detecting-inter-individual-contamination-mismatches-multiomics-next

Detecting Inter-Individual Contamination and Mismatches in Multiomics Next-Generation Sequencing Data

High-throughput processing of patient biosamples by next-generation sequencing and the comparison of molecular data with patient-level and sample-level ...

Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd

In the past decade the Göttingen minipig has gained increasing recognition as animal model in pharmaceutical and safety research because it recapitulates many aspects of human physiology and metabolism. Genome-based comparison of drug targets together with quantitative tissue expression analysis allows rational prediction of pharmacology and cross-reactivity of human drugs in animal models thereby improving drug attrition which is an important challenge in the process of drug development.

Functional analysis and transcriptional output of the Göttingen minipig genome.

The Eastern woodchuck () has been extensively used in research of chronic hepatitis B and liver cancer because its infection with the woodchuck hepatitis virus closely resembles a human hepatitis B virus infection. Development of novel immunotherapeutic approaches requires genetic information on immune pathway genes in this animal model. The woodchuck genome was assembled with a combination of high-coverage whole-genome shotgun sequencing of Illumina paired-end, mate-pair libraries and fosmid pool sequencing. The result is a 2.63 Gigabase (Gb) assembly with a contig N50 of 74.5 kilobases (kb), scaffold N50 of 892 kb, and genome completeness of 99.2%. RNA sequencing (RNA-seq) from seven different tissues aided in the annotation of 30,873 protein-coding genes, which in turn encode 41,826 unique protein products. More than 90% of the genes have been functionally annotated, with 82% of them containing open reading frames. This genome sequence and its annotation will enable further research in chronic hepatitis B and hepatocellular carcinoma and contribute to the understanding of immunological responses in the woodchuck.

The Genome Sequence of the Eastern Woodchuck () - A Preclinical Animal Model for Chronic Hepatitis B.

Endothelial cells (ECs) display remarkable plasticity during development before becoming quiescent and functionally mature. EC maturation is directed by several known transcription factors (TFs), but the specific set of TFs responsible for promoting high-resistance barriers, such as the blood-brain barrier (BBB), have not yet been fully defined. Using expression mRNA data from published studies on ex vivo ECs from the central nervous system (CNS), we predicted TFs that induce high-resistance barrier properties of ECs as in the BBB. We used our previously established method to  generate ECs from human pluripotent stem cells (hPSCs), and then we overexpressed the candidate TFs in hPSC-ECs and measured barrier resistance and integrity using electric cell-substrate impedance sensing, trans-endothelial electrical resistance and FITC-dextran permeability assays. SOX18 and TAL1 were the strongest EC barrier-inducing TFs, upregulating Wnt-related signaling and EC junctional gene expression, respectively, and downregulating EC proliferation-related genes. These TFs were combined with SOX7 and ETS1 that together effectively induced EC barrier resistance, decreased paracellular transport and increased protein expression of tight junctions and induce mRNA expression of several genes involved in the formation of EC barrier and transport. Our data shows identification of a transcriptional network that controls barrier resistance in ECs. Collectively this data may lead to novel approaches for generation of in vitro models of the BBB.

Identification of a combination of transcription factors that synergistically increases endothelial cell barrier resistance.

Microglia are key in the homeostatic well-being of the brain and microglial dysfunction has been implicated in neurodegenerative disorders such as Alzheimer's disease (AD). Due to the many limitations to study microglia  or isolated for large scale drug discovery applications, there is a high need to develop robust and scalable human cellular models of microglia with reliable translatability to the disease. Here, we describe the generation of microglia-like cells from human induced pluripotent stem cells (iPSC) with distinct phenotypes for mechanistic studies in AD. We started out from an established differentiation protocol to generate primitive macrophage precursors mimicking the yolk sac ontogeny of microglia. Subsequently, we tested 36 differentiation conditions for the cells in monoculture where we exposed them to various combinations of media, morphogens, and extracellular matrices. The optimized protocol generated robustly ramified cells expressing key microglial markers. Bulk mRNA sequencing expression profiles revealed that compared to cells obtained in co-culture with neurons, microglia-like cells derived from a monoculture condition upregulate mRNA levels for Triggering Receptor Expressed On Myeloid Cells 2 (TREM2), which is reminiscent to the previously described disease-associated microglia. TREM2 is a risk gene for AD and an important regulator of microglia. The regulatory function of TREM2 in these cells was confirmed by comparing wild type with isogenic TREM2 knock-out iPSC microglia. The TREM2-deficient cells presented with stronger increase in free cytosolic calcium upon stimulation with ATP and ADP, as well as stronger migration towards complement C5a, compared to TREM2 expressing cells. The functional differences were associated with gene expression modulation of key regulators of microglia. In conclusion, we have established and validated a work stream to generate functional human iPSC-derived microglia-like cells by applying a directed and neuronal co-culture independent differentiation towards functional phenotypes in the context of AD. These cells can now be applied to study AD-related disease settings and to perform compound screening and testing for drug discovery.

Alzheimer's Risk Gene TREM2 Determines Functional Properties of New Type of Human iPSC-Derived Microglia.

Microglia, the innate immune cells of the brain, are essential for maintaining homeostasis by their ramified, highly motile processes and for orchestrating the immune response to pathological stimuli. They are implicated in several neurodegenerative diseases like Alzheimer's and Parkinson's disease. One commonality of these diseases is their strong correlation with aging as the highest risk factor and studying age-related alterations in microglia physiology and associated signaling mechanism is indispensable for a better understanding of age-related pathomechanisms. CD22 has been identified as a modifier of microglia phagocytosis in a recent study, but not much is known about the function of CD22 in microglia. Here we show that CD22 surface levels are upregulated in aged versus adult microglia. Furthermore, in the amyloid mouse model PS2APP, Aβ-containing microglia also exhibit increased CD22 signal. To assess the impact of CD22 blockage on microglia morphology and dynamics, we have established a protocol to image microglia process motility in acutely prepared brain slices from CX3CR1-GFP reporter mice. We observed a significant reduction of microglial ramification and surveillance capacity in brain slices from aged versus adult mice. The age-related decrease in surveillance can be restored by antibody-mediated CD22 blockage in aged mice, whereas surveillance in adult mice is not affected by CD22 inhibition. Moreover to complement the results obtained in mice, we show that human iPSC-derived macrophages exhibit an increased phagocytic capacity upon CD22 blockage. Downstream analysis of antibody-mediated CD22 inhibition revealed an influence on BMP and TGFβ associated gene networks. Our results demonstrate CD22 as a broad age-associated modulator of microglia functionality with potential implications for neurodegenerative disorders.

CD22 Blockage Restores Age-Related Impairments of Microglia Surveillance Capacity.

Age-related macular degeneration (AMD), one of the leading causes of blindness worldwide, causes personal suffering and high socioeconomic costs. While there has been progress in the treatments for the neovascular form of AMD, no therapy is yet available for the more common dry form, also known as geographic atrophy. We analysed the retinal tissue in a mouse model of retinal degeneration caused by sodium iodate (NaIO)-induced retinal pigment epithelium (RPE) atrophy to understand the underlying pathology. RNA sequencing (RNA-seq), qRT-PCR, Western blot, immunohistochemistry of the retinas and multiplex ELISA of the mouse serum were applied to find the pathways involved in the degeneration. NaIO caused patchy RPE loss and thinning of the photoreceptor layer. This was accompanied by the increased retinal expression of complement components , ,  and . C1s, C3, CFH and CFB were complement proteins, with enhanced deposition at day 3. C4 was upregulated in retinal degeneration at day 10. Consistently, the transcript levels of proinflammatory , , , ,  and  were increased in the retinas of NaIO mice, but  mRNA was reduced. Macrophages, microglia and gliotic Müller cells could be a cellular source for local retinal inflammatory changes in the NaIO retina. Systemic complement and cytokines/chemokines remained unaltered in this model of NaIO-dependent retinal degeneration. In conclusion, systemically administered NaIO promotes degenerative and inflammatory processes in the retina, which can mimic the hallmarks of geographic atrophy.

Sodium Iodate-Induced Degeneration Results in Local Complement Changes and Inflammatory Processes in Murine Retina.

Bruton's tyrosine kinase (BTK) is an intracellular signaling enzyme that regulates B-lymphocyte and myeloid cell functions. Due to its involvement in both innate and adaptive immune compartments, BTK inhibitors have emerged as a therapeutic option in autoimmune disorders such as multiple sclerosis (MS). Brain-penetrant, small-molecule BTK inhibitors may also address compartmentalized neuroinflammation, which is proposed to underlie MS disease progression. BTK is expressed by microglia, which are the resident innate immune cells of the brain; however, the precise roles of microglial BTK and impact of BTK inhibitors on microglial functions are still being elucidated. Research on the effects of BTK inhibitors has been limited to rodent disease models. This is the first study reporting effects in human microglia.

Article	Year
Functional analysis and transcriptional output of the Göttingen minipig genome. BMC genomics\| PubMed ID: 26573612	2015
The Genome Sequence of the Eastern Woodchuck () - A Preclinical Animal Model for Chronic Hepatitis B. G3 (Bethesda, Md.)\| PubMed ID: 31645421	2019
Identification of a combination of transcription factors that synergistically increases endothelial cell barrier resistance. Scientific reports\| PubMed ID: 32127614	2020
Alzheimer's Risk Gene TREM2 Determines Functional Properties of New Type of Human iPSC-Derived Microglia. Frontiers in immunology\| PubMed ID: 33613545	2020
CD22 Blockage Restores Age-Related Impairments of Microglia Surveillance Capacity. Frontiers in immunology\| PubMed ID: 34140954	2021
Sodium Iodate-Induced Degeneration Results in Local Complement Changes and Inflammatory Processes in Murine Retina. International journal of molecular sciences\| PubMed ID: 34502128	2021
Fenebrutinib, a Bruton's tyrosine kinase inhibitor, blocks distinct human microglial signaling pathways. Journal of neuroinflammation\| PubMed ID: 39465429	2024

Roland Schmucki

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