A single-cell and feeder-free culture system for monkey embryonic stem cells.

Primate pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), hold great potential for research and application in regenerative medicine and drug discovery. To maximize primate PSC potential, a practical system is required for generating desired functional cells and reproducible differentiation techniques. Much progress regarding their culture systems has been reported to date; however, better methods would still be required for their practical use, particularly in industrial and clinical fields. Here we report a new single-cell and feeder-free culture system for primate PSCs, the key feature of which is an originally formulated serum-free medium containing FGF and activin. In this culture system, cynomolgus monkey ESCs can be passaged many times by single-cell dissociation with traditional trypsin treatment and can be propagated with a high proliferation rate as a monolayer without any feeder cells; further, typical PSC properties and genomic stability can be retained. In addition, it has been demonstrated that monkey ESCs maintained in the culture system can be used for various experiments such as in vitro differentiation and gene manipulation. Thus, compared with the conventional culture system, monkey ESCs grown in the aforementioned culture system can serve as a cell source with the following practical advantages: simple, stable, and easy cell maintenance; gene manipulation; cryopreservation; and desired differentiation. We propose that this culture system can serve as a reliable platform to prepare primate PSCs useful for future research and application.

Memory immune responses against pandemic (H1N1) 2009 influenza virus induced by a whole particle vaccine in cynomolgus monkeys carrying Mafa-A1*052:02.

We made an H1N1 vaccine candidate from a virus library consisting of 144 ( = 16 HA×9 NA) non-pathogenic influenza A viruses and examined its protective effects against a pandemic (2009) H1N1 strain using immunologically naïve cynomolgus macaques to exclude preexisting immunity and to employ a preclinical study since preexisting immunity in humans previously vaccinated or infected with influenza virus might make comparison of vaccine efficacy difficult. Furthermore, macaques carrying a major histocompatibility complex class I molecule, Mafa-A1*052:02, were used to analyze peptide-specific CD8(+) T cell responses. Sera of macaques immunized with an inactivated whole particle formulation without addition of an adjuvant showed higher neutralization titers against the vaccine strain A/Hokkaido/2/1981 (H1N1) than did sera of macaques immunized with a split formulation. Neutralization activities against the pandemic strain A/Narita/1/2009 (H1N1) in sera of macaques immunized twice with the split vaccine reached levels similar to those in sera of macaques immunized once with the whole particle vaccine. After inoculation with the pandemic virus, the virus was detected in nasal samples of unvaccinated macaques for 6 days after infection and for 2.67 days and 5.33 days on average in macaques vaccinated with the whole particle vaccine and the split vaccine, respectively. After the challenge infection, recall neutralizing antibody responses against the pandemic virus and CD8(+) T cell responses specific for nucleoprotein peptide NP262-270 bound to Mafa-A1*052:02 in macaques vaccinated with the whole particle vaccine were observed more promptly or more vigorously than those in macaques vaccinated with the split vaccine. These findings demonstrated that the vaccine derived from our virus library was effective for pandemic virus infection in macaques and that the whole particle vaccine conferred more effective memory and broader cross-reactive immune responses to macaques against pandemic influenza virus infection than did the split vaccine.

Triplet pregnancy in a cynomolgus monkey (Macaca fascicularis) after double embryo transfer.

At our research center, cynomolgus monkeys (Macaca fascicularis) are bred by mating or intracytoplasmic sperm injection (ICSI) and embryo transfer. We typically transfer 2 embryos, because the pregnancy rate is better than that for single embryo transfer. In the case we present here, 2 embryos that had been frozen and thawed after ICSI were transplanted into a recipient female macaque, and a multiple pregnancy (3 fetuses) was confirmed. All 3 fetuses were miscarried between days 81 and 85 of pregnancy. One fetus, which was wrapped in the amnion, was expelled along with its own placenta and one other. Because the other placenta had 2 umbilical arteries, 2 fetuses may have shared it. Therefore, we believe this pregnancy was a case of triplets, including a set of twins from an embryo that divided after transfer.

Epidemiology of hepatitis E virus in indoor-captive cynomolgus monkey colony.

A serological survey of hepatitis E virus (HEV) antibody was conducted using 202 adult captive cynomolgus monkeys, who did not show any clinical signs of acute hepatitis. Out of these, 44 monkeys were sero-positive for anti-HEV IgG and all monkeys were negative for anti-HEV IgM. All positive monkeys came from either Vietnam or China, but none from the Philippines, Indonesia, or our facility. Selected 12 monkeys out of positive monkeys from Vietnam, including 9 positive and 3 negative, revealed mostly within the reference ranges for alanine aminotransferase (ALT) and asparatate aminotransferase (AST) by serum biochemistries. Their titers of anti-HEV IgG did not correlate with the concentrations of ALT and AST. Moreover, HEV-RNA could not be detected from any fecal specimens of the 12 monkeys. Thus, monkeys with anti-HEV IgG sero-positive did not seem to be source of the HEV-pollution, because 1) sero-positive monkeys did not excrete HEV-RNA from their feces, and 2) monkeys from the Philippines and Indonesia have remained to be sero-negative for anti-HEV IgG, even if the monkeys were kept in same animal room of our facility. From these results, it could be inferred that primary infection of HEV occurred in the exported countries, but not in our colony. The contamination of HEV in indoor-captive monkeys could be prevented by precise quarantine tests, including ELISA for detecting anti-HEV and RT-PCR for HEV RNA.

Induction of pluripotent stem cells from fetal and adult cynomolgus monkey fibroblasts using four human transcription factors.

Induced pluripotent stem (iPS) cells have the potential to become a universal resource for cell-based therapies in regenerative medicine; however, prior to the use of such iPS cell-based therapies, preclinical assessment of their safety and efficacy is essential. Non-human primates serve as valuable animal models for human diseases or biomedical research; therefore, in this study, we generated cynomolgus monkey iPS cells from adult skin and fetal fibroblast cells by the retrovirally mediated introduction of four human transcription factors: c-Myc, Klf4, Oct3/4, and Sox2 (the so-called "Yamanaka factors"). Twenty to 30 days after the introduction of these factors, several cynomolgus monkey embryonic stem (ES) cell-like colonies appeared on SNL and mouse embryonic fibroblast (MEF) feeder layers. These colonies were picked and cultivated in primate ES medium. Seven iPS cell lines were established, and we detected the expression of pluripotent markers that are also expressed in ES cells. Reverse transcription polymerase chain reaction (PCR) showed that these iPS cells expressed endogenous c-Myc, Klf4, Oct3/4, and Sox2 genes, whereas several transgenes were silenced. Embryoid body and teratoma formation showed that the cynomolgus iPS cells had the developmental potential to differentiate into cells of all three primary germ layers. In summary, we generated cynomolgus monkey iPS cells by retrovirus-mediated transduction of the human transcription factors, c-Myc, Klf4, Oct3/4, and Sox2 into adult cynomolgus monkey skin cells and fetal fibroblasts. The cynomolgus monkey is the most relevant primate model for human disease, and the highly efficient generation of monkey iPS cells would allow investigation of the treatments of various diseases in this model via therapeutic cloning.

Efficacy of single intravenous injection of peramivir against influenza B virus infection in ferrets and cynomolgus macaques.

We evaluated the efficacy of a single intravenous dose peramivir for treatment of influenza B virus infection in ferrets and cynomolgus macaques in the present study. A single dose of peramivir (60 mg/kg of body weight) given to ferrets on 1 day postinfection with influenza B virus significantly reduced median area under the curve (AUC) virus titers (peramivir, 8.3 log(10) 50% tissue culture infective doses [TCID(50)s] · day/ml; control, 10.7 log(10) TCID(50)s · day/ml; P < 0.0001). Furthermore, nasal virus titers on day 2 postinfection in ferrets receiving a single injection of peramivir (30 mg/kg) and AUCs of the body temperature increase in ferrets receiving a single injection of peramivir (30 and 60 mg/kg) were lower than those in ferrets administered oral oseltamivir phosphate (30 and 60 mg/kg/day twice daily for 3 days). In macaques infected with influenza B virus, viral titers in the nasal swab fluid on days 2 and 3 postinfection and body temperature after a single injection of peramivir (30 mg/kg) were lower than those after oral administration of oseltamivir phosphate (30 mg/kg/day for 5 days). The two animal models used in the present study demonstrated that inhibition of viral replication at the early time point after infection was critical in reduction of AUCs of virus titers and interleukin-6 production, resulting in amelioration of symptoms. Our results shown in animal models suggest that the early treatment with a single intravenous injection of peramivir is clinically recommended to reduce symptoms effectively in influenza B virus infection.

Enhancement of corneal endothelium wound healing by Rho-associated kinase (ROCK) inhibitor eye drops.

AIM: To demonstrate the efficacy of Rho-associated kinase (ROCK) inhibitor Y-27632 for corneal endothelial wound healing both in in vitro and in vivo models. METHODS: As an in vitro model, cultivated cynomolgus monkey corneal endothelial cells were scraped to create a linear defect. The wound distance was then determined during a 24-h culture in the presence or absence of 10 µM of Y-27632. As an in vivo model, central corneal endothelium of Japanese white rabbits was damaged by transcorneal freezing, then 10 mM of Y-27632 was applied topically six times daily for 48 h. The wound area of the corneal endothelium was evaluated after 48 h. RESULTS: The mean wound distance in the cultured corneal endothelial cells was significantly shorter in the Y-27632 group than in the control group. In the rabbit model, the mean wound area of the Y-27632 group was significantly smaller than that of the control group. CONCLUSION: This study demonstrated that ROCK inhibitor Y-27632 promotes corneal endothelial wound healing both in in vitro and in vivo.

Establishment of a cynomolgus macaque model of influenza B virus infection.

Pathogenicity of influenza B virus was examined in cynomolgus macaques to establish a macaque model suitable for vaccine and antiviral drug development. We prepared influenza B viruses for inoculation with minimal passages after isolation from patients. Macaques inoculated with influenza B virus showed higher body temperature than that before infection for 6 to 12 days. Virus was detected in nasal, tracheal, and bronchial samples until 6 days after inoculation followed by an increase in neutralizing antibody. High levels of IL-6 and TNF-α in nasal swabs from the infected macaques were correlated with fever. Symptoms and duration of the viral replication would be sufficient to evaluate efficacy of vaccines and antiviral agents. In addition, measurement of immune responses including antibody and cytokine production would provide an immunological rationale in efficacy of vaccines and antiviral agents. The results suggest that cynomolgus macaques are appropriate model animals for research of influenza B virus.

Subcutaneous inoculation of a whole virus particle vaccine prepared from a non-pathogenic virus library induces protective immunity against H7N7 highly pathogenic avian influenza virus in cynomolgus macaques.

Development of H7N7 highly pathogenic avian influenza virus (HPAIV) vaccines is an urgent issue since human cases of infection with this subtype virus have been reported and most humans have no immunity against H7N7 viruses. We made an H7N7 vaccine combining components from an influenza virus library of non-pathogenic type A influenza viruses. Antibody and T cell recall responses specific against the vaccine strain were elicited by subcutaneous inoculation with the whole virus particle vaccine with or without alum as an adjuvant in cynomolgus macaques. No significant difference was observed in magnitude of antibody responses between vaccination with alum and vaccination without alum, though vaccination with alum induced longer recall responses of CD8(+) T cells than did vaccination without alum. After challenge with a subtype of H7N7 HPAIV, the virus was detected in nasal swabs of unvaccinated macaques for 8 days but only for 1 day in the animals vaccinated either with or without alum, although the macaques vaccinated with alum showed elevated body temperature more briefly after infection. These findings demonstrated that this H7N7 HPAIV strain is pathogenic to macaques and that the vaccine conferred protective immunity to macaques against H7N7 HPAIV infection.

Amelioration of pneumonia with Streptococcus pneumoniae infection by inoculation with a vaccine against highly pathogenic avian influenza virus in a non-human primate mixed infection model.

BACKGROUND: Highly pathogenic avian influenza virus (HPAIV) infection has a high mortality rate in humans. Secondary bacterial pneumonia with HPAIV infection has not been reported in human patients, whereas seasonal influenza viruses sometimes enhance bacterial pneumonia, resulting in substantial morbidity and mortality. Therefore, if HPAIV infection were accompanied by bacterial infection, an increase in mortality would be expected. We examined whether a vaccine against HPAIV prevents severe morbidity caused by mixed infection with HPAIV and bacteria. METHODS: H7N7 subtype of HPAIV and Streptococcus pneumoniae were inoculated into cynomolgus macaques with or without vaccination of inactivated whole virus particles. RESULTS: Vaccination against H7N7 HPAIV decreased morbidity caused by HPAIV and pneumonia caused by S. pneumoniae. Bacterial replication in lungs was decreased by vaccination against HPAIV, although the reduction in bacterial colonies was not significant. CONCLUSIONS: Vaccination against HPAIV reduces pneumonia caused by bacterial superinfection and may improve prognosis of HPAIV-infected patients.

MHC class I A loci polymorphism and diversity in three Southeast Asian populations of cynomolgus macaque.

Cynomolgus macaques (Macaca fascicularis, Mafa) have emerged as important animal models for biomedical research, necessitating a more extensive characterization of their major histocompatibility complex polymorphic regions. The current information on the polymorphism or diversity of the polygenetic Mafa class I A loci is limited in comparison to the more commonly studied rhesus macaque Mafa class I A loci. Therefore, in this paper, to better elucidate the degree and types of polymorphisms and genetic differences of Mafa-A1 among three native Southeast Asian populations (Indonesian, Vietnamese, and Filipino) and to investigate how the allele differences between macaques and humans might have evolved to affect their respective immune responses, we identified 83 Mafa-A loci-derived alleles by DNA sequencing of which 66 are newly described. Most alleles are unique to each population, but seven of the most frequent alleles were identical in sequence to some alleles in other macaque species. We also revealed (1) the large and dynamic genetic and structural differences and similarities in allelic variation by analyzing the population allele frequencies, Hardy-Weinberg's equilibrium, heterozygosity, nucleotide diversity profiles, and phylogeny, (2) the difference in genetic structure of populations by Wright's FST statistic and hierarchical analysis of molecular variance, and (3) the different demographic and selection pressures on the three populations by performing Tajima's D test of neutrality. The large level of diversity and polymorphism at the Mafa-A1 was less evident in the Filipino than in the Vietnam or the Indonesian populations, which may have important implications in animal capture, selection, and breeding for medical research.

Cultivated corneal endothelial transplantation in a primate: possible future clinical application in corneal endothelial regenerative medicine.

PURPOSE: To review our attempt to devise a method of cultivated corneal endothelial transplantation using primates in which corneal endothelium, like that of humans, has low proliferative ability. METHODS: Monkey corneal endothelial cells (MCECs) were cultivated, with subcultures grown on collagen type I carriers. The corneal endothelia of 6 eyes of 6 monkeys were scraped intensively, after which cultivated MCEC sheets were inserted into the anterior chamber of 4 eyes. As controls, a collagen sheet without MCECs was transplanted in 1 eye of a monkey, and a suspension of cultivated MCECs was injected into the anterior chamber of 1 eye of another monkey. RESULTS: MCECs produced a confluent monolayer of closely attached hexagonal cells, which expressed both ZO-1 and Na-K adenosine triphosphatase. Early postoperative period MCEC sheets were attached to Descemet membrane, and corneal clarity was recovered. Six months after transplantation, MCEC-transplanted corneas remained clear, and closely attached hexagonal cells were observed. In 1 animal with longer observation, polygonal cells were observed by in vivo specular microscopy at a density of >2000 cells/mm2 and remained >1600 cells/mm2 for < or =2 years. Control eyes showed irreversible bullous keratopathy throughout the observation period. CONCLUSIONS: Cultivated MCECs become attached to the transplanted eye and maintain a clear cornea < or =2 years postoperatively, suggesting that corneal endothelial cells of primates might have proliferative ability in vivo once they have been cultured and proliferated in vitro. Our monkey model constitutes an important step forward for regenerative medicine with possible future application in patients with corneal endothelial dysfunction.

BMP4 induces primitive endoderm but not trophectoderm in monkey embryonic stem cells.

Monkey embryonic stem (ES) cells share similar characteristics to human ES cells and provide a primate model of allotransplantation, which allows to validate efficacy and safety of cell transplantation therapy in regenerative medicine. Bone morphogenetic protein 4 (BMP4) is known to promote trophoblast differentiation in human ES cells in contrast to mouse ES cells where BMP4 synergistically maintains self-renewal with leukemia inhibitory factor (LIF), which represents a significant difference in signal transduction of self-renewal and differentiation between murine and human ES cells. As the similarity of the differentiation mechanism between monkey and human ES cells is of critical importance for their use as a primate model system, we investigated whether BMP4 induces trophoblast differentiation in monkey ES cells. Interestingly, BMP4 did not induce trophoblast differentiation, but instead induced primitive endoderm differentiation. Prominent downregulation of Sox2, which plays a pivotal role not only in pluripotency but also placenta development, was observed in cells treated with BMP4. In addition, upregulation of Hand1, Cdx2, and chorionic gonadotropin beta (CG-beta), which are markers of trophoblast, was not observed. In contrast, BMP4 induced significant upregulation of Gata6, Gata4, and LamininB1, suggesting differentiation into the primitive endoderm, visceral endoderm, and parietal endoderm, respectively. The threshold of BMP4 activity was estimated as about 10 ng/mL. These findings suggest that BMP4 induced differentiation into the primitive endoderm lineage but not into trophoblast in monkey ES cells.

A vaccine prepared from a non-pathogenic H5N1 avian influenza virus strain confers protective immunity against highly pathogenic avian influenza virus infection in cynomolgus macaques.

In order to prepare for the emergence of pandemic influenza viruses, we have established an influenza virus library that contains non-pathogenic influenza A virus strains with 135 combinations of 15 hemagglutinin and 9 neuraminidase subtypes. In this study, we developed a vaccine against H5N1 highly pathogenic avian influenza (HPAI) virus infection in humans using a virus strain selected from the library. We examined its immunogenic potency using cynomolgus macaques as a primate model. Virus antigen-specific antibodies were elicited by intranasal or subcutaneous administration of inactivated whole virus particle vaccines. After challenge with an H5N1 HPAI virus isolate obtained from a Vietnamese patient, the virus was detected only on next day following inoculation in the nasal and/or tracheal swabs of vaccinated macaques that were asymptomatic. On the other hand, the viruses were isolated from nasal and tracheal swabs from non-vaccinated macaques until day 5 and day 7 after inoculation of the H5N1 HPAI virus, respectively. Although six non-vaccinated macaques developed a high body temperature, and two of them lost their appetite after HPAI virus infection, they recovered by the end of the 12-day observation period and did not show the severe symptoms that have been reported in human H5N1 virus infection cases. This demonstrates that the vaccine prepared with the non-pathogenic H5N1 virus from our influenza virus library conferred protective immunity against H5N1 HPAI virus infection to macaques.

Direct development of functionally mature tryptase/chymase double-positive connective tissue-type mast cells from primate embryonic stem cells.

Conditions that influence the selective development or recruitment of connective tissue-type and mucosal-type mast cells (MCs) are not well understood. Here, we report that cynomolgus monkey embryonic stem (ES) cells cocultured with the murine aorta-gonad-mesonephros-derived stromal cell line AGM-S1 differentiated into cobblestone (CS)-like cells by day 10-15. When replated onto fresh AGM-S1 with the addition of stem cell factor, interleukin-6, and Flt3 ligand, these CS-like cells displayed robust growth and generated almost 100% tryptase/chymase double-positive MCs within 3 weeks. At all time points, the percentage of tryptase-positive cells did not exceed that of chymase-positive cells. These ES-derived MCs were CD45+/Kit+/CD31+/CD203c+/HLA-DR- and coexpressed a high-affinity IgE receptor on their surface, which was upregulated after IgE exposure. Electron microscopy showed that they contained many electron dense granules. Moreover, ES-derived MCs responded to stimulation by via IgE and substance P by releasing histamine. These results indicate that ES-derived MCs have the phenotype of functionally mature connective tissue-type MCs. The rapid maturation of ES-derived MCs suggests a unique embryonic pathway in primates for early development of connective tissue-type MCs, which may be independent from the developmental pathway of mucosal-type MCs.

Cultivated corneal endothelial cell sheet transplantation in a primate model.

PURPOSE: To examine the feasibility of cultivated corneal endothelial cell transplantation in a primate model. METHODS: Monkey corneal endothelial cells (MCECs) obtained from three cynomolgus monkeys were cultivated, with subcultures grown on collagen type I carriers for 4 weeks. The corneal endothelium of the right eye of six monkeys was mechanically scraped, after which a cultivated MCEC sheet was brought into the anterior chamber of four eyes and fixed to Descemet's membrane by air. As the control, a collagen sheet without MCECs was transplanted into one eye of one monkey, and a suspension of cultivated MCECs was injected into the anterior chamber in one eye. RESULTS: Cultivated MCECs produced a confluent monolayer of closely attached hexagonal cells that showed both ZO-1 and Na(+)-K(+) ATPase expression. In the early postoperative period MCEC sheets were attached to Descemet's membrane and corneal clarity was recovered. The recovered clarity was accompanied by a decrease in corneal thickness. Fluorescein DiI labeled donor corneal endothelial cells were detected on the host cornea on postoperative day 7. Six months after transplantation MCEC-transplanted corneas remained clear, and hexagonal cells were observed by in vivo specular microscopy with a density of 1992 to 2475 cells/mm(2). Control eyes showed irreversible bullous keratopathy that precluded pachymetry and specular microscopy. CONCLUSIONS: A model of cultivated corneal endothelial transplantation for corneal endothelial dysfunction was established in primates whose corneal endothelial cells have less proliferative capacity in vivo. Our results suggest that this is a useful model for long-term observation in advance of the future clinical application of cultivated corneal endothelial transplantation.

Cloned blastocysts produced by nuclear transfer from somatic cells in cynomolgus monkeys (Macaca fascicularis).

In nonhuman primates (NHPs), there have so far been few reports about nuclear transfer (NT), especially using adult somatic cells. The objective of this study was to determine the developmental competence of NT embryos derived from various somatic cells embryonic stem (ES), amniotic epithelial, cumulus, or fetal fibroblast cells] and the nuclear transfer method, such as electro fusion or piezo microinjection, activation with chemical reagent [ionomycine/6-dimethylaminopurine (DMAP), calcium ionophore A23187/DMAP, or cycloheximide (CHX)] and reprogramming time (1, 2, or 4 h; in this study, the duration from injection or fusion to activation was defined as the reprogramming time). Our results showed that a 1-h reprogramming and activation with ionomycin/DMAP are suitable for NT in monkeys. Developing cleaved embryos up to the six-cell stage was similar among all experiments. However, beyond the eight-cell stage, developmental rates were higher in NT embryos reconstructed with fetal fibroblast cells and amniotic epithelial cells, and we were able to produce NT blastocysts from these cells. Interestingly, electro fusion is sufficient for amniotic epithelial cells and piezo microinjection is better suited for fetal fibroblast cells to produce NT blastocysts, thus suggesting that the best method for somatic cell NT may be different between cell types.

alpha4-Integrin(+) endothelium derived from primate embryonic stem cells generates primitive and definitive hematopoietic cells.

The mechanism of commencement of hematopoiesis in blood islands of the yolk sac and the aorta-gonad-mesonephros (AGM) region during primate embryogenesis remains elusive. In this study, we demonstrated that VE-cadherin(+)CD45(-) endothelial cells derived from nonhuman primate embryonic stem cells are able to generate primitive and definitive hematopoietic cells sequentially, as revealed by immunostaining of floating erythrocytes and colony-forming assay in cultures. Single bipotential progenitors for hematopoietic and endothelial lineages are included in this endothelial cell population. Furthermore, hemogenic activity of these endothelial cells is observed exclusively in the alpha4-integrin(+) subpopulation; bipotential progenitors are 4-fold enriched in this subpopulation. The kinetics of this hemogenic subpopulation is similar to that of hemogenic endothelial cells previously reported in the yolk sac and the AGM region in vivo in that they emerge for only a limited time. We suggest that VE-cadherin(+)CD45(-)alpha4-integrin(+) endothelial cells are involved in primitive and definitive hematopoiesis during primate embryogenesis, though VE-cadherin(-)CD45(-)alpha4-integrin(+) cells are the primary sources for primitive hematopoiesis.

A BAC-based contig map of the cynomolgus macaque (Macaca fascicularis) major histocompatibility complex genomic region.

The construction of a cynomolgus macaque (Macaca fascicularis, Mafa) BAC library for genomic comparison between rhesus and cynomolgus macaques is necessary to promote the cynomolgus macaque as one of the important experimental animals for future medical and biological research. In this paper, we constructed a cynomolgus macaque BAC library and a map of the MHC (Mafa) genomic region for comparison of the genomic organization and nucleotide similarities between the human, the chimpanzee, and the rhesus macaque. The BAC library consists of 221,184 clones with an average insert size of 83 kb, providing a sixfold coverage of the haploid genome. A total of 114 BAC clones and 54 PCR primer sets were used to construct a 4.3-Mb contig of the MHC region. Diversity analysis of genomic sequence from selected subregions of the MHC revealed that the cynomolgus sequence varied compared to rhesus macaque, human, and chimpanzee sequences by 0.48, 4.15, and 4.10%, respectively. From these findings, we conclude that the BAC library and Mafa genomic map are useful tools for genome analysis and will have important applications for comparative genomics and identifying regions of consequence in medical research.

Sequential analysis of alpha- and beta-globin gene expression during erythropoietic differentiation from primate embryonic stem cells.

The temporal pattern of embryonic, fetal, and adult globin expression in the alpha (zeta --> alpha) and beta (epsilon --> gamma and gamma --> beta) clusters were quantitatively analyzed at the transcriptional and translational levels in erythrocytes induced from primate embryonic stem cells in vitro. When vascular endothelial growth factor receptor-2(high) CD34(+) cells were harvested and reseeded onto OP9 stromal cells, two-wave erythropoiesis occurred sequentially. Immunostaining and real-time reverse transcription-polymerase chain reaction analyses of floating mature erythrocytes revealed that globin switches occurred in parallel with the erythropoietic transition. Colony-forming assays showed replacement of primitive clonogenic progenitor cells with definitive cells during culturing. A decline in embryonic zeta- and epsilon-globin expression at the translational level occurred in individual definitive erythroid progenitors. Expression of beta-globin in individual definitive erythroid progenitors was upregulated in the presence of OP9 stromal cells. Thus, this system reproduces early hematopoietic development in vitro and can serve as a model for analyzing the mechanisms of the globin switch in humans.