Rhesus Monkey Embryonic Stem Cells Research Articles

Transcriptional Profiling of Rhesus Monkey Embryonic Stem Cells1

Embryonic stem cells (ESCs) may be able to cure or alleviate the symptoms of various degenerative diseases. However, unresolved issues regarding survival, functionality, and tumor formation mean a prudent approach should be adopted towards advancing ESCs into human clinical trials. The rhesus monkey provides an ideal model organism for developing strategies to prevent immune rejection and test the feasibility, safety, and efficacy of ESC-based medical treatments. Transcriptional profiling of rhesus monkey ESCs provides a foundation for pre-clinical ESC research in this species. In the present study, we used microarray technology, immunocytochemistry, reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time PCR (qPCR) to characterize and transcriptionally profile rhesus monkey ESCs. We identified 367 stemness gene candidates that were highly (>85%) conserved across five different ESC lines. Rhesus monkey ESC lines maintained a pluripotent undifferentiated state over a wide range of POU5F1 (also known as OCT4) expression levels, and comparisons between rhesus monkey, mouse, and human stemness genes revealed five mammalian stemness genes: CCNB1, GDF3, LEFTB, POU5F1, and NANOG. These five mammalian genes are strongly expressed in rhesus monkey, mouse, and human ESCs, albeit only in the undifferentiated state, and represent the core key mammalian stemness factors.

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.

Uniform Adherent Neural Progenitor Populations from Rhesus Embryonic Stem Cells

Rhesus and human embryonic stem cells (ESCs) are similar, making rhesus ESCs an appropriate preclinical allograft model for refining stem cell therapies. Use of rhesus ESC-derived neural progenitors (NPs) in preclinical applications will be enhanced if the neural derivation process is scalable and free from contaminating ESCs or nonneural cells. In this study, we have quantified temporal gene expression changes of rhesus ESC differentiated to uniform NPs using simple feeder-free adherent cultures. NPs exhibited a significant up-regulation of neural-specific genes and a downregulation of pluripotency genes. Additionally, expression of Hu, MAP2, and Tuj1, shows that NPs can form post-mitotic neurons. This study represents a simple and scalable means of producing adherent primate NPs for preclinical testing of neural cell-based therapy.

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.

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.

Genomic imprinting in primate embryos and embryonic stem cells

Embryonic stem (ES) cells hold promise for cell and tissue replacement approaches to treating human diseases. However, long-term in vitro culture and manipulations of ES cells may adversely affect their epigenetic integrity including imprinting. Disruption or inappropriate expression of imprinted genes is associated with several clinically significant syndromes and tumorigenesis in humans. We demonstrated aberrant biallelic expression of IGF2 and H19 in several rhesus monkey ES cell lines while SNRPN and NDN were normally imprinted and expressed from the paternal allele. In contrast, expanded blastocyst-stage embryos, from which these ES cells were derived, exhibited normal paternal expression of IGF2 and maternal expression of H19. To test the possibility that aberrant methylation at an imprinting centre (IC) upstream of H19 accounts for the relaxed imprinting of IGF2 and H19, we performed comprehensive methylation analysis by investigating methylation profiles of CpG sites within the IGF2/H19 IC. Our results demonstrate abnormal hypermethylation within the IGF2/H19 IC in all analysed ES cell lines consistent with biallelic expression of these genes. Cellular overproliferation and tumour formation resulting from tissue or cell transplantation are potential problems that must be addressed before clinical trials of ES cell-based therapy are initiated.

Role of TPO in the Differentiation of Embryonic Stem Cells into Hematopoetic Cells.

Thrombopoetin (TPO) is a primary regulator of megakaryocyte and platelet production. However, studies in c-mpl-deficient mice and in congenital amegakaryocytic thrombocytopenia-patients with non-sense c-mpl mutation who develop pancytopenia during the first years of life suggest that TPO also play an important role on early hematopoesis. We demonstrated that TPO enhances FLK-1 (VEGF-receptor) expression on hemangioblasts during murine embryonic stem cell differentiation in embryoid body-liquid cultures (up to 73%). To extend our studies, we investigated the TPO signaling in FLK-1 positive cells. ES cells at different time point of differentiation showed that TPO enhances c-mpl-, BMP4-, Notch-, HOXB4-, HOXB9-, HOXA10-, Runx1-and CD133- mRNA expression. To investigate mesoderm formation, we also analyzed GATA-4 and T-brachyury mRNA level expression. Interestingly, we found that TPO alone did not increase GATA-4- and T-brachyury- mRNA expression, suggesting that TPO requires other cytokines to form the mesoderm. We also found that TPO could maintain VEGF-A mRNA expression level during differentiation of ES-cells. We hypothesize that VEGF expression together with c-mpl expression is required in hematopoetic differentiation of ES cell. This activity of Tpo was also observed during Rhesus monkey embryonic stem cell differentiation into hematopoetic cell. Only combinations of TPO and VEGF were capable of increasing CD34 positive hematopoietic progenitor cells (up to 8%), but TPO alone failed to induce high levels of CD34+ cell. In addition, analysis of gene expression during hemangioblast development demonstrated that TPO was capable of increasing the expression of VEGF receptors (FLK-1) and TPO receptors (c-mpl) in mice and primates. The in-vitro differentiation of mouse and rhesus monkey ES cells provides an opportunity to better understand the role of TPO in the early stage of hematopoietic development from ES cells to mature hematopoietic cells.

Aberrant Genomic Imprinting in Rhesus Monkey Embryonic Stem Cells

Genomic imprinting involves modification of a gene or a chromosomal region that results in the differential expression of parental alleles. Disruption or inappropriate expression of imprinted genes is associated with several clinically significant syndromes and tumorigenesis in humans. Additionally, abnormal imprinting occurs in mouse embryonic stem cells (ESCs) and in clonally derived animals. Imprinted gene expression patterns in primate ESCs are largely unknown, despite the clinical potential of the latter in the cell-based treatment of human disease. Because of the possible implications of abnormal gene expression to cell or tissue replacement therapies involving ESCs, we examined allele specific expression of four imprinted genes in the rhesus macaque. Genomic and complementary DNA from embryos and ESC lines containing useful single nucleotide polymorphisms were subjected to polymerase chain reaction-based amplification and sequence analysis. In blastocysts, NDN expression was variable indicating abnormal or incomplete imprinting whereas IGF2 and SNRPN were expressed exclusively from the paternal allele and H19 from the maternal allele as expected. In ESCs, both NDN and SNRPN were expressed from the paternal allele while IGF2 and H19 showed loss of imprinting and biallelic expression. In differentiated ESC progeny, these expression patterns were maintained. The implications of aberrant imprinted gene expression to ESC differentiation in vitro and on ESC-derived cell function in vivo after transplantation are unknown.

Cell Cycle Features of Primate Embryonic Stem Cells

Using flow cytometry measurements combined with quantitative analysis of cell cycle kinetics, we show that rhesus monkey embryonic stem cells (ESCs) are characterized by an extremely rapid transit through the G1 phase, which accounts for 15% of the total cell cycle duration. Monkey ESCs exhibit a non-phasic expression of cyclin E, which is detected during all phases of the cell cycle, and do not growth-arrest in G1 after gamma-irradiation, reflecting the absence of a G1 checkpoint. Serum deprivation or pharmacological inhibition of mitogen-activated protein kinase kinase (MEK) did not result in any alteration in the cell cycle distribution, indicating that ESC growth does not rely on mitogenic signals transduced by the Ras/Raf/MEK pathway. Taken together, these data indicate that rhesus monkey ESCs, like their murine counterparts, exhibit unusual cell cycle features in which cell cycle control mechanisms operating during the G1 phase are reduced or absent.

Transplantable Neural Progenitor Populations Derived from Rhesus Monkey Embryonic Stem Cells

Cell-based therapies using embryonic stem cells (ESCs) in the treatment of neural disease will require the generation of homogenous donor neural progenitor (NP) populations. Here we describe an efficient culture system containing hepatocyte growth factor (HGF) and G5 supplement for the production of highly enriched (88.3%+/-8.1%) populations of NPs from rhesus monkey ESCs. Additional purification resulted in NP preparations that were 98% nestin positive. Moreover, NPs, as monolayers or neurospheres, could be maintained for prolonged periods of time in media containing HGF+G5 or G5 alone. In vitro differentiation and in vivo transplantation assays showed that NPs could differentiate into neurons, astrocytes, and oligodendrocytes. The kinds and quantities of differentiated cells derived from NPs were closely correlated with their niches in vivo. Glial differentiation was predominant in periventricular areas, whereas cells migrating into the cortex were mostly neurons. Cell counts showed that 2 months after transplantation, approximately 25% of transplanted NPs survived and 65%-80% of the surviving transplanted cells migrated along the ventricular wall or in a radial fashion. Subcloning demonstrated that several clonal lines derived from NPs expressed nestin and differentiated into three neural lineages in vitro and in rat brains in vivo. In contrast, some subcloned lines showed restricted differentiation both in vitro and in vivo in rat brains. These observations set the stage for obtaining highly enriched NPs and evaluating the efficacy of NP-based transplantation therapy in the nonhuman primate and will provide a platform for probing the molecular mechanisms that control neural induction.

Homologous Feeder Cells Support Undifferentiated Growth and Pluripotency in Monkey Embryonic Stem Cells

In the present study, five homologous feeder cell lines were developed for the culture and maintenance of rhesus monkey embryonic stem cells (rESCs). Monkey ear skin fibroblasts (MESFs), monkey oviductal fibroblasts (MOFs), monkey follicular granulosa fibroblast-like (MFG) cells, monkey follicular granulosa epithelium-like (MFGE) cells, and clonally derived fibroblasts from MESF (CMESFs) were established and compared with the ability of mouse embryonic fibroblasts (MEFs) to support rESC growth. MESF, MOF, MFG, and CMESF cells, but not MFGE cells, were as good as or better than MEFs in supporting undifferentiated growth while maintaining the differentiation potential of the rESCs. In an effort to understand the unique properties of supportive feeder cells, expression levels for a number of candidate genes were examined. MOF, MESF, and MEF cells highly expressed leukemia inhibitory factor, ciliary neurotrophic factor, basic fibroblast growth factor, stem cell factor, transforming growth factor beta1, bone morphogenetic protein 4, and WNT3A, whereas WNT2, WNT4, and WNT5A were downregulated, compared with MFGE cells. Additionally, all monkey feeder cell lines expressed Dkk1 and LRP6, antagonists of the WNT signaling pathway, but not WNT1, WNT8B, or Dkk2. rESCs grown on homologous feeders maintained normal karyotypes, displayed the characteristics of ESCs, including morphology, alkaline phosphatase, Oct4, the cell surface markers stage-specific embryonic antigen (SSEA)-3, SSEA-4, tumor-related antigen (TRA)-1-60, and TRA-1-81, and formed cystic embryoid bodies in vitro that included differentiated cells representing the three major germ layers. These results indicate that the four homologous feeder cell lines can be used to support the undifferentiated growth and maintenance of pluripotency in rESCs.

Thrombopoietin Regulates Differentiation of Rhesus Monkey Embryonic Stem Cells to Hematopoietic Cells

Thrombopoietin (TPO) is the primary regulator of megakaryocyte and platelet production in vitro and in vivo. It supports also survival and proliferation of hematopoietic stem cells. The TPO receptor, c-mpl, is a member of the protooncogene family. Our studies focused on the effect of TPO on proliferation and differentiation of rhesus monkey embryonic stem cells to hematopoietic cells. The rationale of the present investigations was the finding that patients with congenital amegakaryocytic thrombocytopenia (CAMT) reveal c-mpl mutations leading to the development of pancytopenia, suggesting that c-mpl is expressed in early hematopoiesis. Here we demonstrate that rhesus monkey embryonic stem (ES) cells are capable of differentiating into uncommitted progenitor cells, including hemangioblasts (hematopoietic and endothelial precursor cells). The combination of TPO and vascular endothelial growth factor (VEGF) significantly increases the number of hemangioblasts and promotes even differentiation to CD34(+) hematopoietic progenitor cells (up to 8%). In addition, analysis of gene expression during hemangioblast development demonstrates that TPO is capable of increasing the mRNA expression of the VEGF receptor (VEGFR) and its own receptor (c-mpl). The in vitro differentiation of rhesus monkey ES cells provides an opportunity to better understand the mechanism of TPO function in the early stage of hematopoietic development from ES cells to mature hematopoietic cells.

Epithelial–Mesenchymal Transition in Colonies of Rhesus Monkey Embryonic Stem Cells: A Model for Processes Involved in Gastrulation

Rhesus monkey embryonic stem (rhES) cells were grown on mouse embryonic fibroblast (MEF) feeder layers for up to 10 days to form multilayered colonies. Within this period, stem cell colonies differentiated transiently into complex structures with a disc-like morphology. These complex colonies were characterized by morphology, immunohistochemistry, and marker mRNA expression to identify processes of epithelialization as well as epithelial-mesenchymal transition (EMT) and pattern formation. Typically, differentiated colonies were comprised of an upper and a lower ES cell layer, the former growing on top of the layer of MEF cells whereas the lower ES cell layer spread out underneath the MEF cells. Interestingly, in the central part of the colonies, a roundish pit developed. Here the feeder layer disappeared, and upper layer cells seemed to ingress and migrate through the pit downward to form the lower layer while undergoing a transition from the epithelial to the mesenchymal phenotype, which was indicated by the loss of the marker proteins E-cadherin and ZO-1 in the lower layer. In support of this, we found a concomitant 10-fold upregulation of the gene Snail2, which is a key regulator of the EMT process. Conversion of epiblast to mesoderm was also indicated by the regulated expression of the mesoderm marker Brachyury. An EMT is a characteristic process of vertebrate gastrulation. Thus, these rhES cell colonies may be an interesting model for studies on some basic processes involved in early primate embryogenesis and may open new ways to study the regulation of EMT in vitro.

Challenges of Primate Embryonic Stem Cell Research

Embryonic stem (ES) cells hold great promise for treating degenerative diseases, including diabetes, Parkinson's, Alzheimer's, neural degeneration, and cardiomyopathies. This research is controversial to some because producing ES cells requires destroying embryos, which generally means human embryos. However, some of the surplus human embryos available from in vitro fertilization (IVF) clinics may have a high rate of genetic errors and therefore would be unsuitable for ES cell research. Although gross chromosome errors can readily be detected in ES cells, other anomalies such as mitochondrial DNA defects may have gone unrecognized. An insurmountable problem is that there are no human ES cells derived from in vivo-produced embryos to provide normal comparative data. In contrast, some monkey ES cell lines have been produced using in vivo-generated, normal embryos obtained from fertile animals; these can represent a "gold standard" for primate ES cells. In this review, we argue a need for strong research programs using rhesus monkey ES cells, conducted in parallel with studies on human ES and adult stem cells, to derive the maximum information about the biology of normal stem cells and to produce technical protocols for their directed differentiation into safe and functional replacement cells, tissues, and organs. In contrast, ES cell research using only human cell lines is likely to be incomplete, which could hinder research progress, and delay or diminish the effective application of ES cell technology to the treatment of human diseases.

Complex Extracellular Matrices Promote Tissue‐Specific Stem Cell Differentiation

Most cells in tissues contact an extracellular matrix on at least one surface. These complex mixtures of interacting proteins provide structural support and biological signals that regulate cell differentiation and may be important for stem cell differentiation. In this study, we have grown a rhesus monkey embryonic stem cell line in the presence of various extracellular matrix components in monolayer, in a NASA-developed rotating wall vessel bioreactor in vitro, and subcutaneously in vivo. We find that individual components of the extracellular matrix, such as laminin-1 or collagen I, do not influence the growth or morphology of the cells. In contrast, a basement membrane extract, Matrigel, containing multiple extracellular matrix components, induces the cells within 4 days to form immature glandular- and tubular-like structures, many of which contain a lumen with polarized epithelium and microvilli. Such structures were seen in vitro when the cells were grown in the bioreactor and when the cells were injected into mice. These tubular- and glandular-like structures were polarized epithelia based on immunostaining for laminin and cytokeratin. The cell aggregates and tumors also contained additional mixed populations of cells, including mesenchymal cells and neuronal cells, based on immunostaining with vimentin and neuronal markers. An extract of cartilage, containing multiple cartilage matrix components, promoted chondrogenesis in vivo where alcian blue-stained cartilage nodules could be observed. Some of these nodules stained with von Kossa, indicating that they had formed calcified cartilage. We conclude that extracellular matrices can promote the differentiation of embryonic stem cells into differentiated cells and structures that are similar to the tissue from which the matrix is derived. Such preprogramming of cell differentiation with extracellular matrices may be useful in targeting stem cells to repair specific damaged organs.

199 DIFFERENTIATION OF HIGHLY ENRICHED OLIGODENDROCYTE PRECURSORS AND MATURE OLIGODENDROCYTES FROM RHESUS MONKEY EMBRYONIC STEM CELLS

Generating homologous oligodendrocytes are required for studying the molecular mechanisms of oligodendrogliogenesis and for providing donor cells for transplantation therapies. Previous studies have shown that embryonic stem (ES) cells can be induced to generate neural stem cells with many kinds of culture systems; however, few or no oligodendrocytes were obtained from these culture systems. Here we present a simple method containing five steps for obtaining highly enriched oligodendrocyte precursors (75 � 6.8%) and mature oligodendrocytes (81 � 8.6%) from rhesus monkey embryonic stem (rES) cells. We expanded rES cells on a feeder layer of irradiated MESF (ear skin fibroblasts from a one-week-old rhesus monkey), formed embryoid bodies (EBs), promoted Day 9 (3 days in hanging drop and 6 days in suspension) differentiation into highly enriched (90.2 � 6.1%) neural progenitors (NPs) with hepatocyte growth factor (HGF) and G5 supplement [containing 5 ng/mL (bFGF) and 10 ng/mL epidermal growth factor (EGF)], purified NPs with 0.0625% trypsin in 0.04% EDTA (98% of cells were nestin-positive), amplified those progenitors in HGF and G5 media for two months, and then induced oligodendrocyte precursors differentiation in the absence of G5, but in the presence of 20 ng/mL HGF for 2 days. To obtain terminal oligodendrocytes, neurospheres cultured for 2 months were plated on laminin-coated plates for 3 weeks in the presence of HGF. The results showed that differentiated cells expressed myelin basic protein (MBP) and had typical mature oligodendrocyte morphology. Our studies also revealed that HGF significantly increased the NP proliferation speed (P < 0.05) by both decreasing cell apoptosis rate (P < 0.05) and shortening cell cycle time (P < 0.05) in the presence of G5. Additionally, HGF promoted oligodendrocyte maturation by increasing the length and number of branches and the expression of MBP. To test whether the original HGF had similar functions for oligodendrocyte specification, a series of experiments were evaluated by adding HGF or G5 to differentiation or expansion media at different differentiation stages. The results demonstrated that the ability of HGF responsiveness to initiate oligodendrocyte differentiation was regulated by G5 and by HGF alone without G5-induced rES cell differentiation into neurons. Further studies showed that the crucial time point of G5 action was from EBs to NPs; the early addition of HGF to EBs in the presence of G5 increased oligodendrocyte differentiation rate, but was not necessary, and the treatment during the first 2 days was enough to produce a similar effect; and HGF was required for terminal oligodendrocyte differentiation from NPs. Taken together, these results showed that HGF and G5 cooperatively promote rES cell differentiation into highly enriched oligodendrocyte precursors and mature oligodendrocytes.These observations set the method for obtaining highly enriched oligodendrocytes from ES cells in the nonhuman primate for clinical application and provide a platform to probe the molecular mechanisms that control oligodendrocyte differentiation.

Serotonin neurons derived from rhesus monkey embryonic stem cells: similarities to CNS serotonin neurons

We sought an in vitro primate model for serotonin neurons. Rhesus monkey embryonic stem (ES) cell colonies were isolated and differentiated into embryoid bodies (EBs), then transferred to serum-free medium with 1% insulin-transferrin-selenium for 7 days to induce neural precursor cell (NPC) formation. NPCs were cultured in medium with 1% N-2 neural supplement and human fibroblast growth factor 2 (FGF2, 10 ng/ml) for 7 days to stimulate cell proliferation. Lastly, NPCs were dispersed into single cells and cultured without FGF2 for another 7 days to obtain terminal differentiation. Terminal cells were characterized for neuronal and serotonergic markers. Over 95% of the NPCs were immunopositive for nestin and Musashi1. Terminally differentiated cells appeared in both small and large morphologies. Most (>95%) of the mature cells (both small and large) were immunopositive for neuron-specific nuclear protein (NeuN), synaptophysin, microtubule-associated protein (MAP2C), Tau-1, neurofilament 160 (NF-160), beta-tubulin (TujIII), tryptophan hydroxylase (TPH), serotonin, the serotonin reuptake transporter (SERT), estrogen receptor-beta (ERβ), and progestin receptor (PR), but not estrogen receptor-alpha (ERα). Less than 2–3% of cells were positive for tyrosine hydroxylase (TH). Reverse transcriptase polymerase chain reaction (RT-PCR) detected mRNA transcripts for TPH-1, TPH-2, SERT, 5-HT 1A-autoreceptor, ERβ, and PR in the differentiated population. A low level of expression of ERα mRNA was also detected. Quantitative RT-PCR indicated that the relative abundance of TPH-2 mRNA was greater than TPH-1 mRNA. Serotonin as measured by ELISA increased 3-fold in the mature stage compared to the selection and expansion stages. In summary, a remarkably high percentage of cells derived from monkey ES cells exhibited neuronal plus serotonergic markers as well as nuclear steroid receptors similar to primate CNS serotonin neurons, suggesting that these cells may serve as a useful primate model for serotonergic neurons.

Functional endothelial cells derived from rhesus monkey embryonic stem cells

AbstractWe have used rhesus monkey embryonic stem (ES) cells to study endothelial cell development. Rhesus ES cells (R366.4 cell line) exposed to medium containing vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF), and epidermal growth factor (EGF) assumed a relatively uniform endothelial cell morphology and could be propagated and expanded with a consistent phenotype and normal karyotype. When placed in Matrigel, these rhesus ES cell–derived endothelial cells (RESDECs) formed capillary-like structures characteristic of endothelial cells. Immunohistochemical and flow cytometric analysis of RESDECs showed that they take up acetylated low-density lipoprotein (LDL), express CD146, von Willebrand factor, and the integrin αvβ3, and bind the lectin ulex europaeus agglutinin-1. These cells also express the VEGF receptor Flk-1 and secrete VEGF. When introduced in a Matrigel plug implanted subcutaneously in mice, RESDECs formed intact vessels and recruited new endothelial cell growth. In vivo function was demonstrated by coinjection of RESDECs with murine tumor cells subcutaneously into immunocompromised adult mice. RESDECs injected alone did not form measurable tumors. Tumor cells grew more rapidly and had increased vascularization when coinjected with the RESDECs. Immunohistochemical staining demonstrated that the RESDECs participated in forming the tumor neovasculature. RESDECs provide a novel means to examine the mechanisms of endothelial cell development, and may open up new therapeutic strategies.

190FEEDERS FROM RHESUS MONKEY SUPPORT PROLONGED DIFFERENTIATED GROWTH OF RHESUS MONKEY EMBRYONIC STEM CELLS

Previous study demonstrated that feeders are important for growth and differentiation of rhesus monkey embryonic stem cells (rES)(Thomson JA et al., 1995 PNAS \bf 92, 7844–7848). In this study, we developed 5 rhesus monkey feeder cell lines for rES culture: MESF (ear skin fibroblasts from 2-week-old neonatal monkeys), MAF (adult Fallopian tube cells), MGF (adult follicular granulose fibroblast-like cells), MGK (adult follicular granulose kidney-like cells), and MSC (single-cell cloning from MESF), all of which were fibroblast-like cells. Two experiments were designed to investigate rES cell growth and differentiation potentials. In experiment 1, rES (R366.4) was cultured on MESF, MAF, MGF, MGK, and MSC, with MEF (mouse embryonic fibroblasts) as control. In experiment 2, rES was cultured on the mixed feeders: MSC: MGK at 1:1, 7:3, 8:2 and 9:1, depending on the results of experiment 1. Results of experiment 1 showed that the rES underwent undifferentiated growth on MESF, MAF, MGF and MSC (cultured for 15 passages), but not on MGK, with morphology typical of rES on MEFs, and exhibiting positive alkaline phosphatase staining, positive expression of Oct-4 and GAPDH, and negative expression of PAX6, AFP, BMP4 and hCG of rES as well as normal karyotypes. Embryonic bodies (EBs) were formed on Day 7 to 8 from the rES cultured on MESF, MAF, MGF and MSC. The EBs showed positive expression of hCG, AFP and BMP4. Differentiation of neuron, epithelium and muscle cells was observed after 21 days of continuous culture of the rES. The colonies of rES cultured on MESF, MAF, MGF and MSC were higher than on MEF (1.5 to 3 times). Interestingly, expansion speeds and differentiation rates on MSC were less than on MESF. These results also indicated that fibroblast cells, on the one hand, were alone sufficient for supporting prolonged undifferentiated growth of rES, and on the other hand, other mixed cells except fibroblast cells had the ability to promote the growth and differentiation of R366.4 rES. Results of experiment 2 showed that the mixed feeders (MSC:MGK) at 8:2 or 9:1 supported undifferentiated growth of rES but not feeders at 7:3 or 1:1. Our work demonstrated that the rhesus monkey feeders were better able to support undifferentiated growth and maintain differentiation potentials of rES than were MEFs feeders. The rES could grow if the ratio of mixed other cells serving as the feeders exceeded 20%.

Derivation and characterization of monkey embryonic stem cells

Embryonic stem (ES) cell based therapy carries great potential in the treatment of neurodegenerative diseases. However, before clinical application is realized, the safety, efficacy and feasibility of this therapeutic approach must be established in animal models. The rhesus macaque is physiologically and phylogenetically similar to the human, and therefore, is a clinically relevant animal model for biomedical research, especially that focused on neurodegenerative conditions. Undifferentiated monkey ES cells can be maintained in a pluripotent state for many passages, as characterized by a collective repertoire of markers representing embryonic cell surface molecules, enzymes and transcriptional factors. They can also be differentiated into lineage-specific phenotypes of all three embryonic germ layers by epigenetic protocols. For cell-based therapy, however, the quality of ES cells and their progeny must be ensured during the process of ES cell propagation and differentiation. While only a limited number of primate ES cell lines have been studied, it is likely that substantial inter-line variability exists. This implies that diverse ES cell lines may differ in developmental stages, lineage commitment, karyotypic normalcy, gene expression, or differentiation potential. These variables, inherited genetically and/or induced epigenetically, carry obvious complications to therapeutic applications. Our laboratory has characterized and isolated rhesus monkey ES cell lines from in vitro produced blastocysts. All tested cell lines carry the potential to form pluripotent embryoid bodies and nestin-positive progenitor cells. These ES cell progeny can be differentiated into phenotypes representing the endodermal, mesodermal and ectodermal lineages. This review article describes the derivation of monkey ES cell lines, characterization of the undifferentiated phenotype, and their differentiation into lineage-specific, particularly neural, phenotypes. The promises and limitations of primate ES cell-based therapy are also discussed.