Othmar Korn

Divergent reprogramming routes lead to alternative stem-cell states

Pluripotency is defined by the ability of a cell to differentiate to the derivatives of all the three embryonic germ layers: ectoderm, mesoderm and endoderm. Pluripotent cells can be captured via the archetypal derivation of embryonic stem cells or via somatic cell reprogramming. Somatic cells are induced to acquire a pluripotent stem cell (iPSC) state through the forced expression of key transcription factors, and in the mouse these cells can fulfil the strictest of all developmental assays for pluripotent cells by generating completely iPSC-derived embryos and mice. However, it is not known whether there are additional classes of pluripotent cells, or what the spectrum of reprogrammed phenotypes encompasses. Here we explore alternative outcomes of somatic reprogramming by fully characterizing reprogrammed cells independent of preconceived definitions of iPSC states. We demonstrate that by maintaining elevated reprogramming factor expression levels, mouse embryonic fibroblasts go through unique epigenetic modifications to arrive at a stable, Nanog-positive, alternative pluripotent state. In doing so, we prove that the pluripotent spectrum can encompass multiple, unique cell states.

Stemformatics: visualisation and sharing of stem cell gene expression.

Genome-scale technologies are increasingly adopted by the stem cell research community, because of the potential to uncover the molecular events most informative about a stem cell state. These technologies also present enormous challenges around the sharing and visualisation of data derived from different laboratories or under different experimental conditions. Stemformatics is an easy to use, publicly accessible portal that hosts a large collection of exemplar stem cell data. It provides fast visualisation of gene expression across a range of mouse and human datasets, with transparent links back to the original studies. One difficulty in the analysis of stem cell signatures is the paucity of public pathways/gene lists relevant to stem cell or developmental biology. Stemformatics provides a simple mechanism to create, share and analyse gene sets, providing a repository of community-annotated stem cell gene lists that are informative about pathways, lineage commitment, and common technical artefacts. Stemformatics can be accessed at stemformatics.org.

Parallel Simulated Annealing for Materialized View Selection in Data Warehousing Environments

In order to facilitate efficient query processing, the information contained in data warehouses is typically stored as a set of materialized views. Deciding which views to materialize represent a challenge in order to minimize view maintenance and query processing costs. Some existing approaches are applicable only for small problems, which are far from reality. In this paper we introduce a new approach for materialized view selection using Parallel Simulated Annealing (PSA) that selects views from an input Multiple View Processing Plan (MVPP). With PSA, we are able to perform view selection on MVPPs having hundreds of queries and thousands of views. Also, in our experimental study we show that our method provides a significant improvement in the quality of the obtained set of materialized views over existing heuristic and sequential simulated annealing algorithms.

An atypical role for the myeloid receptor Mincle in central nervous system injury.

The C-type lectin Mincle is implicated in innate immune responses to sterile inflammation, but its contribution to associated pathologies is not well understood. Herein, we show that Mincle exacerbates neuronal loss following ischemic but not traumatic spinal cord injury. Loss of Mincle was beneficial in a model of transient middle cerebral artery occlusion but did not alter outcomes following heart or gut ischemia. High functional scores in Mincle KO animals using the focal cerebral ischemia model were accompanied by reduced lesion size, fewer infiltrating leukocytes and less neutrophil-derived cytokine production than isogenic controls. Bone marrow chimera experiments revealed that the presence of Mincle in the central nervous system, rather than recruited immune cells, was the critical regulator of a poor outcome following transient middle cerebral artery occlusion. There was no evidence for a direct role for Mincle in microglia or neural activation, but expression in a subset of macrophages resident in the perivascular niche provided new clues on Mincles role in ischemic stroke.

A molecular classification of human mesenchymal stromal cells

Mesenchymal stromal cells (MSC) are widely used for the study of mesenchymal tissue repair, and increasingly adopted for cell therapy, despite the lack of consensus on the identity of these cells. In part this is due to the lack of specificity of MSC markers. Distinguishing MSC from other stromal cells such as fibroblasts is particularly difficult using standard analysis of surface proteins, and there is an urgent need for improved classification approaches. Transcriptome profiling is commonly used to describe and compare different cell types; however, efforts to identify specific markers of rare cellular subsets may be confounded by the small sample sizes of most studies. Consequently, it is difficult to derive reproducible, and therefore useful markers. We addressed the question of MSC classification with a large integrative analysis of many public MSC datasets. We derived a sparse classifier (The Rohart MSC test) that accurately distinguished MSC from non-MSC samples with >97% accuracy on an internal training set of 635 samples from 41 studies derived on 10 different microarray platforms. The classifier was validated on an external test set of 1,291 samples from 65 studies derived on 15 different platforms, with >95% accuracy. The genes that contribute to the MSC classifier formed a protein-interaction network that included known MSC markers. Further evidence of the relevance of this new MSC panel came from the high number of Mendelian disorders associated with mutations in more than 65% of the network. These result in mesenchymal defects, particularly impacting on skeletal growth and function. The Rohart MSC test is a simple in silico test that accurately discriminates MSC from fibroblasts, other adult stem/progenitor cell types or differentiated stromal cells. It has been implemented in the www.stemformatics.org resource, to assist researchers wishing to benchmark their own MSC datasets or data from the public domain. The code is available from the CRAN repository and all data used to generate the MSC test is available to download via the Gene Expression Omnibus or the Stemformatics resource.

Characterization of Phenotypic and Transcriptional Differences in Human Pluripotent Stem Cells under 2D and 3D Culture Conditions

Human pluripotent stem cells hold great promise for applications in drug discovery and regenerative medicine. Microfluidic technology is a promising approach for creating artificial microenvironments; however, although a proper 3D microenvironment is required to achieve robust control of cellular phenotypes, most current microfluidic devices provide only 2D cell culture and do not allow tuning of physical and chemical environmental cues simultaneously. Here, the authors report a 3D cellular microenvironment plate (3D-CEP), which consists of a microfluidic device filled with thermoresponsive poly(N-isopropylacrylamide)-β-poly(ethylene glycol) hydrogel (HG), which enables systematic tuning of both chemical and physical environmental cues as well as in situ cell monitoring. The authors show that H9 human embryonic stem cells (hESCs) and 253G1 human induced pluripotent stem cells in the HG/3D-CEP system maintain their pluripotent marker expression under HG/3D-CEP self-renewing conditions. Additionally, global gene expression analyses are used to elucidate small variations among different test environments. Interestingly, the authors find that treatment of H9 hESCs under HG/3D-CEP self-renewing conditions results in initiation of entry into the neural differentiation process by induction of PAX3 and OTX1 expression. The authors believe that this HG/3D-CEP system will serve as a versatile platform for developing targeted functional cell lines and facilitate advances in drug screening and regenerative medicine.

Human iPSC-Derived Cerebellar Neurons from a Patient with Ataxia-Telangiectasia Reveal Disrupted Gene Regulatory Networks

Ataxia-telangiectasia (A-T) is a rare genetic disorder caused by loss of function of the ataxia-telangiectasia-mutated kinase and is characterized by a predisposition to cancer, pulmonary disease, immune deficiency and progressive degeneration of the cerebellum. As animal models do not faithfully recapitulate the neurological aspects, it remains unclear whether cerebellar degeneration is a neurodevelopmental or neurodegenerative phenotype. To address the necessity for a human model, we first assessed a previously published protocol for the ability to generate cerebellar neuronal cells, finding it gave rise to a population of precursors highly enriched for markers of the early hindbrain such as EN1 and GBX2, and later more mature cerebellar markers including PTF1α, MATH1, HOXB4, ZIC3, PAX6, and TUJ1. RNA sequencing was used to classify differentiated cerebellar neurons generated from integration-free A-T and control induced pluripotent stem cells. Comparison of RNA sequencing data with datasets from the Allen Brain Atlas reveals in vitro-derived cerebellar neurons are transcriptionally similar to discrete regions of the human cerebellum, and most closely resemble the cerebellum at 22 weeks post-conception. We show that patient-derived cerebellar neurons exhibit disrupted gene regulatory networks associated with synaptic vesicle dynamics and oxidative stress, offering the first molecular insights into early cerebellar pathogenesis of ataxia-telangiectasia.

Corrigendum: Divergent reprogramming routes lead to alternative stem-cell states

This corrects the article DOI: 10.1038/nature14047

The Impact of APP on Alzheimer-like Pathogenesis and Gene Expression in Down Syndrome iPSC-Derived Neurons

Summary Early-onset Alzheimer disease (AD)-like pathology in Down syndrome is commonly attributed to an increased dosage of the amyloid precursor protein (APP) gene. To test this in an isogenic human model, we deleted the supernumerary copy of the APP gene in trisomic Down syndrome induced pluripotent stem cells or upregulated APP expression in euploid human pluripotent stem cells using CRISPRa. Cortical neuronal differentiation shows that an increased APP gene dosage is responsible for increased β-amyloid production, altered Aβ42/40 ratio, and deposition of the pyroglutamate (E3)-containing amyloid aggregates, but not for several tau-related AD phenotypes or increased apoptosis. Transcriptome comparisons demonstrate that APP has a widespread and temporally modulated impact on neuronal gene expression. Collectively, these data reveal an important role for APP in the amyloidogenic aspects of AD but challenge the idea that increased APP levels are solely responsible for increasing specific phosphorylated forms of tau or enhanced neuronal cell death in Down syndrome-associated AD pathogenesis.

Group A Streptococcus M1T1 intracellular infection of primary tonsil epithelial cells dampens levels of secreted IL-8 through the action of SpyCEP

Streptococcus pyogenes (Group A Streptococcus; GAS) commonly causes pharyngitis in children and adults, with severe invasive disease and immune sequelae being an infrequent consequence. The ability of GAS to invade the host and establish infection likely involves subversion of host immune defenses. However, the signaling pathways and innate immune responses of epithelial cells to GAS are not well-understood. In this study, we utilized RNAseq to characterize the inflammatory responses of primary human tonsil epithelial (TEpi) cells to infection with the laboratory-adapted M6 strain JRS4 and the M1T1 clinical isolate 5448. Both strains induced the expression of genes encoding a wide range of inflammatory mediators, including IL-8. Pathway analysis revealed differentially expressed genes between mock and JRS4- or 5448-infected TEpi cells were enriched in transcription factor networks that regulate IL-8 expression, such as AP-1, ATF-2, and NFAT. While JRS4 infection resulted in high levels of secreted IL-8, 5448 infection did not, suggesting that 5448 may post-transcriptionally dampen IL-8 production. Infection with 5448ΔcepA, an isogenic mutant lacking the IL-8 protease SpyCEP, resulted in IL-8 secretion levels comparable to JRS4 infection. Complementation of 5448ΔcepA and JRS4 with a plasmid encoding 5448-derived SpyCEP significantly reduced IL-8 secretion by TEpi cells. Our results suggest that intracellular infection with the pathogenic GAS M1T1 clone induces a strong pro-inflammatory response in primary tonsil epithelial cells, but modulates this host response by selectively degrading the neutrophil-recruiting chemokine IL-8 to benefit infection.