Single-nucleus multi-omics of human stem cell-derived islets identifies deficiencies in lineage specification.

Insulin-producing ß cells created from human pluripotent stem cells have potential as a therapy for insulin-dependent diabetes, but human pluripotent stem cell-derived islets (SC-islets) still differ from their in vivo counterparts. To better understand the state of cell types within SC-islets and identify lineage specification deficiencies, we used single-nucleus multi-omic sequencing to analyse chromatin accessibility and transcriptional profiles of SC-islets and primary human islets. Here we provide an analysis that enabled the derivation of gene lists and activity for identifying each SC-islet cell type compared with primary islets. Within SC-islets, we found that the difference between ß cells and awry enterochromaffin-like cells is a gradient of cell states rather than a stark difference in identity. Furthermore, transplantation of SC-islets in vivo improved cellular identities overtime, while long-term in vitro culture did not. Collectively, our results highlight the importance of chromatin and transcriptional landscapes during islet cell specification and maturation.

Microfluidic device facilitates in vitro modeling of human neonatal necrotizing enterocolitis-on-a-chip.

Necrotizing enterocolitis (NEC) is a deadly gastrointestinal disease of premature infants that is associated with an exaggerated inflammatory response, dysbiosis of the gut microbiome, decreased epithelial cell proliferation, and gut barrier disruption. We describe an in vitro model of the human neonatal small intestinal epithelium (Neonatal-Intestine-on-a-Chip) that mimics key features of intestinal physiology. This model utilizes intestinal enteroids grown from surgically harvested intestinal tissue from premature infants and cocultured with human intestinal microvascular endothelial cells within a microfluidic device. We used our Neonatal-Intestine-on-a-Chip to recapitulate NEC pathophysiology by adding infant-derived microbiota. This model, named NEC-on-a-Chip, simulates the predominant features of NEC, including significant upregulation of proinflammatory cytokines, decreased intestinal epithelial cell markers, reduced epithelial proliferation, and disrupted epithelial barrier integrity. NEC-on-a-Chip provides an improved preclinical model of NEC that facilitates comprehensive analysis of the pathophysiology of NEC using precious clinical samples. This model is an advance toward a personalized medicine approach to test new therapeutics for this devastating disease.

Inhibition of the C-X-C Motif Chemokine 12 (CXCL12) and Its Receptor CXCR4 Reduces Utero-Placental Expression of the VEGF System and Increases Utero-Placental Autophagy.

The placenta, a unique organ that only develops during pregnancy, is essential for nutrient, oxygen, and waste exchange between offspring and mother. Yet, despite its importance, the placenta remains one of the least understood organs and knowledge of early placental formation is particularly limited. Abnormalities in placental development result in placental dysfunction or insufficiency whereby normal placental physiology is impaired. Placental dysfunction is a frequent source of pregnancy loss in livestock, inflicting serious economic impact to producers. Though the underlying causes of placental dysfunction are not well-characterized, initiation of disease is thought to occur during establishment of functional fetal and placental circulation. A comprehensive understanding of the mechanisms controlling placental growth and vascularization is necessary to improve reproductive success in livestock. We propose chemokine C-X-C motif ligand 12 (CXCL12) signaling through its receptor CXCR4 functions as a chief coordinator of vascularization through direct actions on fetal trophoblast and maternal endometrial and immune cells. To investigate CXCL12-CXCR4 signaling on uteroplacental vascular remodeling at the fetal-maternal interface, we utilized a CXCR4 antagonist (AMD3100). On day 12 post-breeding in sheep, osmotic pumps were surgically installed and delivered either AMD3100 or saline into the uterine lumen ipsilateral to the corpus luteum for 14 days. On day 35 of ovine pregnancy, fetal/placental and endometrial tissues were collected, snap-frozen in liquid nitrogen, and uterine horn cross sections were preserved for immunofluorescent analysis. Suppressing CXCL12-CXCR4 at the fetal-maternal interface during initial placental vascularization resulted in diminished abundance of select angiogenic factors in fetal and maternal placenta on day 35. Compared to control, less vascular endothelial growth factor (VEGF) and VEFG receptor 2 (KDR) were observed in endometrium when CXCL12-CXCR4 was diminished. Less VEGF was also evident in fetal placenta (cotyledons) in ewes receiving AMD3100 infusion compared to control. Suppressing CXCL12-CXCR4 at the fetal-maternal interface also resulted in greater autophagy induction in fetal and maternal placenta compared to control, suggestive of CXCL12-CXCR4 impacting cell survival. CXCL12-CXCR4 signaling may govern placental homeostasis by serving as a critical upstream mediator of vascularization and cell viability, thereby ensuring appropriate placental development.

CXCR4 signaling at the fetal-maternal interface may drive inflammation and syncytia formation during ovine pregnancy†.

Early pregnancy features complex signaling between fetal trophoblast cells and maternal endometrium directing major peri-implantation events including localized inflammation and remodeling to establish proper placental development. Proinflammatory mediators are important for conceptus attachment, but a more precise understanding of molecular pathways regulating this process is needed to understand how the endometrium becomes receptive to implantation. Both chemokine ligand 12 (CXCL12) and its receptor CXCR4 are expressed by fetal and maternal tissues. We identified this pair as a critical driver of placental angiogenesis, but their additional importance to inflammation and trophoblast cell survival, proliferation, and invasion imply a role in syncytia formation at the fetal-maternal microenvironment. We hypothesized that CXCL12 encourages both endometrial inflammation and conceptus attachment during implantation. We employed separate ovine studies to (1) characterize endometrial inflammation during early gestation in the ewe, and (2) establish functional implications of CXCL12 at the fetal-maternal interface through targeted intrauterine infusion of the CXCR4 inhibitor AMD3100. Endometrial tissues were evaluated for inflammatory mediators, intracellular signaling events, endometrial modifications, and trophoblast syncytialization using western blotting and immunohistochemistry. Endometrial tissue from ewes receiving CXCR4 inhibitor demonstrated dysregulated inflammation and reduced AKT and NFKB, paired with elevated autophagic activity compared to control. Immunohistochemical observation revealed an impairment in endometrial surface remodeling and diminished trophoblast syncytialization following localized CXCR4 inhibition. These data suggest CXCL12-CXCR4 regulates endometrial inflammation and remodeling for embryonic implantation, and provide insight regarding mechanisms that, when dysregulated, lead to pregnancy pathologies such as intrauterine growth restriction and preeclampsia.

Intrauterine inhibition of chemokine receptor 4 signaling modulates local and systemic inflammation in ovine pregnancy.

PROBLEM: Chemokines help coordinate inflammation within the fetal-maternal microenvironment during gestation. The chemokine CXCL12 signaling through its receptor CXCR4 regulates inflammatory activity, but this phenomenon is not well understood during pregnancy, and there are no reports exploring the role of this pair in peripheral immune tolerance during gestation. Herein, we hypothesize that intrauterine CXCL12-CXCR4 signaling governs local and systemic immunomodulatory dynamics during early gestation in ewes. METHOD OF STUDY: Osmotic pumps were surgically installed for intrauterine infusion of a CXCR4 inhibitor, AMD3100, beginning on day 12 post-breeding in sheep. Endometrial tissues were collected on day 35 of gestation and evaluated for inflammatory potential, Akt pathway activation, and autophagy induction. Demonstrative of peripheral immune activity, levels of select cytokines were assessed in daily blood samples collected throughout the study, as well as in corpus luteum and spleen on day 35. RESULTS: Anti-inflammatory IL10 was primarily localized to endometrial glandular epithelium with lower abundance when CXCR4 was antagonized. Inhibition of CXCR4 at the fetal-maternal interface resulted in less activation of Akt in endometrium, while evidence of autophagy induction was greater. Corpora lutea from ewes receiving intrauterine AMD3100 exhibited lower interferon-gamma (IFNG) expression. Blood inflammatory potential was differentially altered in a temporal fashion throughout infusion. IL10 abundance in spleen was greater following CXCR4 inhibition at the fetal-maternal interface, while IFNG was less. CONCLUSION: Intrauterine CXCL12-CXCR4 signaling governs endometrial and systemic inflammation; disruption of this axis may have detrimental impacts on offspring and maternal health.

CXCR4 signaling at the ovine fetal-maternal interface regulates vascularization, CD34+ cell presence, and autophagy in the endometrium†.

Placenta development is characterized by extensive angiogenesis and vascularization but if these processes are compromised placental dysfunction occurs, which is the underlying cause of pregnancy complications such as preeclampsia and intrauterine growth restriction. Dysregulation of placental angiogenesis has emerged as one of the main pathophysiological features in the development of placental insufficiency and its clinical consequences. The signaling axis initiated by chemokine ligand 12 (CXCL12) and its receptor CXCR4 stimulates angiogenesis in other tissues, and may be central to placental vascularization. We hypothesized that CXCL12-CXCR4 signaling governs the pro-angiogenic placental microenvironment by coordinating production of central angiogenic factors and receptors and regulates endometrial cell survival essential for placental function and subsequent fetal longevity. The CXCR4 antagonist, AMD3100, was used to elucidate the role of CXCL12-CXCR4 signaling regarding uteroplacental vascular remodeling at the fetal-maternal interface. On day 12 postbreeding, osmotic pumps were surgically installed and delivered either AMD3100 or PBS into the uterine lumen ipsilateral to the corpus luteum. On day 20, endometrial tissues were collected, snap-frozen in liquid nitrogen, and uterine horn cross sections preserved for immunofluorescent analysis. In endometrium from ewes receiving AMD3100 infusion, the abundance of select angiogenic factors was diminished, while presence of CD34+ cells increased compared to control ewes. Ewes receiving AMD3100 infusion also exhibited less activation of Akt/mTOR signaling, and elevated LC3B-II, a marker of cellular autophagy in endometrium. This study suggests that CXCL12-CXCR4 signaling governs placental homeostasis by serving as a critical upstream mediator of vascularization and cell viability, thereby ensuring appropriate placental development.