Human Fetal TNF-α-Cytokine-Producing CD4+ Effector Memory T Cells Promote Intestinal Development and Mediate Inflammation Early in Life.

Although the fetal immune system is considered tolerogenic, preterm infants can suffer from severe intestinal inflammation, including necrotizing enterocolitis (NEC). Here, we demonstrate that human fetal intestines predominantly contain tumor necrosis factor-α (TNF-α)+CD4+CD69+ T effector memory (Tem) cells. Single-cell RNA sequencing of fetal intestinal CD4+ T cells showed a T helper 1 phenotype and expression of genes mediating epithelial growth and cell cycling. Organoid co-cultures revealed a dose-dependent, TNF-α-mediated effect of fetal intestinal CD4+ T cells on intestinal stem cell (ISC) development, in which low T cell numbers supported epithelial development, whereas high numbers abrogated ISC proliferation. CD4+ Tem cell frequencies were higher in inflamed intestines from preterm infants with NEC than in healthy infant intestines and showed enhanced TNF signaling. These findings reveal a distinct population of TNF-α-producing CD4+ T cells that promote mucosal development in fetal intestines but can also mediate inflammation upon preterm birth.

Rifaximin stimulates nitrogen detoxification by PXR-independent mechanisms in human small intestinal organoids.

BACKGROUND AND AIMS: Recurrent hepatic encephalopathy (HE) is characterized by hyperammonaemia in combination with neuropsychiatric abnormalities and is treated with lactulose and rifaximin. Rifaximin is a pregnane X receptor (PXR) agonist with low systemic and high intestinal bioavailability. The mechanisms by which it alleviates HE are unclear. We used human small intestinal (hSI) organoids to study whether rifaximin, via PXR activation, affects the epithelial biotransformation machinery, and to gain understanding of its low systemic availability. METHODS: We generated PXR knockdown hSI organoids via lentiviral delivery of short hairpin RNAs. Organoids were cultured for 24 h with rifaximin or rifampicin. RNA-sequencing and metabolomics were performed to analyse gene expression and amino acid metabolism. Luminal rifaximin was quantified by photospectrometry. RESULTS: Treatment of wild-type hSI organoids with rifaximin resulted in >twofold differential expression of 131 genes compared to DMSO. These effects were largely PXR independent and related to amino acid metabolism. Rifaximin decreased expression of glutaminase-2 and increased expression of asparagine synthetase and solute carrier 7A11, thereby increasing intracellular glutamine and asparagine concentrations, indicating active ammonia detoxification. Rifaximin was apically excreted into the lumen in an ATP binding cassette B1 (ABCB1)-dependent manner. CONCLUSIONS: Rifaximin-after uptake into enterocytes-stimulates intracellular nitrogen detoxification by PXR-independent mechanisms. Active apical excretion of rifaximin by ABCB1 into the intestinal lumen explains its low systemic bioavailability. Our study implies that rifaximin, next to modulation of the microbiome, has direct effects on ammonia scavenging in the human small intestinal epithelium.

The CD27 and CD70 costimulatory pathway inhibits effector function of T helper 17 cells and attenuates associated autoimmunity.

T helper 17 (Th17) cells protect against infection but also promote inflammation and autoimmunity. Therefore, the factors that govern Th17 cell differentiation are of special interest. The CD27 and CD70 costimulatory pathway impeded Th17 effector cell differentiation and associated autoimmunity in a mouse model of multiple sclerosis. CD27 or CD70 deficiency exacerbated disease, whereas constitutive CD27 signaling strongly reduced disease incidence and severity. CD27 signaling did not impact master regulators of T helper cell lineage commitment but selectively repressed transcription of the key effector molecules interleukin-17 (IL-17) and the chemokine receptor CCR6 in differentiating Th17 cells. CD27 mediated this repression at least in part via the c-Jun N-terminal kinase (JNK) pathway that restrained IL-17 and CCR6 expression in differentiating Th17 cells. CD27 signaling also resulted in epigenetic silencing of the Il17a gene. Thus, CD27 costimulation via JNK signaling, transcriptional, and epigenetic effects suppresses Th17 effector cell function and associated pathological consequences.

Tissue-specific mutation accumulation in human adult stem cells during life.

The gradual accumulation of genetic mutations in human adult stem cells (ASCs) during life is associated with various age-related diseases, including cancer. Extreme variation in cancer risk across tissues was recently proposed to depend on the lifetime number of ASC divisions, owing to unavoidable random mutations that arise during DNA replication. However, the rates and patterns of mutations in normal ASCs remain unknown. Here we determine genome-wide mutation patterns in ASCs of the small intestine, colon and liver of human donors with ages ranging from 3 to 87 years by sequencing clonal organoid cultures derived from primary multipotent cells. Our results show that mutations accumulate steadily over time in all of the assessed tissue types, at a rate of approximately 40 novel mutations per year, despite the large variation in cancer incidence among these tissues. Liver ASCs, however, have different mutation spectra compared to those of the colon and small intestine. Mutational signature analysis reveals that this difference can be attributed to spontaneous deamination of methylated cytosine residues in the colon and small intestine, probably reflecting their high ASC division rate. In liver, a signature with an as-yet-unknown underlying mechanism is predominant. Mutation spectra of driver genes in cancer show high similarity to the tissue-specific ASC mutation spectra, suggesting that intrinsic mutational processes in ASCs can initiate tumorigenesis. Notably, the inter-individual variation in mutation rate and spectra are low, suggesting tissue-specific activity of common mutational processes throughout life.

Enhanced Collagen Deposition in the Duodenum of Patients with Hyaline Fibromatosis Syndrome and Protein Losing Enteropathy.

Hyaline fibromatosis syndrome (HFS), resulting from ANTXR2 mutations, is an ultra-rare disease that causes intestinal lymphangiectasia and protein-losing enteropathy (PLE). The mechanisms leading to the gastrointestinal phenotype in these patients are not well defined. We present two patients with congenital diarrhea, severe PLE and unique clinical features resulting from deleterious ANTXR2 mutations. Intestinal organoids were generated from one of the patients, along with CRISPR-Cas9 ANTXR2 knockout, and compared with organoids from two healthy controls. The ANTXR2-deficient organoids displayed normal growth and polarity, compared to controls. Using an anthrax-toxin assay we showed that the c.155C>T mutation causes loss-of-function of ANTXR2 protein. An intrinsic defect of monolayer formation in patient-derived or ANTXR2KO organoids was not apparent, suggesting normal epithelial function. However, electron microscopy and second harmonic generation imaging showed abnormal collagen deposition in duodenal samples of these patients. Specifically, collagen VI, which is known to bind ANTXR2, was highly expressed in the duodenum of these patients. In conclusion, despite resistance to anthrax-toxin, epithelial cell function, and specifically monolayer formation, is intact in patients with HFS. Nevertheless, loss of ANTXR2-mediated signaling leads to collagen VI accumulation in the duodenum and abnormal extracellular matrix composition, which likely plays a role in development of PLE.

DGAT2 partially compensates for lipid-induced ER stress in human DGAT1-deficient intestinal stem cells.

Dietary lipids are taken up as FAs by the intestinal epithelium and converted by diacylglycerol acyltransferase (DGAT) enzymes into triglycerides, which are packaged in chylomicrons or stored in cytoplasmic lipid droplets (LDs). DGAT1-deficient patients suffer from vomiting, diarrhea, and protein losing enteropathy, illustrating the importance of this process to intestinal homeostasis. Previously, we have shown that DGAT1 deficiency causes decreased LD formation and resistance to unsaturated FA lipotoxicity in patient-derived intestinal organoids. However, LD formation was not completely abolished in patient-derived organoids, suggesting the presence of an alternative mechanism for LD formation. Here, we show an unexpected role for DGAT2 in lipid metabolism, as DGAT2 partially compensates for LD formation and lipotoxicity in DGAT1-deficient intestinal stem cells. Furthermore, we show that (un)saturated FA-induced lipotoxicity is mediated by ER stress. More importantly, we demonstrate that overexpression of DGAT2 fully compensates for the loss of DGAT1 in organoids, indicating that induced DGAT2 expression in patient cells may serve as a therapeutic target in the future.

Intestinal Failure and Aberrant Lipid Metabolism in Patients With DGAT1 Deficiency.

BACKGROUND & AIMS: Congenital diarrheal disorders are rare inherited intestinal disorders characterized by intractable, sometimes life-threatening, diarrhea and nutrient malabsorption; some have been associated with mutations in diacylglycerol-acyltransferase 1 (DGAT1), which catalyzes formation of triacylglycerol from diacylglycerol and acyl-CoA. We investigated the mechanisms by which DGAT1 deficiency contributes to intestinal failure using patient-derived organoids. METHODS: We collected blood samples from 10 patients, from 6 unrelated pedigrees, who presented with early-onset severe diarrhea and/or vomiting, hypoalbuminemia, and/or (fatal) protein-losing enteropathy with intestinal failure; we performed next-generation sequencing analysis of DNA from 8 patients. Organoids were generated from duodenal biopsies from 3 patients and 3 healthy individuals (controls). Caco-2 cells and patient-derived dermal fibroblasts were transfected or transduced with vectors that express full-length or mutant forms of DGAT1 or full-length DGAT2. We performed CRISPR/Cas9-guided disruption of DGAT1 in control intestinal organoids. Cells and organoids were analyzed by immunoblot, immunofluorescence, flow cytometry, chromatography, quantitative real-time polymerase chain reaction, and for the activity of caspases 3 and 7. RESULTS: In the 10 patients, we identified 5 bi-allelic loss-of-function mutations in DGAT1. In patient-derived fibroblasts and organoids, the mutations reduced expression of DGAT1 protein and altered triacylglycerol metabolism, resulting in decreased lipid droplet formation after oleic acid addition. Expression of full-length DGAT2 in patient-derived fibroblasts restored formation of lipid droplets. Organoids derived from patients with DGAT1 mutations were more susceptible to lipid-induced cell death than control organoids. CONCLUSIONS: We identified a large cohort of patients with congenital diarrheal disorders with mutations in DGAT1 that reduced expression of its product; dermal fibroblasts and intestinal organoids derived from these patients had altered lipid metabolism and were susceptible to lipid-induced cell death. Expression of full-length wildtype DGAT1 or DGAT2 restored normal lipid metabolism in these cells. These findings indicate the importance of DGAT1 in fat metabolism and lipotoxicity in the intestinal epithelium. A fat-free diet might serve as the first line of therapy for patients with reduced DGAT1 expression. It is important to identify genetic variants associated with congenital diarrheal disorders for proper diagnosis and selection of treatment strategies.

An inducible mouse model for microvillus inclusion disease reveals a role for myosin Vb in apical and basolateral trafficking.

Microvillus inclusion disease (MVID) is a rare intestinal enteropathy with an onset within a few days to months after birth, resulting in persistent watery diarrhea. Mutations in the myosin Vb gene (MYO5B) have been identified in the majority of MVID patients. However, the exact pathophysiology of MVID still remains unclear. To address the specific role of MYO5B in the intestine, we generated an intestine-specific conditional Myo5b-deficient (Myo5bfl/fl;Vil-CreERT2) mouse model. We analyzed intestinal tissues and cultured organoids of Myo5bfl/fl;Vil-CreERT2 mice by electron microscopy, immunofluorescence, and immunohistochemistry. Our data showed that Myo5bfl/fl;Vil-CreERT2 mice developed severe diarrhea within 4 d after tamoxifen induction. Periodic Acid Schiff and alkaline phosphatase staining revealed subapical accumulation of intracellular vesicles in villus enterocytes. Analysis by electron microscopy confirmed an almost complete absence of apical microvilli, the appearance of microvillus inclusions, and enlarged intercellular spaces in induced Myo5bfl/fl;Vil-CreERT2 intestines. In addition, we determined that MYO5B is involved not only in apical but also basolateral trafficking of proteins. The analysis of the intestine during the early onset of the disease revealed that subapical accumulation of secretory granules precedes occurrence of microvillus inclusions, indicating involvement of MYO5B in early differentiation of epithelial cells. By comparing our data with a novel MVID patient, we conclude that our mouse model completely recapitulates the intestinal phenotype of human MVID. This includes severe diarrhea, loss of microvilli, occurrence of microvillus inclusions, and subapical secretory granules. Thus, loss of MYO5B disturbs both apical and basolateral trafficking of proteins and causes MVID in mice.

Many inflammatory bowel disease risk loci include regions that regulate gene expression in immune cells and the intestinal epithelium.

BACKGROUND & AIMS: The contribution of genetic factors to the pathogenesis of inflammatory bowel disease (IBD) has been established by twin, targeted-sequencing, and genome-wide association studies. These studies identified many risk loci, and research is underway to identify causal variants. These studies have focused mainly on protein-coding genes. We investigated other functional elements in the human genome, such as regulatory regions. METHODS: Using acetylated histone 3 lysine 27 chromatin immunoprecipitation and sequencing, we identified tens of thousands of potential regulatory regions that are active in intestinal epithelium (primary intestinal crypts and cultured organoids) isolated from resected material and from biopsies collected during ileo-colonoscopies and immune cells (monocytes, macrophages, CD34(+), CD4(+), and CD8(+)). We correlated these regions with susceptibility loci for IBD. RESULTS: We have generated acetylated histone 3 lysine 27 profiles from primary intestinal epithelium and cultured organoids, which we have made publically available. We found that 45 of 163 single nucleotide polymorphisms (SNPs) associated with IBD overlap specifically with active regulatory elements. In addition, by taking strong linkage disequilibrium into account, another 47 IBD-associated SNPs colocalized with active regulatory elements through other SNPs in their vicinity. Altogether, 92 of 163 IBD-associated SNPs correlated with distinct active regulatory elements-a frequency 2.5- to 3.5-fold greater than that expected from random sampling. The variations in these SNPs often create or disrupt known binding motifs; they might affect the binding of transcriptional regulators to alter expression of regulated genes. CONCLUSIONS: In addition to variants in protein coding genes, variants in noncoding DNA regulatory regions that are active in intestinal epithelium and immune cells are potentially involved in the pathogenesis of IBD.

Loss of syntaxin 3 causes variant microvillus inclusion disease.

Microvillus inclusion disease (MVID) is a disorder of intestinal epithelial differentiation characterized by life-threatening intractable diarrhea. MVID can be diagnosed based on loss of microvilli, microvillus inclusions, and accumulation of subapical vesicles. Most patients with MVID have mutations in myosin Vb that cause defects in recycling of apical vesicles. Whole-exome sequencing of DNA from patients with variant MVID showed homozygous truncating mutations in syntaxin 3 (STX3). STX3 is an apical receptor involved in membrane fusion of apical vesicles in enterocytes. Patient-derived organoid cultures and overexpression of truncated STX3 in Caco-2 cells recapitulated most characteristics of variant MVID. We conclude that loss of STX3 function causes variant MVID.

Adult stem cells in the small intestine are intrinsically programmed with their location-specific function.

Differentiation and specialization of epithelial cells in the small intestine are regulated in two ways. First, there is differentiation along the crypt-villus axis of the intestinal stem cells into absorptive enterocytes, Paneth, goblet, tuft, enteroendocrine, or M cells, which is mainly regulated by WNT. Second, there is specialization along the cephalocaudal axis with different absorptive and digestive functions in duodenum, jejunum, and ileum that is controlled by several transcription factors such as GATA4. However, so far it is unknown whether location-specific functional properties are intrinsically programmed within stem cells or if continuous signaling from mesenchymal cells is necessary to maintain the location-specific identity of the small intestine. Using the pure epithelial organoid technique, we show that region-specific gene expression profiles are conserved throughout long-term cultures of both mouse and human intestinal stem cells and correlated with differential Gata4 expression. Furthermore, the human organoid culture system demonstrates that Gata4-regulated gene expression is only allowed in absence of WNT signaling. These data show that location-specific function is intrinsically programmed in the adult stem cells of the small intestine and that their differentiation fate is independent of location-specific extracellular signals. In light of the potential future clinical application of small intestine-derived organoids, our data imply that it is important to generate GATA4-positive and GATA4-negative cultures to regenerate all essential functions of the small intestine.

Constitutive activation of Bruton's tyrosine kinase induces the formation of autoreactive IgM plasma cells.

B-cell receptor (BCR)-mediated signals provide the basis for B-cell differentiation in the BM and subsequently into follicular, marginal zone, or B-1 B-cell subsets. We have previously shown that B-cell-specific expression of the constitutive active E41K mutant of the BCR-associated molecule Bruton's tyrosine kinase (Btk) leads to an almost complete deletion of immature B cells in the BM. Here, we report that low-level expression of the E41K or E41K-Y223F Btk mutants was associated with reduced follicular B-cell numbers and significantly increased proportions of B-1 cells in the spleen. Crosses with 3-83 mu delta and VH81X BCR Tg mice showed that constitutive active Btk expression did not change follicular, marginal zone, or B-1 B-cell fate choice, but resulted in selective expansion or survival of B-1 cells. Residual B cells were hyperresponsive and manifested sustained Ca(2+) mobilization. They were spontaneously driven into germinal center-independent plasma cell differentiation, as evidenced by increased numbers of IgM(+) plasma cells in spleen and BM and significantly elevated serum IgM. Because anti-nucleosome autoantibodies and glomerular IgM deposition were present, we conclude that constitutive Btk activation causes defective B-cell tolerance, emphasizing that Btk signals are essential for appropriate regulation of B-cell activation.

Mice deficient for CD137 ligand are predisposed to develop germinal center-derived B-cell lymphoma.

In the germinal center (GC), B cells proliferate dramatically and diversify their immunoglobulin genes, which increases the risk of malignant transformation. The GC B-cell reaction relies on crosstalk with follicular dendritic cells (FDCs), to which the costimulatory receptor CD137 on FDCs and its ligand on GC B cells potentially contribute. We report that mice deficient for CD137 ligand (CD137L) are predisposed to develop B-cell lymphoma, with an incidence of approximately 60% at 12 months of age. Lymphoma membrane markers were characteristic of GC B cells. Longitudinal histologic analysis identified the GC as site of oncogenic transformation and classified 85% of the malignancies found in approximately 200 mice as GC-derived B-cell lymphoma. To delineate the mechanism underlying lymphomagenesis, gene expression profiles of wild-type and CD137L-deficient GC B cells were compared. CD137L deficiency was associated with enhanced expression of a limited gene set that included Bcl-10 and the GC response regulators Bcl-6, Spi-B, Elf-1, Bach2, and activation-induced cytidine deaminase. Among these are proto-oncogenes that mediate GC B-cell lymphoma development in humans. We conclude that CD137L ordinarily regulates the GC B-cell response and thereby acts as a tumor suppressor.

A Potential Treatment of Congenital Sodium Diarrhea in Patients With Activating GUCY2C Mutations.

INTRODUCTION: Gain-of-function mutations in guanylyl cyclase C (GCC) result in persistent diarrhea with perinatal onset. We investigated a specific GCC inhibitor, SSP2518, for its potential to treat this disorder. METHODS: We investigated the effect of SSP2518 on GCC-mediated intracellular cyclic guanosine monophosphate (cGMP) levels and on GCC-mediated chloride secretion in intestinal organoids from 3 patients with distinct activating GCC mutations and from controls, with and without stimulation of GCC with heat-stable enterotoxin. RESULTS: Patient-derived organoids had significantly higher basal cGMP levels than control organoids, which were lowered by SSP2518 to levels found in control organoids. In addition, SSP2518 significantly reduced cGMP levels and chloride secretion in patient-derived and control organoids (P < 0.05 for all comparisons) after heat-stable enterotoxin stimulation. DISCUSSION: We reported in this study that the GCC inhibitor SSP2518 normalizes cGMP levels in intestinal organoids derived from patients with GCC gain-of-function mutations and markedly reduces cystic fibrosis transmembrane conductance regulator-dependent chloride secretion, the driver of persistent diarrhea.

Bruton's tyrosine kinase cooperates with the B cell linker protein SLP-65 as a tumor suppressor in Pre-B cells.

Expression of the pre-B cell receptor (pre-BCR) leads to activation of the adaptor molecule SLP-65 and the cytoplasmic kinase Btk. Mice deficient for one of these signaling proteins have an incomplete block in B cell development at the stage of large cycling pre-BCR+CD43+ pre-B cells. Our recent findings of defective SLP-65 expression in approximately 50% of childhood pre-B acute lymphoblastic leukemias and spontaneous pre-B cell lymphoma development in SLP-65-/- mice demonstrate that SLP-65 acts as a tumor suppressor. To investigate cooperation between Btk and SLP-65, we characterized the pre-B cell compartment in single and double mutant mice, and found that the two proteins have a synergistic role in the developmental progression of large cycling into small resting pre-B cells. We show that Btk/SLP-65 double mutant mice have a dramatically increased pre-B cell tumor incidence ( approximately 75% at 16 wk of age), as compared with SLP-65 single deficient mice (<10%). These findings demonstrate that Btk cooperates with SLP-65 as a tumor suppressor in pre-B cells. Furthermore, transgenic low-level expression of a constitutive active form of Btk, the E41K-Y223F mutant, prevented tumor formation in Btk/SLP-65 double mutant mice, indicating that constitutive active Btk can substitute for SLP-65 as a tumor suppressor.

The B cell antigen receptor controls integrin activity through Btk and PLCgamma2.

Integrin-mediated adhesion and B cell antigen receptor (BCR) signaling play a critical role in B cell development and function, including antigen-specific B cell differentiation. Here we show that the BCR controls integrin alpha4beta1 (VLA-4)-mediated adhesion of B cells to vascular cell adhesion molecule-1 and fibronectin. Molecular dissection of the underlying signaling mechanism by a combined biochemical, pharmacological, and genetic approach demonstrates that this BCR-controlled integrin-mediated adhesion requires the (consecutive) activation of Lyn, Syk, phosphatidylinositol 3-kinase, Bruton's tyrosine kinase (Btk), phospholipase C (PLC)gamma2, IP3R-mediated Ca2+ release, and PKC. In contrast, activation of mitogen-activated protein kinase kinase (MEK) or extracellular signal-regulated kinase (ERK) is not required, and simultaneous activation of MEK, ERK, and PKB is not sufficient either. Furthermore, Btk is also involved in the control of integrin-mediated adhesion of preB cells. The control of integrin alpha4beta1-mediated B cell adhesion by the BCR involves cytoskeletal reorganization and integrin clustering. These results reveal a novel function for the BCR and Btk, i.e., regulation of integrin alpha4beta1 activity, thereby providing new insights into the control of B cell development and differentiation, as well as into the pathogenesis of the immunodeficiency disease X-linked agammaglobulineamia (XLA).

A Fluorescence-based Assay for Characterization and Quantification of Lipid Droplet Formation in Human Intestinal Organoids.

Dietary lipids are taken up as free fatty acids (FAs) by the intestinal epithelium. These FAs are intracellularly converted into triglyceride (TG) molecules, before they are packaged into chylomicrons for transport to the lymph or into cytosolic lipid droplets (LDs) for intracellular storage. A crucial step for the formation of LDs is the catalytic activity of diacylglycerol acyltransferases (DGAT) in the final step of TG synthesis. LDs are important to buffer toxic lipid species and regulate cellular metabolism in different cell types. Since the human intestinal epithelium is regularly confronted with high concentrations of lipids, LD formation is of great importance to regulate homeostasis. Here we describe a simple assay for the characterization and quantification of LD formation (LDF) upon stimulation with the most common unsaturated fatty acid, oleic acid, in human intestinal organoids. The LDF assay is based on the LD-specific fluorescent dye LD540, which allows for quantification of LDs by confocal microscopy, fluorescent plate reader, or flow cytometry. The LDF assay can be used to characterize LD formation in human intestinal epithelial cells, or to study human (genetic) disorders that affect LD metabolism, such as DGAT1 deficiency. Furthermore, this assay can also be used in a high-throughput pipeline to test novel therapeutic compounds, which restore defects in LD formation in intestinal or other types of organoids.

Intestinal epithelial cell polarity defects in disease: lessons from microvillus inclusion disease.

The intestinal epithelium is a highly organized tissue. The establishment of epithelial cell polarity, with distinct apical and basolateral plasma membrane domains, is pivotal for both barrier formation and for the uptake and vectorial transport of nutrients. The establishment of cell polarity requires a specialized subcellular machinery to transport and recycle proteins to their appropriate location. In order to understand and treat polarity-associated diseases, it is necessary to understand epithelial cell-specific trafficking mechanisms. In this Review, we focus on cell polarity in the adult mammalian intestine. We discuss how intestinal epithelial polarity is established and maintained, and how disturbances in the trafficking machinery can lead to a polarity-associated disorder, microvillus inclusion disease (MVID). Furthermore, we discuss the recent developments in studying MVID, including the creation of genetically manipulated cell lines, mouse models and intestinal organoids, and their uses in basic and applied research.

Women in Translational Medicine: Tools to Break the Glass Ceiling.

Despite the recent movements for female equality and empowerment, few women occupy top positions in scientific decision-making. The challenges women face during their career may arise from societal biases and the current scientific culture. We discuss the effect of such biases at three different levels of the career and provide suggestions to tackle them. At the societal level, gender roles can create a negative feedback loop in which women are discouraged from attaining top positions and men are discouraged from choosing a home-centred lifestyle. This loop can be broken early in life by providing children with female role models that have a work-centred life and opening up the discussion about gender roles at a young age. At the level of hiring, unconscious biases can lead to a preference for male candidates. The introduction of (unbiased) artificial intelligence algorithms and gender champions in the hiring process may restore the balance and give men and women an equal chance. At the level of coaching and evaluation, barriers that women face should be addressed on a personal level through the introduction of coaching and mentoring programmes. In addition, women may play a pivotal role in shifting the perception of scientific success away from bibliometric outcomes only towards a more diverse assessment of quality and societal relevance. Taken together, these suggestions may break the glass ceiling in the scientific world for women; create more gender diversity at the top and improve translational science in medicine.