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We report that a sheep fetal liver provides a microenvironment for generating hematopoietic cells with long-term engrafting capacity and multilineage differentiation potential from human induced pluripotent stem cell (iPSC)–derived hemogenic endothelial cells (HEs). Despite the promise of iPSCs for making any cell types, generating hematopoietic stem and progenitor cells (HSPCs) is still a challenge. We hypothesized that the hematopoietic microenvironment, which exists in fetal liver but is lacking in vitro, turns iPSC-HEs into HSPCs. To test this, we transplanted CD45-negative iPSC-HEs into fetal sheep liver, in which HSPCs first grow. Within 2 months, the transplanted cells became CD45 positive and differentiated into multilineage blood cells in the fetal liver. Then, CD45-positive cells translocated to the bone marrow and were maintained there for 3 years with the capability of multilineage differentiation, indicating that hematopoietic cells with long-term engraftment potential were generated. Moreover, human hematopoietic cells were temporally enriched by xenogeneic donor-lymphocyte infusion into the sheep. This study could serve as a foundation to generate HSPCs from iPSCs.

Macaca fascicularis is a highly advantageous model in which to study human cochlea with regard to both evolutionary proximity and physiological similarity of the auditory system. To better understand the properties of primate cochlear function, we analyzed the genes predominantly expressed in M. fascicularis cochlea. We compared the cochlear transcripts obtained from an adult male M. fascicularis by macaque and human GeneChip microarrays with those in multiple macaque and human tissues or cells and identified 344 genes with expression levels more than 2-fold greater than in the other tissues. These “cochlear signature genes” included 35 genes responsible for syndromic or nonsyndromic hereditary hearing loss. Gene set enrichment analysis revealed groups of genes categorized as “ear development” and “ear morphogenesis” in the top 20 gene ontology categories in the macaque and human arrays, respectively. This dataset will facilitate both the study of genes that contribute to primate cochlear function and provide insight to discover novel genes associated with hereditary hearing loss that have yet to be established using animal models.

Classical- (C-) and atypical L-type bovine spongiform encephalopathy (BSE) prions cause different pathological phenotypes in cattle brains, and the disease-associated forms of each prion protein (PrPSc) has a dissimilar biochemical signature. Bovine C-BSE prions are the causative agent of variant Creutzfeldt-Jakob disease. To date, human infection with L-BSE prions has not been reported, but they can be transmitted experimentally from cows to cynomolgus monkeys (Macaca fascicularis), a non-human primate model. When transmitted to monkeys, C- and L-BSE prions induce different pathological phenotypes in the brain. However, when isolated from infected brains, the two prion proteins (PrPSc) have similar biochemical signatures (i.e., electrophoretic mobility, glycoforms, and resistance to proteinase K). Such similarities suggest the possibility that L-BSE prions alter their virulence to that of C-BSE prions during propagation in monkeys. To clarify this possibility, we conducted bioassays using inbred mice. C-BSE prions with or without propagation in monkeys were pathogenic to mice, and exhibited comparable incubation periods in secondary passage in mice. By contrast, L-BSE prions, either with or without propagation in monkeys, did not cause the disease in mice, indicating that the pathogenicity of L-BSE prions does not converge towards a C-BSE prion type in this primate model. These results suggest that, although C- and L-BSE prions propagated in cynomolgus monkeys exhibit similar biochemical PrPSc signatures and consist of the monkey amino acid sequence, the two prions maintain strain-specific conformations of PrPSc in which they encipher and retain unique pathogenic traits.

Prion diseases are characterized by the prominent accumulation of the misfolded form of a normal cellular protein (PrPSc) in the central nervous system. The pathological features and biochemical properties of PrPScin macaque monkeys infected with the bovine spongiform encephalopathy (BSE) prion have been found to be similar to those of human subjects with variant Creutzfeldt–Jakob disease (vCJD). Non-human primate models are thus ideally suited for performing valid diagnostic tests and determining the efficacy of potential therapeutic agents. In the current study, we developed a highly efficient method forin vitroamplification of cynomolgus macaque BSE PrPSc. This method involves amplifying PrPScby protein misfolding cyclic amplification (PMCA) using mouse brain homogenate as a PrPCsubstrate in the presence of sulfated dextran compounds. This method is capable of amplifying very small amounts of PrPSccontained in the cerebrospinal fluid (CSF) and white blood cells (WBCs), as well as in the peripheral tissues of macaques that have been intracerebrally inoculated with the BSE prion. After clinical signs of the disease appeared in three macaques, we detected PrPScin the CSF by serial PMCA, and the CSF levels of PrPSctended to increase with disease progression. In addition, PrPScwas detectable in WBCs at the clinical phases of the disease in two of the three macaques. Thus, our highly sensitive, novel method may be useful for furthering the understanding of the tissue distribution of PrPScin non-human primate models of CJD.

Nasal administration is an effective route for a needle-free vaccine. However, nasally administered Ags have the potential to reach the CNS directly from the nasal cavity, thus raising safety concerns. In this study, we performed real-time quantitative tracking of a nasal vaccine candidate for botulism, which is a nontoxic subunit fragment of Clostridium botulinum type A neurotoxin (BoHc/A) effective in the induction of the toxin-neutralizing immune response, by using 18F-labeled BoHc/A–positron-emission tomography, an in vivo molecular imaging method. This method provides results that are consistent with direct counting of [18F] radioactivity or the traditional [111In]-radiolabel method in dissected tissues of mice and nonhuman primates. We found no deposition of BoHc/A in the cerebrum or olfactory bulb after nasal administration of 18F-labeled BoHc/A in both animals. We also established a real-time quantitative profile of elimination of this nasal vaccine candidate and demonstrated that it induces highly protective immunity against botulism in nonhuman primates. Our findings demonstrate the efficiency and safety of a nasal vaccine candidate against botulism in mice and nonhuman primates using in vivo molecular imaging.

Adenovirus (Ad) serotype 35 (Ad35) vectors have attracted remarkable attention as alternatives to conventional Ad serotype 5 (Ad5) vectors. In a previous study, we showed that intravenously administered Ad35 vectors exhibited a safer profile than Ad5 vectors in cynomolgus monkeys, which ubiquitously express CD46, an Ad35 receptor, in a pattern similar to that in humans. However, the Ad35 vectors poorly transduced the organs. In this study, we examined the transduction properties of Ad35 vectors after local administration into organs of cynomolgus monkeys. The vectors transduced different types of cells depending on the organ. Hepatocytes and microglia were mainly transduced after the vectors were injected into the liver and cerebrum, respectively. Injection of the vectors into the femoral muscle resulted in the transduction of cells that appeared to be fibroblasts and/or macrophages. Conjunctival epithelial cells showed transgene expression following infusion into the vitreous body of the eyeball. Transgene expression was limited to areas around the injection points in most of the organs. In contrast, Ad35 vector-mediated transgene expression was not detected in any of the organs not injected with Ad35 vectors. These results suggest that Ad35 vectors are suitable for gene delivery by direct administration to organs.

Background. Although directed differentiation of human embryonic stem (ES) cells would enable a ready supply of cells and tissues required for transplantation therapy, the methodology is limited. We have developed a novel method for hematopoietic development from primate ES cells. We first cultured cynomolgus monkey ES cells in vitro and transplanted the cells in vivo into fetal sheep liver, generating sheep with cynomolgus hematopoiesis. Methods. Cynomolgus ES cells were induced to mesodermal cells on murine stromal OP9 cells with multiple cytokines for 6 days. The cells (average 4.8×10 7 cells) were transplanted into fetal sheep in the liver (n=4) after the first trimester (day 55-73, full term 147 days). The animals were delivered at full term, and two of them were intraperitoneally administered with human stem-cell factor (SCF). Results. Cynomolgus hematopoietic progenitor cells were detected in bone marrow at a level of 1% to 2% in all four sheep up to 17 months posttransplant. No teratoma was found in the lambs. After SCF administration, the fractions of cynomolgus hematopoiesis increased by several-fold (up to 13%). Cynomolgus cells were also detected in the circulation, albeit at low levels (

Hematopoietic stem cells in bone marrow can be mobilized into peripheral blood by cytokine administration. Cytokine-mobilized peripheral blood stem cells are of great use in clinical applications. We previously established a modified procedure for the collection of cytokine-mobilized peripheral blood cells from rhesus monkeys (Macaca mulata) using a commercially available apparatus originally developed for human subjects. In this study, we examined the efficacy and safety of this method with even smaller macaques, cynomolgus monkeys (Macaca fascicularis), which are equivalent to human newborns in body weight (mean = 3.3 kg). Using the manufacturer's unmodified protocol (n=6), one monkey died of cardiac failure and three developed severe anemia. In contrast, using our modified procedure (n=6), no such complication was observed in any animal. In addition, the harvested nuclear cell, mononuclear cell and CD34(+) cell counts were significantly higher with the modified method. The modified method should allow safe and efficient collection of cytokine-mobilized peripheral blood cells from non-human primates as small as human newborns in a non-invasive manner.

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 (

It has recently been reported that bone marrow cells can efficiently engraft without marrow conditioning when implanted directly into the bone marrow cavity (intra-bone marrow transplantation, iBMT) in mice. We have successfully examined the efficacy of autologous iBMT in a cynomolgus monkey model in conjuction with an in vivo expansion of transplanted cells by a selective amplifier transgene (Ueda et al., 2004) and provide here the detailed parameters of our iBMT method. We injected retrovirally-marked autologous CD34+ cells directly into the non-conditioned marrow cavity of the femur and humerus after gently irrigating the cavity with saline. This transplant procedure was safely performed without pulmonary embolism. Gene-marked cells were not detectable in the peripheral blood at one hour and one day after iBMT as assessed by sensitive PCR, indicating that iBMT hardly generated a systemic delivery of transplanted cells. On the other hand, 2 to 30% of clonogenic hematopoietic colonies produced from the implanted marrow were gene-marked at 6–12 months after iBMT. Our iBMT method for non-human primates is thus discussed in terms of long-lived hematopoietic stem/progenitor cells, bone marrow niche and long-term engraftment after iBMT without myeloablative conditioning.

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.

A major problem limiting hematopoietic stem cell (HSC) gene therapy is the low efficiency of gene transfer into human HSCs using retroviral vectors. Strategies, which would allow in vivo expansion of gene-modified hematopoietic cells, could circumvent the problem. To this end, we developed a selective amplifier gene (SAG) consisting of a chimeric gene composed of the granulocyte colony-stimulating factor (G-CSF) receptor gene and the estrogen receptor gene hormone-binding domain. We have previously demonstrated that primary bone marrow progenitor cells transduced with the SAG could be expanded in response to estrogen in vitro. In the present study, we evaluated the efficacy of the SAG in the setting of a clinically applicable cynomolgus monkey transplantation protocol. Cynomolgus bone marrow CD34(+) cells were transduced with retroviral vectors encoding the SAG and reinfused into each myeloablated monkey. Three of the six monkeys that received SAG transduced HSCs showed an increase in the levels of circulating progeny containing the provirus in vivo following administration of estrogen or tamoxifen without any serious adverse effects. In one monkey examined in detail, transduced hematopoietic progenitor cells were increased by several-fold (from 5% to 30%). Retroviral integration site analysis revealed that this observed increase was polyclonal and no outgrowth of a dominant single clonal population was observed. These results demonstrate that the inclusion of our SAG in the retroviral construct allows selective in vivo expansion of genetically modified cells by a non-toxic hormone treatment.

Background The green fluorescent protein (GFP) has proven a useful marker in retroviral gene transfer studies targeting hematopoietic stem cells (HSCs) in mice. However, several investigators have reported very low in vivo peripheral blood marking levels in nonhuman primates after transplantation of HSCs transduced with the GFP gene. We retrovirally marked cynomolgus monkey HSCs with the GFP gene, and tracked in vivo marking levels within both bone marrow progenitor cells and mature peripheral blood cells following autologous transplantation after myeloablative conditioning. Methods Bone marrow cells were harvested from three cynomolgus macaques and enriched for the primitive fraction by CD34 selection. CD34+ cells were transduced with one of three retroviral vectors all expressing the GFP gene and were infused after myeloablative total body irradiation (500 cGy × 2). Following transplantation, proviral levels and fluorescence were monitored among clonogenic bone marrow progenitors and mature peripheral blood cells. Results Although 13–37% of transduced cells contained the GFP provirus and 11–13% fluoresced ex vivo, both provirus and fluorescence became almost undetectable in the peripheral blood within several months after transplantation regardless of the vectors used. However, on sampling of bone marrow at multiple time points, significant fractions (5–10%) of clonogenic progenitors contained the provirus and fluoresced ex vivo reflecting a significant discrepancy between GFP gene marking levels within bone marrow cells and their mature peripheral blood progeny. The discrepancy (at least one log) persisted for more than 1 year after transplantation. Since no cytotoxic T lymphocytes against GFP were detected in the animals, an immune response against GFP is an unlikely explanation for the low levels of transduced peripheral blood cells. Administration of granulocyte colony stimulating factor and stem cell factor resulted in mobilization of transduced bone marrow cells detectable as mature granulocyte progeny which expressed the GFP gene, suggesting that transduced progenitor cells in bone marrow could be mobilized into the peripheral blood and differentiated into granulocytes. Conclusions Low levels of GFP-transduced mature cells in the peripheral blood of nonhuman primates may reflect a block to differentiation associated with GFP; this block can be overcome in part by nonphysiological cytokine treatment ex vivo and in vivo. Copyright © 2002 John Wiley & Sons, Ltd.

MazF, an endoribonuclease encoded by Escherichia coli, specifically cleaves the ACA (adenine-cytosine-adenine) sequence of single-stranded RNAs. Conditional expression of MazF under the control of the HIV-1 LTR promoter rendered CD4(+) T cells resistant to HIV-1 replication without affecting cell growth. To investigate the safety, persistence and efficacy of MazF-modified CD4(+) T cells in a nonhuman primate model in vivo, rhesus macaques were infected with a pathogenic simian/human immunodeficiency virus (SHIV) and transplanted with autologous MazF-modified CD4(+) T cells. MazF-modified CD4(+) T cells were clearly detected throughout the experimental period of more than 6 months. The CD4(+) T cell count values increased in all four rhesus macaques. Moreover, the transplantation of the MazF-modified CD4(+) T cells was not immunogenic, and did not elicit cellular or humoral immune responses. These data suggest that the autologous transplantation of MazF-modified CD4(+) T cells in the presence of SHIV is effective, safe and not immunogenic, indicating that this is an attractive strategy for HIV-1 gene therapy.

The quantitative and phenotypic changes of peripheral blood monocytes during the acute stage of simian immunodeficiency virus infection were investigated. We inoculated intravenously three cynomolgus monkeys (Macaca fascicularis) with 100 TCID50 of SIVmac239 and collected whole blood twice a week until 35 days postinoculation. We found that the relative number of monocytes in peripheral blood leukocytes significantly increased at 7-17 days postinoculation. This increase was concomitant with the peak of primary SIV antigenemia. To determine if the monocytes observed during the acute stage were phenotypically altered, they were periodically examined for the expression of surface markers (i.e., CD11b, CD14, CD16, CD29, D32, CD56, CD62L, CD64, CD80, and MHC-II-DR) by flow cytometry. The results showed that the expression levels of CD14 and CD56 on most of the monocytes were remarkably reduced at 7-17 days postinoculation, and a new subpopulation, CD14lowCD16+CD80+ monocytes, was clearly detected at 10 days postinoculation. These results indicate that the phenotypic alteration of peripheral blood monocytes occurs during the primary SIV infection.

Background MazF is an endoribonuclease encoded by Escherichia coli that specifically cleaves the ACA sequence of mRNA. In our previous report, conditional expression of MazF in the HIV-1 LTR rendered CD4+ T lymphocytes resistant to HIV-1 replication. In this study, we examined the in vivo safety and persistence of MazF-transduced cynomolgus macaque CD4+ T cells infused into autologous monkeys. Methodology/Principal Findings The in vivo persistence of the gene-modified CD4+ T cells in the peripheral blood was monitored for more than half a year using quantitative real-time PCR and flow cytometry, followed by experimental autopsy in order to examine the safety and distribution pattern of the infused cells in several organs. Although the levels of the MazF-transduced CD4+ T cells gradually decreased in the peripheral blood, they were clearly detected throughout the experimental period. Moreover, the infused cells were detected in the distal lymphoid tissues, such as several lymph nodes and the spleen. Histopathological analyses of tissues revealed that there were no lesions related to the infused gene modified cells. Antibodies against MazF were not detected. These data suggest the safety and the low immunogenicity of MazF-transduced CD4+ T cells. Finally, gene modified cells harvested from the monkey more than half a year post-infusion suppressed the replication of SHIV 89.6P. Conclusions/Significance The long-term persistence, safety and continuous HIV replication resistance of the mazF gene-modified CD4+ T cells in the non-human primate model suggests that autologous transplantation of mazF gene-modified cells is an attractive strategy for HIV gene therapy.

Objective Hematopoietic stem cells (HSCs) reside in the osteoblastic niche, which consists of osteoblasts. Mesenchymal stromal cells (MSCs) have an ability to differentiate into osteoblasts. Here, using nonhuman primates, we investigated the effects of cotransplantation with MSCs on the engraftment of HSCs after autologous intra-bone marrow transplantation. Materials and Methods From three cynomolgus monkeys, CD34-positive cells (as HSCs) and MSCs were obtained. The former were divided into two equal aliquots and each aliquot was genetically marked with a distinctive retroviral vector to track the in vivo fate. Each HSC aliquot with or without MSCs was autologously injected into the bone marrow (BM) cavity of right or left side, enabling the comparison of in vivo fates of the two HSC grafts in the same body. Results In the three monkeys, CD34 + cells transplanted with MSCs engrafted 4.4, 6.0, and 1.6 times more efficiently than CD34 + cells alone, as assessed by BM colony polymerase chain reaction. In addition, virtually all marked cells detected in the peripheral blood were derived from the cotransplantation aliquots. Notably, colony-forming units derived from the cotransplantation aliquots were frequently detected in BM distant sites from the injection site, implying that cotransplantation with MSCs also restored the ability of gene-marked HSCs to migrate and achieve homing in the distant BM. Conclusion Cotransplantation with MSCs would improve the efficacy of transplantation of gene-modified HSCs in primates, with enhanced engraftment in BM as well as increased chimerism in peripheral blood through migration and homing.

Background In vivo expansion of gene-modified cells would be a promising approach in the field of hematopoietic stem cell gene therapy. To this end, we previously developed a selective amplifier gene (SAG), a chimeric gene encoding the granulocyte colony-stimulating factor (G-CSF) receptor (GCR), as a growth-signal generator and the hormone-binding domain of the steroid receptor as a molecular switch. We have already reported that hematopoietic cells retrovirally transduced with the SAG can be expanded in a steroid-dependent manner in vitro and in vivo in mice and nonhuman primates. In this study, we have developed a new-generation SAG, in which the erythropoietin (EPO) receptor (EPOR) is utilized instead of the steroid receptor as a molecular switch. Methods Two EPO-driven SAGs were constructed, EPORGCR and EPORMpl, containing the GCR and c-Mpl as a signal generator, respectively. First, to compare the steroid-driven and EPO-driven SAGs, Ba/F3 cells were transduced with these SAGs. Next, to examine whether GCR or c-Mpl is the more suitable signal generator of the EPO-driven SAG, human cord blood CD34+ cells were transduced with the two EPO-driven SAGs (EPORMpl and EPORGCR). Finally, we examined the in vivo efficacy of EPORMpl in mice. Irradiated mice were transplanted with EPORMpl-transduced bone marrow cells followed by administration of EPO. Results The EPO-driven SAGs were shown to induce more rapid and potent proliferation of Ba/F3 cells than the steroid-driven SAGs. The EPORMpl induced more efficient EPO-dependent proliferation of the human cord blood CD34+ cells than the EPORGCR in terms of total CD34+ cell, c-Kit+ cell, and clonogenic progenitor cell (CFU-C) numbers. In the transplanted mice the transduced peripheral blood cells significantly increased in response to EPO. Conclusions The new-generation SAGs, especially EPORMpl, are able to efficiently confer an EPO-dependent growth advantage on transduced hematopoietic cells in vitro and in vivo in mice. Copyright © 2003 John Wiley & Sons, Ltd.

One of the most serious problems in applying leukapheresis to human infants is the large extracorporeal blood volume (ECV), resulting in substantial loss of platelets and red blood cells (RBCs). In this study, we developed a safe and effective modified procedure to collect peripheral blood stem cells (PBSCs) from rhesus monkeys (Macaca mulata) using a Baxter CS3000+ Blood Cell Separator (Baxter, Deerfield, IL) with several devices that reduced chamber size and shortened the standard apheresis kit to decrease ECV from 130 to 70 ml. Pump speed was controlled by monitoring hematocrit values and platelet counts during leukapheresis. This system makes it possible to perform safe and effective leukapheresis in rhesus monkeys whose body weight is similar to that of human infants. A total of 12 leukapheresis procedures were performed in nine monkeys and resulted in the collection of sufficient numbers of white blood cells (mean, 1.38 × 109 cells/kg), CD34+ cells (mean, 17.80 × 106 cells/kg), mononuclear cells (mean, 3.67 × 108 cells/kg), and colony forming units (mean, 75.02 × 106 cells/kg) in all cases. In addition, no complications, such as anemia or trombocytopenia, occurred after leukapheresis. This modified leukapheresis procedure will be useful to test new approaches in gene therapy, perform organ transplantation using nonhuman primates, and collect PBSCs from human infants in a noninvasive manner. Our nonhuman primate model provides an important framework for such future clinical studies. J. Clin. Apheresis 18:26–31, 2003. © 2003 Wiley-Liss, Inc.

We have previously reported that CD4/CD8 double-positive (DP) T cells with the resting memory phenotype are present in the periphery of healthy cynomolgus monkeys. In the present study, we performed functional studies on the T cells. The expression of CD4 and CD8 on DP, CD4 single-positive (SP) or CD8 SP T cells was stable in cultures with either mitogen or anti-CD3 antibody stimulation. In spite of lacking CD28 expression, DP T cells showed similar proliferative ability and apoptosis sensitivity to CD4 SP and CD8 SP T cells. DP T cells showed both helper and cytotoxic activities. Although the helper activity of DP T cells was lower than that of CD4 SP T cells, cytotoxic activity was comparable to that of CD8 SP T cells. Fresh DP T cells killed target cells mainly by the perforin-granzyme pathway. In addition, fresh DP T cells expressed a high level of mRNA for IFN-gamma and produced a high level of IFN-gamma when they were activated by anti-CD3 antibody ligation. On the other hand, several expanded DP T cell clones shared TCR V(beta) with expanded CD4 SP T cell clones, strongly suggesting that those two corresponding clones with DP and CD4 SP phenotypes might be derived from the same ancestor T cell. These results showed that the DP T cells are a novel T cell subset with functions overlapping with those of CD4 SP and CD8 SP T cells, and that they might play protective and regulatory roles in secondary immune response in cynomolgus monkeys.

[Background] Mesenchymal stem cells (MSC) have been shown to play critical roles in various in vivo phenomena including osteoblastic differentiation. It has been suggested that, in the bone marrow, hematopoietic stem cells (HSC) reside in osteoblastic niche, which consists of osteoblasts derived from MSC. In mice, previous studies have demonstrated that co-transplantation of MSC improves the engraftment of HSC, especially after transplantation of the cells into the bone marrow cavity directly, namely intra-bone marrow transplantation (iBMT). However, neither the efficacy nor the dynamics such as migration and homing of HSC after iBMT with MSC have been determined in large animals. Here, using non-human primates, we have investigated the effects of co-transplantation of MSC on the engraftment of HSC after autologus iBMT. [Methods] Auto-iBMT of cynomolgus monkeys was performed, using bone marrow stromal cells (as MSC) and CD34-positive cells (as HSC). The latter were divided into two equal aliquots, each of which was genetically marked with a different retroviral vector, G1Na or LNL6. Conditioning of iBMT, TBI or administration of busulfan, was followed by hemi-iBMT; that is, the bone marrow of one side (right or left) of the body was transplanted with one HSC aliquot together with MSC, whereas the other side of the identical body was transplanted with the other HSC aliquot alone. Engraftment of each HSC aliquot was evaluated by colony PCR of bone marrow, as well as by PCR of the genomic DNA obtained from peripheral blood or bone marrow of humerus, femur, and ilium. Both PCR could distinguish the dual markings derived from the two HSC aliquots. [Results] In the first monkey transplanted, we found that the engraftment derived from the co-transplantation aliquot was 4.4-times higher than that derived from the HSC alone aliquot as assessed by colony PCR (48% versus 11%) using the bone marrow samples obtained from the ilium at day 46 post-iBMT. In the second monkey, when the peripheral WBC recovered to 2500–3000/μl after day 28 post-iBMT, 2% of the cells were positive with the retroviral marking derived from the co-transplantation aliquot, although none of them were positive with that derived from the HSC alone aliquot. In addition, colony PCR of the humerus and femur of both sides at day 39 post-iBMT revealed that the engraftment derived from the co-transplantation aliquot was 6.0-times higher than that derived from the HSC alone aliquot. Notably, colony-forming units (CFU) derived from the cotransplantation aliquot were detected in the bone marrow of the opposite side, suggesting that HSC injected into the bone marrow might migrate and achieve homing in the distant bone marrow. [Conclusion] Taken together, these results indicate that, in auto-iBMT of cynomolgus monkeys, co-transplantation of MSC improves the engraftment of HSC, the efficacy of which might be attributable to additional osteoblastic niche, presumably created from co-transplanted MSC.

Sendai virus (SeV) is an enveloped virus with a negative-stranded RNA genome and is a member of the family Paramyxoviridae. SeV-based vectors replicate exclusively in the cytoplasm of transduced cells, and do not go through a DNA phase; therefore, there is no concern about the unwanted integration of foreign sequences into the host chromosomal DNA, which indeed caused a life-threatening disease in a few limited ceases. Human ES cells are expected to have clinical applications as well as to serve as a basic research tool. Foreign genes are often transferred into ES cells for genetic marking or regulating differentiation. We have previously shown that SeV vector can introduce a foreign gene efficiently and stably into cynomolgus monkey ES cells while preserving the pluripotency. The transgene expression, however, should be suppressed in cases of unexpected toxicity or when only a temporal transgene expression is necessary. Since SeV is an RNA virus, we have examined whether anti-RNA virus agents are able to remove the SeV vector from transduced ES cells. The F-defective SeV vector carrying the GFP gene was constructed. Cynomolgus ES cells were transduced with the vector at 10 MOI per cell (transduction efficiency 60%). Fluorescent ES-cell colonies were plucked 1 month post-transduction and propagated in the regular FBS-supplemented medium (GFP-positive cells > 90%). The cells were treated with anti-viral agents which were reported to suppress the replication of SeV; ribavirin (12–20 μM), papaverine (1–100 μM), and/or prostaglandin A1 (PGA1; 0.3–30 μM). The regular culture medium was DMEM/F12 with 15% FBS. The serum-free culture medium was DMEM/F12 with 20% Knockout serum replacement (KSR, Invitrogen). Although the GFP expression in ES cells was suppressed by treatment with either ribavirin or papaverine, the cells showed differentiated phenotypes. Treatment with PGA1 led to the apoptosis of ES cells at lower concentrations than those required to suppress the GFP expression. Treatment with a mixture of these three agents successfully suppressed the GFP expression in the SSEA-4-negative, differentiated population. On the other hand, cultivation of the transduced ES cells in the KSR-supplemented medium, a widely used serum-free recipe for ES cell cultures, resulted in the complete suppression of GFP expression in ES cells in the undifferentiated state. The SeV genome and GFP gene were undetectable even by RNA-PCR. KSR was originally developed to maintain ES cells in an undifferentiated state, and this is the first report about its unexpected antiviral activities. The anti-viral effect of KSR was cancelled by the addition of 15% FBS. KSR, however, had no effect on the SeV-mediated expression of GFP in LLC-MK2, a standard control cell line for SeV transduction. Unfortunately, information on the composition of KSR is not available, and we cannot explain the mechanism by which KSR eradicates the SeV genome. Although none of the antiviral agents alone; ribavirin, papaverine, or PGA1, is effective at suppressing SeV vector-mediated transgene expression in ES cells, a mixture of the three agents may be useful for suppressing the transgene expression after the differentiation of the transduced ES cells. On the other hand, KSR should be useful in terms of eradicating the SeV genome from transduced ES cells as well as maintaining the pluripotency.

The presence of mast cell precursors in peripheral blood was examined. The beige C57BL/6 (bgj/bgj, Chediak-Higashi syndrome) mouse was parabiosed with the normal C57BL/6 mouse. Mast cells containing giant granules and originating in the beige partner appeared in the normal parabiont. A comparable proportion of normal-mouse-type mast cells developed in the beige parabiont as well. In spite of the low radiosensitivity of mature mast cells, irradiation of the normal parabiont reduced the proportion of normal-mouse-type mast cells appearing in the beige partner. It was concluded, therefore, that the precursors of mast cells can migrate through the bloodstream.