Naohiro Terada

Bone marrow cells adopt the phenotype of other cells by spontaneous cell fusion

Recent studies have demonstrated that transplanted bone marrow cells can turn into unexpected lineages including myocytes, hepatocytes, neurons and many others. A potential problem, however, is that reports discussing such ‘transdifferentiation’ in vivo tend to conclude donor origin of transdifferentiated cells on the basis of the existence of donor-specific genes such as Y-chromosome markers. Here we demonstrate that mouse bone marrow cells can fuse spontaneously with embryonic stem cells in culture in vitro that contains interleukin-3. Moreover, spontaneously fused bone marrow cells can subsequently adopt the phenotype of the recipient cells, which, without detailed genetic analysis, might be interpreted as ‘dedifferentiation’ or transdifferentiation.

Regulation of elongation factor 2 kinase by p90RSK1 and p70 S6 kinase

Elongation factor 2 kinase (eEF2k) phosphorylates and inactivates eEF2. Insulin induces dephosphorylation of eEF2 and inactivation of eEF2 kinase, and these effects are blocked by rapamycin, which inhibits the mammalian target of rapamycin, mTOR. However, the signalling mechanisms underlying these effects are unknown. Regulation of eEF2 phosphorylation and eEF2k activity is lost in cells in which phosphoinositide‐dependent kinase 1 (PDK1) has been genetically knocked out. This is not due to loss of mTOR function since phosphorylation of another target of mTOR, initiation factor 4E‐binding protein 1, is not defective. PDK1 is required for activation of members of the AGC kinase family; we show that two such kinases, p70 S6 kinase (regulated via mTOR) and p90RSK1 (activated by Erk), phosphorylate eEF2k at a conserved serine and inhibit its activity. In response to insulin‐like growth factor 1, which activates p70 S6 kinase but not Erk, regulation of eEF2 is blocked by rapamycin. In contrast, regulation of eEF2 by stimuli that activate Erk is insensitive to rapamycin, but blocked by inhibitors of MEK/Erk signalling, consistent with the involvement of p90RSK1.

Hepatic maturation in differentiating embryonic stem cells in vitro

We investigated the potential of mouse embryonic stem (ES) cells to differentiate into hepatocytes in vitro. Differentiating ES cells expressed endodermal‐specific genes, such as α‐fetoprotein, transthyretin, α 1‐anti‐trypsin and albumin, when cultured without additional growth factors and late differential markers of hepatic development, such as tyrosine aminotransferase (TAT) and glucose‐6‐phosphatase (G6P), when cultured in the presence of growth factors critical for late embryonic liver development. Further, induction of TAT and G6P expression was induced regardless of expression of the functional SEK1 gene, which is thought to provide a survival signal for hepatocytes during an early stage of liver morphogenesis. The data indicate that the in vitro ES differentiation system has a potential to generate mature hepatocytes. The system has also been found useful in analyzing the role of growth factors and intracellular signaling molecules in hepatic development.

p70S6 kinase signals cell survival as well as growth, inactivating the pro-apoptotic molecule BAD

Cytokines often deliver simultaneous, yet distinct, cell growth and cell survival signals. The 70-kDa ribosomal protein S6 kinase (p70S6K) is known to regulate cell growth by inducing protein synthesis components. We purified membrane-based p70S6K as a kinase responsible for site-specific phosphorylation of BAD, which inactivates this proapoptotic molecule. Rapamycin inhibited mitochondrial-based p70S6K, which prevented phosphorylation of Ser-136 on BAD and blocked cell survival induced by insulin-like growth factor 1 (IGF-1). Moreover, IGF-1-induced phosphorylation of BAD Ser-136 was abolished in p70S6K-deficient cells. Thus, p70S6K is itself a dual pathway kinase, signaling cell survival as well as growth through differential substrates which include mitochondrial BAD and the ribosomal subunit S6, respectively.

A Heterogeneous Expression Pattern for Nanog in Embryonic Stem Cells

Nanog is a critical homeodomain factor responsible for maintaining embryonic stem (ES) cell self‐renewal and pluripotency. Of interest, Nanog expression is not homogeneous in the conventional culture of murine ES cells. A Nanog‐high population expresses markers for pluripotent ES cells, whereas a Nanog‐low population expresses markers for primitive endoderm, such as Gata6. Since the inner cell mass of early blastocysts has recently been reported to be heterogeneous in terms of Nanog and Gata6 expression, ES cells appear to closely resemble the developing stage from which they originate. We further demonstrate that Nanog can directly repress Gata6 expression through its binding to the proximal promoter region of the Gata6 gene and that overexpression of Nanog reduces heterogeneity during ES cell maintenance. Interestingly, Nanog heterogeneity does not correlate with the heterogeneous expression of stage‐specific embryonic antigen‐1, suggesting that multiple but overlapping levels of heterogeneity may exist in ES cells. These findings provide insight into the factors that control ES cell self‐renewal and the earliest lineage commitment to primitive endoderm while also suggesting methods to promote homogeneity during ES cell maintenance.

Embryonic Stem Cells Proliferate and Differentiate when Seeded into Kidney Scaffolds

The scarcity of transplant allografts for diseased organs has prompted efforts at tissue regeneration using seeded scaffolds, an approach hampered by the enormity of cell types and complex architectures. Our goal was to decellularize intact organs in a manner that retained the matrix signal for differentiating pluripotent cells. We decellularized intact rat kidneys in a manner that preserved the intricate architecture and seeded them with pluripotent murine embryonic stem cells antegrade through the artery or retrograde through the ureter. Primitive precursor cells populated and proliferated within the glomerular, vascular, and tubular structures. Cells lost their embryonic appearance and expressed immunohistochemical markers for differentiation. Cells not in contact with the basement membrane matrix became apoptotic, thereby forming lumens. These observations suggest that the extracellular matrix can direct regeneration of the kidney, and studies using seeded scaffolds may help define differentiation pathways.

Amino Acid-dependent Control of p70s6k INVOLVEMENT OF tRNA AMINOACYLATION IN THE REGULATION

In human T-lymphoblastoid cells, downstream signaling events of mammalian target of rapamycin (mTOR), including the activity of p70s6k and phosphorylation of eukaryotic initiation factor 4E-binding protein 1, were dependent on amino acid concentration in the culture media, whereas other growth-related protein kinases were not. Amino acid-induced p70s6kactivation was completely inhibited by rapamycin but only partially inhibited by wortmannin. Moreover, amino acid concentration similarly affected the p70s6k activity, which was dependent on a rapamycin-resistant mutant (S2035I) of mTOR. These data indicate that mTOR is required for amino acid-dependent activation of p70s6k. The mechanism by which amino acids regulate p70s6k activity was further explored: 1) amino acid alcohols, which inhibit aminoacylation of tRNA by their competitive binding to tRNA synthetases, suppressed p70s6k activity; 2) suppression of p70s6k by amino acid depletion was blocked by cycloheximide or puromycin, which inhibit utilization of aminoacylated tRNA in cells; and 3) in cells having a temperature-sensitive mutant of histidyl tRNA synthetase, p70s6k was suppressed by a transition of cells to a nonpermissible temperature, which was partially restored by addition of high concentrations of histidine. These results indicate that suppression of tRNA aminoacylation is able to inhibit p70s6k activity. Deacylated tRNA may be a factor negatively regulating p70s6k.

Characterization of S6K2, a novel kinase homologous to S6K1

Rapamycin is an immunosuppressant which antagonizes cellular proliferation by inhibiting the function of mTOR. The mTOR : FKBP12 : rapamycin complex blocks G1/S transition by inhibiting downstream targets essential for cell cycle progression. One such target is p70S6k1 (S6K1), a serine/threonine kinase which is inactivated by the mTOR : FKBP12 : rapamycin complex, and which has been linked to translational control by virtue of its ability to phosphorylate the ribosomal protein S6. In the current work, we describe cloning and characterization of a novel S6K1 homolog, p54 S6 kinase 2 (p54S6k2/S6K2). Similar to S6K1, S6K2 is activated by mitogens and by constitutively active PI3K, and is inhibited by rapamycin as well as wortmannin. Differences between activation of S6K1 and S6K2 by PDK1 were observed, suggesting potential differences in the regulation of these homologs. Strikingly, S6K2 activity and S6 phosphorylation were both intact in S6K1−/−ES cell, indicating a possible role for S6K2 in in vivo S6 phosphorylation. Interestingly, we found two isoforms of S6K2 which are localized to distinct cellular compartments; the smaller form resides in the detergent-soluble fraction, whereas the larger form is found in the particulate fraction. Our findings demonstrate the existence of a family of rapamycin-sensitive protein kinases potentially involved in S6 phosphorylation, translational control, and transduction of mTOR signals.

The Grb2/Mek pathway represses nanog in murine embryonic stem cells

ABSTRACT The homeobox gene Nanog is a key intrinsic determinant of self renewal in embryonic stem (ES) cells, and its repression leads ES cells to selectively differentiate into primitive endoderm. Although Nanog repression occurs at the outermost layer of ES cell aggregates independent of the leukemia inhibitory factor (LIF)/STAT3 pathway, it is largely undetermined what external cues and intracellular signals cause the event. Of interest, addition of the tyrosine phosphatase inhibitor, sodium vanadate, selectively repressed Nanog transcription without any detectable changes in upstream transcriptional regulators Oct3/4 and Sox2. Furthermore, sodium vanadate induced primitive endoderm differentiation, even in the inner cells of ES cell aggregates. Expression of Gata6 and Zfp42, two putative downstream Nanog effectors, was also increased and decreased by the addition of sodium vanadate, respectively, but these changes were eliminated by exogenous Nanog expression. The effects of sodium vanadate were abrogated by Grb2 deficiency or by the addition of the Mek inhibitor, PD98059. Indeed, PD98059 prevented Nanog repression induced by ES cell aggregation as well. Furthermore, transfection of a constitutive active Mek mutant into ES cells induced Nanog repression and primitive endoderm differentiation. These data indicate that the Grb2/Mek pathway primarily mediates Nanog gene repression upon ES cell differentiation into primitive endoderm.

Aggregation of embryonic stem cells induces Nanog repression and primitive endoderm differentiation

When embryonic stem cells are allowed to aggregate, the outer layer of the aggregated spheres (referred to as embryoid bodies) differentiates into primitive endoderm. This initial specification of cell lineage facilitates further differentiation of the inner mass of the embryoid bodies. These processes are considered to recapitulate early embryonic development from the blastocyst stage to the egg-cylinder stage. Formation of the primitive endoderm layer in the embryoid bodies was induced solely by aggregation of embryonic stem cells, in the presence of leukemia inhibitory factor/STAT3 and serum/BMP4, which were considered to be sufficient for embryonic stem cell self-renewal. Interestingly, cell aggregation by itself induced Nanog repression at the outer layer, which was essential for aggregation-induced primitive endoderm formation. These data illustrate aggregation-based cell-fate specification during early embryonic development, when downregulation of Nanog plays a crucial role.