Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for investigating the development of early hematopoietic progenitor cells (HPCs). Gene expression in ESCs can be manipulated by several techniques that allow the role for individual molecules in development to be determined. One difficulty is that expression of specific genes often has different phenotypic effects dependent on their temporal expression. This problem can be circumvented by the generation of ESCs that inducibly express a gene of interest using technology such as the doxycycline-inducible transgene system. However, generation of these inducible cell lines is costly and time consuming. Described here is a method for disaggregating ESC-derived embryoid bodies (EBs) into single cell suspensions, retrovirally infecting the cell suspensions, and then reforming the EBs by hanging drop. Downstream differentiation is then evaluated by flow cytometry. Using this protocol, it was demonstrated that exogenous expression of a microRNA gene at the beginning of ESC differentiation blocks HPC generation. However, when expressed in EB derived cells after nascent mesoderm is produced, the microRNA gene enhances hematopoietic differentiation. This method is useful for investigating the role of genes after specific germ layer tissue is derived.     

Differentiation of rhesus embryonic stem cells to neural progenitors and neurons

Embryonic stem (ES) cells are pluripotent cells capable of differentiating into cell lineages derived from all primary germ layers including neural cells. In this study we describe an efficient method for differentiating rhesus monkey ES cells to neural lineages and the subsequent isolation of an enriched population of Nestin and Musashi positive neural progenitor (NP) cells. Upon differentiation, these cells exhibit electrophysiological characteristics resembling cultured primary neurons. Embryoid bodies (EBs) were formed in ES growth medium supplemented with 50% MEDII. After 7 days in suspension culture, EBs were transferred to adherent culture and either differentiated in serum containing medium or expanded in serum free medium. Immunocytochemistry on differentiating cells derived from EBs revealed large networks of MAP-2 and NF200 positive neurons. DAPI staining showed that the center of the MEDII-treated EBs was filled with rosettes. NPs isolated from adherent EB cultures expanded in serum free medium were passaged and maintained in an undifferentiated state by culture in serum free N2 with 50% MEDII and bFGF. Differentiating neurons derived from NPs fired action potentials in response to depolarizing current injection and expressed functional ionotropic receptors for the neurotransmitters glutamate and gamma-aminobutyric acid (GABA). NPs derived in this way could serve as models for cellular replacement therapy in primate models of neurodegenerative disease, a source of neural cells for toxicity and drug testing, and as a model of the developing primate nervous system.     

搅拌式生物反应器培养促进拟胚体的形成及其向心肌细胞分化

目的：摸索搅拌式生物反应器培养小鼠胚胎干细胞（mESC）的最佳条件,建立一种批量制备拟胚体（EB）的方法。方法：研究mESC不同接种密度及生物反应器初始搅拌速度对EB形成的数量和质量的影响,以细菌培养皿中形成的EB为对照,用抗坏血酸诱导其向心肌细胞分化,比较两种培养体系对EB心肌细胞分化潜能的影响,通过免疫荧光染色及RT PCR对ESC来源的心肌细胞进行鉴定。结果：当mESC接种密度为1×105~3×105个/ml,搅拌速度设定为15~30r/min时,搅拌式生物反应器能高效制备出大量相对均一的EB,EB中几乎没有坏死细胞。与细菌培养皿制备的EB相比,生物反应器培养的EB向心肌细胞分化的效率更高,并表达心肌特异性基因。结论：搅拌式生物反应器培养促进EB的形成及其向心肌细胞分化,是一种更为理想的EB培养系统。     

Differentiation of monkey embryonic stem cells into neural lineages

Embryonic stem (ES) cells are self-renewing, pluripotent, and capable of differentiating into all of the cell types found in the adult body. Therefore, they have the potential to replace degenerated or damaged cells, including those in the central nervous system. For ES cell-based therapy to become a clinical reality, translational research involving nonhuman primates is essential. Here, we report monkey ES cell differentiation into embryoid bodies (EBs), neural progenitor cells (NPCs), and committed neural phenotypes. The ES cells were aggregated in hanging drops to form EBs. The EBs were then plated onto adhesive surfaces in a serum-free medium to form NPCs and expanded in serum-free medium containing fibroblast growth factor (FGF)-2 before neural differentiation was induced. Cells were characterized at each step by immunocytochemistry for the presence of specific markers. The majority of cells in complex/cystic EBs expressed antigens (alpha-fetal protein, cardiac troponin I, and vimentin) representative of all three embryonic germ layers. Greater than 70% of the expanded cell populations expressed antigenic markers (nestin and musashi1) for NPCs. After removal of FGF-2, approximately 70% of the NPCs differentiated into neuronal phenotypes expressing either microtubule-associated protein-2C (MAP2C) or neuronal nuclear antigen (NeuN), and approximately 28% differentiated into glial cell types expressing glial fibrillary acidic protein. Small populations of MAP2C/NeuN-positive cells also expressed tyrosine hydroxylase (approximately 4%) or choline acetyltransferase (approximately 13%). These results suggest that monkey ES cells spontaneously differentiate into cells of all three germ layers, can be induced and maintained as NPCs, and can be further differentiated into committed neural lineages, including putative neurons and glial cells.     

Effect of ovarian hormones on survival genes in laser captured serotonin neurons from macaques

We sought the effect of estradiol (E) and progesterone (P) on survival gene expression in laser captured serotonin neurons and in the dorsal raphe region of monkeys with cDNA array analysis. Spayed rhesus macaques were treated with either placebo, E or E + P via Silastic implant for 1 month prior to killing. First, RNA from a small block of midbrain containing the dorsal raphe was hybridized to Rhesus Gene Chips (n = 3/treatment). There was a significant change in 854 probe sets with E +/- P treatment (anova, p < 0.05); however, only 151 probes sets exhibited a twofold or greater change. Twenty-five genes related to cell survival changed significantly. The expression of vascular endothelial growth factor, superoxide dismutase (SOD1), and the caspase inhibitor, BIRC4, was confirmed with quantitative RT-PCR. Then, RNA from laser captured serotonin neurons (n = 2/treatment) was hybridized to Rhesus Gene Chips. There was a significant change in 744 probe sets, but 10 493 probe sets exhibited a twofold or greater change. Pivotal changes in apoptosis and cell cycle pathways included twofold or greater increases in SOD1, IkappaBalpha, Fas apoptotic inhibitory molecule, fibroblast growth factor-receptor 2 (FGFR2), neurotrophic tyrosine kinase receptor 2 (NTRK2), phosphoinositide-3-kinase (p85 subunit), cyclic AMP dependent protein kinase (PKA) (catalytic subunit), calpain 2, and ataxia telangectasia mutated (ATM). Twofold or greater decreases occurred in TNF receptor interacting serine-threonine kinase 1 (RIP1), BH3 interacting domain death agonist (BID), apoptotic peptidase activating factor 1 (Apaf1), caspase recruitment domain 8 (CARD8), apoptosis inducing factor (AIF), Diablo and Cyclins A, B, D, and E. The regulation of SOD1, calpain 2, Diablo, and Cyclin D was confirmed with quantitative RT-PCR (n = 3/treatment). The data indicate that ovarian steroids target the cytokine-signaling pathway, caspase-dependent and -independent pathways and cell cycle proteins to promote serotonin neuron survival.     

Differential expression of mRNAs for PACAP and its receptors during neural differentiation of embryonic stem cells

The expressions of mRNAs for pituitary adenylate cyclase-activating polypeptide (PACAP), vasoactive intestinal peptide (VIP), and their receptors (PAC1, VPAC1 and VPAC2) were examined in the five steps of the in vitro neuronal culture model of embryonic stem (ES) cell differentiation. mRNAs for PACAP, VIP, PAC1 receptor, and VPAC2 receptor were moderately expressed in neural stem cell-enriched cultures, while VPAC1 receptor mRNA was most prominently expressed in embryoid bodies (EBs). The expression of PAC1 receptor mRNA was further upregulated after terminal differentiation into neurons. In contrast, the expressions of PAC1 receptor and PACAP mRNAs were markedly decreased after glial differentiation. These results suggest that this in vitro neuronal culture system will be a useful model for future studies on the functional role of the PACAPergic system during different stages of neuronal development.     

Metabotropic glutamate receptors regulate differentiation of embryonic stem cells into GABAergic neurons

Mouse embryonic stem (ES) cells were stimulated to differentiate either as adherent monolayer cultures in DMEM/F12 supplemented with N2/B27, or as floating embryoid bodies (EBs) exposed to 1 microM retinoic acid (RA) for 4 days, starting from 4 DIV, and subsequently re-plated in DMEM/F12 medium. Adherent monolayer cultures of ES cells expressed mGlu5 receptors throughout the entire differentiation period. Selective pharmacological blockade of mGlu5 receptors with methyl-6-(phenylethynyl)-pyridine (MPEP) (1 microM, added once a day) accelerated the appearance of the neuronal marker, beta-tubulin. In addition, treatment with MPEP increased the number of cells expressing glutamate decarboxylase-65/67 (GAD(65/67)), a marker of GABAergic neurons. In floating EBs, mGlu5 receptors are progressively replaced by mGlu4 receptors. The orthosteric mGlu4/6/7/8 receptor agonist, L-2-amino-4-phosphonobutanoate (L-AP4), or the selective mGlu4 receptor enhancer, PHCCC,--both combined with RA at concentrations of 30 microM--increased the expression of both beta-tubulin and GAD(65/67), inducing the appearance of fully differentiated neurons that released GABA in response to membrane depolarization. We conclude that mGlu receptor subtypes regulate neuronal differentiation of ES cells in a context-dependent manner, and that subtype-selective ligands of these receptors might be used for the optimization of in vitro protocols aimed at producing GABAergic neurons from ES cells.     

Effect of serotonin on the formation of neurons producing gonadotropin-releasing hormone in Wistar rats

The effect of serotonin on the formation of neurons producing gonadotropin-releasing hormone (GnRH) during embryogenesis of Wistar rats was studied. The neurons producing GnRH were detected immunocytochemically on days 18 and 21 of embryonic development and on day 15 of postnatal development of rats with normal serotonin metabolism and rats in which the synthesis of serotonin was inhibited by p-chlorophenylalanine. The total number of GnRH neurons in serotonin deficiency was larger than in the case of its normal metabolism at all developmental stages studied. This is an indirect evidence for the inhibitory effect of serotonin on the formation of GnRH neurons. To confirm the morphogenetic effect of serotonin, we studied the rate of formation of GnRH neurons by injecting bromodeoxyuridine in the formation period of these neurons. It was found that serotonin deficiency had no effect on the time of formation of GnRH neurons: over 97% of neurons formed on days 11 to 15 of embryonic development both in the experimental and control groups. Note that, in serotonin deficiency, the maximum number of GnRH neurons formed one day later than in the normal state. Thus, serotonin inhibits the proliferation of GnRH neuron progenitor cells and thereby has a morphogenetic effect on the development of these neurons.     

The differentiation of hepatocyte-like cells from monkey embryonic stem cells

Embryonic stem cells (ESC) hold great potential for the treatment of liver diseases. Here, we report the differentiation of rhesus macaque ESC along a hepatocyte lineage. The undifferentiated monkey ESC line, ORMES-6, was cultured in an optimal culture condition in an effort to differentiate them into hepatocyte-like cells in vitro. The functional efficacy of the differentiated hepatic cells was evaluated using RT-PCR for the expression of hepatocyte specific genes, and Western blot analysis and immunocytochemistry for hepatic proteins such as alpha-fetoprotein (AFP), albumin and alpha1-antitrypsin (alpha1-AT). Functional assays were performed using the periodic acid schiff (PAS) reaction and ELISA. The final yield of ESC-derived hepatocyte-like cells was measured by flow cytometry for cells that were transduced with a liver-specific lentivirus vector containing the alpha1-AT promoter driving the expression of green fluorescence protein (GFP). The treatment of monkey ESC with an optimal culture condition yielded hepatocyte-like cells that expressed albumin, alpha1-AT, AFP, hepatocyte nuclear factor 3beta, glucose-6-phophatase, and cytochrome P450 genes and proteins as determined by RT-PCR and Western blot analysis. Immunofluorescent staining showed the cells positive for albumin, AFP, and alpha1-AT. PAS staining demonstrated that the differentiated cells showed hepatocyte functional activity. Albumin could be detected in the medium after 20 days of differentiation. Flow cytometry data showed that 6.5 +/- 1.0% of the total differentiated cells were positive for GFP. These results suggest that by using a specific, empirically determined, culture condition, we were able to direct monkey ESC toward a hepatocyte lineage.     

Wnt2 coordinates the commitment of mesoderm to hematopoietic, endothelial, and cardiac lineages in embryoid bodies

Our recent gene expression profiling analyses demonstrated that Wnt2 is highly expressed in Flk1(+) cells, which serve as common progenitors of endothelial cells, blood cells, and mural cells. In this report, we characterize the role of Wnt2 in mesoderm development during embryonic stem (ES) cell differentiation by creating ES cell lines in which Wnt2 was deleted. Wnt2(-/-) embryoid bodies (EBs) generated increased numbers of Flk1(+) cells and blast colony-forming cells compared with wild-type EBs, and had higher Flk1 expression at comparable stages of differentiation. Although Flk1(+) cells were increased, we found that endothelial cell and terminal cardiomyocyte differentiation was impaired, but hematopoietic cell differentiation was enhanced and smooth muscle cell differentiation was unchanged in Wnt2(-/-) EBs. Later stage Wnt2(-/-) EBs had either lower or undetectable expression of endothelial and cardiac genes compared with wild-type EBs. Consistently, vascular plexi were poorly formed and neither beating cardiomyocytes nor alpha-actinin-staining cells were detectable in later stage Wnt2(-/-) EBs. In contrast, hematopoietic cell gene expression was upregulated, and the number of hematopoietic progenitor colonies was significantly enhanced in Wnt2(-/-) EBs. Our data indicate that Wnt2 functions at multiple stages of development during ES cell differentiation and during the commitment and diversification of mesoderm: as a negative regulator for hemangioblast differentiation and hematopoiesis but alternatively as a positive regulator for endothelial and terminal cardiomyocyte differentiation.     

Expression of estrogen receptor-alpha and -beta, glucocorticoid receptor, and progesterone receptor genes in human embryonic stem cells and embryoid bodies

Human embryonic stem cells (hESCs) have the potential to differentiate into various cell types, and the three germ layers in vivo and in vitro. They are therefore useful in transplantation and tissue engineering. Here, we describe the expression patterns of selected steroid receptor mRNAs - estrogen receptor-alpha (ER-alpha), ER-beta, glucocorticoid receptor (GR), and progesterone receptor (PR) - in undifferentiated hESCs and embryoid bodies (EBs) cultured for 2, 4, and 6 d, as assessed by real-time PCR, in order to define the possible influence of steroid hormones on the differentiation of hESCs. These receptor mRNAs were expressed in undifferentiated hESCs and EBs. The expression of PR mRNA only decreased during the differentiation of EBs but not of hESCs. Immunohistochemical analysis gave strong staining of ER-alpha, ER-beta, and GR proteins in the nuclei of hESCs and EBs, whereas PR was not detected. We also examined the potential of these steroid hormones to direct the differentiation of hESCs in vitro. The expression of 11 cell-specific markers representing 3 germ layers and 5 tissue types was used to assess the differentiation of hESCs. We found that certain endodermal marker genes were either only expressed in the estrogen-treated group or their expression was stimulated in that group, suggesting that steroid hormones can control the differentiation of hESCs into various cell types.     

Neurogenic effect of vascular endothelial growth factor during germ layer formation of human embryonic stem cells

Vascular endothelial growth factor (VEGF), a potent mitogen for vascular endothelial cells, has been suggested as a modulator that is involved in neurogenesis as well as angiogenesis. Here, we directly examined the effect of VEGF on neuroectodermal differentiation using human embryonic stem cells (hESCs). VEGF treatment upregulated the expression of neuroectodermal genes (Sox1 and Nestin) during germ layer formation in embryoid bodies (EBs) and efficiently increased the number of neural rosettes expressing both Pax6 and Nestin. The neural progenitors generated from VEGF-treated EBs further differentiated into cells that showed a similar pattern of gene expression observed in the development of dopaminergic neurons upon terminal differentiation. These results support the neurogenic effect of VEGF on hESC differentiation.     

Generation of hybrid hepatocytes by cell fusion from monkey embryoid body cells in the injured mouse liver

Monkey embryonic stem (ES) cells have characteristics that are similar to human ES cells, and might be useful as a substitute model for preclinical research. When embryoid bodies (EBs) formed from monkey ES cells were cultured, expression of many hepatocyte-related genes including cytochrome P450 (Cyp) 3a and Cyp7a1 was observed. Hepatocytes were immunocytochemically observed using antibodies against albumin (ALB), cytokeratin-8/18, and α1-antitrypsin in the developing EBs. The in vitro differentiation potential of monkey ES cells into the hepatic lineage prompted us to examine the transplantability of monkey EB cells. As an initial approach to assess the repopulation potential, we transplanted EB cells into immunodeficient urokinase-type plasminogen activator transgenic mice that undergo liver failure. After transplantation, the hepatocyte colonies expressing monkey ALB were observed in the mouse liver. Fluorescence in-situ hybridization revealed that the repopulating hepatocytes arise from cell fusion between transplanted monkey EB cells and recipient mouse hepatocytes. In contrast, neither cell fusion nor repopulation of hepatocytes was observed in the recipient liver after undifferentiated ES cell transplantation. These results indicate that the differentiated cells in developing monkey EBs, but not contaminating ES cells, generate functional hepatocytes by cell fusion with recipient mouse hepatocytes, and repopulate injured mouse liver.