Generation of cell diversity in the developing brainstem and its modeling in vitro

The intersection of spatial and temporal patterning programs underlies the formation of neural cell type diversity in the developing central nervous system (CNS). Deciphering the molecular mechanisms regulating positional and temporal patterning provides a basic framework for how the developing brain can be functionally assembled, and the growing knowledge about these patterning mechanisms offer a powerful tool to effectively control the differentiation of pluripotent stem cells (PSCs) into different neuronal subtypes of clinical importance. The research described in this thesis is part of a continuing effort to define the molecular mechanisms that modulate the positional and temporal identity of immature neural stem cells (NSCs) and enables these to differentiate into specific subtypes of neurons at defined positions and over specific time-windows in the developing CNS. We have examined the sequential specification of motor neurons (MNs) and serotonergic neurons (5HTNs) by a Nkx2.2+ temporal lineage in the ventral hindbrain (HB) and outlined a three-node timer network that conceptually explains how time can be encoded by NSCs. Additionally, and unexpectedly, we show that timed exposure of retinoic acid (RA) can be applied to effectively pattern human PSC (hPSC)-derived neural progenitors into forebrain (FB), midbrain (MB), and HB regional identities. Based on this finding, we developed novel and robust differentiation protocols for production of mesencephalic dopaminergic (mDA) neurons and 5HTNs of the HB. We show that RA-specified human mDA neurons restore motor function after transplantation into a rat model of Parkinson’s disease (PD), and that mouse and human 5HTNs can be utilized as cellular platforms to screen small molecules for their capacity to modulate serotonin (5-HT) signaling in 5HTNs. In Paper I, we address how time is encoded by NSCs in temporal patterning processes in the CNS. We focused on a region in the ventral HB where NSCs, defined by expression of the transcription factor (TF) Nkx2.2, sequentially generate MNs, 5HTNs and oligodendrocyte precursors (OLPs). Shh signaling induces the initiation of MN production through induction of the MN-determining TF Phox2b while a delayed activation of transforming growth factor β (Tgfβ) suppresses Phox2b, terminates MN production and trigger the birth of late-born 5HTNs (Dias et al., 2014). In Paper I we present a three-node incoherent feed-forward loop (IFFL) circuitry that conceptually explains how time can be measured and set in the Nkx2.2+ lineage. By applying a series of in vivo and in vitro experiments, in combination with computational modeling, we reveal a progressive decline of Gli1-3 transcription and bifunctional Gli2-3 TFs over time. Tgfβ is sensitive to transcriptional repressor forms of Gli proteins (GliR) which prohibit Tgfβ induction by Gli activators (GliA) until GliR has been titrated out. Once activated, the cell non-autonomous activity of Tgfβ counterbalances noise and facilitates a synchronous fate switch of Nkx2.2+ NSCs at the population levels. In Paper II, we show that timed delivery of RA can be effectively applied to regionally pattern hPSCs into FB, MB and HB regional territories, in a manner resembling the previously established activity of WNT signaling. However, while WNT signaling is concentration sensitive, it is the duration of RA exposure that is crucial for regional patterning and the response of cells is relatively insensitive to altered RA concentrations. By combining RA- and Shh-signaling we could robustly direct the differentiation of hPSCs into mDA neurons, whose loss underlie motor deficits in PD. When grafted into the striatum of parkinsonian rats, RA-specified cell preparations engraft, differentiate into functional mDA neurons and relieve motor deficits. These data provide proof-of-concept that RA-based protocol for mDA generation could provide a new and alternative route for cell replacement therapy for PD. In addition to mDA neurons, we show that extended exposure of hPSCs to RA results in efficient generation of cranial human MNs and human 5HTNs, suggesting that RA-based regional patterning can be applied to generate several types of clinically relevant neurons from hPSCs. In Paper III, we utilized protocols developed by Dias et al., 2014 and in Paper II to produce mouse and human 5HTNs, which dysfunction is strongly linked to various neuropsychiatric disorders and are the target of most prescribed antidepressants. After adaptation of protocols to a screenable format and high-content imaging, we performed an unbiased phenotypic screen to identify small molecules that modulate 5-HT signaling in mouse and human 5HTNs. Out of ~5200 annotated small molecules, we identified and confirmed ~200 hits that modulated 5-HT content in mouse neurons with ~70% of these showing a similar phenotypic response on human 5HTNs. Many hits had previously been associated with the monoaminergic system, but many compounds were not obviously connected to 5HTNs. Among those were the muscarinic acetylcholine receptor (mAChR) antagonist oxybutynin which promoted a notable increase of neuronal 5-HT content and which in subsequent secondary assays acted as an inhibitor of monoamine oxidases in 5HTNs. These data provide proof-of-concept that phenotypic screening on stem cell-derived 5HTNs is a powerful tool to identify new types of compounds with potential antidepressant properties

EUNIS Habitat Classification: Expert system, characteristic species combinations and distribution maps of European habitats

Aim: The EUNIS Habitat Classification is a widely used reference framework for European habitat types (habitats), but it lacks formal definitions of individual habitats that would enable their unequivocal identification. Our goal was to develop a tool for assigning vegetation‐plot records to the habitats of the EUNIS system, use it to classify a European vegetation‐plot database, and compile statistically‐derived characteristic species combinations and distribution maps for these habitats. Location: Europe. Methods: We developed the classification expert system EUNIS‐ESy, which contains definitions of individual EUNIS habitats based on their species composition and geographic location. Each habitat was formally defined as a formula in a computer language combining algebraic and set‐theoretic concepts with formal logical operators. We applied this expert system to classify 1,261,373 vegetation plots from the European Vegetation Archive (EVA) and other databases. Then we determined diagnostic, constant and dominant species for each habitat by calculating species‐to‐habitat fidelity and constancy (occurrence frequency) in the classified data set. Finally, we mapped the plot locations for each habitat. Results: Formal definitions were developed for 199 habitats at Level 3 of the EUNIS hierarchy, including 25 coastal, 18 wetland, 55 grassland, 43 shrubland, 46 forest and 12 man‐made habitats. The expert system classified 1,125,121 vegetation plots to these habitat groups and 73,188 to other habitats, while 63,064 plots remained unclassified or were classified to more than one habitat. Data on each habitat were summarized in factsheets containing habitat description, distribution map, corresponding syntaxa and characteristic species combination. Conclusions: EUNIS habitats were characterized for the first time in terms of their species composition and distribution, based on a classification of a European database of vegetation plots using the newly developed electronic expert system EUNIS‐ESy. The data provided and the expert system have considerable potential for future use in European nature conservation planning, monitoring and assessment

Fucose Ameliorates Tritrichomonas sp.-Associated Illness in Antibiotic-Treated Muc2−/− Mice

The mucus layer in the intestine plays a critical role in regulation of host–microbe interactions and maintaining homeostasis. Disruptions of the mucus layer due to genetic, environmental, or immune factors may lead to inflammatory bowel diseases (IBD). IBD frequently are accompanied with infections, and therefore are treated with antibiotics. Hence, it is important to evaluate risks of antibiotic treatment in individuals with vulnerable gut barrier and chronic inflammation. Mice with a knockout of the Muc2 gene, encoding the main glycoprotein component of the mucus, demonstrate a close contact of the microbes with the gut epithelium which leads to chronic inflammation resembling IBD. Here we demonstrate that the Muc2−/− mice harboring a gut protozoan infection Tritrichomonas sp. are susceptible to an antibiotic-induced depletion of the bacterial microbiota. Suppression of the protozoan infection with efficient metronidazole dosage or L-fucose administration resulted in amelioration of an illness observed in antibiotic-treated Muc2−/− mice. Fucose is a monosaccharide presented abundantly in gut glycoproteins, including Mucin2, and is known to be involved in host–microbe interactions, in particular in microbe adhesion. We suppose that further investigation of the role of fucose in protozoan adhesion to host cells may be of great value

A Shh/Gli-driven three-node timer motif controls temporal identity and fate of neural stem cells

How time is measured by neural stem cells during temporal neurogenesis has remained unresolved. By combining experiments and computational modeling, we define a Shh/Gli-driven three-node timer underlying the sequential generation of motor neurons (MNs) and serotonergic neurons in the brainstem. The timer is founded on temporal decline of Gli-activator and Gli-repressor activities established through down-regulation of Gli transcription. The circuitry conforms an incoherent feed-forward loop, whereby Gli proteins not only promote expression of Phox2b and thereby MN-fate but also account for a delayed activation of a self-promoting transforming growth factor–β (Tgfβ) node triggering a fate switch by repressing Phox2b. Hysteresis and spatial averaging by diffusion of Tgfβ counteract noise and increase temporal accuracy at the population level, providing a functional rationale for the intrinsically programmed activation of extrinsic switch signals in temporal patterning. Our study defines how time is reliably encoded during the sequential specification of neurons.ISSN:2375-254

Fucose Ameliorates Tryptophan Metabolism and Behavioral Abnormalities in a Mouse Model of Chronic Colitis

Growing evidence suggests that intestinal mucosa homeostasis impacts immunity, metabolism, the Central Nervous System (CNS), and behavior. Here, we investigated the effect of the monosaccharide fucose on inflammation, metabolism, intestinal microbiota, and social behavior in the Dextran Sulfate Sodium (DSS)-induced chronic colitis mouse model. Our data show that chronic colitis is accompanied by the decrease of the serum tryptophan level and the depletion of the intestinal microbiota, specifically tryptophan-producing E. coli and Bifidobacterium. These changes are associated with defects in the male mouse social behavior such as a lack of preference towards female bedding in an odor preference test. The addition of fucose to the test animals’ diet altered the bacterial community, increased the abundance of tryptophan-producing E. coli, normalized blood tryptophan levels, and ameliorated social behavior deficits. At the same time, we observed no ameliorating effect of fucose on colon morphology and colitis. Our results suggest a possible mechanism by which intestinal inflammation affects social behavior in male mice. We propose fucose as a promising prebiotic, since it creates a favorable environment for the beneficial bacteria that promote normalization of serum tryptophan level and amelioration of the behavioral abnormalities in the odor preference test

A Shh/Gli-driven three-node timer motif controls temporal identity and fate of neural stem cells

How time is measured by neural stem cells during temporal neurogenesis has remained unresolved. By combining experiments and computational modelling, we here define a Shh/Gli-driven three-node timer underlying the sequential generation of motor neurons (MNs) and serotonergic neurons in the brainstem. The timer is founded on temporal decline of Gli-activator and Gli-repressor activities established through downregulation of Gli transcription. The circuitry conforms an incoherent feedforward loop, whereby Gli proteins promote expression of Phox2b and thereby MN-fate, but also account for a delayed activation of a self-promoting Tgfβ-node triggering a fate switch by repressing Phox2b. Hysteresis and spatial averaging by diffusion of Tgfβ counteracts noise and increases temporal accuracy at the population level. Our study defines how time is reliably encoded during the sequential specification of neurons

Delivery of differentiation factors by mesoporous silica particles assists advanced differentiation of transplanted murine embryonic stem cells

Stem cell transplantation holds great hope for the replacement of damaged cells in the nervous system. However, poor long-term survival after transplantation and insufficiently robust differentiation of stem cells into specialized cell types in vivo remain major obstacles for clinical application. Here, we report the development of a novel technological approach for the local delivery of exogenous trophic factor mimetics to transplanted cells using specifically designed silica nanoporous particles. We demonstrated that delivering Cintrofin and Gliafin, established peptide mimetics of the ciliary neurotrophic factor and glial cell line-derived neurotrophic factor, respectively, with these particles enabled not only robust functional differentiation of motor neurons from transplanted embryonic stem cells but also their long-term survival in vivo. We propose that the delivery of growth factors by mesoporous nanoparticles is a potentially versatile and widely applicable strategy for efficient differentiation and functional integration of stem cell derivatives upon transplantation

Robust derivation of transplantable dopamine neurons from human pluripotent stem cells by timed retinoic acid delivery

Stem cell therapies for Parkinson’s disease (PD) have entered first-in-human clinical trials using a set of technically related methods to produce mesencephalic dopamine (mDA) neurons from human pluripotent stem cells (hPSCs). Here, we outline an approach for high-yield derivation of mDA neurons that principally differs from alternative technologies by utilizing retinoic acid (RA) signaling, instead of WNT and FGF8 signaling, to specify mesencephalic fate. Unlike most morphogen signals, where precise concentration determines cell fate, it is the duration of RA exposure that is the key-parameter for mesencephalic specification. This concentration-insensitive patterning approach provides robustness and reduces the need for protocol-adjustments between hPSC-lines. RA-specified progenitors promptly differentiate into functional mDA neurons in vitro, and successfully engraft and relieve motor deficits after transplantation in a rat PD model. Our study provides a potential alternative route for cell therapy and disease modelling that due to its robustness could be particularly expedient when use of autologous- or immunologically matched cells is considered