Norio Nakatsuji

Random integration is one of the more straightforward methods to introduce a transgene into human embryonic stem (ES) cells. However, random integration may result in transgene silencing and altered cell phenotype due to insertional mutagenesis in undefined gene regions. Moreover, reliability of data may be compromised by differences in transgene integration sites when comparing multiple transgenic cell lines. To address these issues, we developed a genetic manipulation strategy based on homologous recombination and Cre recombinase-mediated site-specific integration. First, we performed gene targeting of the hypoxanthine phosphoribosyltransferase 1 (HPRT) locus of the human ES cell line KhES-1. Next, a gene-replacement system was created so that a circular vector specifically integrates into the targeted HPRT locus via Cre recombinase activity. We demonstrate the application of this strategy through the creation of a tetracycline-inducible reporter system at the HPRT locus. We show that reporter gene expression was responsive to doxycycline and that the resulting transgenic human ES cells retain their self-renewal capacity and pluripotency.

Human embryonic stem (hES) cell lines are of great potential in cell transplantation therapy. However, recipients of such allogeneic transplants probably need treatment with immunosuppressants. Recently, Taylor et al. [Lancet 2005;366:2019–2025] proposed banking of hES cell lines and estimated the required number of hES cell lines for beneficial human leukocyte antigen (HLA) matching in the U.K. population. Here, we carried out such an estimation in the Japanese population. We calculated the proportion of patients who can find at least one HLA-matched donor at three loci of HLA-A, -B, and -DR. With a bank of hES cell lines from 170 randomly selected donated embryos, 80% of patients were expected to find at least one hES cell line with a single mismatch at one HLA locus or a better match. Furthermore, 80% of patients are expected to find at least one donor with complete matching at the three HLA loci if parthenogenetic homozygous hES cell lines are established from 55 randomly selected donated oocytes. The relatively low ethnic diversity of the Japanese population may have resulted in a high success rate in beneficial matching. Moreover, parthenogenetic hES cell lines can greatly reduce the number required for a higher degree of HLA matching. Disclosure of potential conflicts of interest is found at the end of this article.

We have established several embryonic stem (ES) cell lines of the cynomolgus monkey. They maintain the normal karyotype and pluripotency in culture for long periods. We obtained government approval and grants to produce human ES cell lines from frozen surplus embryos in April 2002. We have established and characterized three human ES cell lines (KhES-1, KhES-2, KhES-3). We started distribution of human ES cells to other research groups in March 2004. It would be important to produce genetically modified monkey and human ES cells for various purposes. After improvement of the transfection and selection methods, we have produced monkey ES cells with integrated transgenes at efficient and reliable rates. We are also investigating reprogramming of somatic cells into pluripotent stem cells by cell fusion with ES cells. Such reprogramming could be used to produce pluripotent stem cells for each patient without therapeutic cloning, which would raise ethical concerns.

We previously identified a stromal cell-derived inducing activity (SDIA), which induces differentiation of neural cells, including midbrain tyrosine hydroxylase-positive (TH ⁺ ) dopaminergic neurons, from mouse embryonic stem cells. We report here that SDIA induces efficient neural differentiation also in primate embryonic stem cells. Induced neurons contain TH ⁺ neurons at a frequency of 35% and produce a significant amount of dopamine. Interestingly, differentiation of TH ⁺ neurons from undifferentiated embryonic cells occurs much faster in vitro (10 days) than it does in the embryo (≈5 weeks). In addition, 8% of the colonies contain large patches of Pax6 ⁺ -pigmented epithelium of the retina. The SDIA method provides an unlimited source of primate cells for the study of pathogenesis, drug development, and transplantation in degenerative diseases such as Parkinson's disease and retinitis pigmentosa.

Human embryonic stem (ES) cell lines have opened great potential and expectation for cell therapy and regenerative medicine. Monkey and human ES cell lines, which are very similar to each other, have been established from monkey blastocysts and surplus human blastocysts from fertility clinics.Nonhuman primate ES cell lines provide important research tools for basic and applicative research. Firstly, they provide wider aspects of investigation of the regulative mechanisms of stem cells and cell differentiation among primate species. Secondly, their usage does not need clearance or permission from the regulative rules in many countries that are associated with the ethical aspects of human ES cells, although human and nonhuman embryos and fetuses are very similar to each other. Lastly and most importantly, they are indispensable for animal models of cell therapy to test effectiveness, safety, and immunological reaction of the allogenic transplantation in a setting similar to the treatment of human diseases.So far, ES cell lines have been established from rhesus monkey (Macaca mulatta), common marmoset (Callithrix jacchus), and cynomolgus monkey (Macaca fascicularis), using blastocysts produced naturally or by in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). These cell lines seem to have very similar characteristics. They express alkaline phosphatase activity and stage-specific embryonic antigen (SSEA)-4 and, in most cases, SSEA-3. Their pluripotency was confirmed by the formation of embryoid bodies and differentiation into various cell types in culture and also by the formation of teratomas that contained many types of differentiated tissues including derivatives of three germ layers after transplantation into the severe combined immunodeficiency (SCID) mice.The noneffectiveness of the leukemia inhibitory factor (LIF) signal makes culture of primate and human ES cell lines prone to undergo spontaneous differentiation and thus it is difficult to maintain these stem cell colonies. Also, these ES cells are more susceptible to various stresses, causing difficulty with subculturing using enzymatic treatment and cloning from single cells. However, with various improvements in culture methods, it is now possible to maintain stable colonies of monkey ES cells using a serum-free medium and subculturing with trypsin treatment. Under such conditions, cynomolgus monkey ES cell lines can be maintained in an undifferentiated state with a normal karyotype and pluripotency even after prolonged periods of culture over 1 year. Such progress should facilitate many aspects of stem cell research using both nonhuman primate and human ES cell lines.

Human embryonic stem (ES) cells are predicted to be a valuable source for producing ES‐derived therapeutic spare tissues to treat diseases by controlling their growth and differentiation. To understand the regulative mechanisms of their differentiation in vivo and in vitro, ES cells derived from nonhuman primates could be a powerful tool. We established four ES cell lines from cynomolgus monkey (Macaca fascicularis) blastocysts produced by in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). The ES cells were characterized by the expression of specific markers such as alkaline phosphatase and stage‐specific embryonic antigen‐4. They were successfully maintained in an undifferentiated state and with a normal karyotype even after more than 6 months of culture. Pluripotential competence was confirmed by the formation of teratomas containing ectoderm‐, mesoderm‐, and endoderm‐ derivatives after subcutaneous injection into SCID mice. Differentiation to a variety of tissues was identified by immunohistochemical analyses using tissue‐specific antibodies. Therefore, we established pluripotent ES cell lines derived from monkeys that are widely used as experimental animals. These lines could be a useful resource for preclinical stem cell research, including allogenic transplantation into monkey models of disease.

Mouse primordial germ cells (PGCs) migrate from the base of the allantois to the genital ridge. They proliferate both during migration and after their arrival, until initiation of the sex-differentiation of fetal gonads. Then, PGCs enter into the prophase of the first meiotic division in the ovary to become oocytes, while those in the testis become mitotically arrested to become prospermatogonia. Growth regulation of mouse PGCs has been studied by culturing them on feeder cells. They show a limited period of proliferation in vitro and go into growth arrest, which is in good correlation with their developmental changes in vivo. However, in the presence of multiple growth signals, PGCs can restart rapid proliferation and transform into pluripotent embryonic germ (EG) cells. Observation of ectopic germ cells and studies of reaggregate cultures suggested that both male and female PGCs show cell-autonomous entry into meiosis and differentiation into oocytes if they were set apart from the male gonadal environments. Recently, we developed a two-dimensional dispersed culture system in which we can examine transition from the mitotic PGCs into the leptotene stage of the first meiotic division. Such entry into meiosis seems to be programmed in PGCs before reaching the genital ridges and unless it is inhibited by putative signals from the testicular somatic cells.

We aimed to introduce foreign DNA into spermatogenic cells in the testis by injection of the DNA encoding jellyfish fluorescent proteins, green fluorescent protein (GFP) and yellow fluorescent protein (YFP) into the seminiferous tubules and in vivo electroporation. We obtained fluorescent spermatozoa only when using the gene of the YFP protein fused to a mitochondrial localization signal peptide. Intracytoplasmic injection into oocytes of these spermatozoa gave fluorescent fetuses and pups. Almost all of the individuals produced from fluorescent spermatozoa were transgenic. We confirmed integration of the gene into chromosomes and its transmission into offspring. This is the first report of gene transfer into germ cells and subsequent production of transgenic offspring.