Publications by authors named "Thierry Voet"

89 Publications

Enhanced chromatin accessibility contributes to X chromosome dosage compensation in mammals.

Genome Biol 2021 11 1;22(1):302. Epub 2021 Nov 1.

Department of Development and Regeneration, Laboratory of Cellular Reprogramming and Epigenetic Regulation, KU Leuven - University of Leuven, Herestraat 49, 3000, Leuven, Belgium.

Background: Precise gene dosage of the X chromosomes is critical for normal development and cellular function. In mice, XX female somatic cells show transcriptional X chromosome upregulation of their single active X chromosome, while the other X chromosome is inactive. Moreover, the inactive X chromosome is reactivated during development in the inner cell mass and in germ cells through X chromosome reactivation, which can be studied in vitro by reprogramming of somatic cells to pluripotency. How chromatin processes and gene regulatory networks evolved to regulate X chromosome dosage in the somatic state and during X chromosome reactivation remains unclear.

Results: Using genome-wide approaches, allele-specific ATAC-seq and single-cell RNA-seq, in female embryonic fibroblasts and during reprogramming to pluripotency, we show that chromatin accessibility on the upregulated mammalian active X chromosome is increased compared to autosomes. We further show that increased accessibility on the active X chromosome is erased by reprogramming, accompanied by erasure of transcriptional X chromosome upregulation and the loss of increased transcriptional burst frequency. In addition, we characterize gene regulatory networks during reprogramming and X chromosome reactivation, revealing changes in regulatory states. Our data show that ZFP42/REX1, a pluripotency-associated gene that evolved specifically in placental mammals, targets multiple X-linked genes, suggesting an evolutionary link between ZFP42/REX1, X chromosome reactivation, and pluripotency.

Conclusions: Our data reveal the existence of intrinsic compensatory mechanisms that involve modulation of chromatin accessibility to counteract X-to-Autosome gene dosage imbalances caused by evolutionary or in vitro X chromosome loss and X chromosome inactivation in mammalian cells.
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http://dx.doi.org/10.1186/s13059-021-02518-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8558763PMC
November 2021

A Universal Labeling Strategy for Nucleic Acids in Expansion Microscopy.

J Am Chem Soc 2021 Sep 23;143(34):13782-13789. Epub 2021 Aug 23.

Department of Chemistry, KU Leuven, Leuven 3001, Belgium.

Expansion microscopy (ExM) enables the nanoscale imaging of ribonucleic acids (RNAs) on a conventional fluorescence microscope, providing information on the intricate patterns of gene expression at (sub)cellular resolution and within spatial context. To extend the use of such strategies, we examined a series of multivalent reagents that allow the labeling and grafting of deoxyribonucleic acid (DNA) oligonucleotide probes in a unified approach. We show that the reagents are directly compatible with third-generation hybridization chain reaction RNA FISH (fluorescence hybridization) techniques while displaying complete retention of the targeted transcripts. Furthermore, we validate and demonstrate that our labeling method is compatible with multicolor staining. Through oligonucleotide-conjugated antibodies, we demonstrate excellent performance in ×4 ExM and ×10 ExM, achieving a resolution of ∼50 nm in ×10 ExM for both pre- and postexpansion labeling strategies. Our results indicate that our multivalent molecules enable the rapid functionalization of DNA oligonucleotides for ExM.
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http://dx.doi.org/10.1021/jacs.1c05931DOI Listing
September 2021

Evolutionary predictability of genetic versus nongenetic resistance to anticancer drugs in melanoma.

Cancer Cell 2021 Aug 17;39(8):1135-1149.e8. Epub 2021 Jun 17.

Laboratory for Molecular Cancer Biology, Center for Cancer Biology, VIB, Leuven, Belgium; Laboratory for Molecular Cancer Biology, Department of Oncology, KU Leuven, Leuven, Belgium. Electronic address:

Therapy resistance arises from heterogeneous drug-tolerant persister cells or minimal residual disease (MRD) through genetic and nongenetic mechanisms. A key question is whether specific molecular features of the MRD ecosystem determine which of these two distinct trajectories will eventually prevail. We show that, in melanoma exposed to mitogen-activated protein kinase therapeutics, emergence of a transient neural crest stem cell (NCSC) population in MRD concurs with the development of nongenetic resistance. This increase relies on a glial cell line-derived neurotrophic factor-dependent signaling cascade, which activates the AKT survival pathway in a focal adhesion kinase (FAK)-dependent manner. Ablation of the NCSC population through FAK inhibition delays relapse in patient-derived tumor xenografts. Strikingly, all tumors that ultimately escape this treatment exhibit resistance-conferring genetic alterations and increased sensitivity to extracellular signal-regulated kinase inhibition. These findings identify an approach that abrogates the nongenetic resistance trajectory in melanoma and demonstrate that the cellular composition of MRD deterministically imposes distinct drug resistance evolutionary paths.
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http://dx.doi.org/10.1016/j.ccell.2021.05.015DOI Listing
August 2021

Development, maturation, and maintenance of human prostate inferred from somatic mutations.

Cell Stem Cell 2021 07 2;28(7):1262-1274.e5. Epub 2021 Mar 2.

Cancer Genome Project, Wellcome Trust Sanger Institute, Hinxton CB10 1SA, UK; Wellcome - MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge CB2 0AW, UK. Electronic address:

Clonal dynamics and mutation burden in healthy human prostate epithelium are relevant to prostate cancer. We sequenced whole genomes from 409 microdissections of normal prostate epithelium across 8 donors, using phylogenetic reconstruction with spatial mapping in a 59-year-old man's prostate to reconstruct tissue dynamics across the lifespan. Somatic mutations accumulate steadily at ∼16 mutations/year/clone, with higher rates in peripheral than peri-urethral regions. The 24-30 independent glandular subunits are established as rudimentary ductal structures during fetal development by 5-10 embryonic cells each. Puberty induces formation of further side and terminal branches by local stem cells disseminated throughout the rudimentary ducts during development. During adult tissue maintenance, clonal expansions have limited geographic scope and minimal migration. Driver mutations are rare in aging prostate epithelium, but the one driver we did observe generated a sizable intraepithelial clonal expansion. Leveraging unbiased clock-like mutations, we define prostate stem cell dynamics through fetal development, puberty, and aging.
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http://dx.doi.org/10.1016/j.stem.2021.02.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8260206PMC
July 2021

Tissue damage induces a conserved stress response that initiates quiescent muscle stem cell activation.

Cell Stem Cell 2021 06 19;28(6):1125-1135.e7. Epub 2021 Feb 19.

Univ Paris Est Creteil, INSERM, IMRB, F-94010 Creteil, France. Electronic address:

Tissue damage dramatically alters how cells interact with their microenvironment. These changes in turn dictate cellular responses, such as stem cell activation, yet early cellular responses in vivo remain ill defined. We generated single-cell and nucleus atlases from intact, dissociated, and injured muscle and liver and identified a common stress response signature shared by multiple cell types across these organs. This prevalent stress response was detected in published datasets across a range of tissues, demonstrating high conservation but also a significant degree of data distortion in single-cell reference atlases. Using quiescent muscle stem cells as a paradigm of cell activation following injury, we captured early cell activation following muscle injury and found that an essential ERK1/2 primary proliferation signal precedes initiation of the Notch-regulated myogenic program. This study defines initial events in response to tissue perturbation and identifies a broadly conserved transcriptional stress response that acts in parallel with cell-specific adaptive alterations.
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http://dx.doi.org/10.1016/j.stem.2021.01.017DOI Listing
June 2021

Common clonal origin of conventional T cells and induced regulatory T cells in breast cancer patients.

Nat Commun 2021 02 18;12(1):1119. Epub 2021 Feb 18.

RCI Regensburg Centre for Interventional Immunology, University and Department of Hematology/Oncology, University Medical Centre of Regensburg, Regensburg, Germany.

Regulatory CD4 T cells (Treg) prevent tumor clearance by conventional T cells (Tconv) comprising a major obstacle of cancer immune-surveillance. Hitherto, the mechanisms of Treg repertoire formation in human cancers remain largely unclear. Here, we analyze Treg clonal origin in breast cancer patients using T-Cell Receptor and single-cell transcriptome sequencing. While Treg in peripheral blood and breast tumors are clonally distinct, Tconv clones, including tumor-antigen reactive effectors (Teff), are detected in both compartments. Tumor-infiltrating CD4 cells accumulate into distinct transcriptome clusters, including early activated Tconv, uncommitted Teff, Th1 Teff, suppressive Treg and pro-tumorigenic Treg. Trajectory analysis suggests early activated Tconv differentiation either into Th1 Teff or into suppressive and pro-tumorigenic Treg. Importantly, Tconv, activated Tconv and Treg share highly-expanded clones contributing up to 65% of intratumoral Treg. Here we show that Treg in human breast cancer may considerably stem from antigen-experienced Tconv converting into secondary induced Treg through intratumoral activation.
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http://dx.doi.org/10.1038/s41467-021-21297-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7893042PMC
February 2021

Transcriptional characterization of human megakaryocyte polyploidization and lineage commitment.

J Thromb Haemost 2021 05 29;19(5):1236-1249. Epub 2021 Mar 29.

Department of Hematology, University of Cambridge, Cambridge, UK.

Background: Megakaryocytes (MKs) originate from cells immuno-phenotypically indistinguishable from hematopoietic stem cells (HSCs), bypassing intermediate progenitors. They mature within the adult bone marrow and release platelets into the circulation. Until now, there have been no transcriptional studies of primary human bone marrow MKs.

Objectives: To characterize MKs and HSCs from human bone marrow using single-cell RNA sequencing, to investigate MK lineage commitment, maturation steps, and thrombopoiesis.

Results: We show that MKs at different levels of polyploidization exhibit distinct transcriptional states. Although high levels of platelet-specific gene expression occur in the lower ploidy classes, as polyploidization increases, gene expression is redirected toward translation and posttranslational processing transcriptional programs, in preparation for thrombopoiesis. Our findings are in keeping with studies of MK ultrastructure and supersede evidence generated using in vitro cultured MKs. Additionally, by analyzing transcriptional signatures of a single HSC, we identify two MK-biased HSC subpopulations exhibiting unique differentiation kinetics. We show that human bone marrow MKs originate from these HSC subpopulations, supporting the notion that they display priming for MK differentiation. Finally, to investigate transcriptional changes in MKs associated with stress thrombopoiesis, we analyzed bone marrow MKs from individuals with recent myocardial infarction and found a specific gene expression signature. Our data support the modulation of MK differentiation in this thrombotic state.

Conclusions: Here, we use single-cell sequencing for the first time to characterize the human bone marrow MK transcriptome at different levels of polyploidization and investigate their differentiation from the HSC.
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http://dx.doi.org/10.1111/jth.15271DOI Listing
May 2021

PREIMPLANTATION GENETIC TESTING: Single-cell technologies at the forefront of PGT and embryo research.

Reproduction 2020 11;160(5):A19-A31

Laboratory for Cytogenetics and Genome Research, Department of Human Genetics, KU Leuven, Leuven, Belgium.

While chromosomal mosaicism in the embryo was observed already in the 1990s using both karyotyping and FISH technologies, the full extent of this phenomenon and the overall awareness of the consequences of chromosomal instability on embryo development has only come with the advent of sophisticated single-cell technologies. High-throughput techniques, such as DNA microarrays and massive parallel sequencing, have shifted single-cell genome research from evaluating a few loci at a time to the ability to perform comprehensive screening of all 24 chromosomes. The development of genome-wide single-cell haplotyping methods have also enabled for simultaneous detection of single-gene disorders and aneuploidy using a single universal protocol. Today, three decades later haplotyping-based embryo testing is performed worldwide to reliably detect virtually any Mendelian hereditary disease with a known cause, including autosomal-recessive, autosomal-dominant and X-linked disorders. At the same time, these single-cell assays have also provided unique insight into the complexity of embryo genome dynamics, by elucidating mechanistic origin, nature and developmental fate of embryonic aneuploidy. Understanding the impact of postzygotically acquired genomic aberrations on embryo development is essential to determine the still controversial diagnostic value of aneuploidy screening. For that reason, considerable efforts have been put into linking the genetic constitution of the embryo not only to its morphology and implantation potential, but more importantly to its transcriptome using single-cell RNA sequencing. Collectively, these breakthrough technologies have revolutionized single-cell research and clinical practice in assisted reproduction and led to unique discoveries in early embryogenesis.
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http://dx.doi.org/10.1530/REP-20-0102DOI Listing
November 2020

LifeTime and improving European healthcare through cell-based interceptive medicine.

Nature 2020 11 7;587(7834):377-386. Epub 2020 Sep 7.

VIB Technology Watch, Ghent, Belgium.

Here we describe the LifeTime Initiative, which aims to track, understand and target human cells during the onset and progression of complex diseases, and to analyse their response to therapy at single-cell resolution. This mission will be implemented through the development, integration and application of single-cell multi-omics and imaging, artificial intelligence and patient-derived experimental disease models during the progression from health to disease. The analysis of large molecular and clinical datasets will identify molecular mechanisms, create predictive computational models of disease progression, and reveal new drug targets and therapies. The timely detection and interception of disease embedded in an ethical and patient-centred vision will be achieved through interactions across academia, hospitals, patient associations, health data management systems and industry. The application of this strategy to key medical challenges in cancer, neurological and neuropsychiatric disorders, and infectious, chronic inflammatory and cardiovascular diseases at the single-cell level will usher in cell-based interceptive medicine in Europe over the next decade.
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http://dx.doi.org/10.1038/s41586-020-2715-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7656507PMC
November 2020

Heterotypic cell-cell communication regulates glandular stem cell multipotency.

Nature 2020 08 26;584(7822):608-613. Epub 2020 Aug 26.

Laboratory of Stem Cells and Cancer, Université Libre de Bruxelles (ULB), Brussels, Belgium.

Glandular epithelia, including the mammary and prostate glands, are composed of basal cells (BCs) and luminal cells (LCs). Many glandular epithelia develop from multipotent basal stem cells (BSCs) that are replaced in adult life by distinct pools of unipotent stem cells. However, adult unipotent BSCs can reactivate multipotency under regenerative conditions and upon oncogene expression. This suggests that an active mechanism restricts BSC multipotency under normal physiological conditions, although the nature of this mechanism is unknown. Here we show that the ablation of LCs reactivates the multipotency of BSCs from multiple epithelia both in vivo in mice and in vitro in organoids. Bulk and single-cell RNA sequencing revealed that, after LC ablation, BSCs activate a hybrid basal and luminal cell differentiation program before giving rise to LCs-reminiscent of the genetic program that regulates multipotency during embryonic development. By predicting ligand-receptor pairs from single-cell data, we find that TNF-which is secreted by LCs-restricts BC multipotency under normal physiological conditions. By contrast, the Notch, Wnt and EGFR pathways were activated in BSCs and their progeny after LC ablation; blocking these pathways, or stimulating the TNF pathway, inhibited regeneration-induced BC multipotency. Our study demonstrates that heterotypic communication between LCs and BCs is essential to maintain lineage fidelity in glandular epithelial stem cells.
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http://dx.doi.org/10.1038/s41586-020-2632-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7116172PMC
August 2020

Mechanisms of stretch-mediated skin expansion at single-cell resolution.

Nature 2020 08 29;584(7820):268-273. Epub 2020 Jul 29.

Laboratory of Stem Cells and Cancer, Université Libre de Bruxelles, Brussels, Belgium.

The ability of the skin to grow in response to stretching has been exploited in reconstructive surgery. Although the response of epidermal cells to stretching has been studied in vitro, it remains unclear how mechanical forces affect their behaviour in vivo. Here we develop a mouse model in which the consequences of stretching on skin epidermis can be studied at single-cell resolution. Using a multidisciplinary approach that combines clonal analysis with quantitative modelling and single-cell RNA sequencing, we show that stretching induces skin expansion by creating a transient bias in the renewal activity of epidermal stem cells, while a second subpopulation of basal progenitors remains committed to differentiation. Transcriptional and chromatin profiling identifies how cell states and gene-regulatory networks are modulated by stretching. Using pharmacological inhibitors and mouse mutants, we define the step-by-step mechanisms that control stretch-mediated tissue expansion at single-cell resolution in vivo.
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http://dx.doi.org/10.1038/s41586-020-2555-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7116042PMC
August 2020

Interstitial Cell Remodeling Promotes Aberrant Adipogenesis in Dystrophic Muscles.

Cell Rep 2020 05;31(5):107597

Laboratory of Translational Cardiomyology, Department of Development and Regeneration, Stem Cell Research Institute, KU Leuven, 3000 Leuven, Belgium; Human Anatomy Unit, Department of Public Health, Experimental and Forensic Medicine, University of Pavia, 27100 Pavia, Italy. Electronic address:

Fibrosis and fat replacement in skeletal muscle are major complications that lead to a loss of mobility in chronic muscle disorders, such as muscular dystrophy. However, the in vivo properties of adipogenic stem and precursor cells remain unclear, mainly due to the high cell heterogeneity in skeletal muscles. Here, we use single-cell RNA sequencing to decomplexify interstitial cell populations in healthy and dystrophic skeletal muscles. We identify an interstitial CD142-positive cell population in mice and humans that is responsible for the inhibition of adipogenesis through GDF10 secretion. Furthermore, we show that the interstitial cell composition is completely altered in muscular dystrophy, with a near absence of CD142-positive cells. The identification of these adipo-regulatory cells in the skeletal muscle aids our understanding of the aberrant fat deposition in muscular dystrophy, paving the way for treatments that could counteract degeneration in patients with muscular dystrophy.
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http://dx.doi.org/10.1016/j.celrep.2020.107597DOI Listing
May 2020

Defining the Design Principles of Skin Epidermis Postnatal Growth.

Cell 2020 04 6;181(3):604-620.e22. Epub 2020 Apr 6.

Université Libre de Bruxelles, Laboratory of Stem Cells and Cancer, Brussels 1070, Belgium; WELBIO, Université Libre de Bruxelles, Brussels 1070, Belgium. Electronic address:

During embryonic and postnatal development, organs and tissues grow steadily to achieve their final size at the end of puberty. However, little is known about the cellular dynamics that mediate postnatal growth. By combining in vivo clonal lineage tracing, proliferation kinetics, single-cell transcriptomics, and in vitro micro-pattern experiments, we resolved the cellular dynamics taking place during postnatal skin epidermis expansion. Our data revealed that harmonious growth is engineered by a single population of developmental progenitors presenting a fixed fate imbalance of self-renewing divisions with an ever-decreasing proliferation rate. Single-cell RNA sequencing revealed that epidermal developmental progenitors form a more uniform population compared with adult stem and progenitor cells. Finally, we found that the spatial pattern of cell division orientation is dictated locally by the underlying collagen fiber orientation. Our results uncover a simple design principle of organ growth where progenitors and differentiated cells expand in harmony with their surrounding tissues.
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http://dx.doi.org/10.1016/j.cell.2020.03.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7198979PMC
April 2020

Identity-by-state-based haplotyping expands the application of comprehensive preimplantation genetic testing.

Hum Reprod 2020 03;35(3):718-726

Department of Human Genetics, Centre for Human Genetics, University Hospitals Leuven, Leuven 3000, Belgium.

Study Question: Is it possible to haplotype parents using parental siblings to leverage preimplantation genetic testing (PGT) for monogenic diseases and aneuploidy (comprehensive PGT) by genome-wide haplotyping?

Summary Answer: We imputed identity-by-state (IBS) sharing of parental siblings to phase parental genotypes.

What Is Known Already: Genome-wide haplotyping of preimplantation embryos is being implemented as a generic approach for genetic diagnosis of inherited single-gene disorders. To enable the phasing of genotypes into haplotypes, genotyping the direct family members of the prospective parent carrying the mutation is required. Current approaches require genotypes of either (i) both or one of the parents of the affected prospective parent or (ii) an affected or an unaffected child of the couple. However, this approach cannot be used when parents or children are not attainable, prompting an investigation into alternative phasing options.

Study Design, Size, Duration: This is a retrospective validation study, which applied IBS-based phasing of parental haplotypes in 56 embryos derived from 12 PGT families. Genome-wide haplotypes and copy number profiles generated for each embryo using the new phasing approach were compared with the reference PGT method to evaluate the diagnostic concordance.

Participants/materials, Setting, Methods: This study included 12 couples with a known hereditary genetic disorder, participating in the comprehensive PGT program and with at least one parental sibling available (e.g. brother and/or sister). Genotyping data from both prospective parents and the parental sibling(s) were used to perform IBS-based phasing and to trace the disease-associated alleles. The outcome of the IBS-based PGT was compared with the results of the clinically implemented reference haplotyping-based PGT method.

Main Results And The Role Of Chance: IBS-based haplotyping was performed for 12 PGT families. In accordance with the theoretical prediction of allele sharing between sibling pairs, 6 out of 12 (50%) couples or 23 out of 56 embryos could be phased using parental siblings. In families where phasing was possible, haplotype calling in the locus of interest was 100% concordant between the reference PGT method and IBS-based approach using parental siblings.

Large Scale Data: N/A.

Limitations, Reasons For Caution: Phasing of parental haplotypes will only be possible when the disease locus lies in an informative region (categorized as IBS1). Phasing prospective parents using relatives with reduced genetic relatedness as a reference (e.g. siblings) decreases the size and the occurrence of informative IBS1 regions, necessary for haplotype calling. By including more than one extended family member, the chance of obtaining IBS1 coverage in the interrogated locus can be increased. A pre-PGT work-up can define whether the carrier couple could benefit from this approach.

Wider Implications Of The Findings: Phasing by relatives extends the potential of comprehensive PGT, since it allows the inclusion of couples who do not have access to the standard phasing references, such as parents or offspring.

Study Funding/competing Interest(s): The study was funded by the KU Leuven grant (C14/18/092), Research Foundation Flanders (FWO; GA09311N), Horizon 2020 innovation programme (WIDENLIFE, 692065) and Agilent Technologies. J.R.V., T.V. and M.Z.E. are co-inventors of a patent ZL910050-PCT/EP2011/060211-WO/2011/157846 'Methods for haplotyping single-cells' and ZL913096-PCT/EP2014/068315-WO/2015/028576 'Haplotyping and copy number typing using polymorphic variant allelic frequencies' licensed to Agilent Technologies. The other authors have no conflict of interest to declare.
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http://dx.doi.org/10.1093/humrep/dez285DOI Listing
March 2020

Identification of region-specific astrocyte subtypes at single cell resolution.

Nat Commun 2020 03 5;11(1):1220. Epub 2020 Mar 5.

Laboratory of Glia Biology, VIB-KU Leuven Center for Brain and Disease Research, Leuven, Belgium.

Astrocytes, a major cell type found throughout the central nervous system, have general roles in the modulation of synapse formation and synaptic transmission, blood-brain barrier formation, and regulation of blood flow, as well as metabolic support of other brain resident cells. Crucially, emerging evidence shows specific adaptations and astrocyte-encoded functions in regions, such as the spinal cord and cerebellum. To investigate the true extent of astrocyte molecular diversity across forebrain regions, we used single-cell RNA sequencing. Our analysis identifies five transcriptomically distinct astrocyte subtypes in adult mouse cortex and hippocampus. Validation of our data in situ reveals distinct spatial positioning of defined subtypes, reflecting the distribution of morphologically and physiologically distinct astrocyte populations. Our findings are evidence for specialized astrocyte subtypes between and within brain regions. The data are available through an online database (https://holt-sc.glialab.org/), providing a resource on which to base explorations of local astrocyte diversity and function in the brain.
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http://dx.doi.org/10.1038/s41467-019-14198-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7058027PMC
March 2020

Noninvasive prenatal diagnosis by genome-wide haplotyping of cell-free plasma DNA.

Genet Med 2020 05 6;22(5):962-973. Epub 2020 Feb 6.

Center for Human Genetics, KU Leuven, Leuven, Belgium.

Purpose: Whereas noninvasive prenatal screening for aneuploidies is widely implemented, there is an increasing need for universal approaches for noninvasive prenatal screening for monogenic diseases. Here, we present a cost-effective, generic cell-free fetal DNA (cffDNA) haplotyping approach to scan the fetal genome for the presence of inherited monogenic diseases.

Methods: Families participating in the preimplantation genetic testing for monogenic disorders (PGT-M) program were recruited for this study. Two hundred fifty thousand single-nucleotide polymorphisms (SNPs) captured from maternal plasma DNA along with genomic DNA from family members were massively parallel sequenced. Parental genotypes were phased via an available genotype from a close relative, and the fetal genome-wide haplotype and copy number were determined using cffDNA haplotyping analysis based on estimation and segmentation of fetal allele presence in the maternal plasma.

Results: In all families tested, mutational profiles from cffDNA haplotyping are consistent with embryo biopsy profiles. Genome-wide fetal haplotypes are on average 97% concordant with the newborn haplotypes and embryo haplotypes.

Conclusion: We demonstrate that genome-wide targeted capture and sequencing of polymorphic SNPs from maternal plasma cell-free DNA (cfDNA) allows haplotyping and copy-number profiling of the fetal genome during pregnancy. The method enables the accurate reconstruction of the fetal haplotypes and can be easily implemented in clinical practice.
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http://dx.doi.org/10.1038/s41436-019-0748-yDOI Listing
May 2020

In vitro fertilization does not increase the incidence of de novo copy number alterations in fetal and placental lineages.

Nat Med 2019 11 4;25(11):1699-1705. Epub 2019 Nov 4.

Department of Obstetrics and Gynecology, Institute of Clinical Medicine, University of Tartu, Tartu, Estonia.

Although chromosomal instability (CIN) is a common phenomenon in cleavage-stage embryogenesis following in vitro fertilization (IVF), its rate in naturally conceived human embryos is unknown. CIN leads to mosaic embryos that contain a combination of genetically normal and abnormal cells, and is significantly higher in in vitro-produced preimplantation embryos as compared to in vivo-conceived preimplantation embryos. Even though embryos with CIN-derived complex aneuploidies may arrest between the cleavage and blastocyst stages of embryogenesis, a high number of embryos containing abnormal cells can pass this strong selection barrier. However, neither the prevalence nor extent of CIN during prenatal development and at birth, following IVF treatment, is well understood. Here we profiled the genomic landscape of fetal and placental tissues postpartum from both IVF and naturally conceived children, to investigate the prevalence and persistence of large genetic aberrations that probably arose from IVF-related CIN. We demonstrate that CIN is not preserved at later stages of prenatal development, and that de novo numerical aberrations or large structural DNA imbalances occur at similar rates in IVF and naturally conceived live-born neonates. Our findings affirm that human IVF treatment has no detrimental effect on the chromosomal constitution of fetal and placental lineages.
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http://dx.doi.org/10.1038/s41591-019-0620-2DOI Listing
November 2019

Zebrafish MITF-Low Melanoma Subtype Models Reveal Transcriptional Subclusters and MITF-Independent Residual Disease.

Cancer Res 2019 Nov 3;79(22):5769-5784. Epub 2019 Oct 3.

MRC Human Genetics Unit, MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, United Kingdom.

The melanocyte-inducing transcription factor (MITF)-low melanoma transcriptional signature is predictive of poor outcomes for patients, but little is known about its biological significance, and animal models are lacking. Here, we used zebrafish genetic models with low activity of Mitfa (MITF-low) and established that the MITF-low state is causal of melanoma progression and a predictor of melanoma biological subtype. MITF-low zebrafish melanomas resembled human MITF-low melanomas and were enriched for stem and invasive (mesenchymal) gene signatures. MITF-low activity coupled with a p53 mutation was sufficient to promote superficial growth melanomas, whereas BRAF accelerated MITF-low melanoma onset and further promoted the development of MITF-high nodular growth melanomas. Genetic inhibition of MITF activity led to rapid regression; recurrence occurred following reactivation of MITF. At the regression site, there was minimal residual disease that was resistant to loss of MITF activity (termed MITF-independent cells) with very low-to-no MITF activity or protein. Transcriptomic analysis of MITF-independent residual disease showed enrichment of mesenchymal and neural crest stem cell signatures similar to human therapy-resistant melanomas. Single-cell RNA sequencing revealed MITF-independent residual disease was heterogeneous depending on melanoma subtype. Further, there was a shared subpopulation of residual disease cells that was enriched for a neural crest G-like state that preexisted in the primary tumor and remained present in recurring melanomas. These findings suggest that invasive and stem-like programs coupled with cellular heterogeneity contribute to poor outcomes for MITF-low melanoma patients and that MITF-independent subpopulations are an important therapeutic target to achieve long-term survival outcomes. SIGNIFICANCE: This study provides a useful model for MITF-low melanomas and MITF-independent cell populations that can be used to study the mechanisms that drive these tumors as well as identify potential therapeutic options. http://cancerres.aacrjournals.org/content/canres/79/22/5769/F1.large.jpg.
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http://dx.doi.org/10.1158/0008-5472.CAN-19-0037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7116150PMC
November 2019

Activation of Skeletal Stem and Progenitor Cells for Bone Regeneration Is Driven by PDGFRβ Signaling.

Dev Cell 2019 10 19;51(2):236-254.e12. Epub 2019 Sep 19.

Laboratory of Skeletal Cell Biology and Physiology (SCEBP), Skeletal Biology and Engineering Research Center (SBE), Department of Development and Regeneration, KU Leuven, 3000 Leuven, Belgium. Electronic address:

Bone repair and regeneration critically depend on the activation and recruitment of osteogenesis-competent skeletal stem and progenitor cells (SSPCs). Yet, the origin and triggering cues for SSPC propagation and migration remain largely elusive. Through bulk and single-cell transcriptome profiling of fetal osterix (Osx)-expressing cells, followed by lineage mapping, cell tracing, and conditional mouse mutagenesis, we here identified PDGF-PDGFRβ signaling as critical functional mediator of SSPC expansion, migration, and angiotropism during bone repair. Our data show that cells marked by a history of Osx expression, including those arising in fetal or early postnatal periods, represent or include SSPCs capable of delivering all the necessary differentiated progeny to repair acute skeletal injuries later in life, provided that they express functional PDGFRβ. Mechanistically, MMP-9 and VCAM-1 appear to be involved downstream of PDGF-PDGFRβ. Our results reveal considerable cellular dynamism in the skeletal system and show that activation and recruitment of SSPCs for bone repair require functional PDGFRβ signaling.
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http://dx.doi.org/10.1016/j.devcel.2019.08.013DOI Listing
October 2019

Multi-centre evaluation of a comprehensive preimplantation genetic test through haplotyping-by-sequencing.

Hum Reprod 2019 08;34(8):1608-1619

Laboratory for Cytogenetics and Genome Research, Department of Human Genetics, KU Leuven, Leuven, Belgium.

Study Question: Can reduced representation genome sequencing offer an alternative to single nucleotide polymorphism (SNP) arrays as a generic and genome-wide approach for comprehensive preimplantation genetic testing for monogenic disorders (PGT-M), aneuploidy (PGT-A) and structural rearrangements (PGT-SR) in human embryo biopsy samples?

Summary Answer: Reduced representation genome sequencing, with OnePGT, offers a generic, next-generation sequencing-based approach for automated haplotyping and copy-number assessment, both combined or independently, in human single blastomere and trophectoderm samples.

What Is Known Already: Genome-wide haplotyping strategies, such as karyomapping and haplarithmisis, have paved the way for comprehensive PGT, i.e. leveraging PGT-M, PGT-A and PGT-SR in a single workflow. These methods are based upon SNP array technology.

Study Design, Size, Duration: This multi-centre verification study evaluated the concordance of PGT results for a total of 225 embryos, including 189 originally tested for a monogenic disorder and 36 tested for a translocation. Concordance for whole chromosome aneuploidies was also evaluated where whole genome copy-number reference data were available. Data analysts were kept blind to the results from the reference PGT method.

Participants/materials, Setting, Methods: Leftover blastomere/trophectoderm whole genome amplified (WGA) material was used, or secondary trophectoderm biopsies were WGA. A reduced representation library from WGA DNA together with bulk DNA from phasing references was processed across two study sites with the Agilent OnePGT solution. Libraries were sequenced on an Illumina NextSeq500 system, and data were analysed with Agilent Alissa OnePGT software. The embedded PGT-M pipeline utilises the principles of haplarithmisis to deduce haplotype inheritance whereas both the PGT-A and PGT-SR pipelines are based upon read-count analysis in order to evaluate embryonic ploidy. Concordance analysis was performed for both analysis strategies against the reference PGT method.

Main Results And The Role Of Chance: PGT-M analysis was performed on 189 samples. For nine samples, the data quality was too poor to analyse further, and for 20 samples, no result could be obtained mainly due to biological limitations of the haplotyping approach, such as co-localisation of meiotic crossover events and nullisomy for the chromosome of interest. For the remaining 160 samples, 100% concordance was obtained between OnePGT and the reference PGT-M method. Equally for PGT-SR, 100% concordance for all 36 embryos tested was demonstrated. Moreover, with embryos originally analysed for PGT-M or PGT-SR for which genome-wide copy-number reference data were available, 100% concordance was shown for whole chromosome copy-number calls (PGT-A).

Limitations, Reasons For Caution: Inherent to haplotyping methodologies, processing of additional family members is still required. Biological limitations caused inconclusive results in 10% of cases.

Wider Implications Of The Findings: Employment of OnePGT for PGT-M, PGT-SR, PGT-A or combined as comprehensive PGT offers a scalable platform, which is inherently generic and thereby, eliminates the need for family-specific design and optimisation. It can be considered as both an improvement and complement to the current methodologies for PGT.

Study Funding/competing Interest(s): Agilent Technologies, the KU Leuven (C1/018 to J.R.V. and T.V.) and the Horizon 2020 WIDENLIFE (692065 to J.R.V. and T.V). H.M. is supported by the Research Foundation Flanders (FWO, 11A7119N). M.Z.E, J.R.V. and T.V. are co-inventors on patent applications: ZL910050-PCT/EP2011/060211- WO/2011/157846 'Methods for haplotyping single cells' and ZL913096-PCT/EP2014/068315 'Haplotyping and copy-number typing using polymorphic variant allelic frequencies'. T.V. and J.R.V. are co-inventors on patent application: ZL912076-PCT/EP2013/070858 'High-throughput genotyping by sequencing'. Haplarithmisis ('Haplotyping and copy-number typing using polymorphic variant allelic frequencies') has been licensed to Agilent Technologies. The following patents are pending for OnePGT: US2016275239, AU2014345516, CA2928013, CN105874081, EP3066213 and WO2015067796. OnePGT is a registered trademark. D.L., J.T. and R.L.R. report personal fees during the conduct of the study and outside the submitted work from Agilent Technologies. S.H. and K.O.F. report personal fees and other during the conduct of the study and outside the submitted work from Agilent Technologies. J.A. reports personal fees and other during the conduct of the study from Agilent Technologies and personal fees from Agilent Technologies and UZ Leuven outside the submitted work. B.D. reports grants from IWT/VLAIO, personal fees during the conduct of the study from Agilent Technologies and personal fees and other outside the submitted work from Agilent Technologies. In addition, B.D. has a patent 20160275239 - Genetic Analysis Method pending. The remaining authors have no conflicts of interest.
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http://dx.doi.org/10.1093/humrep/dez106DOI Listing
August 2019

Publisher Correction: Self-assembly of embryonic and two extra-embryonic stem cell types into gastrulating embryo-like structures.

Nat Cell Biol 2018 Oct;20(10):1229

Mammalian Embryo and Stem Cell Group, Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, UK.

In the version of this Technical Report originally published, the competing interests statement was missing. The authors declare no competing interests; this statement has now been added in all online versions of the Report.
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http://dx.doi.org/10.1038/s41556-018-0187-zDOI Listing
October 2018

Self-assembly of embryonic and two extra-embryonic stem cell types into gastrulating embryo-like structures.

Nat Cell Biol 2018 08 23;20(8):979-989. Epub 2018 Jul 23.

Mammalian Embryo and Stem Cell Group, Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, UK.

Embryonic stem cells can be incorporated into the developing embryo and its germ line, but, when cultured alone, their ability to generate embryonic structures is restricted. They can interact with trophoblast stem cells to generate structures that break symmetry and specify mesoderm, but their development is limited as the epithelial-mesenchymal transition of gastrulation cannot occur. Here, we describe a system that allows assembly of mouse embryonic, trophoblast and extra-embryonic endoderm stem cells into structures that acquire the embryo's architecture with all distinct embryonic and extra-embryonic compartments. Strikingly, such embryo-like structures develop to undertake the epithelial-mesenchymal transition, leading to mesoderm and then definitive endoderm specification. Spatial transcriptomic analyses demonstrate that these morphological transformations are underpinned by gene expression patterns characteristic of gastrulating embryos. This demonstrates the remarkable ability of three stem cell types to self-assemble in vitro into gastrulating embryo-like structures undertaking spatio-temporal events of the gastrulating mammalian embryo.
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http://dx.doi.org/10.1038/s41556-018-0147-7DOI Listing
August 2018

Publisher Correction: Early lineage segregation of multipotent embryonic mammary gland progenitors.

Nat Cell Biol 2018 Sep;20(9):1099

Laboratory of Stem Cells and Cancer, Université Libre de Bruxelles, Brussels, Belgium.

In the version of this Article originally published, ref. 52 was incorrectly only attributed to its corresponding author, Fre, S., and an older title was used. The correct citation should have been: Lilja, A. M. et al. Clonal analysis of Notch1-expressing cells reveals the existence of unipotent stem cells that retain long-term plasticity in the embryonic mammary gland. Nat. Cell Biol. https://doi.org/10.1038/s41556-018-0108-1 (2018)'. This has now been amended in all online versions of the Article.
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http://dx.doi.org/10.1038/s41556-018-0164-6DOI Listing
September 2018

Toward Minimal Residual Disease-Directed Therapy in Melanoma.

Cell 2018 08 12;174(4):843-855.e19. Epub 2018 Jul 12.

Laboratory for Molecular Cancer Biology, VIB Center for Cancer Biology, KU Leuven, Leuven, Belgium; Department of Oncology, KU Leuven, Leuven, Belgium. Electronic address:

Many patients with advanced cancers achieve dramatic responses to a panoply of therapeutics yet retain minimal residual disease (MRD), which ultimately results in relapse. To gain insights into the biology of MRD, we applied single-cell RNA sequencing to malignant cells isolated from BRAF mutant patient-derived xenograft melanoma cohorts exposed to concurrent RAF/MEK-inhibition. We identified distinct drug-tolerant transcriptional states, varying combinations of which co-occurred within MRDs from PDXs and biopsies of patients on treatment. One of these exhibited a neural crest stem cell (NCSC) transcriptional program largely driven by the nuclear receptor RXRG. An RXR antagonist mitigated accumulation of NCSCs in MRD and delayed the development of resistance. These data identify NCSCs as key drivers of resistance and illustrate the therapeutic potential of MRD-directed therapy. They also highlight how gene regulatory network architecture reprogramming may be therapeutically exploited to limit cellular heterogeneity, a key driver of disease progression and therapy resistance.
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http://dx.doi.org/10.1016/j.cell.2018.06.025DOI Listing
August 2018

A Single-Cell Transcriptome Atlas of the Aging Drosophila Brain.

Cell 2018 08 18;174(4):982-998.e20. Epub 2018 Jun 18.

VIB Center for Brain & Disease Research, KU Leuven, Leuven 3000, Belgium; Department of Human Genetics KU Leuven, Leuven 3000, Belgium. Electronic address:

The diversity of cell types and regulatory states in the brain, and how these change during aging, remains largely unknown. We present a single-cell transcriptome atlas of the entire adult Drosophila melanogaster brain sampled across its lifespan. Cell clustering identified 87 initial cell clusters that are further subclustered and validated by targeted cell-sorting. Our data show high granularity and identify a wide range of cell types. Gene network analyses using SCENIC revealed regulatory heterogeneity linked to energy consumption. During aging, RNA content declines exponentially without affecting neuronal identity in old brains. This single-cell brain atlas covers nearly all cells in the normal brain and provides the tools to study cellular diversity alongside other Drosophila and mammalian single-cell datasets in our unique single-cell analysis platform: SCope (http://scope.aertslab.org). These results, together with SCope, allow comprehensive exploration of all transcriptional states of an entire aging brain.
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http://dx.doi.org/10.1016/j.cell.2018.05.057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6086935PMC
August 2018

Early lineage segregation of multipotent embryonic mammary gland progenitors.

Nat Cell Biol 2018 06 21;20(6):666-676. Epub 2018 May 21.

Laboratory of Stem Cells and Cancer, Université Libre de Bruxelles, Brussels, Belgium.

The mammary gland is composed of basal cells and luminal cells. It is generally believed that the mammary gland arises from embryonic multipotent progenitors, but it remains unclear when lineage restriction occurs and what mechanisms are responsible for the switch from multipotency to unipotency during its morphogenesis. Here, we perform multicolour lineage tracing and assess the fate of single progenitors, and demonstrate the existence of a developmental switch from multipotency to unipotency during embryonic mammary gland development. Molecular profiling and single cell RNA-seq revealed that embryonic multipotent progenitors express a unique hybrid basal and luminal signature and the factors associated with the different lineages. Sustained p63 expression in embryonic multipotent progenitors promotes unipotent basal cell fate and was sufficient to reprogram adult luminal cells into basal cells by promoting an intermediate hybrid multipotent-like state. Altogether, this study identifies the timing and the mechanisms mediating early lineage segregation of multipotent progenitors during mammary gland development.
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http://dx.doi.org/10.1038/s41556-018-0095-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5985933PMC
June 2018

Single-cell sequencing reveals the origin and the order of mutation acquisition in T-cell acute lymphoblastic leukemia.

Leukemia 2018 06 18;32(6):1358-1369. Epub 2018 Apr 18.

Center for Human Genetics, KU Leuven, Leuven, Belgium.

Next-generation sequencing has provided a detailed overview of the various genomic lesions implicated in the pathogenesis of T-cell acute lymphoblastic leukemia (T-ALL). Typically, 10-20 protein-altering lesions are found in T-ALL cells at diagnosis. However, it is currently unclear in which order these mutations are acquired and in which progenitor cells this is initiated. To address these questions, we used targeted single-cell sequencing of total bone marrow cells and CD34CD38 multipotent progenitor cells for four T-ALL cases. Hierarchical clustering detected a dominant leukemia cluster at diagnosis, accompanied by a few smaller clusters harboring only a fraction of the mutations. We developed a graph-based algorithm to determine the order of mutation acquisition. Two of the four patients had an early event in a known oncogene (MED12, STAT5B) among various pre-leukemic events. Intermediate events included loss of 9p21 (CDKN2A/B) and acquisition of fusion genes, while NOTCH1 mutations were typically late events. Analysis of CD34CD38 cells and myeloid progenitors revealed that in half of the cases somatic mutations were detectable in multipotent progenitor cells. We demonstrate that targeted single-cell sequencing can elucidate the order of mutation acquisition in T-ALL and that T-ALL development can start in a multipotent progenitor cell.
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http://dx.doi.org/10.1038/s41375-018-0127-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5990522PMC
June 2018

Single-Cell (Multi)omics Technologies.

Annu Rev Genomics Hum Genet 2018 08 4;19:15-41. Epub 2018 May 4.

Wellcome Sanger Institute, Cambridge CB10 1SA, United Kingdom; email: , ,

Single-cell multiomics technologies typically measure multiple types of molecule from the same individual cell, enabling more profound biological insight than can be inferred by analyzing each molecular layer from separate cells. These single-cell multiomics technologies can reveal cellular heterogeneity at multiple molecular layers within a population of cells and reveal how this variation is coupled or uncoupled between the captured omic layers. The data sets generated by these techniques have the potential to enable a deeper understanding of the key biological processes and mechanisms driving cellular heterogeneity and how they are linked with normal development and aging as well as disease etiology. This review details both established and novel single-cell mono- and multiomics technologies and considers their limitations, applications, and likely future developments.
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http://dx.doi.org/10.1146/annurev-genom-091416-035324DOI Listing
August 2018

Publisher Correction: A TRP channel trio mediates acute noxious heat sensing.

Nature 2018 07;559(7713):E7

Laboratory of Ion Channel Research and TRP Research Platform Leuven (TRPLe), Department of Cellular and Molecular Medicine, University of Leuven, Leuven, Belgium.

In this Letter, the trace is missing in Fig. 1e. This error has been corrected online.
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http://dx.doi.org/10.1038/s41586-018-0100-8DOI Listing
July 2018
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