Genome engineering of isogenic human ES cells to model autism disorders.

Isogenic pluripotent stem cells are critical tools for studying human neurological diseases by allowing one to study the effects of a mutation in a fixed genetic background. Of particular interest are the spectrum of autism disorders, some of which are monogenic such as Timothy syndrome (TS); others are multigenic such as the microdeletion and microduplication syndromes of the 16p11.2 chromosomal locus. Here, we report engineered human embryonic stem cell (hESC) lines for modeling these two disorders using locus-specific endonucleases to increase the efficiency of homology-directed repair (HDR). We developed a system to: (1) computationally identify unique transcription activator-like effector nuclease (TALEN) binding sites in the genome using a new software program, TALENSeek, (2) assemble the TALEN genes by combining golden gate cloning with modified constructs from the FLASH protocol, and (3) test the TALEN pairs in an amplification-based HDR assay that is more sensitive than the typical non-homologous end joining assay. We applied these methods to identify, construct, and test TALENs that were used with HDR donors in hESCs to generate an isogenic TS cell line in a scarless manner and to model the 16p11.2 copy number disorder without modifying genomic loci with high sequence similarity

Dynamics of embryonic stem cell differentiation inferred from single-cell transcriptomics show a series of transitions through discrete cell states

The complexity of gene regulatory networks that lead multipotent cells to acquire different cell fates makes a quantitative understanding of differentiation challenging. Using a statistical framework to analyze single-cell transcriptomics data, we infer the gene expression dynamics of early mouse embryonic stem (mES) cell differentiation, uncovering discrete transitions across nine cell states. We validate the predicted transitions across discrete states using flow cytometry. Moreover, using live-cell microscopy, we show that individual cells undergo abrupt transitions from a naïve to primed pluripotent state. Using the inferred discrete cell states to build a probabilistic model for the underlying gene regulatory network, we further predict and experimentally verify that these states have unique response to perturbations, thus defining them functionally. Our study provides a framework to infer the dynamics of differentiation from single cell transcriptomics data and to build predictive models of the gene regulatory networks that drive the sequence of cell fate decisions during development. DOI: http://dx.doi.org/10.7554/eLife.20487.00

Functional electrical stimulation therapy controlled by a P300-based brain–computer interface, as a therapeutic alternative for upper limb motor function recovery in chronic post-stroke patients. A non-randomized pilot study

IntroductionUp to 80% of post-stroke patients present upper-limb motor impairment (ULMI), causing functional limitations in daily activities and loss of independence. UMLI is seldom fully recovered after stroke when using conventional therapeutic approaches. Functional Electrical Stimulation Therapy (FEST) controlled by Brain–Computer Interface (BCI) is an alternative that may induce neuroplastic changes, even in chronic post-stroke patients. The purpose of this work was to evaluate the effects of a P300-based BCI-controlled FEST intervention, for ULMI recovery of chronic post-stroke patients.MethodsA non-randomized pilot study was conducted, including 14 patients divided into 2 groups: BCI-FEST, and Conventional Therapy. Assessments of Upper limb functionality with Action Research Arm Test (ARAT), performance impairment with Fugl–Meyer assessment (FMA), Functional Independence Measure (FIM) and spasticity through Modified Ashworth Scale (MAS) were performed at baseline and after carrying out 20 therapy sessions, and the obtained scores compared using Chi square and Mann–Whitney U statistical tests ( = 0.05).ResultsAfter training, we found statistically significant differences between groups for FMA (p = 0.012), ARAT (p < 0.001), and FIM (p = 0.025) scales.DiscussionIt has been shown that FEST controlled by a P300-based BCI, may be more effective than conventional therapy to improve ULMI after stroke, regardless of chronicity.ConclusionThe results of the proposed BCI-FEST intervention are promising, even for the most chronic post-stroke patients often relegated from novel interventions, whose expected recovery with conventional therapy is very low. It is necessary to carry out a randomized controlled trial in the future with a larger sample of patients

Functional enhancer elements drive subclass-selective expression from mouse to primate neocortex

Viral genetic tools to target specific brain cell types in humans and non-genetic model organisms will transform basic neuroscience and targeted gene therapy. Here we used comparative epigenetics to identify thousands of human neuronal subclass-specific putative enhancers to regulate viral tools, and 34% of these were conserved in mouse. We established an AAV platform to evaluate cellular specificity of functional enhancers by multiplexed fluorescent in situ hybridization (FISH) and single cell RNA sequencing. Initial testing in mouse neocortex yields a functional enhancer discovery success rate of over 30%. We identify enhancers with specificity for excitatory and inhibitory classes and subclasses including PVALB, LAMP5, and VIP/LAMP5 cells, some of which maintain specificity in vivo or ex vivo in monkey and human neocortex. Finally, functional enhancers can be proximal or distal to cellular marker genes, conserved or divergent across species, and could yield brain-wide specificity greater than the most selective marker genes

Genome engineering of isogenic human ES cells to model autism disorders.

Isogenic pluripotent stem cells are critical tools for studying human neurological diseases by allowing one to study the effects of a mutation in a fixed genetic background. Of particular interest are the spectrum of autism disorders, some of which are monogenic such as Timothy syndrome (TS); others are multigenic such as the microdeletion and microduplication syndromes of the 16p11.2 chromosomal locus. Here, we report engineered human embryonic stem cell (hESC) lines for modeling these two disorders using locus-specific endonucleases to increase the efficiency of homology-directed repair (HDR). We developed a system to: (1) computationally identify unique transcription activator-like effector nuclease (TALEN) binding sites in the genome using a new software program, TALENSeek, (2) assemble the TALEN genes by combining golden gate cloning with modified constructs from the FLASH protocol, and (3) test the TALEN pairs in an amplification-based HDR assay that is more sensitive than the typical non-homologous end joining assay. We applied these methods to identify, construct, and test TALENs that were used with HDR donors in hESCs to generate an isogenic TS cell line in a scarless manner and to model the 16p11.2 copy number disorder without modifying genomic loci with high sequence similarity

All-conjugated block copolymers: from controlled synthesis to chiral expression

In the last two decades, the field of conjugated polymers (CPs) expanded rapidly. Besides the discovery of several controlled chain growth polymerizations and the expansion of the monomer scope, also research toward more complex CPs was performed. Copolymers, e.g. block, gradient and random copolymers, and polymers with different topologies, e.g. hyperbranched, star, circular, graft and toothbrush polymers, were synthesized, but the potential of this field remains large. Because the material properties do not only depend on the molecular structure, but also on the supramolecular structure, also the morphology of CPs is one of the frequently studied topics. For conjugated homopolymers, the aggregation behavior is already well described, but for more complex CPs, like block copolymers, a lot of blind spots remain. Therefore, this dissertation focusses on the controlled synthesis and (chiral) aggregation behavior of conjugated block copolymers. In the first part, the influence of branching of the side chain on the controlled polymerization of 3-alkylthiophenes was investigated. It was found that it reduces the propagation rate constant, leading to remarkably low dispersities. Also the maximal DP that could be obtained in a controlled manner was determined to be 150 and nona-stage polymers were synthesized. Further, it was established that a branched side chain diminishes the long term controlled character of the polymerization and that decomplexation of the Ni-catalyst from the polymer backbone was the main termination reaction. The second part deals with the controlled synthesis and (chiral) aggregation behavior of conjugated triblock copolymers. Triblock copolymers consisting of solely thiophene units, as well as triblock copolymers containing selenophene and (a)chiral thiophene units were synthesized in a controlled manner and by investigating their aggregation behavior, it was found that this is influenced by the order of the blocks. In the third part, block copolymers of electronically different monomers were synthesized. Their chiral aggregation behavior was studied in order to investigate whether block copolymers can be designed to exhibit a set of properties, which cannot be achieved in homopolymers, by transferring properties of one block to the other. In a stepwise approach, a poly(fluorene)-b-poly(thiophene) block copolymer, that exhibits β-phase aggregation and chiral expression (two aggregation features which cannot be obtained together in homopolymers), was designed. In the final part, the influence of the nature and the position of defects on the chiral expression in P3ATs was investigated. Polymers with one head-to-head (HH) or tail-to-tail (TT) defect in predetermined positions and one regioregular polymer were synthesized and their chiral aggregation behavior was compared. It was found that the largest chiral expression was obtained when the defect is situated at the beginning of the polymer chain. A TT defect always decreases the chiral expression, but an HH defect can cause an increased chiral expression compared with a complete regioregular polymer. Also, the effect of mixing polymers with a defect in different positions was investigated. For the polymers with an HH defect, no significant influence was observed, but for the polymers with a TT defect, the chiral expression was relatively lowered or enlarged, depending on the type of mixture that was made. This shows that not only the nature or the position of a defect influences the chiral expression, but also the mixing of polymers with a defect in different positions.status: publishe