Shoen Kume

The Wnt/calcium pathway activates NF-AT and promotes ventral cell fate in Xenopus embryos.

It is thought that inositol-1,4,5-trisphosphate (Ins(1,4,5)P3)-Ca2+ signalling has a function in dorsoventral axis formation in Xenopus embryos; however, the immediate target of free Ca2+ is unclear. The secreted Wnt protein family comprises two functional groups, the canonical Wnt and Wnt/Ca2+ pathways. The Wnt/Ca2+ pathway interferes with the canonical Wnt pathway, but the underlying molecular mechanism is poorly understood. Here, we cloned the complementary DNA coding for the Xenopus homologue of nuclear factor of activated T cells (XNF-AT). A gain-of-function, calcineurin-independent active XNF-AT mutation (CA XNF-AT) inhibited anterior development of the primary axis, as well as Xwnt-8-induced ectopic dorsal axis development in embryos. A loss-of-function, dominant negative XNF-AT mutation (DN XNF-AT) induced ectopic dorsal axis formation and expression of the canonical Wnt signalling target molecules siamois and Xnr3 (ref. 4). Xwnt-5A induced translocation of XNF-AT from the cytosol to the nucleus. These data indicate that XNF-AT functions as a downstream target of the Wnt/Ca2+ and Ins(1,4,5)P3-Ca2+ pathways, and has an essential role in mediating ventral signals in the Xenopus embryo through suppression of the canonical Wnt pathway.

The Xenopus IP3 receptor: Structure, function, and localization in oocytes and eggs

To study the role of the IP3 receptor (IP3R) upon egg activation, cDNA clones encoding IP3R expressed in the Xenopus oocytes were isolated. By analyses of the primary structure and functional expression of the cDNA, Xenopus IP3R (XIP3R) was shown to have an IP3-binding domain and a putative Ca2+ channel region. Immunocytochemical studies revealed polarized distribution of XIP3R in the cytoplasm of the animal hemisphere in a well-organized endoplasmic reticulum-like structure and intensive localization in the perinuclear region of stage VI immature oocytes. In ovulated unfertilized eggs, XIP3R was densely enriched in the cortical region of both hemispheres in addition to its polarized localization. After fertilization, XIP3R showed a drastic change in its distribution in the cortical region. These results imply the predominant role of the XIP3R in both the formation and propagation of Ca2+ waves at fertilization.

Methionine metabolism regulates maintenance and differentiation of human pluripotent stem cells.

Mouse embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are in a high-flux metabolic state, with a high dependence on threonine catabolism. However, little is known regarding amino acid metabolism in human ESCs/iPSCs. We show that human ESCs/iPSCs require high amounts of methionine (Met) and express high levels of enzymes involved in Met metabolism. Met deprivation results in a rapid decrease in intracellular S-adenosylmethionine (SAM), triggering the activation of p53-p38 signaling, reducing NANOG expression, and poising human iPSC/ESCs for differentiation, follow by potentiated differentiation into all three germ layers. However, when exposed to prolonged Met deprivation, the cells undergo apoptosis. We also show that human ESCs/iPSCs have regulatory systems to maintain constant intracellular Met and SAM levels. Our findings show that SAM is a key regulator for maintaining undifferentiated pluripotent stem cells and regulating their differentiation.

Guided Differentiation of Embryonic Stem Cells into Pdx1-Expressing Regional-Specific Definitive Endoderm

The generation of specific lineages of the definitive endoderm from embryonic stem (ES) cells is an important issue in developmental biology, as well as in regenerative medicine. This study demonstrates that ES cells are induced sequentially into regional‐specific gut endoderm lineages, such as pancreatic, hepatic, and other cell lineages, when they are cultured directly on a monolayer of mesoderm‐derived supporting cells. A detailed chronological analysis revealed that Activin, fibroblast growth factor, or bone morphogenetic protein signals are critical at various steps and that additional short‐range signals are required for differentiation into Pdx1‐expressing cells. Under selective culture conditions, definitive endoderm (47%) or Pdx1‐positive pancreatic progenitors (30%) are yielded at a high efficiency. When transplanted under the kidney capsule, the Pdx1‐positive cells further differentiated into all three pancreatic lineages, namely endocrine, exocrine, and duct cells.

Differentiation of mouse and human embryonic stem cells into hepatic lineages

We recently reported a novel method to induce embryonic stem (ES) cells differentiate into an endodermal fate, especially pancreatic, using a supporting cell line. Here we describe the modified culture condition with the addition and withdrawal of secreted growth factors could induce ES cells to selectively differentiate into a hepatic fate efficiently. The signaling of BMP and FGF that have been implicated in hepatic differentiation during normal embryonic development are shown to play pivotal roles in generating hepatic cells from the definitive endoderm derived from ES cells. Moreover, the expression of AFP, Albumin or a biliary molecular marker appeared sequentially thus suggested the differentiation of ES cells recapitulated normal developmental processes of liver. The ES cell‐derived differentiated cells showed evidence of glycogen storage, secreted Albumin, exhibited drug metabolism activities and expressed a set of cytochrome or drug conjugate enzymes, drug transporters specifically expressed in mature hepatocytes. With the same procedure, human ES cells also gave rise to cells with mature hepatocytes’ characteristics. In conclusion, this novel procedure for hepatic differentiation will be useful for elucidation of molecular mechanisms of hepatic fate decision at gut regionalization, and could represent an attractive approach for a surrogate cell source for pharmaceutical studies such as toxicology.

Synthesized basement membranes direct the differentiation of mouse embryonic stem cells into pancreatic lineages

We previously reported that embryonic stem (ES) cells cultured on M15 cells, a mesoderm-derived supportive cell line, were efficiently differentiated towards an endodermal fate, finally adopting the specific lineages of various digestive organs such as the pancreas and liver. We show here that the endoderm-inducing activity of M15 cells is in part mediated through the extracellular matrices, and that laminin α5 is one of the crucial components. In an attempt to establish a feeder-free ES-cell procedure for pancreatic differentiation, we used a synthesized basement membrane (sBM) substratum using an HEK293 cell line stably expressing laminin-511. On the sBM, mouse ES or induced pluripotent stem (iPS) cells sequentially differentiated into the definitive endoderm, pancreatic progenitor cells, and then insulin-expressing pancreatic β-cells in vitro. Knockdown of ES cells with integrin β1 (Itgb1) reduces differentiation towards pancreatic cells. Heparan sulfate proteoglycan 2 (HSPG2) knockdown and heparitinase treatment synergistically decreased the number of Pdx1-expressing cells. These findings indicate that components of the basement membrane have an important role in the differentiation of definitive endoderm lineages. This novel procedure will be useful for the study of pancreatic differentiation of ES or iPS cells and the generation of potential sources of surrogate cells for regenerative medicine.

Efficient Differentiation of Embryonic Stem Cells into Hepatic Cells In Vitro Using a Feeder-Free Basement Membrane Substratum

The endoderm-inducing effect of the mesoderm-derived supportive cell line M15 on embryonic stem (ES) cells is partly mediated through the extracellular matrix, of which laminin α5 is a crucial component. Mouse ES or induced pluripotent stem cells cultured on a synthesized basement membrane (sBM) substratum, using an HEK293 cell line (rLN10-293 cell) stably expressing laminin-511, could differentiate into definitive endoderm and subsequently into pancreatic lineages. In this study, we investigated the differentiation on sBM of mouse and human ES cells into hepatic lineages. The results indicated that the BM components played an important role in supporting the regional-specific differentiation of ES cells into hepatic endoderm. We show here that knockdown of integrin β1 (Itgb1) in ES cells reduced their differentiation into hepatic lineages and that this is mediated through Akt signaling activation. Moreover, under optimal conditions, human ES cells differentiated to express mature hepatocyte markers and secreted high levels of albumin. This novel procedure for inducing hepatic differentiation will be useful for elucidating the molecular mechanisms controlling lineage-specific fates during gut regionalization. It could also represent an attractive approach to providing a surrogate cell source, not only for regenerative medicine, but also for pharmaceutical and toxicologic studies.

Differentiation and characterization of embryonic stem cells into three germ layers.

Embryonic stem cells differentiated on M15 cells have previously been shown to give rise to cells of the mesendodermal and definitive endodermal lineages. Here we demonstrate that neuroectodermal and mesodermal lineages can be derived from ES cells cultured on M15 cells and subsequently subjected to specific culture conditions, as confirmed by the expression of molecular markers. Prospective isolation and microarray analyses showed that neuroectodermal cells expressed anterior-to-posterior, as well as dorso-ventral regional markers, suggesting that this procedure could be used for the induction of cells belonging to a wide variety of neural lineages. Lateral mesoderm and paraxial mesoderm cells were also produced and their gene expression profiles were confirmed by microarray analyses. These results indicate that the M15 cell system provides a valuable tool for generating ES cell-derived lineage-specific cell types belonging to the three germ layers, namely neuroectoderm, mesoderm, and definitive endoderm.

Developmental expression of the inositol 1,4,5-trisphosphate receptor and localization of inositol 1,4,5-trisphosphate during early embryogenesis in Xenopus laevis

To study the role of inositol 1,4,5-trisphosphate (IP3) receptors during early embryogenesis in Xenopus, we examined the temporal-spatial localization of Xenopus IP3 receptor (XIP3R). XIP3R protein is enriched in the animal hemisphere of early cleavage stage embryos and becomes localized in the ectoderm and involuted mesoderm in gastrula stage embryos. Up to tailbud stages, expression of XIP3R is observed in the mesodermal tissues and in most subregions of the central nervous system. A quantitative analysis of endogenous IP3 mass during normal early embryogenesis revealed an increase in IP3 mass first observed at early gastrula stage of 10.5 with an enrichment in the ectoderm throughout the gastrula stages, implying a potential role during gastrulation.

Expression patterns of epiplakin1 in pancreas, pancreatic cancer and regenerating pancreas

Epiplakin1 (Eppk1) is a plakin family gene with its function remains largely unknown, although the plakin genes are known to function in interconnecting cytoskeletal filaments and anchoring them at plasma membrane‐associated adhesive junction. Here we analyzed the expression patterns of Eppk1 in the developing and adult pancreas in the mice. In the embryonic pancreas, Eppk1+/Pdx1+ and Eppk1+/Sox9+ pancreatic progenitor cells were observed in early pancreatic epithelium. Since Pdx1 expression overlapped with that of Sox9 at this stage, these multipotent progenitor cells are Eppk1+/Pdx1+/Sox9+ cells. Then Eppk1 expression becomes confined to Ngn3+ or Sox9+ endocrine progenitor cells, and p48+ exocrine progenitor cells, and then restricted to the duct cells and a cells at birth. In the adult pancreas, Eppk1 is expressed in centroacinar cells (CACs) and in duct cells. Eppk1 is observed in pancreatic intraepithelial neoplasia (PanIN), previously identified as pancreatic ductal adenocarcinoma (PDAC) precursor lesions. In addition, the expansion of Eppk1‐positive cells occurs in a caerulein‐induced acute pancreatitis, an acinar cell regeneration model. Furthermore, in the partial pancreatectomy (Px) regeneration model using mice, Eppk1 is expressed in “ducts in foci”, a tubular structure transiently induced. These results suggest that Eppk1 serves as a useful marker for detecting pancreatic progenitor cells in developing and regenerating pancreas.