Shinya Yasuda

Wnt/β-catenin Signaling in Embryonic Stem Cell Self-renewal and Somatic Cell Reprogramming

Embryonic stem cells and induced pluripotent stem (iPS) cells are characterized by their ability to self-renew and to generate differentiated cells of all three germ layers. This potential makes them an attractive source to address question of developmental and also for use in clinical regenerative medicine. Although the culture conditions to maintain pluripotency and reprogramming technologies have been established, the underlying molecular mechanisms are incompletely understood. Accumulating evidence indicates that the Wnt/β-catenin signaling pathway plays a pivotal role in the maintenance of pluripotency as well as in the process of somatic cell reprogramming. Reciprocally, Wnt/β-catenin signaling also plays a critical role in the lineage decision/commitment process. These dramatically different outcomes upon activation of the Wnt signaling cascade has fueled enormous controversy concerning the role of Wnt signaling in the maintenance of potency and induction of differentiation in stem cells. Here, we discuss and explore the divergent roles of the Wnt signaling pathways based on findings from our lab. Accumulated results from our lab indicate the usage of a critical switching mechanism that regulates the divergent Wnt/catenin transcriptional programs associated with either maintenance of potency or initiation of differentiation.

Role of SOX2 in maintaining pluripotency of human embryonic stem cells

Human embryonic stem cell (ESC) pluripotency is thought to be regulated by several key transcription factors including OCT4, NANOG, and SOX2. Although the functions of OCT4 and NANOG in human ESCs are well defined, that of SOX2 has not been fully characterized. To investigate the role of SOX2, we modulated the level of SOX2 expression in human ESCs. Reduction of SOX2 expression in human ESCs induced trophectodermal and partial endodermal differentiation. Interestingly, CDX2, a typical trophectoderm‐associated gene, was not up‐regulated. In contrast, using the Tet‐on gene inducible system, SOX2 over‐expression in human ESCs induced trophectoderm differentiation accompanied by increased CDX2 expression. Additionally, SOX2 over‐expression resulted in an increase in CGα‐positive cells, which marks later stage trophectoderm development, rather than placental lactogen‐positive cells. Thus, over‐expression as well as repression of SOX2 expression in human ESCs resulted in their differentiation into the trophectoderm lineage. Our data show that SOX2 plays an important role in the maintenance of pluripotency of human ESCs and possibly, trophoblast development.

NANOG maintains self‐renewal of primate ES cells in the absence of a feeder layer

Nanog is a homeodomain transcription factor that is expressed specifically in undifferentiated embryonic stem (ES) cells and has been shown to be essential in the maintenance of pluripotency in mouse ES cells. To examine the function of NANOG in primate ES cells, we generated transgenic monkey ES cell lines expressing three‐ to seven‐fold higher levels of NANOG protein compared to wild‐type ES cells. These NANOG over‐expressing cell lines retained their undifferentiated state in the absence of a feeder layer, as shown by expression of undifferentiated ES cell markers such as alkaline phosphatase (ALP) and OCT‐4. We also demonstrated that in vitro differentiation of transgenic cell lines was mostly restricted to the ectodermal lineage, as examined by reverse transcriptase‐polymerase chain reaction (RT‐PCR). Knockdown experiments using NANOG small interfering (si) RNA resulted in induction of differentiation markers such as AFP, GATA4 and GATA6 for the endoderm and CDX2 for the trophectoderm. These results suggest that NANOG plays a crucial role in maintaining the pluripotent state of primate ES cells.

Wnt Signaling Orchestration with a Small Molecule DYRK Inhibitor Provides Long-Term Xeno-Free Human Pluripotent Cell Expansion

An optimal culture system for human pluripotent stem cells should be fully defined and free of animal components. To date, most xeno‐free culture systems require human feeder cells and/or highly complicated culture media that contain activators of the fibroblast growth factor (FGF) and transforming growth factor‐β (TGFβ) signaling pathways, and none provide for replacement of FGF/TGFβ ligands with chemical compounds. The Wnt/β‐catenin signaling pathway plays an important role in mouse embryonic stem cells in leukemia inhibitory factor‐independent culture; however, the role of Wnt/β‐catenin signaling in human pluripotent stem cell is still poorly understood and controversial because of the dual role of Wnts in proliferation and differentiation. Building on our previous investigations of small molecules modulating Wnt/β‐catenin signaling in mouse embryonic stem cells, we identified a compound, ID‐8, that could support Wnt‐induced human embryonic stem cell proliferation and survival without differentiation. Dual‐specificity tyrosine phosphorylation‐regulated kinase (DYRK) is the target of the small molecule ID‐8. Its role in human pluripotent cell renewal was confirmed by DYRK knockdown in human embryonic stem cells. Using Wnt and the DYRK inhibitor ID‐8, we have developed a novel and simple chemically defined xeno‐free culture system that allows for long‐term expansion of human pluripotent stem cells without FGF or TGFβ activation. These culture conditions do not include xenobiotic supplements, serum, serum replacement, or albumin. Using this culture system, we have shown that several human pluripotent cell lines maintained pluripotency (>20 passages) and a normal karyotype and still retained the ability to differentiate into derivatives of all three germ layers. This Wnt‐dependent culture system should provide a platform for complete replacement of growth factors with chemical compounds.

Specific Direct Small Molecule p300/β-Catenin Antagonists Maintain Stem Cell Potency.

Despite their high degree of identity and even higher homology, the two Kat3 transcriptional coactivators, CBP and p300, have distinct functions, particularly within the Wnt/β-catenin signaling cascade. ICG-001, by directly binding to CBP but not p300, inhibits CBP/β-catenin transcription and has served as an invaluable chemical genomic tool to dissect the Wnt signaling cascade and the divergent roles of these two coactivators. However, to date no direct antagonist of the p300/β-catenin interaction has been reported. We now report the identification and validation of the first highly specific, direct p300/β-catenin antagonists, YH249/250 and their ability to maintain pluripotency in ESC.

Chemically defined and growth-factor-free culture system for the expansion and derivation of human pluripotent stem cells

The large-scale and cost-effective production of quality-controlled human pluripotent stem cells (hPSCs) for use in cell therapy and drug discovery would ideally require a chemically defined xenobiotic-free culture system. Towards the development of such a system, costs associated with the use of recombinant proteins as supplements in basal culture media need to be reduced. Here, we describe a growth-factor-free culture medium that uses just three chemical compounds and a lower number of recombinant proteins than used in commercially available media. We show that the culture medium supports the long-term propagation of hPSCs, as confirmed by karyotype, the expression of pluripotency markers and the capacity to differentiate into cell types derived from the three embryonic germ layers. hPSCs growing in the medium were less dependent on glycolytic pathways than cells grown in medium containing growth factors. Moreover, the medium supported the generation of induced pluripotent stem cells derived from either human dermal fibroblasts or peripheral blood mononuclear cells. Our findings should facilitate the ongoing development of a completely xeno-free, chemically defined, synthetic culture system for hPSCs.A new chemically defined culture medium for the long-term culture of human pluripotent stem cells uses only three chemical compounds and a lower number of recombinant proteins than used in commercially available media.

A case of malignant schwannoma of mandible in von Recklinghausen's disease.

von Recklinghausen病 (以下R病) あるいは神経線維腫症は常染色体遺伝性疾患で3000の出生に対して1人の発現頻度といわれている。カフェオーレ斑, 末梢中枢神経系の神経線維腫症および幼少時より生じる骨格異常が特徴とされる。神経線維腫症は軟組織由来の悪性腫瘍を合併することが知られている。頭頸部腫瘍のなかで軟組織肉腫の発生は少なく, 特に神経線維腫症に合併した悪性神経鞘腫の症例は極めてまれである。今回われわれは, R病の女児に合併した下顎悪性神経鞘腫の1例を経験したので報告する。患者は7歳女児で, 右側下顎部腫脹, 開閉口障害を主訴に紹介来院した。右側下顎枝に直径5cm大の弾性硬の腫瘤を認め, 生検の結果, 悪性神経鞘腫と診断された。外科的切除, 術中放射線照射の後, 化学療法施行されたが約10か月後に局所再発および肺転移を生じ, 初診より1年4か月後に死亡した。