Masashi Tachibana

Repression of the Transcription Factor Th-POK by Runx Complexes in Cytotoxic T Cell Development

Mouse CD4+CD8+ double-positive (DP) thymocytes differentiate into CD4+ helper-lineage cells upon expression of the transcription factor Th-POK but commit to the CD8+ cytotoxic lineage in its absence. We report the redirected differentiation of class I–restricted thymocytes into CD4+CD8– helper-like T cells upon loss of Runx transcription factor complexes. A Runx-binding sequence within the Th-POK locus acts as a transcriptional silencer that is essential for Th-POK repression and for development of CD8+ T cells. Thus, Th-POK expression and genetic programming for T helper cell development are actively inhibited by Runx-dependent silencer activity, allowing for cytotoxic T cell differentiation. Identification of the transcription factors network in CD4 and CD8 lineage choice provides insight into how distinct T cell subsets are developed for regulating the adaptive immune system.

3D spheroid culture of hESC/hiPSC-derived hepatocyte-like cells for drug toxicity testing

Although it is expected that hepatocyte-like cells differentiated from human embryonic stem (ES) cells or induced pluripotent stem (iPS) cells will be utilized in drug toxicity testing, the actual applicability of hepatocyte-like cells in this context has not been well examined so far. To generate mature hepatocyte-like cells that would be applicable for drug toxicity testing, we established a hepatocyte differentiation method that employs not only stage-specific transient overexpression of hepatocyte-related transcription factors but also a three-dimensional spheroid culture system using a Nanopillar Plate. We succeeded in establishing protocol that could generate more matured hepatocyte-like cells than our previous protocol. In addition, our hepatocyte-like cells could sensitively predict drug-induced hepatotoxicity, including reactive metabolite-mediated toxicity. In conclusion, our hepatocyte-like cells differentiated from human ES cells or iPS cells have potential to be applied in drug toxicity testing.

Peroxisome Proliferator-activated Receptor γ-mediated Regulation of Neural Stem Cell Proliferation and Differentiation

Peroxisome proliferator-activated receptor γ (PPARγ) plays an important role in insulin sensitivity, tissue homeostasis, and regulating cellular functions. We found high-level expression of PPARγ in embryo mouse brain and neural stem cells (NSCs), in contrast to extremely low levels in adult mouse brain. Here, we show that PPARγ mediates the proliferation and differentiation of murine NSCs via up-regulation of the epidermal growth factor receptor and activation of the ERK pathway. Cell growth rates of NSCs prepared from heterozygous PPARγ-deficient mouse brains, PPARγ-RNA-silenced NSCs, and PPARγ dominant-negative NSCs were significantly decreased compared with those of wild-type NSCs. Physiological concentrations of PPARγ agonists, rosiglitazone and pioglitazone, stimulated NSC growth, whereas antagonists caused cell death in a concentration-dependent manner via activation of the caspase cascade. The stimulation of cell growth by PPARγ was associated with a rapid activation of the ERK pathway by phosphorylation and up-regulation of epidermal growth factor receptor and cyclin B protein levels. In contrast, activation of PPARγ by agonists inhibited the differentiation of NSCs into neurons. The inhibition of differentiation was associated with an activation of STAT3. These data indicate that PPARγ regulates the development of the central nervous system during early embryogenesis via control of NSC proliferation.

Prediction of interindividual differences in hepatic functions and drug sensitivity by using human iPS-derived hepatocytes

Significance We found that individual cytochrome P450 (CYP) metabolism capacity and drug sensitivity could be predicted by examining them in the primary human hepatocytes–human induced pluripotent stem cells–hepatocyte-like cells (PHH-iPS-HLCs). We also confirmed that interindividual differences of CYP metabolism capacity and drug responsiveness that are due to the diversity of individual single nucleotide polymorphisms in the CYP gene could also be reproduced in the PHH-iPS-HLCs. These findings suggest that interindividual differences in drug metabolism capacity and drug response could be predicted by using HLCs differentiated from human iPS cells. We believe that iPS-HLCs would be a powerful technology not only for accurate and efficient drug development, but also for personalized drug therapy. Interindividual differences in hepatic metabolism, which are mainly due to genetic polymorphism in its gene, have a large influence on individual drug efficacy and adverse reaction. Hepatocyte-like cells (HLCs) differentiated from human induced pluripotent stem (iPS) cells have the potential to predict interindividual differences in drug metabolism capacity and drug response. However, it remains uncertain whether human iPSC-derived HLCs can reproduce the interindividual difference in hepatic metabolism and drug response. We found that cytochrome P450 (CYP) metabolism capacity and drug responsiveness of the primary human hepatocytes (PHH)-iPS-HLCs were highly correlated with those of PHHs, suggesting that the PHH-iPS-HLCs retained donor-specific CYP metabolism capacity and drug responsiveness. We also demonstrated that the interindividual differences, which are due to the diversity of individual SNPs in the CYP gene, could also be reproduced in PHH-iPS-HLCs. We succeeded in establishing, to our knowledge, the first PHH-iPS-HLC panel that reflects the interindividual differences of hepatic drug-metabolizing capacity and drug responsiveness.

The promotion of hepatic maturation of human pluripotent stem cells in 3D co-culture using type I collagen and Swiss 3T3 cell sheets

Hepatocyte-like cells differentiated from human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) are known to be a useful cell source for drug screening. We recently developed an efficient hepatic differentiation method from hESCs and hiPSCs by sequential transduction of FOXA2 and HNF1α. It is known that the combination of three-dimensional (3D) culture and co-culture, namely 3D co-culture, can maintain the functions of primary hepatocytes. However, hepatic maturation of hESC- or hiPSC-derived hepatocyte-like cells (hEHs or hiPHs, respectively) by 3D co-culture systems has not been examined. Therefore, we utilized a cell sheet engineering technology to promote hepatic maturation. The gene expression levels of hepatocyte-related markers (such as cytochrome P450 enzymes and conjugating enzymes) and the amount of albumin secretion in the hEHs or hiPHs, which were 3D co-cultured with the Swiss 3T3 cell sheet, were significantly up-regulated in comparison with those in the hEHs or hiPHs cultured in a monolayer. Furthermore, we found that type I collagen synthesized in Swiss 3T3 cells plays an important role in hepatic maturation. The hEHs or hiPHs that were 3D co-cultured with the Swiss 3T3 cell sheet would be powerful tools for medical applications, such as drug screening.

Critical role of P1-Runx1 in mouse basophil development.

Runx1(P1N/P1N) mice are deficient in the transcription factor distal promoter-derived Runt-related transcription factor 1 (P1-Runx1) and have a > 90% reduction in the numbers of basophils in the BM, spleen, and blood. In contrast, Runx1(P1N/P1N) mice have normal numbers of the other granulocytes (neutrophils and eosinophils). Although basophils and mast cells share some common features, Runx1(P1N/P1N) mice have normal numbers of mast cells in multiple tissues. Runx1(P1N/P1N) mice fail to develop a basophil-dependent reaction, IgE-mediated chronic allergic inflammation of the skin, but respond normally when tested for IgE- and mast cell-dependent passive cutaneous anaphylaxis in vivo or IgE-dependent mast cell degranulation in vitro. These results demonstrate that Runx1(P1N/P1N) mice exhibit markedly impaired function of basophils, but not mast cells. Infection with the parasite Strongyloides venezuelensis and injections of IL-3, each of which induces marked basophilia in wild-type mice, also induce modest expansions of the very small populations of basophils in Runx1(P1N/P1N) mice. Finally, Runx1(P1N/P1N) mice have normal numbers of the granulocyte progenitor cells, SN-Flk2(+/-), which can give rise to all granulocytes, but exhibit a > 95% reduction in basophil progenitors. The results of the present study suggest that P1-Runx1 is critical for a stage of basophil development between SN-Flk2(+/-) cells and basophil progenitors.

RNA interfering approach for clarifying the PPARγ pathway using lentiviral vector expressing short hairpin RNA

Peroxisome proliferator‐activated receptor γ (PPARγ) plays a central role in adipocyte differentiation and insulin sensitivity. Although PPARγ also appears to regulate diverse cellular processes in other cell types such as lymphocytes, the detailed mechanisms remain unclear. In this study, we established a lentivirus‐mediated short hairpin RNA expression system and identified a potent short hairpin RNA which suppresses PPARγ expression, resulting in marked inhibition of preadipocyte‐to‐adipocyte differentiation in 3T3‐L1 cells. Our PPARγ‐knockdown method will serve to clarify the PPARγ pathway in various cell types in vivo and in vitro, and will facilitate the development of therapeutic applications for a variety of diseases.

Long-Term Self-Renewal of Human ES/iPS-Derived Hepatoblast-like Cells on Human Laminin 111-Coated Dishes

Summary The establishment of self-renewing hepatoblast-like cells (HBCs) from human pluripotent stem cells (PSCs) would realize a stable supply of hepatocyte-like cells for medical applications. However, the functional characterization of human PSC-derived HBCs was not enough. To purify and expand human PSC-derived HBCs, human PSC-derived HBCs were cultured on dishes coated with various types of human recombinant laminins (LN). Human PSC-derived HBCs attached to human laminin-111 (LN111)-coated dish via integrin alpha 6 and beta 1 and were purified and expanded by culturing on the LN111-coated dish, but not by culturing on dishes coated with other laminin isoforms. By culturing on the LN111-coated dish, human PSC-derived HBCs were maintained for more than 3 months and had the ability to differentiate into both hepatocyte-like cells and cholangiocyte-like cells. These expandable human PSC-derived HBCs would be manageable tools for drug screening, experimental platforms to elucidate mechanisms of hepatoblasts, and cell sources for hepatic regenerative therapy.

Runx1/Cbfβ2 Complexes Are Required for Lymphoid Tissue Inducer Cell Differentiation at Two Developmental Stages

Hematopoietic lymphoid tissue inducer (LTi) cells are essential for the development of secondary lymphoid tissues including lymph nodes and Peyer’s patches. Two transcription factors, the helix-loop-helix inhibitor Id2 and the retinoic acid-related orphan receptor γt (Rorγt), have been shown to be crucial for LTi cell development. However, it remains unclear how the specification of multipotent hematopoietic progenitor cells toward the LTi lineage is programmed. In this study, we report impaired lymphoid tissue organogenesis in mice in which the function of Runx1/Cbfβ transcription factor complexes was attenuated by the loss of either the distal promoter-derived Runx1 or Cbfβ2 variant protein. We found that LTi progenitors in fetal liver, defined previously as a lineage marker-negative α4β7 integrin (α4β7)+ IL-7R α-chain (IL-7Rα)+ population, can be subdivided into Rorγt-expressing IL-7Rαhigh cells and nonexpressing IL-7Rαmid cells. Whereas Id2 and Rorγt are required to direct α4β7+IL-7Rαmid cells to become α4β7+IL-7Rαhigh cells, Runx1/Cbfβ2 complexes are necessary for the emergence of α4β7+IL-7Rαmid cells. In addition, the loss of Cbfβ2, but not P1-Runx1, resulted in an inefficient upregulation of Rorγt in residual α4β7+IL-7Rα+ LTi cells at anlagen. Our results thus revealed that Runx1/Cbfβ2 complexes regulate the differentiation of LTi cells at two stages: an early specification of hematopoietic progenitors toward the LTi lineage and a subsequent activation of Rorγt expression at anlagen.

Inhibition of peroxisome proliferator-activated receptor gamma activity in esophageal carcinoma cells results in a drastic decrease of invasive properties.

Esophageal cancer is difficult to treat because of its rapid progression, and more effective therapeutic approaches are needed. The PPARγ is a nuclear receptor superfamily member that is expressed in many cancers. PPARγ expression is a feature of esophageal cancer cell lines, and in the present investigation, the PPARγ antagonists T0070907 and GW9662 could induce loss of invasion but could not induce growth reduction or apoptosis at low concentrations (<10 mM). A high concentration of antagonists (50 µM) inhibited cell growth and induced apoptosis, but these effects did not explain our result at the low concentration. Morphological change, decreased expression of the cell signaling pathway and inhibition of cancer cell invasion were observed in the low concentration. This suggested that PPARγ antagonists inhibited esophageal cancer cell invasion as well as cell adherence, most likely due to alteration in the FAK–MAPK pathway, and this was independent of apoptosis. These results suggested that PPARγ plays an important role in cancer cell invasion and that it might be a novel target for therapy of esophageal cancer. (Cancer Sci 2006; 97: 854–860)