2i Medium Research Articles

G1 length dictates H3K27me3 landscapes.

Stem cells have lower facultative heterochromatin as defined by trimethylation of histone H3 lysine 27 (H3K27me3) compared to differentiated cells. However, the mechanisms underlying these differential H3K27me3 levels remain elusive. Because H3K27me3 levels are diluted two-fold in every round of replication and then restored through the rest of the cell cycle, we reasoned that the cell cycle length could be a key regulator of total H3K27me3 levels. Here, we propose that a fast cell cycle restricts H3K27me3 levels in stem cells. To test this model, we determined changes to H3K27me3 levels in mESCs globally and at specific loci upon G1 phase lengthening - accomplished by thymidine block or growth in the absence of serum (with the "2i medium"). H3K27me3 levels in mESC increase with G1 arrest when grown in serum and in 2i medium. Additionally, we observed via CUT&RUN and ChIP-seq that regions that gain H3K27me3 in G1 arrest and 2i media overlap, supporting our model of cell cycle length as a critical regulator of the stem cell epigenome and cellular identity. Furthermore, we demonstrate the inverse effect - that G1 shortening in differentiated cells results in a loss of H3K27me3 levels. Finally, in tumor cells with extreme H3K27me3 loss, lengthening of the G1 phase leads to H3K27me3 recovery despite the presence of the dominant negative, sub-stoichiometric H3.1K27M mutation. Our results indicate that G1 length is an essential determinant of H3K27me3 landscapes across diverse cell types.

Culture bovine prospermatogonia with 2i medium.

Germplasm cryopreservation and expansion of gonocytes/prospermatogonia or spermatogonial stem cells (SSCs) are important; however, it's difficult in cattle. Since inhibitors of Mek1/2 and Gsk3β (2i) can enhance pluripotency maintenance, effects of 2i-based medium on the cultivation of bovine prospermatogonia from the cryopreserved tissues were examined. The testicular tissues of newborn bulls were well cryopreserved. High mRNA levels of prospermatogonium/SSC markers (PLZF, GFRα-1) and pluripotency markers (Oct4/Pouf5, Sox2, Nanog) were detected and the PLZF+ /GFRα-1+ prospermatogonia were consistently identified immunohistochemically in the seminiferous cords. Using differential plating and Percoll-based centrifugation, 41.59% prospermatogonia were enriched and they proliferated robustly in 2i medium. The 2i medium boosted mRNA abundances of Pouf5, Sox2, Nanog, GFRα-1, PLZF, anti-apoptosis gene Bcl2, LIF receptor gene LIFR and enhanced PLZF protein expression, but suppressed mRNA expressions of spermatogonial differentiation marker c-kit and pro-apoptotic gene Bax, in the cultured prospermatogonia. It also alleviated H2 O2 -induced apoptosis of the enriched cells and decreased histone H3 lysine (K9) trimethylation (H3K9me3) and its methylase Suv39h1/2 mRNA level in the cultured seminiferous cords. Overall, 2i medium improves the cultivation of bovine prospermatogonia isolated from the cryopreserved testes, by inhibiting Suv39h1/2-mediated H3K9me3 through Mek1/2 and Gsk3β signalling, evidencing successful cryopreservation and expansion of bovine germplasm.

3D Synthetic Microstructures Fabricated by Two‐Photon Polymerization Promote Homogeneous Expression of NANOG and ESRRB in Mouse Embryonic Stem Cells

AbstractThe development of in vitro models, which accurately recapitulate early embryonic development, is one of the fundamental challenges in stem cell research. Most of the currently employed approaches involve the culture of embryonic stem cells (ESCs) on 2D surfaces. However, the monolayer nature of these cultures does not permit cells to grow and proliferate in realistic 3D microenvironments, as in an early embryo. In this paper, novel 3D synthetic microstructure arrays, fabricated by two‐photon polymerization photolithography, are utilized to mimic tissue‐specific architecture, enabling cell‐to‐matrix interaction and cell‐to‐cell communication in vitro. Mouse ESCs (mESCs) are able to grow and proliferate on these structures and maintain their pluripotent state. Furthermore, the 3D microstructure arrays are integrated into a microscopy slide allowing the evaluation of the expression of key pluripotency factors at the single cell level. Comparing 2D and 3D surfaces, mESCs grown in serum + leukemia inhibitory factor on 3D microstructures exhibit a stronger signal intensity of three pluripotency markers—homeobox protein NANOG, octamer‐binding transcription factor 4, and estrogen‐related receptor beta (ESRRB)—and more homogenous expression of NANOG and ESRRB, than cells cultivated in 2i medium, demonstrating that 3D microstructures capture naïve pluripotency in vitro. Thus, the slide affords a novel and unique tool to model and study early development.

Mapping the Effects of Genetic Variation on Chromatin State and Gene Expression Reveals Loci That Control Ground State Pluripotency.

Mouse embryonic stem cells (mESCs) cultured in the presence of LIF occupy a ground state with highly active pluripotency-associated transcriptional and epigenetic circuitry. However, ground state pluripotency in some inbred strain backgrounds is unstable in the absence of ERK1/2 and GSK3 inhibition. Using an unbiased genetic approach, we dissect the basis of this divergent response to extracellular cues by profiling gene expression and chromatin accessibility in 170 genetically heterogeneous mESCs. We map thousands of loci affecting chromatin accessibility and/or transcript abundance, including 10 QTL hotspots where genetic variation at a single locus coordinates the regulation of genes throughout the genome. For one hotspot, we identify a single enhancer variant ∼10 kb upstream of Lifr associated with chromatin accessibility and mediating a cascade of molecular events affecting pluripotency. We validate causation through reciprocal allele swaps, demonstrating the functional consequences of noncoding variation in gene regulatory networks that stabilize pluripotent states invitro.

Generation of Human-Induced Pluripotent Stem Cells from Peripheral Blood Mononuclear Cells using Small-Molecule Compound VC6TFZ

BACKGROUND: Induced pluripotent stem cells (iPSCs) were generated from somatic cells through reprogramming process. Peripheral blood mononuclear cells (PBMNCs) were an attractive source cells due to the ease of accessibility, need minimal invasive procedure, and can be stored frozen. Small-molecule compound VC6TFZ has been successfully reprogrammed iPSCs from mouse fibroblast, but it has not been proven in human.
 AIM: This study aims to determine whether the small-molecule compound VC6TFZ can induce pluripotency of PBMNC to generate iPSCs.
 METHODS: Mononuclear cells were isolated from peripheral venous blood using centrifugation gradient density method. Mononuclear cells were cultured for 6 days in expansion medium and 48 h using hanging drop method. Pluripotency induction process using small-molecule compound VC6TFZ was done in 14 days then the medium changed to 2i medium for 7 days. Identification of iPSCs based on colony morphology and expression of pluripotency marker OCT4 and SOX2.
 RESULTS: Colonies appeared on day 9 of reprogramming process. These colonies had round, large, and cobble stone morphology like embryonic stem cell. These colonies had positive expression of pluripotency markers OCT4 and SOX2. All experimental groups had significantly higher expression of OCT4 and SOX2 than control group.
 CONCLUSION: Small-molecule compound VC6TFZ could induce pluripotency of human PBMNC to generate iPSCs.

Non-invasive detection of DNA methylation states in carcinoma and pluripotent stem cells using Raman microspectroscopy and imaging

DNA methylation plays a critical role in the regulation of gene expression. Global DNA methylation changes occur in carcinogenesis as well as early embryonic development. However, the current methods for studying global DNA methylation levels are invasive and require sample preparation. The present study was designed to investigate the potential of Raman microspectroscopy and Raman imaging as non-invasive, marker-independent and non-destructive tools for the detection of DNA methylation in living cells. To investigate global DNA methylation changes, human colon carcinoma HCT116 cells, which were hypomorphic for DNA methyltransferase 1, therefore showing a lower global DNA methylation (DNMT1−/− cells), were compared to HCT116 wildtype cells. As a model system for early embryogenesis, murine embryonic stem cells were adapted to serum-free 2i medium, leading to a significant decrease in DNA methylation. Subsequently, 2i medium -adapted cells were compared to cells cultured in serum-containing medium. Raman microspectroscopy and imaging revealed significant differences between high- and low-methylated cell types. Higher methylated cells demonstrated higher relative intensities of Raman peaks, which can be assigned to the nucleobases and 5-methylcytosine. Principal component analysis detected distinguishable populations of high- and low-methylated samples. Based on the provided data we conclude that Raman microspectroscopy and imaging are suitable tools for the real-time, marker-independent and artefact-free investigation of the DNA methylation states in living cells.

Wnt3a Activates the WNT-YAP/TAZ Pathway to Sustain CDX2 Expression in Bovine Trophoblast Stem Cells.

Trophoblast stem cells (TSCs), the precursors of placental cells, are effective for studying placental formation in vitro. Using a dual inhibition (2i) medium and mixed L-Wnt3a/mouse embryonic fibroblast feeder cells, we previously established the bovine trophoblast cell line BTS-1. In this study, we used bovine fetal fibroblasts and added Wnt3a to the 2i medium to establish another bovine TSC line (BTSW). BTSW cells expressed pluripotency markers, including NANOG, SOX2, OCT4, TRA-1-60, TRA-1-81, SSEA4, CDH1, and KRT18, and TSC markers CDX2, TEAD4, and ESRRB. Methylation sequencing of the promoter regions of NANOG, OCT4, and CDX2 revealed no significant differences between BTS-1 and BTSW cells. Removal of Wnt3a from the culture medium resulted in downregulation (p < 0.05) of NANOG, OCT4, CDX2, and TSC marker genes, and upregulation of TSC differentiation markers, including MASH2, GCM1, and PAG. Western blotting indicated activation of the WNT-YAP/TAZ signaling pathway in BTS-1 and BTSW cells, consequently activating TEAD4 transcription. However, this pathway was not activated in BCFF cells, an established bovine embryonic stem-like cell line that expresses OCT4, SOX2, and NANOG, but not CDX2. Thus, Wnt3a may play a critical role in bovine TSC maintenance by activating and regulating CDX2 expression through the WNT-YAP/TAZ signaling pathway.

Inductive and Selective Effects of GSK3 and MEK Inhibition on Nanog Heterogeneity in Embryonic Stem Cells.

SummaryEmbryonic stem cells (ESCs) display heterogeneous expression of pluripotency factors such as Nanog when cultured with serum and leukemia inhibitory factor (LIF). In contrast, dual inhibition of the signaling kinases GSK3 and MEK (2i) converts ESC cultures into a state with more uniform and high Nanog expression. However, it is so far unclear whether 2i acts through an inductive or selective mechanism. Here, we use continuous time-lapse imaging to quantify the dynamics of death, proliferation, and Nanog expression in mouse ESCs after 2i addition. We show that 2i has a dual effect: it both leads to increased cell death of Nanog low ESCs (selective effect) and induces and maintains high Nanog levels (inductive effect) in single ESCs. Genetic manipulation further showed that presence of NANOG protein is important for cell viability in 2i medium. This demonstrates complex Nanog-dependent effects of 2i treatment on ESC cultures.

Derivation of Stem Cell Lines from Mouse Preimplantation Embryos.

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines retain the ability to either self-renew or differentiate under specific conditions. Due to these properties, mESC are a useful tool in regenerative medicine, disease modeling, and tissue engineering studies. This article describes a simple protocol to obtain mESC lines with high derivation efficiencies (60-80%) by culturing blastocysts from permissive mouse strains on feeder cells in defined medium supplemented with leukemia inhibitory factor. The protocol can also be applied to efficiently derive mESC lines from non-permissive mouse strains, by the simple addition of a cocktail of two small-molecule inhibitors to the derivation medium (2i medium). Detailed procedures on the preparation and culture of feeder cells, collection and culture of mouse embryos, and derivation and culture of mESC lines are provided. This protocol does not require specialized equipment and can be carried out in any laboratory with basic mammalian cell culture expertise.

Stat3 phosphorylation is required for embryonic stem cells ground state maintenance in 2i culture media.

Embryonic stem cells (ES cells) can be maintained its undifferentiated state with feeder cells or LIF, which can activate Jak/Stat3 pathway. Recently, it has been reported a new culture condition comprising serum-free medium with ERK and GSK3β inhibitors (2i) could drive ES cells into a state of pluripotency more like inner cell mass (ICM) in mouse blastocysts called ground state. However, although 2i could sustain ES cells self-renewal, LIF is routinely added. The roles of Stat3 activation are still unclear now. Here we investigated whether Jak/Stat3 might also contribute to the induction of ground state pluripotency. We introduced a lentiviral construct with 7-repeat Stat3-binding sequence to drive Renilla luciferase into ES cells, which can be used as a reporter to detect Stat3 activation by noninvasive bioluminescence imaging. Using this ES cells, we investigated the role of Stat3 activation in ground state maintenance. The results showed that Stat3 could be activated by 2i. Stattic, a chemical inhibitor of Stat3 phosphorylation, could effectively inhibit Stat3 activation in ES cells. When Stat3 activation was suppressed, ground state related genes were down regulated, and ES cells could not be maintained the ground state pluripotency even in 2i medium. All of these results indicate Stat3 activation is required in ground state maintenance.

Significant differences of function and expression of microRNAs between ground state and serum-cultured pluripotent stem cells

Serum- and 2i-cultured embryonic stem cells (ESCs) show different epigenetic landscapes and transcriptomic profiles. The difference in the function and expression of microRNAs (miRNAs) between these two states remains unclear. Here, we showed that 2i- and serum-cultured ESCs exhibited distinctive miRNA expression profiles with >100 miRNAs differentially expressed, and the expression changes were largely due to transcriptional regulation. We further characterized the function of miRNAs differentially expressed under two conditions and found that ESCs exhibited higher degree of dependency on miRNAs for rapid proliferation; since Dgcr8−/− or Dicer1−/− but not wild-type ESCs showed slower growth rate and more accumulation in the G1 phase under 2i than serum condition. More interestingly, introduction of various self-renewal-silencing miRNAs in wild-type or Dgcr8−/− ESCs failed to silence the self-renewal in 2i medium, but regained the ability to silence the self-renewal upon the addition of serum. Our findings reveal significant differences in the expression and function of miRNAs between serum- and 2i-cultured ESCs.

Characterization of goat inner cell mass derived cells in double kinase inhibition condition

The identification of small molecular inhibitors, which were reported to promote the derivation of mouse and human embryonic stem cells (ESCs), provides a potential strategy for the derivation of domesticated ungulate ESCs. In present study, goat inner cell mass (ICM) derived cells in the double inhibition (2i) condition, in which, mitogen-activated protein kinase kinase (MAP2K) and glycogen synthase kinase 3 (GSK3) were inhibited by PD0325901 and BIO respectively, were characterized. The results showed that goat ICM derived cells in 2i medium adding leukaemia inhibitor factor (LIF) possessed a mouse ES-like morphology. But these cells had much compromised proliferation capacity, resulting in difficulty in expansion. In 2i alone medium, goat ICM derived cells possessed primate ES-like morphology. These cells expressed pluripotent markers and could differentiate into derivatives of three germ layers in vitro. However, these cells could not be proliferated in long-term (persisted for 15 passages) because of spontaneously neural differentiation. Additionally, goat ICM derived cells could be inducing differentiated into neural lineage in vitro. Although goat ESCs could not be established in PD0325901 and BIO alone medium, this derivation condition provides a useful research system to find signaling molecular those regulate early embryonic development and pluripotency in goat.

DNA (De)Methylation: The Passive Route to Naïvety?

Mouse pluripotent embryonic stem (ES) cells can exist in distinct yet interchangeable epigenetic states dictated by their culture environment. Previous reports have shown that naïve pluripotent cells grown in the presence of 2i are characterised by global DNA hypomethylation and changes in the abundance and distribution of histone modifications. New research provides insights regarding how this might be achieved.

Fast and Efficient Transfection of Mouse Embryonic Stem Cells Using Non-Viral Reagents.

Reliable and efficient DNA and RNA transfection methods are required when studying the role of individual genes in mouse pluripotent stem cells. However, these cells usually grow in tight clusters and are therefore more difficult to transfect than many other cell lines. We have found that transfection is especially challenging when mouse embryonic stem (mES) cells are cultured in the newly described 2i medium, which is based on two chemical inhibitors of differentiation pathways. In the present study we have performed a side-by-side comparison of commercially available, non-viral transfection reagents with regard to their ability to deliver plasmid DNA and siRNA into adherent and/or trypsinized mES cells cultured in 2i medium, assessing transfection rates, plasmid gene expression, siRNA mediated knockdown of Oct4 and viability. Finally, we present a fast and efficient method for transfection of trypsinized mES cells using the liposomal-based Lipofectamine 2000. With only a five-minute long transfection time we obtained at least 85 % transfected cells with 80 % maintained viability. Moreover, this protocol saves up to a day of experimental time since the cells are in suspension at the time of transfection, which allows for immediately re-plating into the appropriate format. This fast, simplified and highly efficient transfection method will be valuable for both basic research and high-throughput applications.Electronic supplementary materialThe online version of this article (doi:10.1007/s12015-016-9673-5) contains supplementary material, which is available to authorized users.

Proteome Analysis of Ground State Pluripotency

The differentiation potential of pluripotent embryonic stem cells (ESCs) can be manipulated via serum and medium conditions for direct cellular development or to maintain a naïve ground state. The self-renewal state of ESCs can thus be induced by adding inhibitors of mitogen activated protein kinase (MAPK) and glycogen synthase kinase-3 (Gsk3), known as 2 inhibitors (2i) treatment. We have used a shotgun proteomics approach to investigate differences in protein expressions between 2i- and serum-grown mESCs. The results indicated that 164 proteins were significantly upregulated and 107 proteins downregulated in 2i-grown cells compared to serum. Protein pathways in 2i-grown cells with the highest enrichment were associated with glycolysis and gluconeogenesis. Protein pathways related to organ development were downregulated in 2i-grown cells. In serum-grown ESCs, protein pathways involved in integrin and focal adhesion, and signaling proteins involved in the actin cytoskeleton regulation were enriched. We observed a number of nuclear proteins which were mostly involved in self-renewal maintenance and were expressed at higher levels in 2i compared to serum - Dnmt1, Map2k1, Parp1, Xpo4, Eif3g, Smarca4/Brg1 and Smarcc1/Baf155. Collectively, the results provided an insight into the key protein pathways used by ESCs in the ground state or metastable conditions through 2i or serum culture medium, respectively.

Reprogramming mature terminally differentiated adipocytes to induced pluripotent stem cells

Mature adipocytes are terminally differentiated somatic cells. Here, we report the successful generation of induced pluripotent stem (iPS) cells from mouse mature adipocytes by forced expression of six transcription factors (Oct4, Sox2, c-Myc, Klf4, Rarγ, and Lrh1) with a piggyBac transposon-based strategy. The resulting iPS cells were pluripotent as evidenced by the fact that they stained positive for alkaline phosphatase, expressed high levels of key pluripotency markers including Oct4, Nanog, and SSEA1, and remained pluripotent on a 2i media. In vitro differentiation of the iPS cells showed that the cell derivatives of all three germ layers could be readily obtained through formation of embryoid bodies. Most importantly, these adipocyte-derived iPS cells were capable of producing chimera with high frequencies when reintroduced into early-stage embryos and transmitted through the germ line. This study demonstrates that the new six-factor reprogramming technology facilitates the reset of the terminally differentiated adipocytes to the ground state of pluripotency, enabling us to fully explore the potential of mature adipocytes as a viable cell source for regenerative medicine.

ETS-related Transcription Factors ETV4 and ETV5 Are Involved in Proliferation and Induction of Differentiation-associated Genes in Embryonic Stem (ES) Cells

The pluripotency and self-renewal capacity of embryonic stem (ES) cells is regulated by several transcription factors. Here, we show that the ETS-related transcription factors Etv4 and Etv5 (Etv4/5) are specifically expressed in undifferentiated ES cells, and suppression of Oct3/4 results in down-regulation of Etv4/5. Simultaneous deletion of Etv4 and Etv5 (Etv4/5 double knock-out (dKO)) in ES cells resulted in a flat, epithelial cell-like appearance, whereas the morphology changed into compact colonies in a 2i medium (containing two inhibitors for GSK3 and MEK/ERK). Expression levels of self-renewal marker genes, including Oct3/4 and Nanog, were similar between wild-type and dKO ES cells, whereas proliferation of Etv4/5 dKO ES cells was decreased with overexpression of cyclin-dependent kinase inhibitors (p16/p19, p15, and p57). A differentiation assay revealed that the embryoid bodies derived from Etv4/5 dKO ES cells were smaller than the control, and expression of ectoderm marker genes, including Fgf5, Sox1, and Pax3, was not induced in dKO-derived embryoid bodies. Microarray analysis demonstrated that stem cell-related genes, including Tcf15, Gbx2, Lrh1, Zic3, and Baf60c, were significantly repressed in Etv4/5 dKO ES cells. The artificial expression of Etv4 and/or Etv5 in Etv4/5 dKO ES cells induced re-expression of Tcf15 and Gbx2. These results indicate that Etv4 and Etv5, potentially through regulation of Gbx2 and Tcf15, are involved in the ES cell proliferation and induction of differentiation-associated genes in ES cells.

PRDM14 maintains pluripotency of embryonic stem cells through TET-mediated active DNA demethylation

Pluripotency and self-renewal of mouse embryonic stem cells (ESCs) depend on a network of transcription factors maintained by exogenous leukaemia inhibitory factor (LIF). PR-domain containing transcriptional regulator 14 (PRDM14), is essential for maintenance of ESC self-renewal when the cells are cultured in serum plus LIF, but not in 2i medium plus LIF. Here, we show that pluripotency of ESCs is maintained by enforced expression of PRDM14 at a high level, as observed in ESCs in 2i plus LIF and developing primordial germ cells in the absence of LIF. Constitutive expression of PRDM14 represses de novo DNA methylation in pluripotency-associated genes, resulting in the maintenance of gene expression after withdrawal of LIF, while also repressing the upregulation of differentiation markers. Further, knockdown of Tet1/Tet2 and administration of base excision repair (BER) pathway inhibitors impairs the PRDM14-induced resistance of ESCs to differentiation. We conclude that, in the absence of LIF, PRDM14 governs the retention of pluripotency-associated genes through the regulation of TET functions in the BER-mediated active demethylation pathway, while acting to exert TET-independent transcriptional repressive activity of several differentiation markers.

Single-cell genome-wide bisulfite sequencing for assessing epigenetic heterogeneity.

We report a single-cell bisulfite sequencing method (scBS-Seq) capable of accurately measuring DNA methylation at up to 48.4% of CpGs. We observed that ESCs grown in serum or 2i both display epigenetic heterogeneity, with “2i-like” cells present in serum cultures. In silico integration of 12 individual mouse oocyte datasets largely recapitulates the whole DNA methylome, making scBS-Seq a versatile tool to explore DNA methylation in rare cells and heterogeneous populations.

Stochastic NANOG fluctuations allow mouse embryonic stem cells to explore pluripotency.

Heterogeneous expression of the transcription factor NANOG has been linked to the existence of various functional states in pluripotent stem cells. This heterogeneity seems to arise from fluctuations of Nanog expression in individual cells, but a thorough characterization of these fluctuations and their impact on the pluripotent state is still lacking. Here, we have used a novel fluorescent reporter to investigate the temporal dynamics of NANOG expression in mouse embryonic stem cells (mESCs), and to dissect the lineage potential of mESCs at different NANOG states. Our results show that stochastic NANOG fluctuations are widespread in mESCs, with essentially all expressing cells showing fluctuations in NANOG levels, even when cultured in ground-state conditions (2i media). We further show that fluctuations have similar kinetics when mESCs are cultured in standard conditions (serum plus leukemia inhibitory factor) or ground-state conditions, implying that NANOG fluctuations are inherent to the pluripotent state. We have then compared the developmental potential of low-NANOG and high-NANOG mESCs, grown in different conditions, and confirm that mESCs are more susceptible to enter differentiation at the low-NANOG state. Further analysis by gene expression profiling reveals that low-NANOG cells have marked expression of lineage-affiliated genes, with variable profiles according to the signalling environment. By contrast, high-NANOG cells show a more stable expression profile in different environments, with minimal expression of lineage markers. Altogether, our data support a model in which stochastic NANOG fluctuations provide opportunities for mESCs to explore multiple lineage options, modulating their probability to change functional state.