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Cerebral organoids exhibit broad regional heterogeneity accompanied by limited cortical cellular diversity despite the tremendous upsurge in derivation methods, suggesting inadequate patterning of early neural stem cells (NSCs). Here we show that a short and early Dual SMAD and WNT inhibition course is necessary and sufficient to establish robust and lasting cortical organoid NSC identity, efficiently suppressing non-cortical NSC fates, while other widely used methods are inconsistent in their cortical NSC-specification capacity. Accordingly, this method selectively enriches for outer radial glia NSCs, which cyto-architecturally demarcate well-defined outer sub-ventricular-like regions propagating from superiorly radially organized, apical cortical rosette NSCs. Finally, this method culminates in the emergence of molecularly distinct deep and upper cortical layer neurons, and reliably uncovers cortex-specific microcephaly defects. Thus, a short SMAD and WNT inhibition is critical for establishing a rich cortical cell repertoire that enables mirroring of fundamental molecular and cyto-architectural features of cortical development and meaningful disease modelling.

DNA and Histone 3 Lysine 27 methylation typically function as repressive modifications and operate within distinct genomic compartments. In mammals, the majority of the genome is kept in a DNA methylated state, whereas the Polycomb repressive complexes regulate the unmethylated CpG-rich promoters of developmental genes. In contrast to this general framework, the extra-embryonic lineages display non-canonical, globally intermediate DNA methylation levels, including disruption of local Polycomb domains. Here, to better understand this unusual landscape’s molecular properties, we genetically and chemically perturbed major epigenetic pathways in mouse trophoblast stem cells. We find that the extra-embryonic epigenome reflects ongoing and dynamic de novo methyltransferase recruitment, which is continuously antagonized by Polycomb to maintain intermediate, locally disordered methylation. Despite its disorganized molecular appearance, our data point to a highly controlled equilibrium between counteracting repressors within extra-embryonic cells, one that can seemingly persist indefinitely without bistable features typically seen for embryonic forms of epigenetic regulation.

In this chapter, we will discuss the applications of CRISPR/Cas9 in the context of clinical diagnostics. We will provide an overview of existing methods and their use cases in the diagnostic field. Special attention will be given to selective sequencing approaches using third-generation sequencing and PAM-site requirements. As target sequences in an AT-rich environment cannot easily be accessed by the commercially available SpCas9 due to rarity of NGG PAM-sites, new enzymes such as ScCas9 with PAM-site requirements of NNG will be highlighted. Original research on CRISPR/Cas9 systems to determine molecular glioma markers by enriching regions of interest will be discussed in the context of potential future applications in clinical diagnostics.

The intraoperative diagnosis of brain tumors remains a clinical challenge despite recent technological advances. The current clinical practice differentiates non-surgical brain tumors from those preferably treated with cytoreductive surgery employing intraoperative frozen section diagnostics. A detailed molecular diagnosis required for this classification task within the timeframe of a routine neurosurgical procedure is currently unavailable. We have analyzed a clinical cohort of several brain tumor entities using Nanopore long-read sequencing on two Oxford Nanopore Technologies sequencing platforms (MinION, PromethION). Since currently available molecular cancer classifiers such as the DKFZ methylation profiling classifier cannot be readily adapted to real-time sequencing analysis, we implemented a novel algorithm (MethyLYZR) to predict the underlying cancer type. Publicly available Illumina Infinium array data were used to train the classifier to distinguish 91 brain tumor classes. For validation of classification accuracy, we conducted a comprehensive validation strategy. Both nanopore platforms could sequence more than 5,000 pre-selected CpG within less than 20 minutes for most of our samples. When combining an optimized library preparation protocol with the time used for sequencing the minimal number of CpGs needed for classification, we saw sample-to-answer times of less than 1 hour – in many cases within 45 minutes - from receiving a fresh biopsy to a robust cancer type prediction. Comparing actual and predicted diagnoses resulted in a favorable error rate, indicating potentially highly clinical validity. Our real-time based molecular diagnostic algorithm enables, in most cases, a reliable diagnostic call within the timeframe of a typical neuro-oncological surgery. MethyLYZR as a predictive tool may allow us to adjust the surgical strategy and deliver the prognosis to our patients right after surgery, thus allowing for as-of-yet unexplored opportunities for the intraoperative application of individualized therapeutic modalities.

Prader-Willi syndrome (PWS) is associated with severe hyperphagia, a specific behavioral phenotype and a high risk for developing psychotic episodes. Despite intense research, how genes within the PWS locus contribute to the phenotype remains elusive. In this study, we sequenced the whole genomes of 20 individuals with PWS using long-read nanopore sequencing by Oxford Nanopore Technologies (ONT). We demonstrate that ONT sequencing can resolve the PWS locus by determining the genetic subtype of PWS. Furthermore, we identified several novel structural variants (SV, >30bp) common in all PWS individuals. We are the first to show that the opioid system and the nociceptin/orphanin FQ system may be affected in PWS due to SVs in OPRM1 and OPRL1. Furthermore, we demonstrate that individuals with PWS, especially those with psychosis, exhibit a high burden of SVs in loci with known associations with bipolar disorder, schizophrenia and autism spectrum disorder. Our results challenge the current hypothesis that the PWS phenotype can be mainly explained by the loss of paternally expressed genes on chr15q11.2-13.

The northern white rhinoceros (NWR; Ceratotherium simum cottoni) is functionally extinct, with only two females remaining alive. Efforts to rescue the NWR have inspired the exploration of unconventional conservation methods, including the generation of artificial gametes from induced pluripotent stem cells and somatic cell nuclear transfer. To enable the technologies required for these approaches, we used complementary sequencing and mapping methods to generate a NWR chromosome-level reference genome that meets or exceeds the metrics proposed by the Vertebrate Genome Project. It represents 40 autosomes, an X and a partially-resolved Y chromosome, and the mitochondrial genome. We compared the NWR reference genome to the southern white rhinoceros (SWR) population that has been physically separated from the NWR for tens of thousands of years. Using short-read data from the SWR and optical mapping, we found that the two populations are very similar on both the chromosome level and mitochondrial genome level. The results of this study are encouraging for the efforts underway to rescue the NWR.

Two isogenic hiPSC lines, ZIPi013-B and ZIPi013-E, were generated by reprogramming fetal dermal fibroblasts with episomal vectors. Previously, the same fetal fibroblasts were reprogrammed multiple times in a study comparing other reprogramming methods. As a consequence, the genomes have been sequenced multiple times. Both new cell lines offer the opportunity to study basic stem cell biology and model human disease. They can be applied as reference cell lines for creating isogenic clones bearing disease mutations on a well-characterized genomic background, as both cell lines have demonstrated excellent differentiation capacity in multiple labs. Resource table Unique stem cell lines identifier ZIPi013-B ZIPi013-E Alternative names of stem cell lines ZIP13K2 (ZIPi013-B) ZIP13K5 (ZIPi013-E) Institution Zentrum fur Integrative Psychiatrie gGmbH, Kiel, Germany Contact information of distributor PD Dr. Franz-Josef Muller, franz-josef.mueller@uksh.de Type of cell lines iPSC Origin human Cell Source Fibroblasts Clonality Clonal Method of reprogramming Transgene free, episomal Multiline rationale Isogenic clones Gene modification NO Type of modification N/A Associated disease N/A Gene/locus N/A Method of modification N/A Name of transgene or resistance N/A Inducible/constitutive system N/A Date archived/stock date Stock date ZIPi013-B 8th December 2017, stock date ZIPi013-E 12th December 2017 Cell line repository/bank N/A Ethical approval https://www.sciencellonline.com/technical-support/ethical-statement.html Ethikkommission der medizinischen Fakultat der Christian-Albrechts-Universitat zu Kiel, approval number A145/11

The embryonic stem cell test (EST) represents the only validated and accepted in vitro system for the detection and classification of compounds according to their developmental and reproductive teratogenic potency. The widespread implementation of the EST, however, in particular for routine application in pharmaceutical development, has not been achieved so far. Several drawbacks still limit the high-throughput screening of potential drug candidates in this format: The long assay period, the use of non-homogeneous viability assays, the low throughput analysis of marker protein expression and the compatibility of the assay procedures to automation. We have therefore introduced several advancements into the EST workflow: A reduction of the assay period, an introduction of homogeneous viability assays, and a straightforward analysis of marker proteins by flow cytometry and high content imaging to assess the impact of small molecules on differentiation capacity. Most importantly, essential parts of the assay procedure have been adapted to lab automation in 96-well format, thus enabling the interrogation of several compounds in parallel. In addition, extensive investigations were performed to explore the predictive capacity of this next-generation EST, by testing a set of well-known embryotoxicants that encompasses the full range of chemical-inherent embryotoxic potencies possible. Due to these significant improvements, the augmented workflow provides a basis for a sensitive, more rapid, and reproducible high throughput screening compatible platform to predict in vivo developmental toxicity from in vitro data which paves the road towards application in an industrial setting. Graphical abstract•The embryonic stem cell test to predict teratogenicity was made automation-compatible.•Several key improvements to the assay procedure have been introduced to increase performance.•The workflow was adapted to human iPS cells and isogenic fibroblast donor cells. Electronic supplementary material The online version of this article (10.1007/s10565-020-09538-0) contains supplementary material, which is available to authorized users.

DNA methylation plays a critical role during development, particularly in repressing retrotransposons. The mammalian methylation landscape is dependent on the combined activities of the canonical maintenance enzyme Dnmt1 and the de novo Dnmts, 3a and 3b. Here, we demonstrate that Dnmt1 displays de novo methylation activity in vitro and in vivo with specific retrotransposon targeting. We used whole-genome bisulfite and long-read Nanopore sequencing in genetically engineered methylation-depleted mouse embryonic stem cells to provide an in-depth assessment and quantification of this activity. Utilizing additional knockout lines and molecular characterization, we show that the de novo methylation activity of Dnmt1 depends on Uhrf1, and its genomic recruitment overlaps with regions that enrich for Uhrf1, Trim28 and H3K9 trimethylation. Our data demonstrate that Dnmt1 can catalyze DNA methylation in both a de novo and maintenance context, especially at retrotransposons, where this mechanism may provide additional stability for long-term repression and epigenetic propagation throughout development., Nature Structural & Molecular Biology, 28 (7), ISSN:1545-9993, ISSN:1545-9985

Mitochondrial disorders causing neurodegeneration in childhood are genetically heterogeneous, and the underlying genetic etiology remains unknown in many affected individuals. We identified biallelic variants in PMPCB in individuals of four families including one family with two affected siblings with neurodegeneration and cerebellar atrophy. PMPCB encodes the catalytic subunit of the essential mitochondrial processing protease (MPP), which is required for maturation of the majority of mitochondrial precursor proteins. Mitochondria isolated from two fibroblast cell lines and induced pluripotent stem cells derived from one affected individual and differentiated neuroepithelial stem cells showed reduced PMPCB levels and accumulation of the processing intermediate of frataxin, a sensitive substrate for MPP dysfunction. Introduction of the identified PMPCB variants into the homologous S. cerevisiae Mas1 protein resulted in a severe growth and MPP processing defect leading to the accumulation of mitochondrial precursor proteins and early impairment of the biogenesis of iron-sulfur clusters, which are indispensable for a broad range of crucial cellular functions. Analysis of biopsy materials of an affected individual revealed changes and decreased activity in iron-sulfur cluster-containing respiratory chain complexes and dysfunction of mitochondrial and cytosolic Fe-S cluster-dependent enzymes. We conclude that biallelic mutations in PMPCB cause defects in MPP proteolytic activity leading to dysregulation of iron-sulfur cluster biogenesis and triggering a complex neurological phenotype of neurodegeneration in early childhood.

Mammalian cells stably maintain high levels of DNA methylation despite expressing both positive (DNMT3A/B) and negative (TET1–3) regulators. Here, we used wildtype and TET triple knockout human embryonic stem cells (ESCs), generated DNMT3-null as well as TET and DNMT3-null pentuple knockouts, and compared methylation patterns using whole genome bisulfite sequencing (WGBS). The greatest impact on global methylation levels was observed in DNMT3-deficient cells, including reproducible focal demethylation at thousands of normally methylated loci. This demethylation depends upon TET expression and only occurs when both DNMT3s are absent. Dynamic loci are enriched for hydroxymethylcytosine and overlap with subsets of putative somatic enhancers that are methylated in ESCs and can be activated upon differentiation. We observe similar dynamics in mouse ESCs that were less frequenct in epiblast stem cells (EpiSCs) and scarce in somatic tissues, suggesting a conserved pluripotency-linked mechanism. Taken together, our data reveal tightly regulated competition between DNMT3s and TETs at thousands of somatic regulatory sequences within pluripotent cells.

Nanopore DNA sequencing enables the sequence determination of single DNA molecules up to 10,000 times longer than currently permitted by second-generation sequencing platforms. Nanopore sequencing gives real-time access to sequencing data and enables the detection of epigenetic modifications. This unique feature set is poised to foster the development of novel biomedical applications previously deemed unfeasible. Nanopore sequencing is based on picoampere scale measurement of current modulated by DNA or RNA polymers traveling through a nanometer opening between two compartments. Each of the five canonical nucleobases (A, T, G, C, U) has a characteristic electrical resistance, which ultimately enables the determination of the precise base sequence. However, a substantial computational effort is required to resolve the underlying sequence from a time-warped and noisy stream of digitized current measurements. Recently, a wide range of digital signal analysis and machine learning methods have been developed for Nanopore sequencing applications. Clinically relevant questions, such as the quantification of short repetitive DNA sequences remain an unresolved challenge to current generic, state-of-the-art nanopore data analysis methods. We believe realistic simulation of the signal stream can be instrumental in the development of tailored algorithms for such novel biomedical applications. Based on our work with the Oxford Nanopore Technologies MinION and PromethION platform, we have developed Nanopore SimulatION, a software package for the in silico generation of realistic, raw-signal-level data. Nanopore SimulatION starts from a reference genome in conjunction with a configuration and model file derived from real-world nanopore sequencing experiments as input. To validate our algorithm, we have sequenced custom synthetic DNA, and in so doing have generated a “ground-truth” data set potentially useful for downstream algorithm development. Additionally, we demonstrate Nanopore SimulatION‘ s utility for method development in typical clinical use cases. Supplementary examples, raw data obtained from our synthetic DNA sequencing experiments and the software are available under the open Mozilla Public license at https://github.com/crohrandt/.

Expansions of short tandem repeats are genetic variants that have been implicated in neuropsychiatric and other disorders but their assessment remains challenging with current molecular methods. Here, we developed a Cas12a-based enrichment strategy for nanopore sequencing that, combined with a new algorithm for raw signal analysis, enables us to efficiently target, sequence and precisely quantify repeat numbers as well as their DNA methylation status. Taking advantage of these single molecule nanopore signals provides therefore unprecedented opportunities to study pathological repeat expansions.

Expansions of short tandem repeats are genetic variants that have been implicated in several neuropsychiatric and other disorders, but their assessment remains challenging with current polymerase-based methods1-4. Here we introduce a CRISPR-Cas-based enrichment strategy for nanopore sequencing combined with an algorithm for raw signal analysis. Our method, termed STRique for short tandem repeat identification, quantification and evaluation, integrates conventional sequence mapping of nanopore reads with raw signal alignment for the localization of repeat boundaries and a hidden Markov model-based repeat counting mechanism. We demonstrate the precise quantification of repeat numbers in conjunction with the determination of CpG methylation states in the repeat expansion and in adjacent regions at the single-molecule level without amplification. Our method enables the study of previously inaccessible genomic regions and their epigenetic marks.

Miller-Dieker syndrome (MDS) is caused by a heterozygous deletion of chromosome 17p13.3 involving the genes LIS1 and YWHAE (coding for 14.3.3ε) and leads to malformations during cortical development. Here, we used patient-specific forebrain-type organoids to investigate pathological changes associated with MDS. Patient-derived organoids are significantly reduced in size, a change accompanied by a switch from symmetric to asymmetric cell division of ventricular zone radial glia cells (vRGCs). Alterations in microtubule network organization in vRGCs and a disruption of cortical niche architecture, including altered expression of cell adhesion molecules, are also observed. These phenotypic changes lead to a non-cell-autonomous disturbance of the N-cadherin/β-catenin signaling axis. Reinstalling active β-catenin signaling rescues division modes and ameliorates growth defects. Our data define the role of LIS1 and 14.3.3ε in maintaining the cortical niche and highlight the utility of organoid-based systems for modeling complex cell-cell interactions in vitro.

Pluripotency is a term in cell biology describing a unique state present in distinct stem cell lines, which were either established from the inner cell mass of the mammalian embryo or derived from somatic cells that have been reprogrammed to induced pluripotent stem cells. Pluripotent stem cells are continuously self-renewing, and their differentiation capacity enables them to develop into all derivatives of the three germ layers of a gastrulating embryo (endoderm, ectoderm, mesoderm). Both human embryonic stem cells (hESC) and human-induced pluripotent stem cells (hiPSC) are virtually indistinguishable, at least based on their global RNA expression patterns. Yet, after these in vitro cell cultures have been generated, the cell lines’ pluripotent properties may change considerably on the genetic and/or epigenetic level as a consequence of long-term propagation. Among other unphysiological changes, cell lines might acquire aneuploidies, loose physiological imprinting marks, or develop differentiation biases favoring one cell lineage over the other. As a result, stem cell researchers have to continuously monitor each stem cell line’s integrity, transcriptional profile, and functional properties. Regulatory transcription factors, protein-protein interactions, and signaling networks govern the pluripotent state. As a consequence, emerging small- and large-scale perturbations to these gene regulatory networks mediate the outlined unfavorable changes to the pluripotent phenotype. Here, we describe a reliable bioinformatic framework called PluriTest for confirmation and assessment of pluripotency as an animal-free, fast, and inexpensive way based on genome-wide transcriptional RNA profiles from microarrays. Additionally, we discuss future developments using RNA expression profiling for pluripotency assessment.

Assessing pluripotency in human cells is inherently an intractable problem. In animal systems, pluripotency can be verified through direct means: pluripotent stem cells can be introduced into an developing embryo and thus the cellular developmental potential of any given in vitro preparation can be directly determined by observing the amount of chimaerism or viability of organisms partially or fully derived from in vitro stem cells ( Nagy, Rossant et al ., 1993 ). For obvious ethical reasons such stringent experimentation is impossible in the human system; thus indirect evidence through biomarkers and correlative measures of differentiation potential are the next best level of evidence for human stem cell pluripotency. Many if not most stem cell markers that are widely used for predicting pluripotency in human cells are chosen because of evidence from other species. Yet there are hard limitations to this approach because of two main obstacles: first, using only few markers will erroneously validate potentially abnormal stem cells when these irregular preparations express the limited set of markers similar to normal stem cells. Second, in spite of some striking orthologous similarities, the transfer of mechanistic insight from animals to the human systems for fine-grained determination of pluripotent features is problematic. There are species-specific differences in the cultured pluripotent phenotypes and significant evolutionary structural variations in the transcription network wiring of human and animal cells. We have developed a pragmatic, purely data-driven approach toward empirically defining the “normal” human pluripotent state via interrogation of large scale datasets of genome-wide somatic and pluripotent expression profiles ( Muller, Schuldt et al ., 2011 ). Models derived from this dataset can be used to rapidly, confidently and inexpensively assess the pluripotency of human cells through web-based analysis of microarray data from new stem cell preparations. In this article, we will introduce the reader into some of the underlying concepts of the tool we call “PluriTest”, demonstrate a real world use case, and discuss the potential as well as the limitations of our approach.

The introduction of stem cell-associated molecular factors into human patient-derived cells allows for their reprogramming in the laboratory environment. As a result, human induced pluripotent stem cells (hiPSC) can now be reprogrammed epigenetically without disruption of their overall genomic integrity. For patients with neurodegenerative diseases characterized by progressive loss of functional neurons, the ability to reprogram any individual's cells and drive their differentiation toward susceptible neuronal subtypes holds great promise. Apart from applications in regenerative medicine and cell replacement–based therapy, hiPSCs are increasingly used in preclinical research for establishing disease models and screening for drug toxicities. The rapid developments in this field prompted us to review recent progress toward the applications of stem cell technologies for movement disorders. We introduce reprogramming strategies and explain the critical steps in the differentiation of hiPSCs to clinical relevant subtypes of cells in the context of movement disorders. We summarize and discuss recent discoveries in this field, which, based on the rapidly expanding basic science literature as well as upcoming trends in personalized medicine, will strongly influence the future therapeutic options available to practitioners working with patients suffering from such disorders. © 2014 International Parkinson and Movement Disorder Society