Chimeric contribution of human extended pluripotent stem cells to monkey embryos ex vivo.

Interspecies chimera formation with human pluripotent stem cells (hPSCs) represents a necessary alternative to evaluate hPSC pluripotency in vivo and might constitute a promising strategy for various regenerative medicine applications, including the generation of organs and tissues for transplantation. Studies using mouse and pig embryos suggest that hPSCs do not robustly contribute to chimera formation in species evolutionarily distant to humans. We studied the chimeric competency of human extended pluripotent stem cells (hEPSCs) in cynomolgus monkey (Macaca fascicularis) embryos cultured ex vivo. We demonstrate that hEPSCs survived, proliferated, and generated several peri- and early post-implantation cell lineages inside monkey embryos. We also uncovered signaling events underlying interspecific crosstalk that may help shape the unique developmental trajectories of human and monkey cells within chimeric embryos. These results may help to better understand early human development and primate evolution and develop strategies to improve human chimerism in evolutionarily distant species.

Decorin suppresses tumor lymphangiogenesis: A mechanism to curtail cancer progression.

The complex interplay between malignant cells and the cellular and molecular components of the tumor stroma is a key aspect of cancer growth and development. These tumor-host interactions are often affected by soluble bioactive molecules such as proteoglycans. Decorin, an archetypical small leucine-rich proteoglycan primarily expressed by stromal cells, affects cancer growth in its soluble form by interacting with several receptor tyrosine kinases (RTK). Overall, decorin leads to a context-dependent and protracted cessation of oncogenic RTK activity by attenuating their ability to drive a prosurvival program and to sustain a proangiogenic network. Through an unbiased transcriptomic analysis using deep RNAseq, we identified that decorin down-regulated a cluster of tumor-associated genes involved in lymphatic vessel (LV) development when systemically delivered to mice harboring breast carcinoma allografts. We found that Lyve1 and Podoplanin, two established markers of LVs, were markedly suppressed at both the mRNA and protein levels, and this suppression correlated with a significant reduction in tumor LVs. We further identified that soluble decorin, but not its homologous proteoglycan biglycan, inhibited LV sprouting in an ex vivo 3D model of lymphangiogenesis. Mechanistically, we found that decorin interacted with vascular endothelial growth factor receptor 3 (VEGFR3), the main lymphatic RTK, and its activity was required for the decorin-mediated block of lymphangiogenesis. Finally, we identified that Lyve1 was in part degraded via decorin-evoked autophagy in a nutrient- and energy-independent manner. These findings implicate decorin as a biological factor with antilymphangiogenic activity and provide a potential therapeutic agent for curtailing breast cancer growth and metastasis.

Ex vivo reconstitution of fetal oocyte development in humans and cynomolgus monkeys.

In vitro oogenesis is key to elucidating the mechanism of human female germ-cell development and its anomalies. Accordingly, pluripotent stem cells have been induced into primordial germ cell-like cells and into oogonia with epigenetic reprogramming, yet further reconstitutions remain a challenge. Here, we demonstrate ex vivo reconstitution of fetal oocyte development in both humans and cynomolgus monkeys (Macaca fascicularis). With an optimized culture of fetal ovary reaggregates over three months, human and monkey oogonia enter and complete the first meiotic prophase to differentiate into diplotene oocytes that form primordial follicles, the source for oogenesis in adults. The cytological and transcriptomic progressions of fetal oocyte development in vitro closely recapitulate those in vivo. A comparison of single-cell transcriptomes among humans, monkeys, and mice unravels primate-specific and conserved programs driving fetal oocyte development, the former including a distinct transcriptomic transformation upon oogonia-to-oocyte transition and the latter including two active X chromosomes with little X-chromosome upregulation. Our study provides a critical step forward for realizing human in vitro oogenesis and uncovers salient characteristics of fetal oocyte development in primates.

Partial male-to-female reprogramming of mouse fetal testis by Sertoli cell ablation.

Temporal transcription profiles of fetal testes with Sertoli cell ablation were examined in 4-day culture using a diphtheria toxin (DT)-dependent cell knockout system in AMH-TRECK transgenic (Tg) mice. RNA analysis revealed that ovarian-specific genes, including Foxl2, were ectopically expressed in DT-treated Tg testis explants initiated at embryonic days 12.5-13.5. FOXL2-positive cells were ectopically observed in two testicular regions: near the testicular surface epithelia and around its adjacent mesonephros. The surface FOXL2-positive cells, together with ectopic expression of Lgr5 and Gng13 (markers of ovarian cords), were derived from the testis epithelia/subepithelia, whereas another FOXL2-positive population was the 3ßHSD-negative stroma near the mesonephros. In addition to high expression of Fgfr1/Fgfr2 and heparan sulfate proteoglycan (a reservoir for FGF ligand) in these two sites, exogenous FGF9 additives repressed DT-dependent Foxl2 upregulation in Tg testes. These findings imply retention of Foxl2 inducibility in the surface epithelia and peri-mesonephric stroma of the testicular parenchyma, in which certain paracrine signals, including FGF9 derived from fetal Sertoli cells, repress feminization in these two sites of the early fetal testis.

Breast cancer patient-derived whole-tumor cell culture model for efficient drug profiling and treatment response prediction.

Breast cancer (BC) is a complex disease comprising multiple distinct subtypes with different genetic features and pathological characteristics. Although a large number of antineoplastic compounds have been approved for clinical use, patient-to-patient variability in drug response is frequently observed, highlighting the need for efficient treatment prediction for individualized therapy. Several patient-derived models have been established lately for the prediction of drug response. However, each of these models has its limitations that impede their clinical application. Here, we report that the whole-tumor cell culture (WTC) ex vivo model could be stably established from all breast tumors with a high success rate (98 out of 116), and it could reassemble the parental tumors with the endogenous microenvironment. We observed strong clinical associations and predictive values from the investigation of a broad range of BC therapies with WTCs derived from a patient cohort. The accuracy was further supported by the correlation between WTC-based test results and patients' clinical responses in a separate validation study, where the neoadjuvant treatment regimens of 15 BC patients were mimicked. Collectively, the WTC model allows us to accomplish personalized drug testing within 10 d, even for small-sized tumors, highlighting its potential for individualized BC therapy. Furthermore, coupled with genomic and transcriptomic analyses, WTC-based testing can also help to stratify specific patient groups for assignment into appropriate clinical trials, as well as validate potential biomarkers during drug development.

Skinny deeping: Uncovering immune cell behavior and function through imaging techniques.

As the largest organ of the body, the skin is a key barrier tissue with specialized structures where ongoing immune surveillance is critical for protecting the body from external insults. The innate immune system acts as first-responders in a coordinated manner to react to injury or infections, and recent developments in intravital imaging techniques have made it possible to delineate dynamic immune cell responses in a spatiotemporal manner. We review here key studies involved in understanding neutrophil, dendritic cell and macrophage behavior in skin and further discuss how this knowledge collectively highlights the importance of interactions and cellular functions in a systems biology manner. Furthermore, we will review emerging imaging technologies such as high-content proteomic screening, spatial transcriptomics and three-dimensional volumetric imaging and how these techniques can be integrated to provide a systems overview of the immune system that will further our current knowledge and lead to potential exciting discoveries in the upcoming decades.

Spatial determinates of effector and memory CD8+ T cell fates.

The lymph node plays a critical role in mounting an adaptive immune response to infection, clearance of foreign pathogens, and cancer immunosurveillance. Within this complex structure, intranodal migration is vital for CD8+ T cell activation and differentiation. Combining tissue clearing and volumetric light sheet fluorescent microscopy of intact lymph nodes has allowed us to explore the spatial regulation of T cell fates. This has determined that short-lived effector (TSLEC ) are imprinted in peripheral lymph node interfollicular regions, due to CXCR3 migration. In contrast, stem-like memory cell (TSCM ) differentiation is determined in the T cell paracortex. Here, we detail the inflammatory and chemokine regulators of spatially restricted T cell differentiation, with a focus on how to promote TSCM . We propose a default pathway for TSCM differentiation due to CCR7-directed segregation of precursors away from the inflammatory effector niche. Although volumetric imaging has revealed the consequences of intranodal migration, we still lack knowledge of how this is orchestrated within a complex chemokine environment. Toward this goal, we highlight the potential of combining microfluidic chambers with pre-determined complexity and subcellular resolution microscopy.

Imaging leukocyte migration through afferent lymphatics.

Afferent lymphatics mediate the transport of antigen and leukocytes, especially of dendritic cells (DCs) and T cells, from peripheral tissues to draining lymph nodes (dLNs). As such they play important roles in the induction and regulation of adaptive immunity. Over the past 15 years, great advances in our understanding of leukocyte trafficking through afferent lymphatics have been made through time-lapse imaging studies performed in tissue explants and in vivo, allowing to visualize this process with cellular resolution. Intravital imaging has revealed that intralymphatic leukocytes continue to actively migrate once they have entered into lymphatic capillaries, as a consequence of the low flow conditions present in this compartment. In fact, leukocytes spend considerable time migrating, patrolling and interacting with the lymphatic endothelium or with other intralymphatic leukocytes within lymphatic capillaries. Cells typically only start to detach once they arrive in downstream-located collecting vessels, where vessel contractions contribute to enhanced lymph flow. In this review, we will introduce the biology of afferent lymphatic vessels and report on the presumed significance of DC and T cell migration via this route. We will specifically highlight how time-lapse imaging has contributed to the current model of lymphatic trafficking and the emerging notion that - besides transport - lymphatic capillaries exert additional roles in immune modulation.

Whole blood stimulation as a tool for studying the human immune system.

The human immune system is best accessible via tissues and organs not requiring major surgical intervention, such as blood. In many circumstances, circulating immune cells correlate with an individual's health state and give insight into physiological and pathophysiological processes. Stimulating whole blood ex vivo is a powerful tool to investigate immune responses. In the context of clinical research, the applications of whole blood stimulation include host immunity, disease characterization, diagnosis, treatment, and drug development. Here, we summarize different setups and readouts of whole blood assays and discuss applications for preclinical research and clinical practice. Finally, we propose combining whole blood stimulation with high-throughput technologies, such as single-cell RNA-sequencing, to comprehensively analyze the human immune system for the identification of biomarkers, therapeutic interventions as well as companion diagnostics.

Aster repulsion drives short-ranged ordering in the Drosophila syncytial blastoderm.

Biological systems are highly complex, yet notably ordered structures can emerge. During syncytial stage development of the Drosophila melanogaster embryo, nuclei synchronously divide for nine cycles within a single cell, after which most of the nuclei reach the cell cortex. The arrival of nuclei at the cortex occurs with remarkable positional order, which is important for subsequent cellularisation and morphological transformations. Yet, the mechanical principles underlying this lattice-like positional order of nuclei remain untested. Here, using quantification of nuclei position and division orientation together with embryo explants, we show that short-ranged repulsive interactions between microtubule asters ensure the regular distribution and maintenance of nuclear positions in the embryo. Such ordered nuclear positioning still occurs with the loss of actin caps and even the loss of the nuclei themselves; the asters can self-organise with similar distribution to nuclei in the wild-type embryo. The explant assay enabled us to deduce the nature of the mechanical interaction between pairs of nuclei. We used this to predict how the nuclear division axis orientation changes upon nucleus removal from the embryo cortex, which we confirmed in vivo with laser ablation. Overall, we show that short-ranged microtubule-mediated repulsive interactions between asters are important for ordering in the early Drosophila embryo and minimising positional irregularity.

pH regulates hematopoietic stem cell potential via polyamines.

Upon ex vivo culture, hematopoietic stem cells (HSCs) quickly lose potential and differentiate into progenitors. The identification of culture conditions that maintain the potential of HSCs ex vivo is therefore of high clinical interest. Here, we demonstrate that the potential of murine and human HSCs is maintained when cultivated for 2 days ex vivo at a pH of 6.9, in contrast to cultivation at the commonly used pH of 7.4. When cultivated at a pH of 6.9, HSCs remain smaller, less metabolically active, less proliferative and show enhanced reconstitution ability upon transplantation compared to HSC cultivated at pH 7.4. HSCs kept at pH 6.9 show an attenuated polyamine pathway. Pharmacological inhibition of the polyamine pathway in HSCs cultivated at pH 7.4 with DFMO mimics phenotypes and potential of HSCs cultivated at pH 6.9. Ex vivo exposure to a pH of 6.9 is therefore a positive regulator of HSC function by reducing polyamines. These findings might improve HSC short-term cultivation protocols for transplantation and gene therapy interventions.

TRKing down drug resistance in NTRK fusion-positive cancers†.

In a recent issue of The Journal of Pathology, Chen and colleagues established novel patient-derived ex vivo models of NTRK fusion-positive soft tissue sarcoma to characterize resistance mechanisms against targeted therapy with tyrosine kinase inhibitors. Prolonged exposure to escalating concentrations of the tyrosine kinase inhibitor, entrectinib, ultimately led to the occurrence of resistant clones that harbored an inactivating mutation in the NF2 gene, not previously described in this context, accompanied by increased PI3K/AKT/mTOR and Ras/Raf/MEK/ERK signaling. Finally, an inhibitor screen identified, among others, MEK and mTOR inhibitors as potential combination agents. © 2024 The Pathological Society of Great Britain and Ireland.

Acquired NF2 mutation confers resistance to TRK inhibition in an ex vivo LMNA::NTRK1-rearranged soft-tissue sarcoma cell model.

Genomic rearrangements of the neurotrophic receptor tyrosine kinase genes (NTRK1, NTRK2, and NTRK3) are the most common mechanism of oncogenic activation for this family of receptors, resulting in sustained cancer cell proliferation. Several targeted therapies have been approved for tumours harbouring NTRK fusions and a new generation of TRK inhibitors has already been developed due to acquired resistance. We established a patient-derived LMNA::NTRK1-rearranged soft-tissue sarcoma cell model ex vivo with an acquired resistance to targeted TRK inhibition. Molecular profiling of the resistant clones revealed an acquired NF2 loss of function mutation that was absent in the parental cell model. Parental cells showed continuous sensitivity to TRK-targeted treatment, whereas the resistant clones were insensitive. Furthermore, resistant clones showed upregulation of the MAPK and mTOR/AKT pathways in the gene expression based on RNA sequencing data and increased sensitivity to MEK and mTOR inhibitor therapy. Drug synergy was seen using trametinib and rapamycin in combination with entrectinib. Medium-throughput drug screening further identified small compounds as potential drug candidates to overcome resistance as monotherapy or in combination with entrectinib. In summary, we developed a comprehensive model of drug resistance in an LMNA::NTRK1-rearranged soft-tissue sarcoma and have broadened the understanding of acquired drug resistance to targeted TRK therapy. Furthermore, we identified drug combinations and small compounds to overcome acquired drug resistance and potentially guide patient care in a functional precision oncology setting. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Segmentation of supragranular and infragranular layers in ultra-high-resolution 7T ex vivo MRI of the human cerebral cortex.

Accurate labeling of specific layers in the human cerebral cortex is crucial for advancing our understanding of neurodevelopmental and neurodegenerative disorders. Building on recent advancements in ultra-high-resolution ex vivo MRI, we present a novel semi-supervised segmentation model capable of identifying supragranular and infragranular layers in ex vivo MRI with unprecedented precision. On a dataset consisting of 17 whole-hemisphere ex vivo scans at 120 $\mu $m, we propose a Multi-resolution U-Nets framework that integrates global and local structural information, achieving reliable segmentation maps of the entire hemisphere, with Dice scores over 0.8 for supra- and infragranular layers. This enables surface modeling, atlas construction, anomaly detection in disease states, and cross-modality validation while also paving the way for finer layer segmentation. Our approach offers a powerful tool for comprehensive neuroanatomical investigations and holds promise for advancing our mechanistic understanding of progression of neurodegenerative diseases.

Hematopoietic stem cell gene therapy improves outcomes in a clinically relevant mouse model of multiple sulfatase deficiency.

Multiple sulfatase deficiency (MSD) is a severe, lysosomal storage disorder caused by pathogenic variants in the gene SUMF1, encoding the sulfatase modifying factor formylglycine-generating enzyme. Patients with MSD exhibit functional deficiencies in all cellular sulfatases. The inability of sulfatases to break down their substrates leads to progressive and multi-systemic complications in patients, similar to those seen in single-sulfatase disorders such as metachromatic leukodystrophy and mucopolysaccharidoses IIIA. Here, we aimed to determine if hematopoietic stem cell transplantation with ex vivo SUMF1 lentiviral gene therapy could improve outcomes in a clinically relevant mouse model of MSD. We first tested our approach in MSD patient-derived cells and found that our SUMF1 lentiviral vector improved protein expression, sulfatase activities, and glycosaminoglycan accumulation. In vivo, we found that our gene therapy approach rescued biochemical deficits, including sulfatase activity and glycosaminoglycan accumulation, in affected organs of MSD mice treated post-symptom onset. In addition, treated mice demonstrated improved neuroinflammation and neurocognitive function. Together, these findings suggest that SUMF1 HSCT-GT can improve both biochemical and functional disease markers in the MSD mouse.

Development and IND-enabling studies of a novel Cas9 genome-edited autologous CD34+ cell therapy to induce fetal hemoglobin for sickle cell disease.

Sickle cell disease (SCD) is a common, severe genetic blood disorder. Current pharmacotherapies are partially effective and allogeneic hematopoietic stem cell transplantation is associated with immune toxicities. Genome editing of patient hematopoietic stem cells (HSCs) to reactivate fetal hemoglobin (HbF) in erythroid progeny offers an alternative potentially curative approach to treat SCD. Although the FDA released guidelines for evaluating genome editing risks, it remains unclear how best to approach pre-clinical assessment of genome-edited cell products. Here, we describe rigorous pre-clinical development of a therapeutic γ-globin gene promoter editing strategy that supported an investigational new drug application cleared by the FDA. We compared γ-globin promoter and BCL11A enhancer targets, identified a potent HbF-inducing lead candidate, and tested our approach in mobilized CD34+ hematopoietic stem progenitor cells (HSPCs) from SCD patients. We observed efficient editing, HbF induction to predicted therapeutic levels, and reduced sickling. With single-cell analyses, we defined the heterogeneity of HbF induction and HBG1/HBG2 transcription. With CHANGE-seq for sensitive and unbiased off-target discovery followed by targeted sequencing, we did not detect off-target activity in edited HSPCs. Our study provides a blueprint for translating new ex vivo HSC genome editing strategies toward clinical trials for treating SCD and other blood disorders.

SARS-CoV2 infection in whole lung primarily targets macrophages that display subset-specific responses.

Deciphering the initial steps of SARS-CoV-2 infection, that influence COVID-19 outcomes, is challenging because animal models do not always reproduce human biological processes and in vitro systems do not recapitulate the histoarchitecture and cellular composition of respiratory tissues. To address this, we developed an innovative ex vivo model of whole human lung infection with SARS-CoV-2, leveraging a lung transplantation technique. Through single-cell RNA-seq, we identified that alveolar and monocyte-derived macrophages (AMs and MoMacs) were initial targets of the virus. Exposure of isolated lung AMs, MoMacs, classical monocytes and non-classical monocytes (ncMos) to SARS-CoV-2 variants revealed that while all subsets responded, MoMacs produced higher levels of inflammatory cytokines than AMs, and ncMos contributed the least. A Wuhan lineage appeared to be more potent than a D614G virus, in a dose-dependent manner. Amidst the ambiguity in the literature regarding the initial SARS-CoV-2 cell target, our study reveals that AMs and MoMacs are dominant primary entry points for the virus, and suggests that their responses may conduct subsequent injury, depending on their abundance, the viral strain and dose. Interfering on virus interaction with lung macrophages should be considered in prophylactic strategies.

Using Organoids to Tap Mammary Gland Diversity for Novel Insight.

This article offers a comprehensive perspective on the transformative role of organoid technology on mammary gland biology research across a diverse array of mammalian species.The mammary gland's unique development and regenerative capabilities render this organ an ideal model for studying developmental evolution, stem cell behavior, and regenerative processes. The discussion extends to the use of cross-species mammary organoids to address key biological inquiries in evolution, tissue regeneration, cancer research, and lactation, highlighting the limitations of traditional mouse models and the benefits of incorporating a more diverse range of animal models.Advances in organoid biology have been critical in overcoming ethical and practical constraints of in-vivo studies, especially in human research. The generation of human and mouse mammary organoids that faithfully recapitulate in-vivo tissues marks a significant stride in this field. Parallel capabilities are now emerging for other mammals, as well.Utilizing mammary organoids from various species has the potential to make invaluable contributions to our understanding of mammary gland biology, with implications for regenerative medicine, cancer research, and lactation studies, thereby contributing to advancements in human health, agriculture, and nutrition science.

Ex-Vivo 13C NMR Spectroscopy of Rodent Brain: TNF Restricts Neuronal Utilization of Astrocyte-Derived Metabolites.

Tumor necrosis factor (TNF) has well-established roles in neuroinflammatory disorders, but the effect of TNF on the biochemistry of brain cells remains poorly understood. Here, we microinjected TNF into the brain to study its impact on glial and neuronal metabolism (glycolysis, pentose phosphate pathway, citric acid cycle, pyruvate dehydrogenase, and pyruvate carboxylase pathways) using 13C NMR spectroscopy on brain extracts following intravenous [1,2-13C]-glucose (to probe glia and neuron metabolism), [2-13C]-acetate (probing astrocyte-specific metabolites), or [3-13C]-lactate. An increase in [4,5-13C]-glutamine and [2,3-13C]-lactate coupled with a decrease in [4,5-13C]-glutamate was observed in the [1,2-13C]-glucose-infused animals treated with TNF. As glutamine is produced from glutamate by astrocyte-specific glutamine synthetase the increase in [4,5-13C]-glutamine reflects increased production of glutamine by astrocytes. This was confirmed by infusion with astrocyte substrate [2-13C]-acetate. As lactate is metabolized in the brain to produce glutamate, the simultaneous increase in [2,3-13C]-lactate and decrease in [4,5-13C]-glutamate suggests decreased lactate utilization, which was confirmed using [3-13C]-lactate as a metabolic precursor. These results suggest that TNF rearranges the metabolic network, disrupting the energy supply chain perturbing the glutamine-glutamate shuttle between astrocytes and the neurons. These insights pave the way for developing astrocyte-targeted therapeutic strategies aimed at modulating effects of TNF to restore metabolic homeostasis in neuroinflammatory disorders.

Macrophage-derived extracellular vesicles alter cardiac recovery and metabolism in a rat heart model of donation after circulatory death.

Conditions to which the cardiac graft is exposed during transplantation with donation after circulatory death (DCD) can trigger the recruitment of macrophages that are either unpolarized (M0) or pro-inflammatory (M1) as well as the release of extracellular vesicles (EV). We aimed to characterize the effects of M0 and M1 macrophage-derived EV administration on post-ischaemic functional recovery and glucose metabolism using an isolated rat heart model of DCD. Isolated rat hearts were subjected to 20 min aerobic perfusion, followed by 27 min global, warm ischaemia or continued aerobic perfusion and 60 min reperfusion with or without intravascular administration of EV. Four experimental groups were compared: (1) no ischaemia, no EV; (2) ischaemia, no EV; (3) ischaemia with M0-macrophage-dervied EV; (4) ischaemia with M1-macrophage-derived EV. Post-ischaemic ventricular and metabolic recovery were evaluated. During reperfusion, ventricular function was decreased in untreated ischaemic and M1-EV hearts, but not in M0-EV hearts, compared to non-ischaemic hearts (p < 0.05). In parallel with the reduced functional recovery in M1-EV versus M0-EV ischaemic hearts, rates of glycolysis from exogenous glucose and oxidative metabolism tended to be lower, while rates of glycogenolysis and lactate release tended to be higher. EV from M0- and M1-macrophages differentially affect post-ischaemic cardiac recovery, potentially by altering glucose metabolism in a rat model of DCD. Targeted EV therapy may be a useful approach for modulating cardiac energy metabolism and optimizing graft quality in the setting of DCD.