A central role for TFIID in the pluripotent transcription circuitry.

Embryonic stem (ES) cells are pluripotent and characterized by open chromatin and high transcription levels, achieved through auto-regulatory and feed-forward transcription factor loops. ES-cell identity is maintained by a core of factors including Oct4 (also known as Pou5f1), Sox2, Klf4, c-Myc (OSKM) and Nanog, and forced expression of the OSKM factors can reprogram somatic cells into induced pluripotent stem cells (iPSCs) resembling ES cells. These gene-specific factors for RNA-polymerase-II-mediated transcription recruit transcriptional cofactors and chromatin regulators that control access to and activity of the basal transcription machinery on gene promoters. How the basal transcription machinery is involved in setting and maintaining the pluripotent state is unclear. Here we show that knockdown of the transcription factor IID (TFIID) complex affects the pluripotent circuitry in mouse ES cells and inhibits reprogramming of fibroblasts. TFIID subunits and the OSKM factors form a feed-forward loop to induce and maintain a stable transcription state. Notably, transient expression of TFIID subunits greatly enhanced reprogramming. These results show that TFIID is critical for transcription-factor-mediated reprogramming. We anticipate that, by creating plasticity in gene expression programs, transcription complexes such as TFIID assist reprogramming into different cellular states.

FGF signalling inhibits neural induction in human embryonic stem cells.

Human embryonic stem cells (hESCs) can exit the self-renewal programme, through the action of signalling molecules, at any given time and differentiate along the three germ layer lineages. We have systematically investigated the specific roles of three signalling pathways, TGFß/SMAD2, BMP/SMAD1, and FGF/ERK, in promoting the transition of hESCs into the neuroectoderm lineage. In this context, inhibition of SMAD2 and ERK signalling served to cooperatively promote exit from hESC self-renewal through the rapid downregulation of NANOG and OCT4. In contrast, inhibition of SMAD1 signalling acted to maintain SOX2 expression and prevent non-neural differentiation via HAND1. Inhibition of FGF/ERK upregulated OTX2 that subsequently induced the neuroectodermal fate determinant PAX6, revealing a novel role for FGF2 in indirectly repressing PAX6 in hESCs. Combined inhibition of the three pathways hence resulted in highly efficient neuroectoderm formation within 4 days, and subsequently, FGF/ERK inhibition promoted rapid differentiation into peripheral neurons. Our study assigns a novel, biphasic role to FGF/ERK signalling in the neural induction of hESCs, which may also have utility for applications requiring the rapid and efficient generation of peripheral neurons.

Smed-SmB, a member of the LSm protein superfamily, is essential for chromatoid body organization and planarian stem cell proliferation.

Planarians are an ideal model system to study in vivo the dynamics of adult pluripotent stem cells. However, our knowledge of the factors necessary for regulating the 'stemness' of the neoblasts, the adult stem cells of planarians, is sparse. Here, we report on the characterization of the first planarian member of the LSm protein superfamily, Smed-SmB, which is expressed in stem cells and neurons in Schmidtea mediterranea. LSm proteins are highly conserved key players of the splicing machinery. Our study shows that Smed-SmB protein, which is localized in the nucleus and the chromatoid body of stem cells, is required to safeguard the proliferative ability of the neoblasts. The chromatoid body, a cytoplasmatic ribonucleoprotein complex, is an essential regulator of the RNA metabolism required for the maintenance of metazoan germ cells. However, planarian neoblasts and neurons also rely on its functions. Remarkably, Smed-SmB dsRNA-mediated knockdown results in a rapid loss of organization of the chromatoid body, an impairment of the ability to post-transcriptionally process the transcripts of Smed-CycB, and a severe proliferative failure of the neoblasts. This chain of events leads to a quick depletion of the neoblast pool, resulting in a lethal phenotype for both regenerating and intact animals. In summary, our results suggest that Smed-SmB is an essential component of the chromatoid body, crucial to ensure a proper RNA metabolism and essential for stem cell proliferation.

Transcription Factors Active in the Anterior Blastema of Schmidtea mediterranea.

Regeneration, the restoration of body parts after injury, is quite widespread in the animal kingdom. Species from virtually all Phyla possess regenerative abilities. Human beings, however, are poor regenerators. Yet, the progress of knowledge and technology in the fields of bioengineering, stem cells, and regenerative biology have fostered major advancements in regenerative medical treatments, which aim to regenerate tissues and organs and restore function. Human induced pluripotent stem cells can differentiate into any cell type of the body; however, the structural and cellular complexity of the human tissues, together with the inability of our adult body to control pluripotency, require a better mechanistic understanding. Planarians, with their capacity to regenerate lost body parts thanks to the presence of adult pluripotent stem cells could help providing such an understanding. In this paper, we used a top-down approach to shortlist blastema transcription factors (TFs) active during anterior regeneration. We found 44 TFs-31 of which are novel in planarian-that are expressed in the regenerating blastema. We analyzed the function of half of them and found that they play a role in the regeneration of anterior structures, like the anterior organizer, the positional instruction muscle cells, the brain, the photoreceptor, the intestine. Our findings revealed a glimpse of the complexity of the transcriptional network governing anterior regeneration in planarians, confirming that this animal model is the perfect playground to study in vivo how pluripotency copes with adulthood.

Studies on acceptance, evaluation and impact of the Cologne program "Research and Medical Studies".

Introduction: The curricular implementation of events (or programs) for science-related training in human medicine has been on the agenda of the medical faculties since the publication of the Federal-State Working Group [1]. The Medical Faculty of the University of Cologne developed and established a systematic, longitudinal science curriculum together with the start of the model curriculum in human medicine in 2003. Here, we investigate the questions of whether the described (para-) curricular elements are accepted by students and lecturers and how they are evaluated, especially by students. In addition, we investigate whether selected parameters can be used to demonstrate changes in the students' scientific activities. Project description: The program "Research and Medical Studies" (RaMS) consists of several components: these elements of the mandatory curricular (Scientific Projects, SP) and optional components (Research in Medical Studies (RiMS), Research Track (RT), Research Fair Cologne (RFC)) are described here. Results were recorded at various levels: Likert Scale evaluation of the event's elements were collected as satisfaction parameters from the studentsProcess data on participation in the voluntary events were collected and evaluated as absolute and relational figures (WS 12/13-SS 17). Data on the outcome of the RaMS program were collected: Type of scientific projects in the academic years 2011/12-2014/15), number and type of available projects offered at the RFC (in the years 2011-18) and number of student research funding applications in a comparison of the periods 2010-13 vs. 2014-17). Results: The students' acceptance of mandatory and paracurricular courses of the RaMS program is pleasingly high, which is not surprising, at least in the case of the voluntary courses. The participation of students in RiMS, RT and RFC is satisfactory for voluntary courses. In the case of the RT, with certified participation of approximately 47% of all registrations (corresponding to 10% of the total cohort), this is comparable to similar programs. It can be shown that the number of experimental science projects has more than doubled over time in parallel with the development of RaMS. The average number of provided projects according to the RFC is 42 (which corresponds to a placement rate of approx. 1:4). The number of successful student applications for a research support grant during the period the measures were implemented has doubled. Discussion and conclusion: The RaMS program shows a route for the implementation of the SP required by the next licensing regulations in medical education, which was initially supported and expanded solitarily, later by further elements (RiMS), also in the sense of a science-based career development (RT, RFC). The student acceptance and the measured success, in the form of successful participation in the Research Track, increased choice of experimental projects, significant increase of submitted as well as approved research grants and the high project placement rate of the Research Fair, encourage the further development of the program, which is indicated in the conclusion.

An induction gene trap screen in neural stem cells reveals an instructive function of the niche and identifies the splicing regulator sam68 as a tenascin-C-regulated target gene.

Neural stem cells (NSCs) reside in a niche that abounds in extracellular matrix (ECM) molecules. The ECM glycoprotein tenascin-C (Tnc) that occurs in more than 25 isoforms represents a major constituent of the privileged NSC milieu. To understand its role for NSCs, the induction gene trap technology was successfully applied to mouse embryonic NSCs, and a library of more than 500 NSC lines with independent gene trap vector integrations was established. Our pilot screen identified Sam68 as a target of Tnc signaling in NSCs. The Tnc-mediated downregulation of Sam68, which we found expressed at low levels in the niche along with Tnc, was independently confirmed on the protein level. Sam68 is a multifunctional RNA-binding protein, and its potential significance for cultured NSCs was studied by overexpression. Increased Sam68 levels caused a marked reduction in NSC cell proliferation. In addition, Sam68 is a signal-dependent regulator of alternative splicing, and its overexpression selectively increased the larger Tnc isoforms, whereas a mutated phosphorylation-deficient Sam68 variant did not. This emphasizes the importance of Sam68 for NSC biology and implicates an instructive rather than a purely permissive role for Tnc in the neural stem cell niche.

Generic wound signals initiate regeneration in missing-tissue contexts.

Despite the identification of numerous regulators of regeneration in different animal models, a fundamental question remains: why do some wounds trigger the full regeneration of lost body parts, whereas others resolve by mere healing? By selectively inhibiting regeneration initiation, but not the formation of a wound epidermis, here we create headless planarians and finless zebrafish. Strikingly, in both missing-tissue contexts, injuries that normally do not trigger regeneration activate complete restoration of heads and fin rays. Our results demonstrate that generic wound signals have regeneration-inducing power. However, they are interpreted as regeneration triggers only in a permissive tissue context: when body parts are missing, or when tissue-resident polarity signals, such as Wnt activity in planarians, are modified. Hence, the ability to decode generic wound-induced signals as regeneration-initiating cues may be the crucial difference that distinguishes animals that regenerate from those that cannot.

Interaction of AF4 wild-type and AF4.MLL fusion protein with SIAH proteins: indication for t(4;11) pathobiology?

The human AF4 (ALL-1 fused gene on chromosome 4) gene (4q11) is recurrently involved in reciprocal translocations to the MLL (mixed lineage leukemia) gene (11q23), correlated with high-risk acute lymphoblastic leukemia (ALL) in infants and early childhood. The t(4;11) translocation is one of the most frequent MLL translocations known today. In general, MLL translocations are the result of an illegitimate recombination process leading to reciprocal fusions of unrelated translocation partner (TP) genes with the MLL gene. Owing to the constant presence of the derivative (11) product, it was hypothesised that only MLL.TP fusion genes are responsible for the leukemogenic process. This concept has been successfully tested for some known MLL fusions, while other MLL fusions failed. Here, we demonstrate growth-transforming potential of AF4 wild-type and the AF4.MLL fusion protein. The underlying oncogenic mechanism involves the two E3 ubiquitin ligases SIAH1 and SIAH2, the N-terminal portion of AF4 and the protection of the AF4.MLL fusion protein against proteosomal degradation.

Derivation and expansion using only small molecules of human neural progenitors for neurodegenerative disease modeling.

Phenotypic drug discovery requires billions of cells for high-throughput screening (HTS) campaigns. Because up to several million different small molecules will be tested in a single HTS campaign, even small variability within the cell populations for screening could easily invalidate an entire campaign. Neurodegenerative assays are particularly challenging because neurons are post-mitotic and cannot be expanded for implementation in HTS. Therefore, HTS for neuroprotective compounds requires a cell type that is robustly expandable and able to differentiate into all of the neuronal subtypes involved in disease pathogenesis. Here, we report the derivation and propagation using only small molecules of human neural progenitor cells (small molecule neural precursor cells; smNPCs). smNPCs are robust, exhibit immortal expansion, and do not require cumbersome manual culture and selection steps. We demonstrate that smNPCs have the potential to clonally and efficiently differentiate into neural tube lineages, including motor neurons (MNs) and midbrain dopaminergic neurons (mDANs) as well as neural crest lineages, including peripheral neurons and mesenchymal cells. These properties are so far only matched by pluripotent stem cells. Finally, to demonstrate the usefulness of smNPCs we show that mDANs differentiated from smNPCs with LRRK2 G2019S are more susceptible to apoptosis in the presence of oxidative stress compared to wild-type. Therefore, smNPCs are a powerful biological tool with properties that are optimal for large-scale disease modeling, phenotypic screening, and studies of early human development.

Heterogeneity of planarian stem cells in the S/G2/M phase.

The planarian adult stem cell (pASC) population has a specific molecular signature and can be easily visualized and isolated by flow cytometry. However, the lack of antibodies against specific surface markers for planarian cells prevents a deeper analysis of specific cell populations. Here, if we describe the results of the immunoscreening of pASC plasma membrane proteins (PMPs). A novel papain-based method for planarian cell dissociation enabling both high yield and improved cell viability was used to generate single cell preparations for PMP purification. PMPs were used for intraperitoneal immunization of mice and thus about 1000 hybridoma clones were generated and screened. Supernatants collected from the hybridoma clones were first screened by ELISA and then by live immuno-staining. About half of these supernatants stained all the planarian cells, whereas the other half specifically labeled a subfraction thereof. A detailed analysis of two hybridoma supernatants revealed that large subfractions of the X1, X2 and Xin populations differentially express specific membrane markers. Quantitative PCR data disclosed a correlation between the immunostaining results and the expression of markers of the early and late progeny, also for those pASCs in the S/G2/M phase of the cell cycle (X1 population). Thus, about two thirds of the cycling pASCs showed a specific membrane signature coupled with the expression of markers hitherto considered to be restricted to differentiating, post-mitotic progeny. In summary, a library of 66 monoclonal antibodies against planarian PMPs was generated. The analysis of two of the clones generated revealed that a subset of cells of the X1 population expresses early and late progeny markers, which might indicate that these cells are committed while still proliferating. The findings demonstrate the usefulness of our PMP antibody library for planarian research.

Direct reprogramming of fibroblasts into neural stem cells by defined factors.

Recent studies have shown that defined sets of transcription factors can directly reprogram differentiated somatic cells to a different differentiated cell type without passing through a pluripotent state, but the restricted proliferative and lineage potential of the resulting cells limits the scope of their potential applications. Here we show that a combination of transcription factors (Brn4/Pou3f4, Sox2, Klf4, c-Myc, plus E47/Tcf3) induces mouse fibroblasts to directly acquire a neural stem cell identity-which we term as induced neural stem cells (iNSCs). Direct reprogramming of fibroblasts into iNSCs is a gradual process in which the donor transcriptional program is silenced over time. iNSCs exhibit cell morphology, gene expression, epigenetic features, differentiation potential, and self-renewing capacity, as well as in vitro and in vivo functionality similar to those of wild-type NSCs. We conclude that differentiated cells can be reprogrammed directly into specific somatic stem cell types by defined sets of specific transcription factors.