) was observed after five passages in the presence of SC1. Furthermore, SC1 caused no overt toxicity on mouse ES cells.