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Induced pluripotent stem (iPS) cells established by introduction of the transgenes POU5F1 (also known as Oct3/4), SOX2, KLF4 and c-MYC have competence similar to embryonic stem (ES) cells. iPS cells generated from cynomolgus monkey somatic cells by using genes taken from the same species would be a particularly important resource, since various biomedical investigations, including studies on the safety and efficacy of drugs, medical technology development, and research resource development, have been performed using cynomolgus monkeys. In addition, the use of xenogeneic genes would cause complicating matters such as immune responses when they are expressed. In this study, therefore, we established iPS cells by infecting cells from the fetal liver and newborn skin with amphotropic retroviral vectors containing cDNAs for the cynomolgus monkey genes of POU5F1, SOX2, KLF4 and c-MYC. Flat colonies consisting of cells with large nuclei, similar to those in other primate ES cell lines, appeared and were stably maintained. These cell lines had normal chromosome numbers, expressed pluripotency markers and formed teratomas. We thus generated cynomolgus monkey iPS cell lines without the introduction of ecotropic retroviral receptors or other additional transgenes by using the four allogeneic transgenes. This may enable detailed analysis of the mechanisms underlying the reprogramming. In conclusion, we showed that iPS cells could be derived from cynomolgus monkey somatic cells. To the best of our knowledge, this is the first report on iPS cell lines established from cynomolgus monkey somatic cells by using genes from the same species.

The successful engraftment of genetically modified hematopoietic stem cells (HSCs) without toxic conditioning is a desired goal for HSC gene therapy. To this end, we have examined the combination of intrabone marrow transplantation (iBMT) and in vivo expansion by a selective amplifier gene (SAG) in a nonhuman primate model. The SAG is a chimeric gene consisting of the erythropoietin (EPO) receptor gene (as a molecular switch) and c-Mpl gene (as a signal generator). Cynomolgus CD34+ cells were retrovirally transduced with or without SAG and returned into the femur and humerus following irrigation with saline without prior conditioning. After iBMT without SAG, 2–30% of colony-forming cells were gene marked over 1 year. The marking levels in the peripheral blood, however, remained low (

We evaluated 9.4-T magnetic resonance (MR) microimaging in assessing normal and defective bone development in mouse embryos. For this purpose, we performed 9.4-T MR microimaging on developing bones in normal embryos, and also in Runx2/Cbfa1-/- embryos with severely defective bone development. MR images were compared with the histological and histochemical features of these fetal bones. MR microimaging delineate successfully the normal long bone development in embryos. The T1- and T2-weighted MR microimaging demonstrated chondrocyte maturation in different regions of growing cartilage, such as epiphysis, physis, hypertrophic cartilage, and zone of provisional calcification. These developmental changes were detectable in as early as E14.5 embryos. The MR microimaging clearly demonstrated defective bone development in Runx2/Cbfa1-/- embryos. The femur from E18.5 homozygous Runx2/Cbfa1-/- embryos lacked MR signal intensity patterns including the hypertrophic cartilage, which are characteristic of the bone from the age-matched Runx2/Cbfa1+/+ embryos. Interestingly, however, the tibia from the same mutants was associated with MR signal patterns indicative of hypertrophic cartilage but not of the primary spongiosa and ossifying perichondrium, suggesting that bone development is differently regulated in these two long bones. On the other hand, the bones from heterozygous Runx2/Cbfa1+/- embryos exhibited an MR phenotype intermediate between the Runx2/Cbfa1+/+ and Runx2/Cbfa1-/- embryos; the primary spongiosa and ossifying perichondrium formation occurred normally even in the absence of preceding organized maturation of chondrocytes, a phenotype that was not detected by histological examinations. We concluded that MR microimaging is useful in assessing the bone development.

Fas ligand (FasL) cDNAs were cloned and sequenced from cynomolgus, rhesus, and pig-tailed monkeys. The 840-bp cDNAs were identical among these three species of monkeys except for one nucleotide. The deduced 280 amino acids were completely identical and displayed 97% homology with human FasL (hFasL). Recombinant soluble FasL obtained from COS cells transfected with cynomolgus monkey FasL (cm-FasL) cDNA induced apoptosis in cells displaying human or cynomolgus monkey Fas-expressing cells. Several anti-human FasL monoclonal antibodies (mAbs) were able to neutralize the cytotoxic activity of monkey FasL, and a combination of mAbs was selected to obtain the most sensitive detection of monkey soluble FasL (sFasL) under sandwich enzyme-linked immunosorbent assay (ELISA). Plasma from normal monkey did not contain detectable levels of sFasL, whereas plasma from monkeys acutely infected with simian immunodeficiency virus (SIV) displayed increased levels of sFasL.

We previously developed “selective amplifier genes (SAGs)” which confer a growth advantage to transduced cells. The SAG is a chimeric gene encoding the G-CSF receptor (GCR) and the estrogen or tamoxifen (Tm) receptor and is able to expand transduced hematopoietic cells by treatment with estrogen or Tm. In the current study, we examined the in vitro efficacy of modified SAGs containing the thrombopoietin (TPO) receptor (c-Mpl) gene instead of GCR as a more potent signal generator. In addition, we constructed various mutant Mpl-type SAGs to abolish the responsiveness to endogenous TPO while retaining Tm-dependency. When Ba/F3 cells were retrovirally transduced with the Mpl-type SAGs, the cells showed Tm- and TPO-dependent growth even without IL-3. The Mpl-type SAGs induced more potent proliferation of Ba/F3 and cynomolgus CD34+ cells than the GCR-type SAG. One mutant Mpl-type SAG (ΔGCRMplTmR) successfully lost the responsiveness to TPO without affecting the Tm-dependence.

We have previously reported that CD4+CD8+ double-positive (DP) T cells with a resting memory phenotype exist in a substantial proportion of peripheral blood lymphocytes of adult cynomolgus macaques. In this study, we examined the effects of simian immunodeficiency virus of macaque (SIVmac) infection on DP T cells. In vitro, SIVmac239 nef-open (239) and its nef-deletion mutant replicated well in both CD4+CD8- and DP T cells. However, when the macaques were infected with 239, DP, but not CD4+CD8-, T cells were transiently increased in parallel with cell activation and viral replication, followed by depletion within 1 month postinfection. Interestingly, the nef gene was required for depletion but not for the increase and activation of DP T cells. These data suggest that the pathogenic SIV infection may downmodulate production and/or blood circulation of DP T cells by a Nef function-related mechanism(s) different from that for the depletion of CD4+CD8- T cells.