Monkey Embryonic Stem Research Articles

Transcriptome and Proteome Analyses of Human, Monkey and Mouse Embryonic Stem Cells During Embryoid Body-Based Differentiation

We have applied proteomics and transcriptomics approaches to investigate the molecular mechanisms that control embryonic stem cell (ESC) self-renewal and differentiation. We analyzed total, nuclear and membrane proteomes of differentiated and undifferentiated human, mouse and monkey embryonic stem cells. The trancriptome of proliferating and differentiating human ESCs has also been analyzed using a microarray approach. Comparative analyses across species and organelles revealed several proteins and mechanisms emerged as key participants in stem cells proliferation and differentiation. Microarray and Real-Time analyses of ESCs also resulted in identification of several new mechanisms. The results of transcriptome and proteome data will be compared and the methods to maximize the benefit of the integration of transcriptome and proteome data will be discussed.

Embryonic stem cells as a source of photoreceptors for retinal transplantation

Evaluation of: Osakada F, Ikeda H, Mandai M et al. Toward the generation of rod and cone photoreceptors from mouse, monkey and human embryonic stem cells. Nat. Biotechnol. 26(2), 215–224 (2008).Retinitis pigmentosa is a degenerative disease causing blindness in later life. More than 30 genes have been linked to this hereditary disease. Heterogeneity of such magnitude in retinitis pigmentosa represents a major impediment to the development of therapeutics, hence mutation-independent approaches have to be exploited. The loss of retinal neurons is generally regarded as the irreversible cause, and the end-stage, of blindness. Any strategy to restore sight in these cases would almost certainly require cell replacement or transplantation. The paper under evaluation offers methods for in vitro differentiation of murine, monkey and human embryonic stem cells into photoreceptor-like cells. This is an important step forward in the generation of cells apt for retinal transplants.

サル胚性幹細胞からNeural Stem Spheresへの分化誘導初期過程のプロテオーム解析から得られた新知見

サル胚性幹細胞からNeural Stem Spheresへの分化誘導初期過程のプロテオーム解析から得られた新知見

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.

The protein expression profile of cynomolgus monkey embryonic stem cells in two-dimensional gel electrophoresis: a successful identification of multiple proteins using human databases

Global gene and protein expression analyses have had great impacts on scientific progresses in this new era of bioinformatics. Although studies using murine and human materials can fully exploit the large volume of their databases, there are quite a few inconveniences for an investigation on non-human primate materials due to still insufficient data collections. Here we examined the availability of human databases for the protein identification process using the two-dimensional electrophoresis-based proteomic study in cynomolgus monkey embryonic stem (ES) cells. Querying public human protein databases, we successfully identified multiple protein spots via mass spectrometric analysis using MALDI-TOF apparatus. The results of the protein identification were confirmed by western blotting using polyclonal antibodies raised against human epitopes. Interestingly, the results of western blotting further identified the existence of previously unreported multiple isoforms of common proteins including glycolytic pathway enzymes. Thus, combined analyses of the mass spectrometry querying the Homo sapience databases and the western blotting using polyclonal antibodies is highly effective in determining protein expressions in monkey cells. Our success in obtaining a draft protein expression profile of cynomolgus monkey ES cells will contribute to the promotion of non-human primate ES cell researches.

α4-Integrin+ Endothelium Derived from Primate Embryonic Stem Cells Generates Both 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. We previously showed the development of both primitive and definitive hematopoiesis when cynomolgus monkey embryonic stem cells were co-cultured with OP9 stromal cells. In this study, we examined the hematopoietic potential of endothelial cells developing in our coculture system and demonstrated that VE-cadherin+CD45− endothelial cells derived from embryonic stem cells were able to generate primitive and definitive hematopoietic cells sequentially, as revealed by immunostaining of floating erythrocytes and colony-forming assay in cultures. All floating erythrocytes which emerged initially expressed ε- and ζ-globins, while β-globin expression was hardly detected. The percentage of floating erythrocytes positive for β-globin gradually increased thereafter, and almost all erythrocytes were positive by day 40. Meanwhile, expression of ε- and ζ-globins declined gradually. Clonal analysis revealed that single bipotential cells for hematopoietic and endothelial lineages were included in this endothelial cell population. Hemogenic activity of endothelial cells was observed exclusively in the α4-integrin+ subpopulation. RT-PCR data showed that Runx1, a transcriptional factor associated with definitive hematopoiesis, was expressed in the hemogenic α4-integrin+ subpopulation, but not the non-hemogenic α4-integrin− subpopulation among embryonic stem cell-derived endothelial cells. The kinetics of this hemogenic subpopulation was similar to that of hemogenic endothelial cells previously reported in the yolk sac and the AGM region in vivo, in that they emerged only for a limited time. On the other hand, VE-cadherin−CD45−α4-integrin+ cells gave rise to more primitive erythrocytes than VE-cadherin+CD45−α4-integrin+ cells, but hardly contributed to definitive hematopoiesis. These results indicate that VE-cadherin+CD45−α4-integrin+ endothelial cells generate primitive and definitive hematopoietic cells sequentially, while VE-cadherin−CD45−α4-integrin+ cells are primary sources for primitive hematopoiesis. It seems that precursors of primitive and definitive erythropoiesis arise simultaneously but that the definitive precursors require a period of maturation before they differentiated into blood cells. We suggest that a subset of endothelial cells is involved in primitive as well as definitive hematopoiesis during primate embryogenesis, and that α4-integrin marks the hemogenic subpopulation in primates.

Isolation and Characterization of Novel Rhesus Monkey Embryonic Stem Cell Lines

ESCs are important as research subjects since the mechanisms underlying cellular differentiation, expansion, and self-renewal can be studied along with differentiated tissue development and regeneration in vitro. Furthermore, human ESCs hold promise for cell and tissue replacement approaches to treating human diseases. The rhesus monkey is a clinically relevant primate model that will likely be required to bring these clinical applications to fruition. Monkey ESCs share a number of properties with human ESCs, and their derivation and use are not affected by bioethical concerns. Here, we summarize our experience in the establishment of 18 ESC lines from rhesus monkey preimplantation embryos generated by the application of the assisted reproductive technologies. The newly derived monkey ESC lines were maintained in vitro without losing their chromosomal integrity, and they expressed markers previously reported present in human and monkey ESCs. We also describe initial efforts to compare the pluripotency of ESC lines by expression profiling, chimeric embryo formation, and in vitro-directed differentiation into endodermal, mesodermal, and ectodermal lineages.

Molecular Cloning and Function of Oct-3 Isoforms in Cynomolgus Monkey Embryonic Stem Cells

Oct-3 is a key molecule for maintaining self-renewal in mouse embryonic stem (ES) cells. The function of Oct-3 in ES cells of other species, however, especially primate ES cells, is not clear. In the present study, we cloned two splicing isoforms of Oct-3, Oct-3A and Oct-3B, from cynomolgus monkey ES cells, and found that they have high homology to human Oct-3A and Oct-3B. To examine their function, Oct-3A and Oct-3B were overexpressed in cynomolgus monkey ES cells. Transient Oct-3A overexpression induced ES cell differentiation into endodermal and mesodermal lineages and disrupted proliferation of undifferentiated monkey ES cells. In contrast, Oct-3B overexpression did not induce differentiation of monkey ES cells. These findings indicate that a certain Oct-3A expression level has an important role in sustaining self-renewal in non-human primate ES cells.

Cynomolgus monkey embryonic stem cell lines express green fluorescent protein

We successfully established cynomolgus monkey embryonic stem (cES) cell lines expressing enhanced green fluorescent protein (GFP) by introducing a GFP-encoding gene under cytomegalovirus immediate early enhancer (CMVIE) promoter regulation into cES cells. The cells maintained the ability of in vitro differentiation toward ectodermal, mesodermal, and endodermal lineages, and produced teratomas composed of tissues derived from the three embryonic germ layers when transplanted into severe combined immunodeficient disease mice. GFP expression was also observed in the differentiated cells. These GFP-expressing cES cell lines are considered useful for basic research, including cell transplantation.

Stable Maintenance of Monkey Embryonic Stem Cells in the Absence of bFGF

Monkey embryonic stem (ES) cells are useful tools in preclinical studies of gene therapy and tissue engineering as well as in primate developmental biology. However, their maintenance is not easy, requiring addition of bFGF to the medium. 1 Herein, we have described a stable, cost-effective method that does not require bFGF. We used a high-density (1 to 1.5 × 10 5 cells/cm 2) of mouse embryonic fibroblasts (MEF) as feeder cells to successfully maintain undifferentiated monkey ES cells for 2 years (∼150 passages). Furthermore, these ES cells were competent for electroporation of enhanced green fluorescent protein (EGFP) and subsequent drug selection procedures. We were able to establish EGFP-expressing cell lines using this culture condition. These cell lines expressed undifferentiated markers, such as alkaline phosphatase, SSEA-4, TRA-60, and TRA-81. In addition, strong EGFP expression was observed after differentiation into cardiomyocytes, neurons, or adipocytes, suggesting that these cell lines are a useful tool to study cell transplantation. 1,2 This method simplifies the culture of monkey ES cells.

Primate embryonic stem cells create their own niche while differentiating in three-dimensional culture systems.

Rhesus monkey embryonic stem cells (ESCs) (R366.4), cultured on a three-dimensional (3D) collagen matrix with or without human neonatal foreskin fibroblasts (HPI.1) as feeder cells, or embedded in the collagen matrix, formed complex tubular or spherical gland-like structures and differentiated into phenotypes characteristic of neural, epithelial and endothelial lineages. Here, we analysed the production of endogenous extracellular matrix (ECM) proteins, cell-cell adhesion molecules, cell-surface receptors, lectins and their glycoligands, by differentiating ESCs, forming a micro-environment, a niche, able to positively influence cell behaviour. The expression of some of these molecules was modulated by HPI.1 cells while others were unaffected. We hypothesized that both soluble factors and the niche itself were critical in directing growth and/or differentiation of ESCs in this 3D environment. Creating such an appropriate experimental 3D micro-environment, further modified by ESCs and modulated by exogenous soluble factors, may constitute a template for adequate culture systems in developmental biology studies concerning differentiation of stem cells.

Identification and Characterization of Hemoangiogenic Progenitors During Cynomolgus Monkey Embryonic Stem Cell Differentiation

We identified intermediate-stage progenitor cells that have the potential to differentiate into hematopoietic and endothelial lineages from nonhuman primate embryonic stem (ES) cells. Sequential fluorescence-activated cell sorting and immunostaining analyses showed that when ES cells were cultured in an OP9 coculture system, both lineages developed after the emergence of two hemoangiogenic progenitor-bearing cell fractions, namely, vascular endothelial growth factor receptor (VEGFR)-2(high) CD34(-) and VEGFR-2(high) CD34(+) cells. Exogenous vascular endothelial growth factor increased the proportion of VEGFR-2(high) cells, particularly that of VEGFR-2(high) CD34(+) cells, in a dose-dependent manner. Although either population of VEGFR-2(high) cells could differentiate into primitive and definitive hematopoietic cells (HCs), as well as endothelial cells (ECs), the VEGFR-2(high) CD34(+) cells had greater hemoangiogenic potential. Both lineages developed from VEGFR-2(high) CD34(-)or VEGFR-2(high) CD34(+) precursor at the single-cell level, which strongly supports the existence of hemangioblasts in these cell fractions. Thus, this culture system allows differentiation into the HC and EC lineages to be defined by surface markers. These observations should facilitate further studies both on early developmental processes and on regeneration therapies in human.

Generation and Characterization of Functional Cardiomyocytes from Rhesus Monkey Embryonic Stem Cells

Embryonic stem cells (ESCs) from mice and humans (hESCs) have been shown to be able to efficiently differentiate toward cardiomyocytes (CMs). Because murine ESCs and hESCs do not allow for establishment of pre-clinical allogeneic transplantation models, the aim of our study was to generate functional CMs from rhesus monkey ESCs (rESCs). Although formation of ectodermal and neuronal/glial cells appears to be the default pathway of the rESC line R366.4, we were able to change this commitment and to direct generation of endodermal/mesodermal cells and further differentiation toward CMs. Differentiation of rESCs resulted in an average of 18% of spontaneously contracting embryoid bodies (EBs) from rESCs. Semiquantitative reverse transcription-polymerase chain reaction analyses demonstrated expression of marker genes typical for endoderm, mesoderm, cardiac mesoderm, and CMs, including brachyury, goosecoid, Tbx-5, Tbx-20, Mesp1, Nkx2.5, GATA-4, FOG-2, Mlc2a, MLC2v, ANF, and alpha-MHC in rESC-derived CMs. Immunohistological and ultrastructural studies showed expression of CM-typical proteins, including sarcomeric actinin, troponin T, titin, connexin 43, and cross-striated muscle fibrils. Electrophysiological studies by means of multielectrode arrays revealed evidence of functionality, electrical coupling, and beta-adrenergic signaling of the generated CMs. This is the first study demonstrating generation of functional CMs derived from rESCs. In contrast to hESCs, rESCs allow for establishment of pre-clinical allogeneic transplantation models. Moreover, rESC-derived CMs represent a cell source for the development of high-throughput assays for cardiac safety pharmacology.

Monkey Embryonic Stem Cells Differentiate into AdipocytesIn Vitro

Production of functional adipocytes is important in adipocyte research and regenerative medicine. In this paper, we describe the differentiation of monkey embryonic stem (ES) cells into insulin-responsive adipocytes. Treatment of embryoid body (EB) outgrowth with adipogenic hormones induced the expression of adipocyte-specific genes, such as PPARgamma, C/EBPalpha, aP2, insulin receptor, and GLUT4. Expression of adipocytokines, leptin and adiponectin, was also detected. Furthermore, translocation of GLUT4 was observed by insulin stimulation in differentiated adipocytes. These results suggested that monkey ES cells can be a useful tool for studying adipogenesis in primate.

Improved Safety of Hematopoietic Transplantation with Monkey Embryonic Stem Cells in the Allogeneic Setting

Cynomolgus monkey embryonic stem cell (cyESC)-derived in vivo hematopoiesis was examined in an allogeneic transplantation model. cyESCs were induced to differentiate into the putative hematopoietic precursors in vitro, and the cells were transplanted into the fetal cynomolgus liver at approximately the end of the first trimester (n = 3). Although cyESC-derived hematopoietic colony-forming cells were detected in the newborns (4.1%-4.7%), a teratoma developed in all newborns. The risk of tumor formation was high in this allogeneic transplantation model, given that tumors were hardly observed in immunodeficient mice or fetal sheep that had been xeno-transplanted with the same cyESC derivatives. It turned out that the cyESC-derived donor cells included a residual undifferentiated fraction positive for stage-specific embryonic antigen (SSEA)-4 (38.2% +/- 10.3%) despite the rigorous differentiation culture. When an SSEA-4-negative fraction was transplanted (n = 6), the teratoma was no longer observed, whereas the cyESC-derived hematopoietic engraftment was unperturbed (2.3%-5.0%). SSEA-4 is therefore a clinically relevant pluripotency marker of primate embryonic stem cells (ESCs). Purging pluripotent cells with this surface marker would be a promising method of producing clinical progenitor cell preparations using human ESCs.

Development of Tryptase and Chymase Double Positive Mature Mast Cells from Cynomolgus Monkey Embryonic Stem Cells

Development of Tryptase and Chymase Double Positive Mature Mast Cells from Cynomolgus Monkey Embryonic Stem Cells

Primate Embryonic Stem Cell-Derived Neuronal Progenitors Transplanted into Ischemic Brain

Transplantation of stem cells has the possibility of restoring neural functions after stroke damage. Therefore, we transplanted neuronal progenitors generated from monkey embryonic stem (ES) cells into the ischemic mouse brain to test this possibility. Monkey ES cells were caused to differentiate into neuronal progenitors by the stromal cell-derived inducing activity method. Focal cerebral ischemia was induced by occluding the middle cerebral artery by the intraluminal filament technique. The donor cells were transplanted into the ischemic lateral striatum at 24 h after the start of reperfusion. The cells transplanted into the ischemic brain became located widely around the ischemic area, and, moreover, the transplanted cells differentiated into various types of neurons and glial cells. Furthermore, at 28 days after the transplantation, over 10 times more cells in the graft were labeled with Fluorogold (FG) by stereotactic focal injection of FG into the anterior thalamus and substantia nigra on the grafted side when compared with the number at 14 days. From these results we confirmed the survival and differentiation of, as well as network formation by, monkey ES-cell-derived neuronal progenitors transplanted into the ischemic mouse brain.

Promoted differentiation of cynomolgus monkey ES cells into hepatocyte-like cells by co-culture with mouse fetal liver-derived cells

To explore whether a co-culture of cynomolgus monkey embryonic stem (cES) cells with embryonic liver cells could promote their differentiation into hepatocytes. Mouse fetal liver-derived cells (MFLCs) were prepared as adherent cells from mouse embryos on embryonic d (ED) 14, after which undifferentiated cES cells were co-cultured with MFLCs. The induction of cES cells along a hepatic lineage was examined in MFLC-assisted differentiation, spontaneous differentiation, and growth factors (GF) and chemicals-induced differentiations (GF-induced differentiation) using retinoic acid, leukemia inhibitory factor (LIF), FGF2, FGF4, hepatocyte growth factor (HGF), oncostatin M (OSM), and dexamethasone. The mRNA expression of alpha-fetoprotein, albumin (ALB), alpha-1-antitrypsin, and hepatocyte nuclear factor 4alpha was observed earlier in the differentiating cES cells co-cultured with MFLCs, as compared to cES cells undergoing spontaneous differentiation and those subjected to GF-induced differentiation. The expression of cytochrome P450 7a1, a possible marker for embryonic endoderm-derived mature hepatocytes, was only observed in cES cells that had differentiated in a co-culture with MFLCs. Further, the disappearance of Oct3/4, a representative marker of an undifferentiated state, was noted in cells co-cultured with MFLCs, but not in those undergoing spontaneous or GF-induced differentiation. Immunocytochemical analysis revealed an increased ratio of ALB-immunopositive cells among cES cells co-cultured with MFLCs, while glycogen storage and urea synthesis were also demonstrated. MFLCs showed an ability to induce cES cells to differentiate toward hepatocytes. The co-culture system with MFLCs is a useful method for induction of hepatocyte-like cells from undifferentiated cES cells.

Feeder layer- and serum-free culture of rhesus monkey embryonic stem cells

The common culture system of rhesus monkey embryonic stem (rES) cells depends largely on feeder cells and serum, which limits the research and application of rES cells. This study reports a feeder layer-free and serum-free system for culture of rES cells. rES cells could be cultured through at least 22 passages on laminin in medium supplemented with serum replacement (SR), basic fibroblast growth factor (bFGF) and transforming growth factor β1 (TGFβ1), and maintained stable proliferation rates and normal karyotypes, while displaying all the embryonic stem cell characteristics including morphology, alkaline phosphatase (AKP), Oct-4, cell surface markers SSEA-3, SSEA-4, TRA-1–60 and TRA-1–81, and formed cystic embryoid bodies in vitro. In addition, the studies showed that TGFβ1, bFGF and laminin are necessary for maintaining the undifferentiated growth of rES cells in long-term culture. Moreover, withdrawal of TGFβ1 increased the differentiation rate by decreasing the expression of integrins. Therefore, this system would provide a well-defined culture system for rES cells, and would facilitate research into self-renewal and differentiation mechanisms of rES cells.

In Vitro expression of natriuretic peptides in cardiomyocytes differentiated from monkey embryonic stem cells

Functional characterization of ES cell-derived cardiomyocytes is important for differentiation control and application to the cell therapy. One of the crucial functions of cardiomyocytes is a production of atrial and brain natriuretic peptides (ANP and BNP, respectively), which have important endocrine, autocrine, and paracrine functions. In this study, we focused on the functional aspect of the cardiomyocytes differentiated from monkey ES cells in vitro and investigated the expression of ANP and BNP. Spontaneously contracting cells showed nodal-like action potentials, and expression of ANP and BNP by RT-PCR and immunocytochemistry. Interestingly, ANP and BNP expressions were detected as immunoreactive granules in the perinuclear area and these signals appeared to co-localize with trans-Golgi network. These findings suggest that monkey ES cells were able to differentiate into cardiomyocytes with functional characteristics in vitro and therefore can be used as a useful model to study mechanisms and functions in early cardiogenesis.