Octamer binding proteins confer transcriptional activity in early mouse embryogenesis.

Oct4 and Oct5 are two mouse maternally expressed proteins binding to the octamer motif. Both are found in unfertilized oocytes and embryonic stem cells, whereas Oct4 is also found in primordial germ cells. In this study, the activity of the octamer motif was analysed in two embryonic stem cell lines containing Oct4 and Oct5, the teratocarcinoma-derived cell line F9 and the blastocyst-derived cell line D3. It is known that oligomerization of the octamer motif creates a powerful B-cell specific enhancer. As shown here, this oligomerized transcriptional element is also a very strong enhancer in F9 and D3 embryonic stem cells. After differentiation of the stem cells, both enhancer activity and the amount of the octamer binding proteins decrease. An intact octamer stimulates heterologous promoters in embryonic stem cells, whereas mutations in the octamer motif abolish transcriptional stimulation and binding of the octamer factors. The use of transgenic embryos demonstrates transcriptional activation in the inner cell mass but not in the trophoblast of blastocysts. The results indicate that Oct4 and Oct5 are active early in mouse development

A family of octamer-specific proteins present during mouse embryogenesis: Evidence for germline-specific expression of an Oct factor.

We have analysed various adult organs and different developmental stages of mouse embryos for the presence of octamer-binding proteins. A variety of new octamer-binding proteins were identified in addition to the previously described Oct1 and Oct2. Oct1 is ubiquitously present in murine tissues, in agreement with cell culture data. Although Oct2 has been described as a B-cell-specific protein, similar complexes were also found with extracts from brain, kidney, embryo and sperm. In embryo and brain at least two other proteins, Oct3 and Oct7, are present. A new microextraction procedure allowed the detection of two maternally expressed octamer-binding proteins, Oct4 and Oct5. Both proteins are present in unfertilized oocytes and embryonic stem cells, the latter containing an additional protein, Oct6. Whereas Oct4 was not found in sperm or testis, it is expressed in male and female primordial germ cells. Therefore Oct4 expression is specific for the female germline at later stages of germ cell development. Our results indicate that a family of octamer-binding proteins is present during mouse development and is differentially expressed during early embryogenesis. Protease clipping experiments of Oct4 and Oct1 suggest that both proteins contain similar DNA-binding domains

Effects of erythropoietin in murine-induced pluripotent cell-derived panneural progenitor cells

Induced cell fate changes by reprogramming of somatic cells offers an efficient strategy to generate autologous pluripotent stem (iPS) cells from any adult cell type. The potential of iPS cells to differentiate into various cell types is well established, however the efficiency to produce functional neurons from iPS cells remains modest. Here, we generated panneural progenitor cells (pNPCs) from mouse iPS cells and investigated the effect of the neurotrophic growth factor erythropoietin (EPO) on their survival, proliferation and neurodifferentiation. Under neural differentiation conditions, iPS-derived pNPCs gave rise to microtubule-associated protein-2 positive neuronlike cells (34% to 43%) and platelet-derived growth factor receptor positive oligodendrocytelike cells (21% to 25%) while less than 1% of the cells expressed the astrocytic marker glial fibrillary acidic protein. Neuronlike cells generated action potentials and developed active presynaptic terminals. The pNPCs expressed EPO receptor (EPOR) mRNA and displayed functional EPOR signaling. In proliferating cultures, EPO (0.1–3 U/mL) slightly improved pNPC survival but reduced cell proliferation and neurosphere formation in a concentration-dependent manner. In differentiating cultures EPO facilitated neurodifferentiation as assessed by the increased number of β-III-tubulin positive neurons. Our results show that EPO inhibits iPS pNPC self-renewal and promotes neurogenesis

Natural formation of chloro- and bromoacetone in salt lakes of Western Australia

Western Australia is a semi-/arid region known for saline lakes with a wide range of geochemical parameters (pH 2.5-7.1, Cl- 10-200 g L-1. This study reports on the haloacetones chloro- and bromoacetone in air over 6 salt lake shorelines. Significant emissions of chloroacetone (up to 0.2 µmol m-2 h-1) and bromoacetone (up to 1. 5 µmol m-2 h-1) were detected, and a photochemical box model was employed to evaluate the contribution of their atmospheric formation from the olefinic hydrocarbons propene and methacrolein in the gas phase. The measured concentrations could not explain the photochemical halogenation reaction, indicating a strong hitherto unknown source of haloacetones. Aqueous-phase reactions of haloacetones, investigated in the laboratory using humic acid in concentrated salt solutions, were identified as alternative formation pathway by liquid-phase reactions, acid catalyzed enolization of ketones, and subsequent halogenation. In order to verify this mechanism, we made measurements of the Henry's law constants, rate constants for hydrolysis and nucleophilic exchange with chloride, UV-spectra and quantum yields for the photolysis of bromoacetone and 1,1-dibromoacetone in the aqueous phase. We suggest that heterogeneous processes induced by humic substances in the quasi-liquid layer of the salt crust, particle surfaces and the lake water are the predominating pathways for the formation of the observed haloacetones

Hirnorganoide – Modellsysteme des menschlichen Gehirns

This is the final version. Available from Deutsche Akademie der Naturforscher Leopoldina via the DOI in this record. Hirnorganoide sind Gewebestrukturen aus dem Labor, die Teile der Hirnfunktion imitieren. Sie eröffnen als vereinfachtes Modellsystem einen experimentellen Zugang zu Fragen rund um die Entwicklung und die Funktion des menschlichen Gehirns. Während die Forschung an menschlichen lebenden Gehirnen aus ethischen Gründen enge Grenzen hat und Tiermodelle viele Fragen nur bedingt beantworten können, bieten Hirnorganoide neue Forschungsmöglichkeiten. In der Stellungnahme „Hirnorganoide ‒ Modellsysteme des menschlichen Gehirns“ der Nationalen Akademie der Wissenschaften Leopoldina beschreiben Wissenschaftlerinnen und Wissenschaftler die Möglichkeiten dieses Forschungsgebietes und erörtern, ob es aus ethischen oder juristischen Gründen stärker reguliert werden sollte

Human haematopoietic stem cells express Oct4 pseudogenes and lack the ability to initiate Oct4 promoter-driven gene expression

The transcription factor Oct4 is well defined as a key regulator of embryonic stem (ES) cell pluripotency. In recent years, the role of Oct4 has purportedly extended to the self renewal and maintenance of multipotency in adult stem cell (ASC) populations. This profile has arisen mainly from reports utilising reverse transcription-polymerase chain reaction (RT-PCR) based methodologies and has since come under scrutiny following the discovery that many developmental genes have multiple pseudogenes associated with them. Six known pseudogenes exist for Oct4, all of which exhibit very high sequence homology (three >97%), and for this reason the generation of artefacts may have contributed to false identification of Oct4 in somatic cell populations. While ASC lack a molecular blueprint of transcription factors proposed to be involved with 'stemness' as described for ES cells, it is not unreasonable to assume that similar gene patterns may exist. The focus of this work was to corroborate reports that Oct4 is involved in the regulation of ASC self-renewal and differentiation, using a combination of methodologies to rule out pseudogene interference. Haematopoietic stem cells (HSC) derived from human umbilical cord blood (UCB) and various differentiated cell lines underwent RT-PCR, product sequencing and transfection studies using an Oct4 promoter-driven reporter. In summary, only the positive control expressed Oct4, with all other cell types expressing a variety of Oct4 pseudogenes. Somatic cells were incapable of utilising an exogenous Oct4 promoter construct, leading to the conclusion that Oct4 does not appear involved in the multipotency of human HSC from UCB

Neural Stem Cells Achieve and Maintain Pluripotency without Feeder Cells

Background: Differentiated cells can be reprogrammed into pluripotency by transduction of four defined transcription factors. Induced pluripotent stem cells (iPS cells) are expected to be useful for regenerative medicine as well as basic research. Recently, the report showed that mouse embryonic fibroblasts (MEF) cells are not essential for reprogramming. However, in using fibroblasts as donor cells for reprogramming, individual fibroblasts that had failed to reprogram could function as feeder cells. Methodology/Principal Finding: Here, we show that adult mouse neural stem cells (NSCs), which are not functional feeder cells, can be reprogrammed into iPS cells using defined four factors (Oct4, Sox2, Klf4, and c-Myc) under feeder-free conditions. The iPS cells, generated from NSCs expressing the Oct4-GFP reporter gene, could proliferate for more than two months (passage 20). Generated and maintained without feeder cells, these iPS cells expressed pluripotency markers (Oct4 and Nanog), the promoter regions of Oct4 and Nanog were hypomethylated, could differentiated into to all three germ layers in vitro, and formed a germline chimera. These data indicate that NSCs can achieve and maintain pluripotency under feeder-free conditions. Conclusion/Significance: This study suggested that factors secreted by feeder cells are not essential in the initial/early stages of reprogramming and for pluripotency maintenance. This technology might be useful for a human system, as

Genome editing reveals a role for OCT4 in human embryogenesis.

Despite their fundamental biological and clinical importance, the molecular mechanisms that regulate the first cell fate decisions in the human embryo are not well understood. Here we use CRISPR-Cas9-mediated genome editing to investigate the function of the pluripotency transcription factor OCT4 during human embryogenesis. We identified an efficient OCT4-targeting guide RNA using an inducible human embryonic stem cell-based system and microinjection of mouse zygotes. Using these refined methods, we efficiently and specifically targeted the gene encoding OCT4 (POU5F1) in diploid human zygotes and found that blastocyst development was compromised. Transcriptomics analysis revealed that, in POU5F1-null cells, gene expression was downregulated not only for extra-embryonic trophectoderm genes, such as CDX2, but also for regulators of the pluripotent epiblast, including NANOG. By contrast, Pou5f1-null mouse embryos maintained the expression of orthologous genes, and blastocyst development was established, but maintenance was compromised. We conclude that CRISPR-Cas9-mediated genome editing is a powerful method for investigating gene function in the context of human development.DW was supported by the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre Programme. NK was supported by the University of Oxford Clarendon Fund. AB was supported by a British Heart Foundation PhD Studentship (FS/11/77/39327). LV was supported by core grant funding from the Wellcome Trust and Medical Research Council (PSAG028). J-SK was supported by the Institute for Basic Science (IBS-R021-D1). Work in the KKN and JMAT labs was supported by the Francis Crick Institute which receives its core funding from Cancer Research UK, the UK Medical Research Council, and the Wellcome Trust (FC001120 and FC001193)

Generation of Healthy Mice from Gene-Corrected Disease-Specific Induced Pluripotent Stem Cells

Using the murine model of tyrosinemia type 1 (fumarylacetoacetate hydrolase [FAH] deficiency; FAH−/− mice) as a paradigm for orphan disorders, such as hereditary metabolic liver diseases, we evaluated fibroblast-derived FAH−/−-induced pluripotent stem cells (iPS cells) as targets for gene correction in combination with the tetraploid embryo complementation method. First, after characterizing the FAH−/− iPS cell lines, we aggregated FAH−/−-iPS cells with tetraploid embryos and obtained entirely FAH−/−-iPS cell–derived mice that were viable and exhibited the phenotype of the founding FAH−/− mice. Then, we transduced FAH cDNA into the FAH−/−-iPS cells using a third-generation lentiviral vector to generate gene-corrected iPS cells. We could not detect any chromosomal alterations in these cells by high-resolution array CGH analysis, and after their aggregation with tetraploid embryos, we obtained fully iPS cell–derived healthy mice with an astonishing high efficiency for full-term development of up to 63.3%. The gene correction was validated functionally by the long-term survival and expansion of FAH-positive cells of these mice after withdrawal of the rescuing drug NTBC (2-(2-nitro-4-fluoromethylbenzoyl)-1,3-cyclohexanedione). Furthermore, our results demonstrate that both a liver-specific promoter (transthyretin, TTR)-driven FAH transgene and a strong viral promoter (from spleen focus-forming virus, SFFV)-driven FAH transgene rescued the FAH-deficiency phenotypes in the mice derived from the respective gene-corrected iPS cells. In conclusion, our data demonstrate that a lentiviral gene repair strategy does not abrogate the full pluripotent potential of fibroblast-derived iPS cells, and genetic manipulation of iPS cells in combination with tetraploid embryo aggregation provides a practical and rapid approach to evaluate the efficacy of gene correction of human diseases in mouse models