Koichi Saeki

Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome.

Induced pluripotent stem cells (iPSC) have been generated from somatic cells by introducing reprogramming factors. Integration of foreign genes into the host genome is a technical hurdle for the clinical application. Here, we show that Sendai virus (SeV), an RNA virus and carries no risk of altering host genome, is an efficient solution for generating safe iPSC. Sendai-viral human iPSC expressed pluripotency genes, showed demethylation characteristic of reprogrammed cells. SeV-derived transgenes were decreased during cell division. Moreover, viruses were able to be easily removed by antibody-mediated negative selection utilizing cell surface marker HN that is expressed on SeV-infected cells. Viral-free iPSC differentiated to mature cells of the three embryonic germ layers in vivo and in vitro including beating cardiomyocytes, neurons, bone and pancreatic cells. Our data demonstrated that highly-efficient, non-integrating SeV-based vector system provides a critical solution for reprogramming somatic cells and will accelerate the clinical application.

Oxidation-triggered c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein (MAP) kinase pathways for apoptosis in human leukaemic cells stimulated by epigallocatechin-3-gallate (EGCG): a distinct pathway from those of chemically induced and receptor-mediated apoptosis

We investigated intracellular signalling pathways for apoptosis induced by epigallocatechin-3-gallate (EGCG) as compared with those induced by a toxic chemical substance (etoposide, VP16) or the death receptor ligand [tumour necrosis factor (TNF)]. EGCG as well as VP16 and TNF induced activation of two apoptosis-regulating mitogen-activated protein (MAP) kinases, namely c-Jun N-terminal kinase (JNK) and p38 MAP kinase, in both human leukaemic U937 and OCI-AML1a cells. In U937 cells, the apoptosis and activation of caspases-3 and -9 induced by EGCG but not VP16 and TNF were inhibited with SB203580, a specific inhibitor of p38, while those induced by EGCG and VP16 but not TNF were inhibited with SB202190, a rather broad inhibitor of JNK and p38. In contrast, the EGCG-induced apoptosis in OCI-AML1a cells was resistant to SB203580 but not to SB202190. Unlike TNF, EGCG did not induce the activation of nuclear factor-kappaB but rather induced the primary activation of caspase-9. N -Acetyl-L-cysteine (NAC) almost completely abolished apoptosis induced by EGCG under conditions in which the apoptosis induced by VP16 or TNF was not affected. The JNK/p38 activation by EGCG was also potently inhibited by NAC, whereas those by VP16 and TNF were either not or only minimally affected by NAC. In addition, dithiothreitol also suppressed both apoptosis and JNK/p38 activation by EGCG, and EGCG-induced activation of MAP kinase kinase (MKK) 3/6, MKK4 and apoptosis-regulating kinase 1 (ASK1) was suppressed by NAC. Dominant negative ASK1, MKK6, MKK4 and JNK1 potently inhibited EGCG-induced cell death. EGCG induced an intracellular increase in reactive oxygen species and GSSG, both of which were also inhibited by NAC, and the decreased synthesis of glutathione rendered the cell susceptible to EGCG-induced apoptosis. Taken together these results strongly suggest that EGCG executed apoptotic cell death via an ASK1, MKK and JNK/p38 cascade which is triggered by NAC-sensitive intracellular oxidative events in a manner distinct from chemically induced or receptor-mediated apoptosis.

A Feeder‐Free and Efficient Production of Functional Neutrophils from Human Embryonic Stem Cells

A novel, feeder‐free hematopoietic differentiation protocol was established for highly efficient production of neutrophils from human embryonic stem cells (hESCs). For the induction of differentiation, spheres were generated in the presence of serum and cytokine cocktail and subjected to attachment culture on gelatin‐coated plates. After approximately 2 weeks, a sac‐like structure filled with abundant round cells emerged at the center of flattened spheres. After cutting off this sac‐like structure, round cells actively proliferated, either floating in the supernatant or associated weakly with the adherent cells. Almost all of these round cells were CD45‐positive hematopoietic cells with myeloid phagocytic markers (CD33 and CD11b), and approximately 30%–50% of the round cells were mature neutrophils, as judged from morphology, cytochemical characteristics (myeloperoxidase and neutrophil alkaline phosphatase), and neutrophil‐specific cell surface markers (CD66b, CD16b, and GPI‐80). In addition, hESC‐derived neutrophils had chemotactic capacity in response to the bacterial chemotactic peptide formyl‐methionyl‐leucyl‐phenylalanine and neutrophil‐specific chemokine interleukin (IL)‐8. Using “semipurified” neutrophils migrated to IL‐8, both phagocytic and respiratory burst activities were demonstrated. Finally, it was shown that hESC‐derived neutrophils had chemotactic activity in vivo in a murine air‐pouch inflammatory model. The present results indicate successful induction of functional mature neutrophils from hESCs via highly efficient feeder‐free differentiation culture system of human hematopoietic cells. STEM CELLS 2009;27:59–67

Feeder-Free and Serum-Free Production of Hepatocytes, Cholangiocytes, and Their Proliferating Progenitors from Human Pluripotent Stem Cells: Application to Liver-Specific Functional and Cytotoxic Assays

We have established a serum- and feeder-free culture system for the efficient differentiation of multifunctional hepatocytes from human embryonic stem (ES) cells and three entirely different induced pluripotent stem (iPS) cells (including vector/transgene-free iPS cells generated using Sendai virus vector) without cell sorting and gene manipulation. The differentiation-inducing protocol consisted of a first stage; endoderm induction, second stage; hepatic initiation, and third stage; hepatic maturation. At the end of differentiation culture, hepatocytes induced from human pluripotent stem cells expressed hepatocyte-specific proteins, such as α-fetoprotein, albumin, α1 antitrypsin and cytochrome P450 (CYP3A4), at similar or higher levels compared with three control human hepatocyte or hepatic cell lines. These human iPS/ES cell-derived hepatocytes also showed mature hepatocyte functions: indocyanine green dye uptake (≈ 30%), storage of glycogen (>80%) and metabolic activity of CYP3A4. Furthermore, they produced a highly sensitive hepatotoxicity assay system for D-galactosamine as determined by the extracellular release of hepatocyte-specific enzymes. Hepatoprotective prostaglandin E1 attenuated this toxicity. Interestingly, bile duct-specific enzymes were also detected after drug treatment, suggesting the presence of bile-duct epithelial cells (cholangiocytes) in our culture system. Electron microscopic studies confirmed the existence of cholangiocytes, and an immunostaining study proved the presence of bipotential hepatoblasts with high potential for proliferation. Differentiated cells were transferrable onto new dishes, on which small-sized proliferating cells with hepatocyte markers emerged and expanded. Thus, our differentiation culture system provides mature functional hepatocytes, cholangiocytes, and their progenitors with proliferative potential from a wide variety of human pluripotent stem cells.

Identification of Human Neutrophils during Experimentally Induced Inflammation in Mice with Transplanted CD34+ Cells from Human Umbilical Cord Blood

Nonobese diabetic/severe combined immunodeficiency/γ chainnull (NOG) mice are excellent recipients for xenotransplantation and have been especially valuable for the evaluation of human hematopoietic stem cell (HSC) activities. Because human hematopoietic cells that developed in this mouse were mainly lymphoid cells and not myeloid cells, mature human myeloid cells such as neutrophils were hardly detectable in peripheral blood. We demonstrated that human neutrophils accumulated by means of a zymosan-induced air pouch inflammation technique could be identified with a fluorescence-activated cell sorter in NOG mice with transplanted CD34+ cells from human umbilical cord blood, which were putative hematopoietic progenitor cells including HSC. Our results indicate that human neutrophils with a chemotactic capacity can develop from human hematopoietic progenitor cells in vivo, suggesting that our system may be a useful tool for the evaluation of human HSC activities.

Distinct involvement of cAMP-response element-dependent transcriptions in functional and morphological maturation during retinoid-mediated human myeloid differentiation

We evaluated the involvement of cyclic adenosine monophosphate‐response element (CRE)‐dependent transcriptions in all‐trans retinoic acid (ATRA)‐induced myeloid differentiation using human monoblastic U937 cells. ATRA treatment caused an increment in the CRE‐dependent transcription activity and induced a wide variety of differentiation phenotypes including functional and morphological maturation. Indeed, ATRA treatment induced the expression of CCAAT/enhancer‐binding protein β (C/EBPβ), a CRE‐dependent transcription factor important in monocytic differentiation, and the inhibition of CRE‐enhancer activity by the expression of a dominant‐negative CRE‐binding protein (dn‐CREB) abolished the induction of C/EBPβ. Functional maturation, such as the enhancement of cell adhesion and respiratory burst activity, was dramatically suppressed by the expression of dn‐CREB. In addition, the differentiation‐dependent induction of an adhesion molecule (CD11b), the phagocyte oxidase required for respiratory burst, and the transcription factor PU.1 responsible for phagocyte oxidase induction were all abolished by dn‐CREB. Surprisingly, morphological maturation, including nuclear convolution and ctoplasmic vacuolar formation, was augmented by dn‐CREB. Under the same conditions, the differentiation‐associated cell‐growth arrest was not affected by the expression of dn‐CREB. Our results clearly indicate that CRE‐driven transcription plays at least three distinct roles during myeloid differentiation: It stimulates functional maturation but suppresses morphological maturation and has no effects on cell‐growth arrest.

A feeder-free hematopoietic differentiation system with generation of functional neutrophils from feeder- and cytokine-free primate embryonic stem cells.

We have established a novel feeder- and recombinant cytokine-free culture system for the maintenance of primate embryonic stem (ES) cells along with a feeder-free hematopoietic differentiation protocol for high efficiency CD45-positive cell production. In our system, cynomolgus monkey ES cells were properly maintained in an undifferentiated state with high immature marker expressions and teratoma-producing activities. Embryoid bodies (EBs) were generated in the presence of serum and cytokine cocktail and subjected to attachment culture on gelatin-coated plates. After about 2 weeks, a sac-like structure filled with abundant round cells emerged at the center of flattened EB. Then total cells were collected and transferred onto new gelatin-coated plates, where cells were firmly attached and actively proliferated to confluence. After another few days culture, abundant floating cells were detected in the culture supernatant. These cells expressed high levels of CD45 (>90%), while adherent cells expressed low levels of CD45 (<10%). The former consisted of various differentiated stages of myeloid cells from immature myeloblasts to mature polymorphonuclear neutrophils and macrophages. Although the percentages of neutrophils varied between 10 to 20 depending on experiments, their mature phenotype was reproducibly confirmed by specific staining and functional assays. Our protocol provides the minimum essence for primate ES cell maintenance and hematopoietic differentiation that is beneficial from economical and clinical points of view.

In Vitro and In Silico Studies of the Molecular Interactions of Epigallocatechin-3-O-gallate (EGCG) with Proteins That Explain the Health Benefits of Green Tea

Green tea has been shown to have beneficial effects on many diseases such as cancer, obesity, inflammatory diseases, and neurodegenerative disorders. The major green tea component, epigallocatechin-3-O-gallate (EGCG), has been demonstrated to contribute to these effects through its anti-oxidative and pro-oxidative properties. Furthermore, several lines of evidence have indicated that the binding affinity of EGCG to specific proteins may explain its mechanism of action. This review article aims to reveal how EGCG-protein interactions can explain the mechanism by which green tea/EGCG can exhibit health beneficial effects. We conducted a literature search, using mainly the PubMed database. The results showed that several methods such as dot assays, affinity gel chromatography, surface plasmon resonance, computational docking analyses, and X-ray crystallography have been used for this purpose. These studies have provided evidence to show how EGCG can fit or occupy the position in or near functional sites and induce a conformational change, including a quaternary conformational change in some cases. Active site blocking, steric hindrance by binding of EGCG near an active site or induced conformational change appeared to cause inhibition of enzymatic activity and other biological activities of proteins, which are related to EGCG’s biological oligomer and formation of their toxic aggregates, leading to the prevention of neurodegenerative diseases and amyloidosis. In conclusion, these studies have provided useful information on the action of green tea/catechins and would lead to future studies that will provide further evidence for rational EGCG therapy and use EGCG as a lead compound for drug design.