Using CRISPR barcoding as a molecular clock to capture dynamic processes at single-cell resolution.

Germline Competent Embryonic Stem Cells Derived from Rat Blastocysts

A minority of mouse embryonic stem cells (ESCs) display totipotent features resembling 2‐cell stage embryos and are known as 2‐cell‐like (2C‐like) cells. However, how ESCs transit into this 2C‐like state remains largely unknown. Here, we report that the overexpression of negative elongation factor A (Nelfa), a maternally provided factor, enhances the conversion of ESCs into 2C‐like cells in chemically defined conditions, while the deletion of endogenous Nelfa does not block this transition. We also demonstrate that Nelfa overexpression significantly enhances somatic cell reprogramming efficiency. Interestingly, we found that the co‐overexpression of Nelfa and Bcl2 robustly activates the 2C‐like state in ESCs and endows the cells with dual cell fate potential. We further demonstrate that Bcl2 overexpression upregulates endogenous Nelfa expression and can induce the 2C‐like state in ESCs even in the absence of Nelfa. Our findings highlight the importance of BCL2 in the regulation of the 2C‐like state and provide insights into the mechanism underlying the roles of Nelfa and Bcl2 in the establishment and regulation of the totipotent state in mouse ESCs.

MicroRNAs (miRNAs), a group of noncoding RNAs, function as post-transcriptional gene regulators and control the establishment, self-renewal and differentiation of stem cells. Vitamin C has been recognized as a reprogramming enhancer because of its ability to induce a blastocyst-like state in embryonic stem cells (ESCs). However, knowledge on the regulation of miRNAs by vitamin C in ESCs is limited. In this study, we found that vitamin C induced miRNA expression, particularly of ESC-specific miRNAs. Moreover, vitamin C maintained the miRNA expression of the Dlk1-Dio3 imprinting region. The miRNAs in this region contain identical seed sequences, which target a class of genes, including Kdm6b, Klf13, and Sox6, and are mainly related to cell differentiation and development. These genes were significantly downregulated by vitamin C. Notably, miR-143 promoted self-renewal of mouse ESCs and suppressed expression of the de novo methyltransferase gene Dnmt3a. Knockdown of miR-143 by use of its inhibitor counteracted the vitamin C-induced reduction in Dnmt3a expression, showing that vitamin C repressed Dnmt3a expression via miR-143. Vitamin C also promoted DNA demethylation, including of pluripotency gene promoters (Tbx3, Tcl1, and Esrrb) and ESC-specific miRNA promoters (miR-290-295 and miR-17-92 clusters), and DNA hydroxymethylation, including of the intergenic differentially methylated region of the Dlk1-Dio3 region. These results strongly suggested that vitamin C promoted widespread DNA demethylation in gene promoters by modulating epigenetic modifiers, including Dnmt3a, which activated pluripotency genes and ESC-specific miRNAs. Then, differentiation and development genes were repressed by ESC-enriched miRNAs, which maintained the stem cell state.

Pluripotent embryonic stem cells (ESCs) are capable of differentiating into cell types belonging to all three germ layers within the body, which makes them an interesting and intense field of research. Inefficient specific differentiation and contamination with unwanted cell types are the major issues in the use of ESCs in regenerative medicine. Lineage-specific progenitors generated from ESCs could be utilized to circumvent the issue. We demonstrate here that sustained activation of the Wnt pathway (using Wnt3A or an inhibitor of glycogen synthase kinase 3beta) in multiple mouse and human ESCs results in meso/endoderm-specific differentiation. Using monolayer culture conditions, we have generated multipotential "mesendodermal progenitor clones" (MPC) from mouse ESCs by sustained Wnt pathway activation. MPCs express increased levels of meso/endodermal and mesendodermal markers and exhibit a stable phenotype in culture over a year. The MPCs have enhanced potential to differentiate along endothelial, cardiac, vascular smooth muscle, and skeletal lineages than undifferentiated ESCs. In conclusion, we demonstrate that the Wnt pathway activation can be utilized to generate lineage-specific progenitors from ESCs, which can be further differentiated into desired organ-specific cells.

The Transcriptionally Permissive Chromatin State of Embryonic Stem Cells Is Acutely Tuned to Translational Output.

The pluripotent state between human and mouse embryonic stem cells is different. Pluripotent state of human embryonic stem cells (ESCs) is believed to be primed and is similar with that of mouse epiblast stem cells (EpiSCs), which is different from the naïve state of mouse ESCs. Human ESCs could be converted into a naïve state through exogenous expression of defined transcription factors (Hanna et al., 2010). Here we report a rapid conversion of human ESCs to mouse ESC-like naïve states only by modifying the culture conditions. These converted human ESCs, which we called mhESCs (mouse ESC-like human ESCs), have normal karyotype, allow single cell passage, exhibit domed morphology like mouse ESCs and express some pluripotent markers similar with mouse ESCs. Thus the rapid conversion established a naïve pluripotency in human ESCs like mouse ESCs, and provided a new model to study the regulation of pluripotency.

Streptavidin affinity purification of protein complexes, in combination with in vivo biotinylation of critical transcription factors, has contributed to the analysis of the pluripotent state in mouse embryonic stem (ES) cells and made it possible to construct a protein‐protein interaction network.This has facilitated discovery of novel pluripotency factors and a better understanding of stem cell pluripotency. Here we describe detailed procedures for an in vivo biotinylation system setup in mouse ES cells, and affinity purification of multi‐protein complexes using in vivo biotinylation. In addition, we present a protocol employing SDS‐PAGE fractionation to reduce sample complexity prior to submission for mass spectrometry (MS) protein identification. Curr. Protoc. Stem Cell Biol. 11:1B.5.1‐1B.5.17. © 2009 by John Wiley & Sons, Inc.

In gene regulation, proteins function as members of protein complexes to recognize chromosomal target DNA loci. In dissecting the pluripotent state in mouse embryonic stem (mES) cells, we have used in vivo biotinylation of critical transcription factors for affinity purification of protein complexes and chromatin immunoprecipitation (ChIP)-on-chip for target identification, respectively. Here, we describe detailed procedures for such studies to dissect protein-protein and protein-DNA interactions in mES cells. Specifically, the following three procedures will be described: (i) in vivo biotinylation system setup in mES cells; (ii) affinity purification of multiprotein complexes by one-step streptavidin capture and tandem anti-FLAG/streptavidin affinity purification; (iii) biotin-mediated ChIP (bioChIP). The system setup takes approximately 50 d to complete, and it takes another approximately 15 d and approximately 3 d to perform affinity purification of protein complexes and bioChIP, respectively.

In dissecting the pluripotent state in mouse embryonic stem (ES) cells, we have employed in vivo biotinylation of critical transcription factors for streptavidin affinity purification of protein complexes and constructed a protein-protein interaction network. This has facilitated discovery of novel pluripotency factors and a better understanding of stem cell pluripotency. Here we describe detailed procedures for in vivo biotinylation system setup in mouse ES cells, and affinity purification of multi-protein complexes using in vivo biotinylation. In addition, we present a protocol employing SDS-PAGE fractionation to reduce sample complexity prior to submission for mass spectrometry (MS) protein identification.

Mouse embryonic stem (ES) cells can be maintained in an undifferentiated state in the presence of leukemia inhibitory factor (LIF), a member of the interleukin-6 cytokine family. In other mammals, this is not possible with LIF alone. Chicken ES-like cells (blastodermal cells) have only been cultured with mouse LIF because chicken LIF was not available. However the culture system is imperfect and chicken ES-like cells equivalent to mouse ES cells were not observed. In the present study, we cloned the cDNA-encoding chicken LIF using mRNA subtraction and RACE methodology. The chicken LIF cDNA encodes a protein with approximately 40% sequence identity to mouse LIF. It has 211 amino acids including a putative N-terminal signal peptide of 24 residues. Chicken blastodermal cells were cultured in the presence of bacterially expressed chicken LIF or mouse LIF. The expression of alkaline phosphatase and embryonal carcinoma cell monoclonal antibody-1 and stage-specific embryonic antigen-1 and the activation of STAT3 were examined, all of which are indices of the undifferentiated state. Exposure in the blastodermal cells to recombinant chicken LIF but not to mouse LIF maintained the expression of these various markers. After 9 days of incubation, the blastodermal cells formed cystic embryoid bodies in the presence of mouse LIF but not in the presence of recombinant chicken LIF. We conclude that chicken LIF is able to maintain chicken ES cell cultures in the undifferentiated state.

Mouse embryonic stem cells (ESCs) consist of a rare population of heterogeneous 2-cell-like cells (2CLCs). These cells transiently recapitulate the transcriptional and epigenetic features of the 2-cell embryos, serving as a unique model for studying totipotency acquisition and embryonic development. Accumulating evidence has demonstrated that transcription factors and epigenetic modifications exert crucial functions in the transition of ESCs to 2CLCs. However, the roles of RNA modification in the regulation of the 2C-like state remain elusive. Using a DUX-induced 2CLCs system, we examine N6-methyladenosine (m6A) modification landscape transcriptome-wide and observe dynamic regulation of m6A during DUX-driven 2C-like reprogramming. Notably, many core 2C transcripts like Dux and Zscan4 are highly methylated. We identify the m6A reader protein YTHDF2 as a critical regulator of 2C-like state. Depletion of YTHDF2 facilitates robust expression of 2C-signature genes and ESCs-to-2CLCs transition. Intriguingly, YTHDF2 binds to a subset of m6A-modified 2C transcripts and promotes their decay. We further demonstrate that YTHDF2 suppresses the 2C-like program in a manner that is dependent on both m6A and the DUX-ZSCAN4 molecular circuit. Mechanistically, YTHDF2 interacts with CNOT1, a key component of the RNA deadenylase complex. Consistently, silencing of CNOT1 upregulates the 2C program and promotes ESCs-to-2CLCs transition. Collectively, our findings reveal novel insights into the epitranscriptomic regulation of the 2C-like state in mouse ESCs.

Mechanism of SB431542 in inhibiting mouse embryonic stem cell differentiation

Decision letter: Single-cell RNA sequencing of the Strongylocentrotus purpuratus larva reveals the blueprint of major cell types and nervous system of a non-chordate deuterostome

Cross-activation of FGF, NODAL, and WNT pathways constrains BMP-signaling-mediated induction of the totipotent state in mouse embryonic stem cells

BackgroundRenal cell carcinoma (RCC) is a prevalent and aggressive form of kidney cancer, with variable response rates to checkpoint blockade immunotherapies. While high T cell infiltration often indicates a favorable...