Live isolation of naïve ESCs via distinct glucose metabolism and stored glycogen

Naïve and primed pluripotent stem cells recapitulate the peri- and post-implantation development, respectively. Thus, investigation of distinct traits between each pluripotent stem cell type would shed light on early embryonic processes. Herein, by screening a fluorescent probe library, we found that intracellular glycogen led to specific reactivity to CDg4, a glycogen fluorescence sensor, in both human and mouse naïve embryonic stem cells (ESCs). The requirement of constant inhibition of Gsk3β as well as high oxidative phosphorylation (OxPHOS) in naïve compared to primed ESCs was closely associated to high level of intracellular glycogen in naïve ESCs. Both capacity of OxPHOS and stored glycogen, rescued naïve ESCs by transient inhibition of glycolysis, which selectively eliminated primed ESCs. Additionally, naïve ESCs with active OxPHOS were enriched from a mixture with primed ESCs by high reactivity to ATP-Red1, a mitochondrial ATP fluorescence probe. These results indicate the active OxPHOS and high intracellular glycogen as a novel “biomarker” delineating metabolic remodeling during the transition of naïve pluripotency.     

The primitive growth factor NME7AB induces mitochondrially active naïve-like pluripotent stem cells

Naïve pluripotent stem cells (PSCs) display a distinctive phenotype when compared to their “primed” counterparts, including, but not limited to, increased potency to differentiate and more robust mitochondrial respiration. The cultivation and maintenance of naïve PSCs have been notoriously challenging, requiring the use of complex cytokine cocktails. NME7_AB is a newly discovered embryonic stem cell growth factor that is expressed exclusively in the first few days of human blastocyst development. It has been previously reported that growing primed induced PSCs (iPSCs) in bFGF-depleted medium with NME7_AB as the only added growth factor facilitates the regression of these cells to their naïve state. Here, we confirm this regression by demonstrating the reactivation of mitochondrial function in the induced naïve-like PSCs and increased ATP production in these cells, as compared to that in primed iPSCs.     

Primed for pluripotency

To date, pluripotent epiblast stem cells (EpiSCs) had only been derived from postimplantation mouse embryos. In this issue of Cell Stem Cell, Najm et al. (2011) demonstrate that EpiSCs can be routinely derived from preimplantation embryos, showing that both human and mouse blastocysts can produce the same class of primed pluripotent cells.     

A short G1 phase is an intrinsic determinant of naïve embryonic stem cell pluripotency

A short G1 phase is a characteristic feature of mouse embryonic stem cells (ESCs). To determine if there is a causal relationship between G1 phase restriction and pluripotency, we made use of the Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) reporter system to FACS-sort ESCs in the different cell cycle phases. Hence, the G1 phase cells appeared to be more susceptible to differentiation, particularly when ESCs self-renewed in the naïve state of pluripotency. Transitions from ground to naïve, then from naïve to primed states of pluripotency were associated with increased durations of the G1 phase, and cyclin E-mediated alteration of the G1/S transition altered the balance between self-renewal and differentiation. LIF withdrawal resulted in a lengthening of the G1 phase in naïve ESCs, which occurred prior to the appearance of early lineage-specific markers, and could be reversed upon LIF supplementation. We concluded that the short G1 phase observed in murine ESCs was a determinant of naïve pluripotency and was partially under the control of LIF signaling.     

Dose response kinetics of CD8 lymphocytes from young animals transfused into old animals and challenged with influenza

Transfusion of autologous leukocytes after prolonged storage has been proposed as a means of rejuvenating the immune system of older individuals. The rationale for this approach is that age related immune decline is associated with a diminished pool of naïve T cells following atrophy of the thymus and reduction in thymic output. The presence of high levels of naïve T cells within the blood of young individuals could provide a boost to the immune system of an older “self” through a rejuvenation of the naïve T cell pool. However what remains unresolved is whether the cells could be incorporated effectively into the T cell pool of the host and whether effectors could be generated. Using CD45 congenic mice in our experiments we show that the transfusion of young donor cells into older congenic host animals leads to their successful incorporation into the peripheral T cell pool. When the recipients were challenged with influenza virus, specific effector CD8 cells were generated which were of both host and donor origin. We found no relationship between the number of responder cells of donor origin at the time of assay and the number of cells injected.     

Mitochondria in early mammalian development

The role of mitochondria as central determinants of development competence of oocytes and preimplantation stage embryos is considered in the context of the diverse activities these organelles have in normal cell function. Stage- and cell-cycle-specific mitochondrial translocations and redistributions are described with respect to mechanisms of cytoplasmic remodeling that may establish domains of autonomous regulation of mitochondrial function and activity during early development. The functions of mitochondria as intracellular signaling elements, as regulators of signaling pathways, and oxygen sensors in differentiated cells are suggested to have similar capacities during mammalian oogenesis and early embryogenesis. Questions concerning the numerical size of the oocyte mitochondrial complement, the energy required to support normal preovulatory oogenesis and preimplantation embryogenesis, and the regulation of mitochondrial activity by extrinsic and intrinsic factors are addressed with respect the potential they may have for new investigational approaches to study the origin of the differential developmental competence of human oocytes and preimplantation stage embryos.     

Reconciling the different roles of Gsk3β in “naïve” and “primed” pluripotent stem cells

Signaling pathways orchestrated by PI3K/Akt, Raf/Mek/Erk and Wnt/β-catenin are known to play key roles in the self-renewal and differentiation of pluripotent stem cells. The serine/threonine protein kinase Gsk3β has roles in all three pathways, making its exact function difficult to decipher. Consequently, conflicting reports have implicated Gsk3β in promoting self-renewal, while others suggest that it performs roles in the activation of differentiation pathways. Different thresholds of Gsk3β activity also have different biological effects on pluripotent cells, making this situation even more complex. Here, we describe a further level of complexity that is most apparent when comparing “naïve” murine and “primed” human pluripotent stem cells. In naïve cells, Gsk3β activity is restrained by PI3K/Akt, but when released from inhibitory signals it antagonizes self-renewal pathways by targeting pluripotency factors such as Myc and Nanog. This situation also applies in primed cells, but, in addition, a separate pool of Gsk3β is required to suppress canonical Wnt signaling. These observations suggest that different Gsk3β-protein complexes shift the balance between naïve and primed pluripotent cells and identify fundamental differences in their cell signaling. Altogether, these findings have important implications for the mechanisms underpinning the establishment of different pluripotent cell states and for the control of self-renewal and differentiation.     

Precise recapitulation of methylation change in early cloned embryos

Change of DNA methylation during preimplantation development is very dynamic, which brings this term to the most attractive experimental target for measuring the capability of cloned embryo to reprogram its somatic genome. However, one weak point is that the preimplantation stage carries little information on genomic sequences showing a site-specific re-methylation after global demethylation; these sequences, if any, may serve as an advanced subject to test how exactly the reprogramming/programming process is recapitulated in early cloned embryos. Here, we report a unique DNA methylation change occurring at bovine neuropeptide galanin gene sequence. The galanin gene sequence in early bovine embryos derived by in vitro fertilization (IVF) maintained a undermethylated status till the morula stage. By the blastocyst, certain CpG sites became methylated specifically, which may be an epigenetic sign for the galanin gene to start a differentiation programme. The same sequence was moderately methylated in somatic donor cell and, after transplanted into an enucleated oocyte by nuclear transfer (NT), came rapidly demethylated to a completion, and then, at the blastocyst stage, re-methylated at exactly the same CpG sites, as observed so in normal blastocysts. The precise recapitulation of normal methylation reprogramming and programming at the galanin gene sequence in bovine cloned embryos gives a cue for the potential of cloned embryo to superintend the epigenetic states of foreign genome, even after global demethylation.     

Inducible CRISPR genome editing platform in naive human embryonic stem cells reveals JARID2 function in self-renewal

To easily edit the genome of naïve human embryonic stem cells (hESC), we introduced a dual cassette encoding an inducible Cas9 into the AAVS1 site of naïve hESC (iCas9). The iCas9 line retained karyotypic stability, expression of pluripotency markers, differentiation potential, and stability in 5iLA and EPS pluripotency conditions. The iCas9 line induced efficient homology–directed repair (HDR) and non-homologous end joining (NHEJ) based mutations through CRISPR-Cas9 system. We utilized the iCas9 line to study the epigenetic regulator, PRC2 in early human pluripotency. The PRC2 requirement distinguishes between early pluripotency stages, however, what regulates PRC2 activity in these stages is not understood. We show reduced H3K27me3 and pluripotency markers in JARID2 2iL-I-F hESC mutants, indicating JARID2 requirement in maintenance of hESC 2iL-I-F state. These data suggest that JARID2 regulates PRC2 in 2iL-I-F state and the lack of PRC2 function in 5iLA state may be due to lack of sufficient JARID2 protein.     

Induction of protective immunity by primed B-1 cells in Toxoplasma gondii -infected B cell-deficient mice

We examined the role of B‐1 cells in protection against Toxoplasma gondii infection using B cell‐deficient mice (μMT mice). We found that primed but not naïve B‐1 cells from wild‐type C57BL/6 mice protected B cell‐deficient recipients from challenge infection. All μMT mice transferred with primed B‐1 cells survived more than 5 months after T. gondii infection, whereas 100% of μMT mice transferred with naïve B‐1 cells succumbed by 18 days after infection. Additionally, high expression of both T help (Th) 1‐ and Th2‐type cytokines and a high level of nitric oxide production were observed in T. gondii‐infected μMT mice transferred with primed B‐1 cells. Thus, it was clearly demonstrated that B‐1 cells play an important role in host protection against T. gondii infection in μMT mice.     

The transition of mouse pluripotent stem cells from the naïve to the primed state requires Fas signaling through 3-O sulfated heparan sulfate structures recognized by the HS4C3 antibody

The characteristics of pluripotent embryonic stem cells of human and mouse are different. The properties of human embryonic stem cells (hESCs) are similar to those of mouse epiblast stem cells (mEpiSCs), which are in a later developmental pluripotency state, the so-called “primed state” compared to mouse embryonic stem cells (mESCs) which are in a naïve state. As a result of the properties of the primed state, hESCs proliferate slowly, cannot survive as single cells, and can only be transfected with genes at low efficiency. Generating hESCs in the naïve state is necessary to overcome these problems and allow their application in regenerative medicine. Therefore, clarifying the mechanism of the transition between the naïve and primed states in pluripotent stem cells is important for the establishment of stable methods of generating naïve state hESCs. However, the signaling pathways which contribute to the transition between the naïve and primed states are still unclear. In this study, we carried out induction from mESCs to mEpiSC-like cells (mEpiSCLCs), and observed an increase in the activation of Fas signaling during the induction. The expression of Fgf5, an epiblast marker, was diminished by inhibition of Fas signaling using the caspase-8 and -3 blocking peptides, IETD and DEVD, respectively. Furthermore, during the induction, we observed increased expression of 3-O sulfated heparan sulfate (HS) structures synthesized by HS 3-O-sulfotransferase (3OST), which are recognized by the HS4C3 antibody (HS4C3-binding epitope). Knockdown of 3OST-5 reduced Fas signaling and the potential for the transition to mEpiSCLCs. This indicates that the HS4C3-binding epitope is necessary for the transition to the primed state. We propose that Fas signaling through the HS4C3-binding epitope contributes to the transition from the naïve state to the primed state.     

MicroRNA‐125b modulates inflammatory chemokine CCL4 expression in immune cells and its reduction causes CCL4 increase with age

Chemokines play a pivotal role in regulating the immune response through a tightly controlled expression. Elevated levels of inflammatory chemokines commonly occur with aging but the mechanism underlying this age‐associated change is not fully understood. Here, we report the role of microRNA‐125b (miR‐125b) in regulating inflammatory CC chemokine 4 (CCL4) expression in human immune cells and its altered expression with aging. We first analyzed the mRNA level of CCL4 in eight different types of immune cells including CD4 and CD8 T‐cell subsets (naïve, central and effector memory), B cells and monocytes in blood from both young (≤42 years) and old (≥70 years) adults. We observed that monocytes and naïve CD8 T cells expressed higher levels of CCL4 and exhibited an age‐related increase in CCL4. We then found the level of miR‐125b was inversely correlated with the level of CCL4 in these cells, and the level of miR‐125b was reduced in monocytes and naïve CD8 T cells of the old compared to the young adults. Knock‐down of miR‐125b by shRNA in monocytes and naïve CD8 T cells led to an increase of CCL4 protein, whereas enhanced miR‐125b expression by transfection in naïve CD8 T cells resulted in a reduction of the CCL4 mRNA and protein in response to stimulation. Finally, we demonstrated that miR‐125b action requires the ‘seed’ sequence in 3′UTR of CCL4. Together these findings demonstrated that miR‐125b is a negative regulator of CCL4 and its reduction is partially responsible for the age‐related increase of CCL4.     

Differential localization of TGF-beta 2 in mouse preimplantation and early postimplantation development

The localization of transforming growth factor type beta 2 (TGF-beta 2) has been followed during preimplantation and early postimplantation murine development using an anti-peptide antibody that specifically recognizes TGF-beta 2. The staining pattern showed that TGF-beta 2 is expressed from the four-cell stage onward and is differentially regulated as cells diverge to various lineages. High levels of staining were found in the trophectoderm of the blastocyst but no staining was observed in the inner cell mass. During postimplantation development the primitive and embryonic ectoderm also lacked detectable staining while visceral endoderm stained well. Parietal endoderm cells also showed positive staining reaction although to a lesser extent than visceral endoderm cells. These findings were confirmed in model systems of the embryo, namely, embryonal carcinoma and embryonic stem cells differentiated to to cells with either visceral or parietal endoderm characteristics. The possible regulatory role of this factor in early embryogenesis is discussed.     

DNA methylation,retroviruses, and embryogenesis

By exposing preimplantation embryos to Moloney leukemia virus (M-MuLV), we have previously derived substrains of mice designated as Mov-1-Mov-13 which genetically transmit the virus from one generation to the next. In some of the substrains the inserted viral genome becomes activated at specific stages of embryogenesis and the available evidence suggests that these viral genomes are developmentally regulated. To investigate the effect of cellular differentiation on virus expression, M-MuLV was introduced either into preimplantation or postimplantation mouse embryos or into embryonal carcinoma (EC) cells. Whereas preimplantation embryos or EC cells are not permissive for virus expression, efficient replication occurred in postimplantation embryos or in differentiated cell lines. The viral genomes introduced into early embryonal cells were highly methylated and noninfcctious when analyzed in the adult. In contrast, viral genomes introduced into postimplantation embryos or into differentiated cells remained unmethylated and were infectious in a transfection assay. These results demonstrate an efficient de novo methylation activity which appears to be involved in repression of genes introduced into pluripotent embryonal cells and which is not observed in cells of the postimplantation embryo or in differentiated cells in tissue culture.