Frank Edenhofer

Direct Conversion of Fibroblasts into Stably Expandable Neural Stem Cells

Recent advances have suggested that direct induction of neural stem cells (NSCs) could provide an alternative to derivation from somatic tissues or pluripotent cells. Here we show direct derivation of stably expandable NSCs from mouse fibroblasts through a curtailed version of reprogramming to pluripotency. By constitutively inducing Sox2, Klf4, and c-Myc while strictly limiting Oct4 activity to the initial phase of reprogramming, we generated neurosphere-like colonies that could be expanded for more than 50 passages and do not depend on sustained expression of the reprogramming factors. These induced neural stem cells (iNSCs) uniformly display morphological and molecular features of NSCs, such as the expression of Nestin, Pax6, and Olig2, and have a genome-wide transcriptional profile similar to that of brain-derived NSCs. Moreover, iNSCs can differentiate into neurons, astrocytes, and oligodendrocytes. Our results demonstrate that functional NSCs can be generated from somatic cells by factor-driven induction.

Cargo-dependent mode of uptake and bioavailability of TAT-containing proteins and peptides in living cells

Cell‐penetrating peptides (CPPs) are capable of introducing a wide range of cargoes into living cells. Descriptions of the internalization process vary from energy‐independent cell penetration of membranes to endocytic uptake. To elucidate whether the mechanism of entry of CPP constructs might be influenced by the properties of the cargo, we used time lapse confocal microscopy analysis of living mammalian cells to directly compare the uptake of the well‐studied CPP TAT fused to a protein (>50 amino acids) or peptide (<50 amino acids) cargo. We also analyzed various constructs for their subcellular distribution and mobility after the internalization event. TAT fusion proteins were taken up largely into cytoplasmic vesicles whereas peptides fused to TAT entered the cell in a rapid manner that was dependent on membrane potential. Despite their accumulation in the nucleolus, photobleaching of TAT fusion peptides revealed their mobility. The bioavailability of internalized TAT peptides was tested and confirmed by the strong inhibitory effect on cell cycle progression of two TAT fusion peptides derived from the tumor suppressor p21WAF/Cip and DNA Ligase I measured in living cells.—Tünnemann, G., Martin, R. M., Haupt, S., Patsch, C., Edenhofer, F., Cardoso, M. C. Cargo‐dependent mode of uptake and bioavailability of TAT‐containing proteins and peptides in living cells. FASEB J. 20, 1775–1784 (2006)

Ability of the hydrophobic FGF and basic TAT peptides to promote cellular uptake of recombinant Cre recombinase: A tool for efficient genetic engineering of mammalian genomes

Conditional mutagenesis is a powerful tool to analyze gene functions in mammalian cells. The site-specific recombinase Cre can be used to recombine loxP-modified alleles under temporal and spatial control. However, the efficient delivery of biologically active Cre recombinase to living cells represents a limiting factor. In this study we compared the potential of a hydrophobic peptide modified from Kaposi fibroblast growth factor with a basic peptide derived from HIV-TAT to promote cellular uptake of recombinant Cre. We present the production and characterization of a Cre protein that enters mammalian cells and subsequently performs recombination with high efficiency in a time- and concentration-dependent manner. Histidine-tagged Cre recombinase transduced inefficiently unless fused to a nuclear localization signal (NLS). Fusion of NLS-Cre to the fibroblast growth factor transduction peptide did not improve the transducibility, whereas fusion with the TAT peptide significantly enhanced cellular uptake and subsequent recombination. More than 95% recombination efficiency in fibroblast cells, as well as murine embryonic stem cells, was achieved after His-TAT-NLS-Cre transduction. Efficient recombination could also be obtained in primary splenocytes ex vivo. We expect that application of His-TAT-NLS-Cre, which can be produced readily in large quantities from a bacterial source, will expand our abilities to manipulate mammalian genomes.

Sox2 Is Essential for Formation of Trophectoderm in the Preimplantation Embryo

Background In preimplantation mammalian development the transcription factor Sox2 (SRY-related HMG-box gene 2) forms a complex with Oct4 and functions in maintenance of self-renewal of the pluripotent inner cell mass (ICM). Previously it was shown that Sox2−/− embryos die soon after implantation. However, maternal Sox2 transcripts may mask an earlier phenotype. We investigated whether Sox2 is involved in controlling cell fate decisions at an earlier stage. Methods and Findings We addressed the question of an earlier role for Sox2 using RNAi, which removes both maternal and embryonic Sox2 mRNA present during the preimplantation period. By depleting both maternal and embryonic Sox2 mRNA at the 2-cell stage and monitoring embryo development in vitro we show that, in the absence of Sox2, embryos arrest at the morula stage and fail to form trophectoderm (TE) or cavitate. Following knock-down of Sox2 via three different short interfering RNA (siRNA) constructs in 2-cell stage mouse embryos, we have shown that the majority of embryos (76%) arrest at the morula stage or slightly earlier and only 18.7–21% form blastocysts compared to 76.2–83% in control groups. In Sox2 siRNA-treated embryos expression of pluripotency associated markers Oct4 and Nanog remained unaffected, whereas TE associated markers Tead4, Yap, Cdx2, Eomes, Fgfr2, as well as Fgf4, were downregulated in the absence of Sox2. Apoptosis was also increased in Sox2 knock-down embryos. Rescue experiments using cell-permeant Sox2 protein resulted in increased blastocyst formation from 18.7% to 62.6% and restoration of Sox2, Oct4, Cdx2 and Yap protein levels in the rescued Sox2-siRNA blastocysts. Conclusion and Significance We conclude that the first essential function of Sox2 in the preimplantation mouse embryo is to facilitate establishment of the trophectoderm lineage. Our findings provide a novel insight into the first differentiation event within the preimplantation embryo, namely the segregation of the ICM and TE lineages.

Novel reporter mouse reveals constitutive and inflammatory expression of IFN-beta in vivo.

Type I IFN is a major player in innate and adaptive immune responses. Besides, it is involved in organogenesis and tumor development. Generally, IFN responses are amplified by an autocrine loop with IFN-β as the priming cytokine. However, due to the lack of sensitive detection systems, where and how type I IFN is produced in vivo is still poorly understood. In this study, we describe a luciferase reporter mouse, which allows tracking of IFN-β gene induction in vivo. Using this reporter mouse, we reveal strong tissue-specific induction of IFN-β following infection with influenza or La Crosse virus. Importantly, this reporter mouse also allowed us to visualize that IFN-β is expressed constitutively in several tissues. As suggested before, low amounts of constitutively produced IFN might maintain immune cells in an activated state ready for a timely response to pathogens. Interestingly, thymic epithelial cells were the major source of IFN-β under noninflammatory conditions. This relatively high constitutive expression was controlled by the NF Aire and might influence induction of tolerance or T cell development.

Inhibition of Notch Signaling in Human Embryonic Stem Cell-Derived Neural Stem Cells Delays G1/S Phase Transition and Accelerates Neuronal Differentiation In Vitro and In Vivo

The controlled in vitro differentiation of human embryonic stem cells (hESCs) and other pluripotent stem cells provides interesting prospects for generating large numbers of human neurons for a variety of biomedical applications. A major bottleneck associated with this approach is the long time required for hESC‐derived neural cells to give rise to mature neuronal progeny. In the developing vertebrate nervous system, Notch signaling represents a key regulator of neural stem cell (NSC) maintenance. Here, we set out to explore whether this signaling pathway can be exploited to modulate the differentiation of hESC‐derived NSCs (hESNSCs). We assessed the expression of Notch pathway components in hESNSCs and demonstrate that Notch signaling is active under self‐renewing culture conditions. Inhibition of Notch activity by the γ‐secretase inhibitor N‐[N‐(3,5‐difluorophenacetyl)‐L‐alanyl]‐S‐phenylglycine t‐butyl ester (DAPT) in hESNSCs affects the expression of human homologues of known targets of Notch and of several cell cycle regulators. Furthermore, DAPT‐mediated Notch inhibition delays G1/S‐phase transition and commits hESNSCs to neurogenesis. Combined with growth factor withdrawal, inhibition of Notch signaling results in a marked acceleration of differentiation, thereby shortening the time required for the generation of electrophysiologically active hESNSC‐derived neurons. This effect can be exploited for neural cell transplantation, where transient Notch inhibition before grafting suffices to promote the onset of neuronal differentiation of hESNSCs in the host tissue. Thus, interference with Notch signaling provides a tool for controlling human NSC differentiation both in vitro and in vivo. STEM CELLS 2010;28:955–964

Peroxisome Proliferator-Activated Receptor γ Control of Dendritic Cell Function Contributes to Development of CD4+ T Cell Anergy

There is increasing evidence that dendritic cell (DC) immunogenicity is not only positively regulated by ligands of pattern recognition receptors, but also negatively by signals that prevent DC activation and full functional maturation. Depending on their activation status, DCs can induce either immunity or tolerance. In this study, we provide molecular evidence that the transcription factor peroxisome proliferator-activated receptor γ (PPARγ) is a negative regulator of DC maturation and function. Sustained PPARγ activation in murine DCs reduced maturation-induced expression of costimulatory molecules and IL-12, and profoundly inhibited their capacity to prime naive CD4+ T cells in vitro. Using PPARγ-deficient DCs, generated by Cre-mediated ablation of the PPARγ gene, agonist-mediated suppression of maturation-induced functional changes were abrogated. Moreover, absence of PPARγ increased DC immunogenicity, suggesting a constitutive regulatory function of PPARγ in DCs. Adoptive transfer of PPARγ-activated Ag-presenting DCs induced CD4+ T cell anergy, characterized by impaired differentiation resulting in absent Th1 and Th2 cytokine production and failure of secondary clonal expansion upon restimulation. Collectively, our data support the notion that PPARγ is an efficient regulator of DC immunogenicity that may be exploited to deliberately target CD4+ T cell-mediated immune responses.

Site-specific recombination in human embryonic stem cells induced by cell-permeant Cre recombinase

The biomedical application of human embryonic stem (hES) cells will increasingly depend on the availability of technologies for highly controlled genetic modification. In mouse genetics, conditional mutagenesis using site-specific recombinases has become an invaluable tool for gain- and loss-of-function studies. Here we report highly efficient Cre-mediated recombination of a chromosomally integrated loxP-modified allele in hES cells and hES cell–derived neural precursors by protein transduction. Recombinant modified Cre recombinase protein translocates into the cytoplasm and nucleus of hES cells and subsequently induces recombination in virtually 100% of the cells. Cre-transduced hES cells maintain the expression of pluripotency markers as well as the capability of differentiating into derivatives of all three germ layers in vitro and in vivo. We expect this technology to provide an important technical basis for analyzing complex genetic networks underlying human development as well as generating highly purified, transplantable hES cell–derived cells for regenerative medicine.

A thermoresponsive and chemically defined hydrogel for long-term culture of human embryonic stem cells

Cultures of human embryonic stem cell typically rely on protein matrices or feeder cells to support attachment and growth, while mechanical, enzymatic or chemical cell dissociation methods are used for cellular passaging. However, these methods are ill defined, thus introducing variability into the system, and may damage cells. They also exert selective pressures favouring cell aneuploidy and loss of differentiation potential. Here we report the identification of a family of chemically defined thermoresponsive synthetic hydrogels based on 2-(diethylamino)ethyl acrylate, which support long-term human embryonic stem cell growth and pluripotency over a period of 2–6 months. The hydrogels permitted gentle, reagent-free cell passaging by virtue of transient modulation of the ambient temperature from 37 to 15 °C for 30 min. These chemically defined alternatives to currently used, undefined biological substrates represent a flexible and scalable approach for improving the definition, efficacy and safety of human embryonic stem cell culture systems for research, industrial and clinical applications.

Runx genes are direct targets of Scl/Tal1 in the yolk sac and fetal liver

Transcription factors such as Scl/Tal1, Lmo2, and Runx1 are essential for the development of hematopoietic stem cells (HSCs). However, the precise mechanisms by which these factors interact to form transcriptional networks, as well as the identity of the genes downstream of these regulatory cascades, remain largely unknown. To this end, we generated an Scl(-/-) yolk sac cell line to identify candidate Scl target genes by global expression profiling after reintroduction of a TAT-Scl fusion protein. Bioinformatics analysis resulted in the identification of 9 candidate Scl target transcription factor genes, including Runx1 and Runx3. Chromatin immunoprecipitation confirmed that both Runx genes are direct targets of Scl in the fetal liver and that Runx1 is also occupied by Scl in the yolk sac. Furthermore, binding of an Scl-Lmo2-Gata2 complex was demonstrated to occur on the regions flanking the conserved E-boxes of the Runx1 loci and was shown to transactivate the Runx1 element. Together, our data provide a key component of the transcriptional network of early hematopoiesis by identifying downstream targets of Scl that can explain key aspects of the early Scl(-/-) phenotype.