The differentiation of embryonic stem cells (ESCs) into neurons and glial cells represents a promising cell-based therapy for neurodegenerative diseases. Because the rhesus macaque is physiologically and phylogenetically similar to humans, it is a clinically relevant animal model for ESC research. In this study, the pluripotency and neural differentiation potential of a rhesus monkey ESC line (ORMES6) was investigated. ORMES6 was derived from an in vitro produced blastocyst, which is the same way human ESCs have been derived. ORMES6 stably expressed the embryonic transcription factors POU5F1 (Oct4), Sox2 and NANOG. Stage-specific embryonic antigen 4 (SSEA 4) and the glycoproteins TRA-1-60 and TRA-1-81 were also expressed. The embryoid bodies (EBs) formed from ORMES6 ESCs spontaneously gave rise to cells of three germ layers. After exposure to basic fibroblast growth factor (bFGF) for 14–16 days, columnar rosette cells formed in the EB outgrowths. Sox2, microtubule-associated protein (MAP2), β-tublinIII and glial fibrillary acidic protein (GFAP) genes and Nestin, FoxD3, Pax6 and β-tublinIII antigens were expressed in the rosette cells. Oct4 and NANOG expression were remarkably down-regulated in these cells. After removal of bFGF from the medium, the rosette cells differentiated along neural lineages. The differentiated cells expressed MAP2, β-tublinIII, Neuro D and GFAP genes. Most differentiated cells expressed early neuron-specific antigen β-tublinIII (73±4.7%) and some expressed intermediate neuron antigen MAP2 (18±7.2%). However, some differentiated cells expressed the glial cell antigens A2B5 (7.17%±1.2%), GFAP (4.93±1.9%), S100 (7±3.5%) and O4 (0.2±70.2%). The rosette cells were transplanted into the striatum of immune-deficient NIHIII mice. The cells persisted for approximately 2 weeks and expressed Ki67, NeuN, MAP2 and GFAP. These results demonstrate that the rhesus monkey ESC line ORMES6 retains the pluripotent characteristics of ESCs and can be efficiently induced to differentiate along neural lineages.
Embryonic stem cell; Rhesus monkey; Neurogenesis; Neural lineage differentiation
Totipotent cells in early embryos are progenitors of all stem cells and are capable of developing into a whole organism, including extraembryonic tissues such as placenta. Pluripotent cells in the inner cell mass (ICM) are the descendants of totipotent cells and can differentiate into any cell type of a body except extraembryonic tissues. The ability to contribute to chimeric animals upon reintroduction into host embryos is the key feature of murine totipotent and pluripotent cells. Here, we demonstrate that rhesus monkey embryonic stem cells (ESCs) and isolated ICMs fail to incorporate into host embryos and develop into chimeras. However, chimeric offspring were produced following aggregation of totipotent cells of the 4-cell embryos. These results provide insights into the species-specific nature of primate embryos and suggest that a chimera assay using pluripotent cells may not be feasible.
During preimplantation development, the embryo must establish totipotency and enact the earliest differentiation choices, processes that involve extensive chromatin modification. To identify novel developmental regulators, we screened for genes that are preferentially transcribed in the pluripotent inner cell mass (ICM) of the mouse blastocyst. Genes that encode chromatin remodeling factors were prominently represented in the ICM, including Chd1l, a member of the Snf2 gene family. Chd1l is developmentally regulated and expressed in embryonic stem (ES) cells, but its role in development has not been investigated. Here we show that inhibiting Chd1l protein production by microinjection of antisense morpholinos causes arrest prior to the blastocyst stage. Despite this important function in vivo, Chd1l is non-essential for cultured ES cell survival, pluripotency, or differentiation, suggesting that Chd1l is vital for events in embryos that are distinct from events in ES cells. Our data reveal a novel role for the chromatin remodeling factor Chd1l in the earliest cell divisions of mammalian development.
Chd1l; ALC1; Preimplantation development; ICM; ES cells; Chromatin remodeling
Monoparental parthenotes represent a potential source of histocompatible stem cells that should be isogenic with the oocyte donor and therefore suitable for use in cell or tissue replacement therapy. We generated five rhesus monkey parthenogenetic embryonic stem cell (PESC) lines with stable, diploid female karyotypes that were morphologically indistinguishable from biparental controls, expressed key pluripotent markers, and generated cell derivatives representative of all three germ layers following in vivo and in vitro differentiation. Interestingly, high levels of heterozygosity were observed at the majority of loci that were polymorphic in the oocyte donors. Some PESC lines were also heterozygous in the major histocompatibility complex region, carrying haplotypes identical to those of the egg donor females. Expression analysis revealed transcripts from some imprinted genes that are normally expressed from only the paternal allele. These results indicate that limitations accompanying the potential use of PESC-derived phenotypes in regenerative medicine, including aberrant genomic imprinting and high levels of homozygosity, are cell line-dependent and not always present. PESC lines were derived in high enough yields to be practicable, and their derivatives are suitable for autologous transplantation into oocyte donors or could be used to establish a bank of histocompatible cell lines for a broad spectrum of patients.
Embryonic stem cells; Parthenogenetic; Meiotic recombination; Imprinting; Histocompatible
Parthenogenetic embryonic stem cells (PESCs) may have future utilities in cell replacement therapies since they are closely related to the female from which the activated oocyte was obtained. Furthermore, the avoidance of parthenogenetic development in mammals provides the most compelling rationale for the evolution of genomic imprinting, and the biological process of parthenogenesis raises complex issues regarding differential gene expression.
METHODS AND RESULTS
We describe here homozygous rhesus monkey PESCs derived from a spontaneously duplicated, haploid oocyte genome. Since the effect of homozygosity on PESCs pluripotency and differentiation potential is unknown, we assessed the similarities and differences in pluripotency markers and developmental potential by in vitro and in vivo differentiation of homozygous and heterozygous PESCs. To understand the differences in gene expression regulation between parthenogenetic and biparental embryonic stem cells (ESCs), we conducted microarray analysis of genome-wide mRNA profiles of primate PESCs and ESCs derived from fertilized embryos using the Affymetrix Rhesus Macaque Genome array. Several known paternally imprinted genes were in the highly down-regulated group in PESCs compared with ESCs. Furthermore, allele-specific expression analysis of other genes whose expression is also down-regulated in PESCs, led to the identification of one novel imprinted gene, inositol polyphosphate-5-phosphatase F (INPP5F), which was exclusively expressed from a paternal allele.
Our findings suggest that PESCs could be used as a model for studying genomic imprinting, and in the discovery of novel imprinted genes.
pluripotent stem cells; parthenogenesis; imprinting; homozygosity
BACKGROUND: Embryonic stem (ES) cells are lines of cells that are isolated from blastocysts. The murine ES cells were demonstrated to be true pluripotent cells as they differentiate into all embryonic lineages. Yet, in vitro differentiation of rhesus ES cells was somewhat inconsistent and disorganized. The recent isolation of human ES cells calls for exploring their pluripotential nature. MATERIALS AND METHODS: Human ES cells were grown in suspension to induce their differentiation into embryoid bodies (EBs). The differentiation status of the human ES cells and EBs was analyzed by following the expression pattern of several lineage-specific molecular markers using reverse transcription polymerase chain reaction (RT-PCR) and in situ hybridization. RESULTS: Here we report the induction in vitro of cystic embryoid bodies from human ES cells. Our findings demonstrate induction of expression of cell-specific genes during differentiation of the human ES cells into EBs. In the human EBs, we could show a characteristic regional expression of embryonic markers specific to different cellular lineages, namely, zeta-globin (mesoderm), neurofilament 68Kd (ectoderm), and alpha-fetoprotein (endoderm). Moreover, we present a synchronously pulsing embryoid body that expresses the myocardium marker alpha-cardiac actin. In addition, dissociating the embryoid bodies and plating the cells as monolayers results in multiple morphologies, among them cells with neuronal appearance that express neurofilament 68Kd chain. CONCLUSION: Human ES cells can reproducibly differentiate in vitro into EBs comprising the three embryonic germ layers. The ability to induce formation of human embryoid bodies that contain cells of neuronal, hematopoietic and cardiac origins will be useful in studying early human embryonic development as well as in transplantation medicine.
We recently demonstrated that somatic cells from adult primates could be reprogrammed into a pluripotent state by somatic cell nuclear transfer. However, the low efficiency with donor cells from one monkey necessitated the need for large oocyte numbers. Here, we demonstrate nearly threefold higher blastocyst development and embryonic stem (ES) cell derivation rates with different nuclear donor cells. Two ES cell lines were isolated using adult female rhesus macaque skin fibroblasts as nuclear donors and oocytes retrieved from one female, following a single controlled ovarian stimulation. In addition to routine pluripotency tests involving in vitro and in vivo differentiation into various somatic cell types, primate ES cells derived from reprogrammed somatic cells were also capable of contributing to cells expressing markers of germ cells. Moreover, imprinted gene expression, methylation, telomere length, and X-inactivation analyses were consistent with accurate and extensive epigenetic reprogramming of somatic cells by oocyte-specific factors.
Reprogramming; Nuclear transfer; Embryonic stem cells; Primates
The timing of the origins of fetal alcohol syndrome have been difficult to determine, in part because of the challenge associated with in vivo studies of the peri-implantation stage of embryonic development. Because embryonic stem cells (ESCs) are derived from blastocyst stage embryos, they are used as a model for early embryo development.
Rhesus monkey ESC lines (ORMES-6 and -7) were treated with 0, 0.01%, 0.1%, or 1.0% ethanol, 1.0% ethanol with estradiol or 0.00025% acetaldehyde with or without estradiol for 4 weeks.
Although control ESCs remained unchanged, abnormal morphology of ESCs in the ethanol and acetaldehyde treatment groups was observed before 2 weeks of treatment. Immunofluorescence staining of key pluripotency markers ( TRA-1- 81 and alkaline phosphatase) indicated a loss of ESC pluripotency in the 1.0% ethanol group. ORMES-7 was more sensitive to effects of ethanol than ORMES-6.
Estradiol appeared to increase sensitivity to ethanol in the ORMES-6 and -7 cell line. The morphological changes and labeling for pluripotency, proliferation and apoptosis demonstrated that ethanol affects how these early cells develop in culture, their differentiation state in particular. The effects of ethanol may be mediated in part through metabolic pathways regulating acetaldehyde formation, and while potentially accentuated by estradiol in some individuals, how remains to be determined.
Primate; fetal alcohol syndrome; embryo development
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 α-fetoprotein (AFP), albumin and α1-antitrypsin (α1-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 α1-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, α1-AT, AFP, hepatocyte nuclear factor 3β, 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 α1-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.
Sox2 is a key gene that controls transcriptional networks required for pluripotency. The role of Sox2 in the developmental transition of a highly differentiated oocyte to totipotent blastomeres of the early preimplantation embryo, however, is not known. We report that Sox2, which is localized in the nucleus, is first zygotically expressed during the 2-cell stage and that its expression dramatically increases between the morula and blastocyst stages. Injecting a cRNA encoding Sox2 into 1-cell embryos resulted in overexpression of SOX2 by approximately 70% and developmental arrest at the 2-cell stage, whereas injecting cRNAs encoding Pou5f1, Myc (also known as c-Myc), or Klf4 has little effect on the ability of 2-cell embryos to cleave to the 4-cell stage. Global transcription assessed by bromo uridine triphosphate incorporation is reduced by approximately 15%, and transcript profiling revealed that approximately 15% of zygotically expressed genes are dramatically repressed in 2-cell embryos overexpressing SOX2. Furthermore, overexpressing a dominant-negative SOX2 perturbs reprogramming of gene expression in 2-cell embryos, though to a much lesser extent than that observed following overexpression of SOX2, and leads to developmental failure after the 2-cell stage but before the 8-cell stage. Results of these experiments implicate Sox2 as a critical transcriptional regulator in the oocyte-to-embryo transition that entails formation of totipotent blastomeres and indicate that the amount of Sox2 is critical for successful execution of this transition.
Overexpression of SOX2 or a dominant-negative form inhibits embryo development beyond the 2-cell stage by perturbing the reprogramming of gene expression during genome activation that is necessary for continued development.
chromatin; early development; embryo; gene expression; transcriptional regulation
The genetic mechanisms governing human pre-implantation embryo development and the in vitro counterparts, human embryonic stem cells (hESCs), still remain incomplete. Previous global genome studies demonstrated that totipotent blastomeres from day-3 human embryos and pluripotent inner cell masses (ICMs) from blastocysts, display unique and differing transcriptomes. Nevertheless, comparative gene expression analysis has revealed that no significant differences exist between hESCs derived from blastomeres versus those obtained from ICMs, suggesting that pluripotent hESCs involve a new developmental progression. To understand early human stages evolution, we developed an undifferentiation network signature (UNS) and applied it to a differential gene expression profile between single blastomeres from day-3 embryos, ICMs and hESCs. This allowed us to establish a unique signature composed of highly interconnected genes characteristic of totipotency (61 genes), in vivo pluripotency (20 genes), and in vitro pluripotency (107 genes), and which are also proprietary according to functional analysis. This systems biology approach has led to an improved understanding of the molecular and signaling processes governing human pre-implantation embryo development, as well as enabling us to comprehend how hESCs might adapt to in vitro culture conditions.
Pluripotent stem cells produce tissue-specific lineages through programmed acquisition of sequential gene expression patterns that function as a blueprint for organ formation. As embryonic stem cells respond concomitantly to diverse signaling pathways during differentiation, extraction of a pro-cardiogenic network would offer a roadmap to streamline cardiac progenitor output.
Methods and Results
To resolve gene ontology priorities within precursor transcriptomes, cardiogenic subpopulations were here generated according to either growth factor guidance or stage-specific biomarker sorting. Innate expression profiles were independently delineated through unbiased systems biology mapping, and cross-referenced to filter transcriptional noise unmasking a conserved progenitor motif (55 up- and 233 down-regulated genes). The streamlined pool of 288 genes organized into a core biological network that prioritized the “Cardiovascular Development” function. Recursive in silico deconvolution of the cardiogenic neighborhood and associated canonical signaling pathways identified a combination of integrated axes, CXCR4/SDF-1, Flk-1/VEGF and BMP2r/BMP2, predicted to synchronize cardiac specification. In vitro targeting of the resolved triad in embryoid bodies accelerated expression of Nkx2.5, Mef2C and cardiac-MHC, enhanced beating activity, and augmented cardiogenic yield.
Transcriptome-wide dissection of a conserved progenitor profile thus revealed functional highways that coordinate cardiogenic maturation from a pluripotent ground state. Validating the bioinformatics algorithm established a strategy to rationally modulate cell fate, and optimize stem cell-derived cardiogenesis.
By sequentially applying sonic hedgehog (C25II) and CHIR99021 (GSK3β inhibitor) to induce the midbrain floor plate progenitors and fibroblast growth factor 8 (FGF8) to promote dopaminergic differentiation in a chemically defined medium, we have established a robust system for generation of midbrain dopamine (DA) neurons from human and rhesus monkey embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). We found that CHIR99021 specifies diencephalon to hindbrain fates in a concentration-dependent manner and only a narrow concentration range of CHIR99021 at a particular window is necessary to induce the midbrain floor plate progenitors, expressing Corin, En1, FoxA2 and Lmx1a. FGF8 enhances the dopaminergic fate of the progenitors, thus generating DA neurons with midbrain characteristics, including expression of TH, Lmx1a/b, FoxA2, FoxP1, Nurr1 and En1 as well as typical electrophysiological properties. More than half of these DA neurons expressed A9 DA neuron markers Girk2 and ALDH1a1. The new strategy will allow generation of enriched populations of functional midbrain DA neurons from both human and monkey PSCs for disease modeling, drug testing, and potential cell therapy.
Parkinson’s disease; drug discovery; neural patterning; transplantation
Mammalian embryos at the blastocyst stage have three major lineages, which in culture can give rise to embryonic stem (ES) cells from the inner cell mass or epiblast, trophoblast stem cells from the trophectoderm, and primitive endoderm stem cells. None of these stem cells is totipotent, because they show gene expression profiles characteristic of their sources and usually contribute only to the lineages of their origins in chimeric embryos. It is unknown whether embryos prior to the blastocyst stage can be cultivated towards totipotent stem cell cultures. Medaka is an excellent model for stem cell research. This laboratory fish has generated diploid and even haploid ES cells from the midblastula embryo with ~2000 cells. Here we report in medaka that dispersed cells from earlier embryos can survive, proliferate and attach in culture. We show that even 32-cells embryos can be dissociated into individual cells capable of producing continuously growing ES-like cultures. Our data point to the possibility to derive stable cell culture from cleavage embryos in this organism.
cleavage; morula; blastula; stem cell culture; totipotency
Pluripotency, a characteristic of cells in the inner cell mass of the mammalian preimplantation blastocyst as well as of embryonic stem cells, is defined as the ability of a cell to generate all of the cell types of an organism. A group of transcription factors is essential for the establishment and maintenance of the pluripotent state. Recent studies have demonstrated that differentiated somatic cells could be reverted to a pluripotent state by the overexpression of a set of transcription factors, further highlighting the significance of transcription factors in the control of pluripotency. Among these factors, a member of the POU transcription factor family, Oct4, is central to the machinery governing pluripotency. Oct4 is highly expressed in pluripotent cells and becomes silenced upon differentiation. Interestingly, the precise expression level of Oct4 determines the fate of embryonic stem cells. Therefore, to control the expression of Oct4 precisely, a variety of regulators function at multiple levels, including transcription, translation of mRNA and post-translational modification. Additionally, in cooperation with Sox2, Nanog and other members of the core transcriptional regulatory circuitry, Oct4 activates both protein-coding genes and noncoding RNAs necessary for pluripotency. Simultaneously, in association with transcriptional repressive complexes, Oct4 represses another set of targets involved in developmental processes. Importantly, Oct4 can re-establish pluripotency in somatic cells, and proper reprogramming of Oct4 expression is indispensable for deriving genuine induced pluripotent stem cell lines. In the past several years, genome-wide identification of Oct4 target genes and Oct4-centered protein interactomes has been reported, indicating that Oct4 exerts tight control over pluripotency regulator expression and protects embryonic stem cells in an undifferentiated state. Nevertheless, further investigation is required to fully elucidate the underlying molecular mechanisms through which Oct4 maintains and reinitiates pluripotency. Systemic and dynamic exploration of the protein complexes and target genes associated with Oct4 will help to elucidate the role of Oct4 more comprehensively.
In mammals, the first cell fate decision is initialized by cell polarization at the 8- to 16-cell stage of the preimplantation embryo. At this stage, outside cells adopt a trophectoderm (TE) fate, whereas the inside cell population gives rise to the inner cell mass (ICM). Prior to implantation, transcriptional interaction networks and epigenetic modifications divide the extraembryonic and embryonic fate irrevocably. Here, we report that extraembryonic trophoblast stem cell (TSC) lines are converted to induced pluripotent stem cells (TSC-iPSCs) by overexpressing Oct4, Sox2, Klf4, and cMyc. Methylation studies and gene array analyses indicated that TSC-iPSCs had adopted a pluripotent potential. The rate of conversion was lower than those of somatic reprogramming experiments, probably due to the unique genetic network controlling extraembryonic lineage fixation. Both in vitro and in vivo, TSC-iPSCs differentiated into tissues representing all three embryonic germ layers, indicating that somatic cell fate could be induced. Finally, TSC-iPSCs chimerized the embryo proper and contributed to the germ line of mice, indicating that these cells had acquired full somatic differentiation potential. These results lead to a better understanding of the molecular processes that govern the first lineage decision in mammals.
The first week of human pre-embryo development is characterized by the induction of totipotency and then pluripotency. The understanding of this delicate process will have far reaching implication for in vitro fertilization and regenerative medicine. Human mature MII oocytes and embryonic stem (ES) cells are both able to achieve the feat of cell reprogramming towards pluripotency, either by somatic cell nuclear transfer or by cell fusion, respectively. Comparison of the transcriptome of these two cell types may highlight genes that are involved in pluripotency initiation.
Based on a microarray compendium of 205 samples, we compared the gene expression profile of mature MII oocytes and human ES cells (hESC) to that of somatic tissues. We identified a common oocyte/hESC gene expression profile, which included a strong cell cycle signature, genes associated with pluripotency such as LIN28 and TDGF1, a large chromatin remodelling network (TOP2A, DNMT3B, JARID2, SMARCA5, CBX1, CBX5), 18 different zinc finger transcription factors, including ZNF84, and several still poorly annotated genes such as KLHL7, MRS2, or the Selenophosphate synthetase 1 (SEPHS1). Interestingly, a large set of genes was also found to code for proteins involved in the ubiquitination and proteasome pathway. Upon hESC differentiation into embryoid bodies, the transcription of this pathway declined. In vitro, we observed a selective sensitivity of hESC to the inhibition of the activity of the proteasome.
These results shed light on the gene networks that are concurrently overexpressed by the two human cell types with somatic cell reprogramming properties.
The mammalian totipotent and pluripotent lineage exhibits genome-wide dynamics in respect to DNA methylation content. The first phase of global DNA demethylation and de novo remethylation occurs during preimplantation development and gastrulation, respectively, while the second phase occurs in primordial germ cells and primary oocytes/prospermatogonia, respectively. These dynamics are indicative of a comprehensive epigenetic resetting or reprogramming of the genome in preparation for major differentiation events. To gain further insight into the mechanisms driving DNA methylation dynamics and other types of epigenetic modification, we performed an RNA expression microarray analysis of fetal prospermatogonia at the stage when they are undergoing rapid de novo DNA remethylation. We have identified a number of highly or specifically expressed genes which could be important for determining epigenetic change in prospermatogonia. These data provide a useful resource in the discovery of molecular pathways involved in epigenetic reprogramming in the mammalian germ line.
epigenetics; DNA methylation; prospermatogonium; oogonium; pachytene spermatocyte; microarray
Induced pluripotent Huntington's disease monkey stem cells (rHD-iPSCs) were established by the overexpression of rhesus macaque transcription factors (Oct4, Sox2, and Klf4) in transgenic Huntington's monkey skin fibroblasts. The rHD-iPSCs were pluripotent and capable of differentiating into neuronal cell types in vitro and developed teratoma in immune compromised mice. We also demonstrated the upregulation of endogenous Oct4 and Sox2 after successful reprogramming to pluripotency in rHD-iPSCs, which was not expressed in skin fibroblasts. rHD-iPSCs also developed cellular features comparable to Huntington's disease (HD), including the accumulation of mutant huntingtin (htt) aggregate and the formation of intranuclear inclusions (NIs) paralleling neural differentiation in vitro. Induced pluripotent stem cells from transgenic HD monkeys open a new era of nonhuman primate modeling of human diseases. rHD-iPSCs that develop key HD cellular features and parallel neural differentiation can be a powerful platform for investigating the developmental impact on HD pathogenesis and developing new therapies, which can be evaluated in HD monkeys from whom the rHD-iPSCs were derived.
The interplay of transcription factors and epigenetic modifiers, including histone modifications, DNA methylation and microRNAs during development is essential for the acquisition of specific cell fates. Here we review the epigenetic “programming” of stem cells into oligodendrocytes, by analyzing three sequential stages of lineage progression. The first transition from pluripotent stem cell to neural precursor is characterized by repression of pluripotency genes and restriction of the lineage potential to the neural fate. The second transition from multipotential precursor to oligodendrocyte progenitor is associated with the progressive loss of plasticity and the repression of neuronal and astrocytic genes. The last step of differentiation of oligodendrocyte progenitors into myelin-forming cells is defined by a model of de-repression of myelin genes.
The rhesus monkey embryonic stem cell line 366.4 differentiates into serotonin neurons. We examined the genetic cascade during differentiation and compared ESC-derived serotonin neurons to adult monkey serotonin neurons. RNA was extracted from ESC colonies, embryoid bodies (EBs), neurospheres in selection (N1) and proliferation stages (N2), differentiated serotonin neurons (N3) and from laser captured (LC) serotonin neurons of spayed female macaques treated with placebo, estrogen (E), progesterone (P) or E+P. The RNA was labeled and hybridized to Rhesus Monkey Affymetrix Gene Chips (n=1 per stage and 2 per animal treatment). Gene expression was examined with GeneSifter software. 545 genes that were related to developmental processes showed a 3-fold or greater change between stages. TGFβ, Wnt, VEGF and Hedgehog signaling pathways showed the highest percent of probe set changes during differentiation. Genes in the categories (a) homeobox binding and transcription factors, (b) growth factors and receptors, (c) brain and neural specific factors and (d) serotonin specific factors are reported. Pivotal genes were confirmed with quantitative RT-PCR. In the serotonin developmental cascade, FGFR2 was robustly expressed at each stage. GATA3 was robustly expressed in EBs. Sonic hedgehog (Shh), PTCH (Shh-R), and Fev1 transcription factor expression coincided with the induction of serotonin specific marker genes during N1-selection. A majority of the examined genes were expressed in adult serotonin neurons. However, in the ESC-derived neurons, there was significant over-representation of probe sets related to cell cycle, axon guidance & dorso-ventral axis formation. This analysis suggests that the 366.4 cell line possesses cues for serotonin differentiation at early stages of differentiation, but that ESC-derived serotonin neurons are still immature.
embryonic stem cells; development; serotonin; macaques; Affymetrix array; gene expression; dorsal raphe
The trophoblast cell lineage is specified early at the blastocyst stage, leading to the emergence of the trophectoderm and the pluripotent cells of the inner cell mass. Using a double mRNA amplification technique and a comparison with transcriptome data on pluripotent stem cells, placenta, germinal and adult tissues, we report here some essential molecular features of the human mural trophectoderm. In addition to genes known for their role in placenta (CGA, PGF, ALPPL2 and ABCG2), human trophectoderm also strongly expressed Laminins, such as LAMA1, and the GAGE Cancer/Testis genes. The very high level of ABCG2 expression in trophectoderm, 7.9-fold higher than in placenta, suggests a major role of this gene in shielding the very early embryo from xenobiotics. Several genes, including CCKBR and DNMT3L, were specifically up-regulated only in trophectoderm, indicating that the trophoblast cell lineage shares with the germinal lineage a transient burst of DNMT3L expression. A trophectoderm core transcriptional regulatory circuitry formed by 13 tightly interconnected transcription factors (CEBPA, GATA2, GATA3, GCM1, KLF5, MAFK, MSX2, MXD1, PPARD, PPARG, PPP1R13L, TFAP2C and TP63), was found to be induced in trophectoderm and maintained in placenta. The induction of this network could be recapitulated in an in vitro trophoblast differentiation model.
Electronic supplementary material
The online version of this article (doi:10.1007/s12015-011-9301-3) contains supplementary material, which is available to authorized users.
Pluripotent stem cells; Transcription factor; Transcriptional network; Transcriptome; Trophectoderm
Pluripotent stem cells that are capable of differentiating into different cell types and develop robust hallmark cellular features are useful tools for clarifying the impact of developmental events on neurodegenerative diseases such as Huntington's disease. Additionally, a Huntington's cell model that develops robust pathological features of Huntington's disease would be valuable for drug discovery research.
To test this hypothesis, a pluripotent Huntington's disease monkey hybrid cell line (TrES1) was established from a tetraploid Huntington's disease monkey blastocyst generated by the fusion of transgenic Huntington's monkey skin fibroblast and a wild-type non-transgenic monkey oocyte. The TrES1 developed key Huntington's disease cellular pathological features that paralleled neural development. It expressed mutant huntingtin and stem cell markers, was capable of differentiating to neural cells, and developed teratoma in severely compromised immune deficient (SCID) mice. Interestingly, the expression of mutant htt, the accumulation of oligomeric mutant htt and the formation of intranuclear inclusions paralleled neural development in vitro , and even mutant htt was ubiquitously expressed. This suggests the development of Huntington's disease cellular features is influenced by neural developmental events.
Huntington's disease cellular features is influenced by neural developmental events. These results are the first to demonstrate that a pluripotent stem cell line is able to mimic Huntington's disease progression that parallels neural development, which could be a useful cell model for investigating the developmental impact on Huntington's disease pathogenesis.
The generation of induced pluripotent stem cells (iPSCs) opens up the possibility for personalized cell therapy. Here, we show that transplanted autologous rhesus monkey iPSC-derived neural progenitors survive for up to 6 months and differentiate into neurons, astrocytes, and myelinating oligodendrocytes in the brains of MPTP-induced hemiparkinsonian rhesus monkeys with a minimal presence of inflammatory cells and reactive glia. This finding represents a significant step toward personalized regenerative therapies.
Mammalian preimplantation embryos provide an excellent opportunity to study temporal and spatial gene expression in whole mount in situ hybridization (WISH). However, large-scale studies are made difficult by the size of the embryos (∼60 μm diameter) and their fragility. We have developed a chamber system that allows parallel processing of embryos without the aid of a microscope. We first selected 91 candidate genes that were transcription factors highly expressed in blastocysts, and more highly expressed in embryonic (ES) than in trophoblast (TS) stem cells. We then used the WISH to identify 48 genes expressed predominantly in the ICM and to follow several of these genes in all seven preimplantation stages. The ICM-predominant expressions of these genes suggest their involvement in the pluripotency of embryonic cells. This system provides a useful tool to a systematic genome-scale analysis of preimplantation embryos.
preimplantation embryo; whole mount in situ hybridization; High-throughput screen; hybridization chamber; ICM; TE