Makoto Motono

Prolonged maturation culture favors a reduction in the tumorigenicity and the dopaminergic function of human ESC-derived neural cells in a primate model of Parkinson's disease

For the safe clinical application of embryonic stem cells (ESCs) for neurological diseases, it is critical to evaluate the tumorigenicity and function of human ESC (hESC)‐derived neural cells in primates. We have herein, for the first time, compared the growth and function of hESC‐derived cells with different stages of neural differentiation implanted in the brains of primate models of Parkinsons disease. We herein show that residual undifferentiated cells expressing ESC markers present in the cell preparation can induce tumor formation in the monkey brain. In contrast, a cell preparation matured by 42‐day culture with brain‐derived neurotrophic factor/glial cell line‐derived neurotrophic factor (BDNF/GDNF) treatment did not form tumors and survived as primarily dopaminergic (DA) neurons. In addition, the monkeys with such grafts showed behavioral improvement for at least 12 months. These results support the idea that hESCs, if appropriately matured, can serve as a source for DA neurons without forming any tumors in a primate brain. STEM CELLS 2012;30:935–945

Production of human erythropoietin by chimeric chickens

The use of transgenic avian allows cost effective and safe production of pharmaceutical proteins. Here, we report the successful production of chimeric chickens expressing human erythropoietin (hEpo) using a high-titer retroviral vector. The hEpo expressed by transgenic hens accumulated abundantly in egg white and had N- and O-linked carbohydrates. While attachment of terminal sialic acid and galactose was incomplete, portions of N- and O-linked carbohydrates were present. In vitro biological activity of egg white-hEpo was comparable to that produced by recombinant CHO cells.

Production of transgenic chickens from purified primordial germ cells infected with a lentiviral vector

Replication-defective retroviral or lentiviral vectors have been used for the production of transgenic animals. Chicken primordial germ cells (PGCs) are the precursors for ova and spermatozoa. Here, we describe the production of transgenic chickens via a germline transmission system using PGCs infected with a replication-defective lentiviral vector. PGCs were sorted with a fluorescence-activated cell sorter based on the expression of stage-specific embryonic antigen-1 from 2.5- and 5.5-day embryos. PGCs from both stages of embryo were infected with a lentiviral vector at a similar efficiency in vitro. PGCs were then transferred into the bloodstream of 2.5-day recipient embryos. The efficiency with which the PGCs were delivered and settled in the gonads was lower for PGCs from 5.5-day embryos than those from 2.5-day embryos when a limited number of PGCs was transferred, while the difference was not obvious upon the transfer of increased number of cells. Using a high number of 5.5-day PGCs infected with a lentiviral vector, transgenic chimeras (G(0)) with an acceptable efficiency for germline transmission were obtained. G(0) female chickens produced transgenic progeny (G(1)) with higher efficiency compared to G(0) male chickens. In G(1) transgenic chickens obtained by this method, enhanced green fluorescent protein was effectively expressed under the control of the actin promoter.

Production of recombinant tumor necrosis factor receptor/Fc fusion protein by genetically manipulated chickens.

We previously reported the production of recombinant proteins using genetically manipulated chickens and quails. In this study, we constructed a retroviral vector encoding an expression cassette for a fusion protein of the extracellular domain of the human tumor necrosis factor (TNF) receptor 2 and Fc region of human IgG1 (TNFR/Fc), which is expected as an effective drug for inflammatory diseases such as rheumatoid arthritis. The concentrated viral vector was injected into developing chicken embryos. The chickens that hatched stably produced TNFR/Fc in the serum and egg yolk for six months. It appears that the fused protein is transported and accumulated into yolk from the serum, which is mediated by the Fc receptor. The protein purified from the yolk and serum inhibited the cytotoxic activity of TNF-* toward L929 cells, indicating that the protein produced by the chickens is biologically active. These results indicate the effectiveness of the recovery of Fc-fused proteins from the yolk of genetically manipulated chickens.

Genetic modification of a chicken expression system for the galactosylation of therapeutic proteins produced in egg white

As a tool for large scale production of recombinant proteins, chickens have advantages such as high productivity and low breeding costs compared to other animals. We previously reported the production of erythropoietin, the tumor necrosis factor receptor fused to an Fc fragment, and an Fc-fused single-chain Fv antibody in eggs laid by genetically manipulated chickens. In egg white, however, the incomplete addition of terminal sugars such as sialic acid and galactose was found on N-linked glycans of exogenously expressed proteins. This could be a draw back to the use of transgenic chickens since the loss of these terminal sugars may affect the functions and stability of recombinant proteins purified from chicken egg white for pharmaceutical usage. To overcome this problem, we studied galactosyltransferase (GalT) activity in the magnum where the majority of egg-white proteins are secreted. In the magnum, lower β1,4-GalT1 expression and poor galactose-transfer activity were observed. Thus, we supposed that the lack of GalT1 activity may partly cause the incomplete glycosylation of egg-white proteins, and generated genetically manipulated chickens expressing GalT1 by retrovirus-mediated gene transfer. In a Golgi fraction prepared from magnum cells of the genetically manipulated chickens, significant GalT activity was detected. The series of analyses revealed a considerable improvement in the galactosylation of native egg-white proteins as well as an exogenously expressed single-chain Fv antibody fused to an Fc fragment. We conclude that chickens with genetically modified GalT activity in the magnum could be an attractive platform for producing galactosylated therapeutics.

Chicken oviduct-specific expression of transgene by a hybrid ovalbumin enhancer and the Tet expression system.

We generated genetically manipulated chickens and quail by infecting them with a retroviral vector expressing the human growth hormone under the control of chicken ovalbumin promoter/enhancer up to -3861 bp from the transcriptional start site. The growth hormone was expressed in an oviduct-specific manner and was found in egg white, although its level was low. The DNA sequence of the integrated form of the viral vector in the packaging cells was shown to be truncated and contained only the sequence spanning -3861 to -1569 bp. This represented only the DNase I hypersensitive site (DHS) III of the 4 DHSs and lacked the proximal promoter of the ovalbumin control region. We found several TATA-like and other promoter motifs of approximately -1800 bp and considered that these promoter motifs and DHS III may cause weak but oviduct-specific expression of the growth hormone. To prove this hypothesis and apply this system to oviduct-specific expression of the transgene, the truncated regulatory sequence was fused to an artificial transactivator-promoter system. In this system, initial weak but oviduct-specific expression of the Tet activator from the promoter element in the ovalbumin control sequence triggered a self-amplifying cycle of expression. DsRed was specifically expressed in oviduct cells of genetically manipulated chickens using this system. Furthermore, deletion of a short region possibly containing the promoter elements (-2112 to -1569 bp) completely abrogated oviduct-specific expression. Taken together, these results suggest that weak expression of this putative promoter causes oviduct-specific expression of the transgene.

Organization of the capsule biosynthesis gene locus of the oral streptococcus Streptococcus anginosus

The capsular polysaccharide (CPS) of the important oral streptococcus Streptococcus anginosus, which causes endocarditis, and the genes for its synthesis have not been clarified. In this study, we investigated the gene locus required for CPS synthesis in S. anginosus. Southern hybridization using the cpsE gene of the well-characterized bacterium S. agalactiae revealed that there is a similar gene in the genome of S. anginosus. By using the colony hybridization technique and inverse PCR, we isolated the CPS synthesis (cps) genes of S. anginosus. This gene cluster consisted of genes containing typical regulatory genes, cpsA-D, and glycosyltransferase genes coding for glucose, rhamnose, N-acetylgalactosamine, and galactofuranose transferases. Furthermore, we confirmed that the cps locus is required for CPS synthesis using a mutant strain with a defective cpsE gene. The cps cluster was found to be located downstream the nrdG gene, which encodes ribonucleoside triphosphate reductase activator, as is the case in other oral streptococci such as S. gordonii and S. sanguinis. However, the location of the gene cluster was different from those of S. pneumonia and S. agalactiae.

WNT-C59, a Small-Molecule WNT Inhibitor, Efficiently Induces Anterior Cortex That Includes Cortical Motor Neurons From Human Pluripotent Stem Cells

The recapitulation of human neural development in a controlled, defined manner from pluripotent stem cells (PSCs) has considerable potential for studies of human neural development, circuit formation and function, and the construction of in vitro models of neurological diseases. The inhibition of Wnt signaling, often by the recombinant protein DKK1, is important for the induction of cortical neurons. Here, we report a novel differentiation method using a small‐molecule WNT inhibitor, WNT‐C59 (C59), to efficiently induce human anterior cortex. We compared two types of small molecules, C59 and XAV939 (XAV), as substitutes for DKK1 to induce cortical neurons from PSCs in serum‐free embryoid body‐like aggregate culture. DKK1 and XAV inhibited only the canonical pathway of Wnt signaling, whereas C59 inhibited both the canonical and noncanonical pathways. C59 efficiently induced CTIP2+/COUP‐TF1− cells, which are characteristic of the cells found in the anterior cortex. In addition, when grafted into the cortex of adult mice, the C59‐induced cells showed abundant axonal fiber extension toward the spinal cord. These results raise the possibility of C59 contributing to cell replacement therapy for motor neuron diseases or insults.

Sterilization of Chicken Primordial Germ Cells

It is important to introduce exogenous genes into germ cells for making transgenic bird efficiently. Primordial germ cells (PGCs) are the progenitor cells for the gametes. If PGCs that have been transfected in vitro are efficiently migrated into the gonads of recipient birds, transgenic progeny can be easily obtained. This is enhanced by depletion of recipient PGCs prior to the transfer of transfected PGCs. By now several methods have been reported to deplete endogenous PGCs, though efficiency of each methods is not directly compared. Avian PGCs are located in the central region of area pellucida in the blastodermal stage of embryo and sensitive to irradiation of X ray or ultraviolet (UV). We have compared methods using UV, γ irradiation, and removal of cells from the center of area pellucida to reduce the number of PGCs in chicken embryos. By careful depletion of the cells of area pellucida, we have obtained a high depletion efficiency of PGCs with relatively low damage to embryos.