ER stress and lipid imbalance drive diabetic embryonic cardiomyopathy in an organoid model of human heart development.

Congenital heart defects are the most prevalent human birth defects, and their incidence is exacerbated by maternal health conditions, such as diabetes during the first trimester (pregestational diabetes). Our understanding of the pathology of these disorders is hindered by a lack of human models and the inaccessibility of embryonic tissue. Using an advanced human heart organoid system, we simulated embryonic heart development under pregestational diabetes-like conditions. These organoids developed pathophysiological features observed in mouse and human studies before, including ROS-mediated stress and cardiomyocyte hypertrophy. scRNA-seq revealed cardiac cell-type-specific dysfunction affecting epicardial and cardiomyocyte populations and alterations in the endoplasmic reticulum and very-long-chain fatty acid lipid metabolism. Imaging and lipidomics confirmed these findings and showed that dyslipidemia was linked to fatty acid desaturase 2 mRNA decay dependent on IRE1-RIDD signaling. Targeting IRE1 or restoring lipid levels partially reversed the effects of pregestational diabetes, offering potential preventive and therapeutic strategies in humans.

ER stress and lipid imbalance drive embryonic cardiomyopathy in a human heart organoid model of pregestational diabetes.

Congenital heart defects constitute the most common birth defect in humans, affecting approximately 1% of all live births. The incidence of congenital heart defects is exacerbated by maternal conditions, such as diabetes during the first trimester. Our ability to mechanistically understand these disorders is severely limited by the lack of human models and the inaccessibility to human tissue at relevant stages. Here, we used an advanced human heart organoid model that recapitulates complex aspects of heart development during the first trimester to model the effects of pregestational diabetes in the human embryonic heart. We observed that heart organoids in diabetic conditions develop pathophysiological hallmarks like those previously reported in mouse and human studies, including ROS-mediated stress and cardiomyocyte hypertrophy, among others. Single cell RNA-seq revealed cardiac cell type specific-dysfunction affecting epicardial and cardiomyocyte populations, and suggested alterations in endoplasmic reticulum function and very long chain fatty acid lipid metabolism. Confocal imaging and LC-MS lipidomics confirmed our observations and showed that dyslipidemia was mediated by fatty acid desaturase 2 (FADS2) mRNA decay dependent on IRE1-RIDD signaling. We also found that the effects of pregestational diabetes could be reversed to a significant extent using drug interventions targeting either IRE1 or restoring healthy lipid levels within organoids, opening the door to new preventative and therapeutic strategies in humans.

Modeling the Effects of Maternal Diabetes on the Developing Human Heart Using Pluripotent Stem Cell-Derived Heart Organoids.

Congenital heart defects (CHD) constitute the most common type of birth defect in humans. Maternal diabetes during the first trimester of pregnancy (pregestational diabetes, or PGD) is one of the most prominent factors contributing to CHD, and is present in a significant population of female patients with diabetes in reproductive age. PGD is challenging to manage clinically due to the extreme sensitivity of the developing embryo to glucose oscillations, and constitutes a critical health problem for the mother and the fetus. The prevalence of PGD-induced CHD is increasing due to the ongoing diabetes epidemic. While studies using animal models and cells in culture have demonstrated that PGD alters critical cellular and developmental processes, the mechanisms remain obscure, and it is unclear to what extent these models recapitulate PGD-induced CHD in humans. Clinical practice precludes direct studies in developing human embryos, further highlighting the need for physiologically relevant models. To bypass many of these technical and ethical limitations, we describe here a human pluripotent stem cell (hPSC)-based method to generate developmentally relevant self-organizing human heart organoids. By using glucose and insulin to mimic the diabetic environment that the embryo faces in PGD, this system allows modeling critical features of PGD in a human system with relevant physiology, structure, and cell types. The protocol starts with the generation of hPSC-derived embryoid bodies in a 96-well plate, followed by a small molecule-based three-step Wnt activation/inhibition/activation strategy. Organoids are then differentiated under healthy (normal insulin and glucose) and diabetic conditions (high insulin and glucose) over time, allowing for the study of the effects of pregestational diabetes on the developing human heart. We also provide an immunofluorescence protocol for comparing, characterizing, and analyzing the differences between the healthy and diabetic organoids, and comment on additional steps for preparing the organoids for analysis by other techniques after differentiation. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of hPSC-derived embryoid bodies Basic Protocol 2: Differentiation of EBs into heart organoids under healthy and diabetes-like conditions Basic Protocol 3: Immunofluorescence and organoid preparation for other assays.

Expression of ABC transporters during syncytialization in preeclampsia.

Preeclampsia complicates 2-8% of pregnancies and is associated with prematurity and intrauterine growth restriction. Cholesterol and sterol transport is a key function of the placenta and it is elicited through ATP binding cassette (ABC) transporters. ABCA1 expression changes during trophoblast cell fusion, a process required to form the placental syncytium that enables maternal-fetal nutrient transfer. ABCA1 expression is dysregulated in preeclamptic placentas. But whether ABC transporters expression during trophoblast fusion is disrupted in preeclampsia remains unknown. We investigated if cholesterol and sterol ABC transporters are altered in term and preterm preeclampsia placentas and during human cytotrophoblast syncytialization. Human placental biopsies were collected from healthy term (≥37 weeks; n = 11) and term preeclamptic (≥36 6/7 weeks; n = 8) and pre-term preeclamptic (28-35 weeks; n = 8) pregnancies. Both, protein and mRNA expression for ABCA1, ABCG1, ABCG5, and ABCG8 were evaluated. Primary cytotrophoblasts isolated from a subset of placentas were induced to syncytialize for 96 h and ABCA1, ABCG1 and ABCG8 mRNA expression evaluated at 0 h and 96 h. Protein and gene expression of ABC transporters were not altered in preeclamptic placentas. In the healthy Term group, ABCA1 expression was similar before and after syncytialization. After 96 h of syncytialization, mRNA expression of ABCA1 and ABCG1 increased significantly, while ABCG8 decreased significantly in term-preeclampsia, but not pre-term preeclampsia. While placental expression of ABCA1 and ABCG1 remained unaltered in term preeclampsia, the disruption in their dynamic expression pattern during cytotrophoblast syncytialization suggests that cholesterol transport may contribute to the pathophysiologic role of the placenta in preeclampsia.

Gestational microbiome: metabolic perturbations and developmental programming.

A growing body of research suggests that alterations to the human microbiome are associated with disease states, including obesity and diabetes. During pregnancy, these disease states are associated with maternal microbial dysbiosis. This review discusses the current literature regarding the typical maternal and offspring microbiome as well as alterations to the microbiome in the context of obesity, type 2 diabetes mellitus, and gestational diabetes mellitus. Furthermore, this review outlines the proposed mechanisms linking associations between the maternal microbiome in the aforementioned disease states and offspring microbiome. Additionally, this review highlights associations between alterations in offspring microbiome and postnatal health outcomes.

Plasma Inorganic Pyrophosphate Deficiency Links Multiparity to Cardiovascular Disease Risk.

Epidemiological studies indicate that elevated alkaline phosphatase activity is associated with increased cardiovascular disease risk. Other epidemiological data demonstrate that mothers giving multiple childbirths (multipara) are also at increased risk of developing late-onset cardiovascular disease. We hypothesized that these two associations stem from a common cause, the insufficient plasma level of the ectopic mineralization inhibitor inorganic pyrophosphate, which is a substrate of alkaline phosphatase. As alkaline phosphatase activity is elevated in pregnancy, we hypothesized that pyrophosphate concentrations decrease gestationally, potentially leading to increased maternal vascular calcification and cardiovascular disease risk in multipara. We investigated plasma pyrophosphate kinetics pre- and postpartum in sheep and at term in humans and demonstrated its shortage in pregnancy, mirroring alkaline phosphatase activity. Next, we tested whether multiparity is associated with increased vascular calcification in pseudoxanthoma elasticum patients, characterized by low intrinsic plasma pyrophosphate levels. We demonstrated that these patients had increased vascular calcification when they give birth multiple times. We propose that transient shortages of pyrophosphate during repeated pregnancies might contribute to vascular calcification and multiparity-associated cardiovascular disease risk threatening hundreds of millions of healthy women worldwide. Future trials are needed to assess if gestational pyrophosphate supplementation might be a suitable prophylactic treatment to mitigate maternal cardiovascular disease risk in multiparous women.

Elimination of "kitome" and "splashome" contamination results in lack of detection of a unique placental microbiome.

BACKGROUND: A placental microbiome, which may be altered in gestational diabetes mellitus (GDM), has been described. However, publications raising doubts about the existence of a placental microbiome that is different than contaminants in DNA extraction kits and reagents ("kitomes") have emerged. The aims of this study were to confirm the existence of a placental microbiome distinct from contaminants and determine if it is altered in GDM mothers. RESULTS: We first enrolled normal weight, obese and GDM mothers (N = 17) at term elective cesarean section delivery in a pilot case control study. Bacterial DNA was extracted from placental parenchyma, maternal and cord blood, maternal vaginal-rectal swabs, and positive and negative controls with the standard Qiagen/MoBio Power Soil kit. Placentas had significantly higher copies of bacterial 16S rRNA genes than negative controls, but the placental microbiome was similar in all three groups and could not be distinguished from contaminants in blank controls. To determine the source and composition of the putative placental bacterial community identified in the pilot study, we expanded the study to 10 subjects per group (N = 30) and increased the number and variety of negative controls (N = 53). We modified our protocol to use an ultraclean DNA extraction kit (Qiagen QIAamp UCP with Pathogen Lysis Tube S), which reduced the "kitome" contamination, but we were still unable to distinguish a placental microbiome from contaminants in negative controls. We noted microbial DNA from the high biomass vaginal-rectal swabs and positive controls in placental and negative control samples and determined that this resulted from close proximity well-to-well cross contamination or "splashome". We eliminated this source of contamination by repeating the sequencing run with a minimum of four wells separating high biomass from low biomass samples. This reduced the reads of bacterial 16S rRNA genes in placental samples to insignificant numbers. CONCLUSIONS: We identified the problem of well-to-well contamination ("splashome") as an additional source of error in microbiome studies of low biomass samples and found a method of eliminating it. Once "kitome" and "splashome" contaminants were eliminated, we were unable to identify a unique placental microbiome.

Monitoring age-related susceptibility of young mice to oral Salmonella enterica serovar Typhimurium infection using an in vivo murine model.

Neonates and young children are acutely susceptible to infections by gastrointestinal bacterial pathogens, such as Salmonella enterica serovar Typhimurium (S. typhimurium). To reveal age-related differences in susceptibility to this pathogen, we used in vivo bioluminescence imaging (BLI) to monitor the progression of infection in neonatal (1-wk-old), suckling (2-wk-old), juvenile (4-wk-old), and adult (6-wk-old) BALB/c mice. Mice were orally infected with various doses of a bioluminescent-labeled wild-type or mutant S. typhimurium strain, and progression of infection was monitored by BLI for 2 wks. We found that neonatal and suckling mice were more susceptible to the wild-type strain at inoculum sizes 4 and 2 log(10)'s lower for neonatal and suckling mice, respectively, than those for adult mice. At the lower inocula, newborn mice showed disseminated systemic infection as indicated by the pattern of photon emission assessed by BLI, whereas no bioluminescent signals were detectable in adult mice. In addition, an orgA(-) mutant strain of S. typhimurium with reduced virulence in adult mice produced systemic infection in newborn, suckling, and juvenile mice. Furthermore, as low as 3 log(10) CFU could be detected by BLI in tissue. The present study demonstrates that susceptibility to S. typhimurium infection decreases with age. Also, we established that BLI can be used to monitor the progression of infection in mice. Thus, this model of age-related susceptibility to S. typhimurium using BLI can be used to advance our understanding of the mechanisms involved in newborn susceptibility to infection.

Salivary cortisol as indicators of pain in preterm infants: a pilot study.

Assessment and management of pain in preterm infants is critical and complicated. The addition of salivary cortisol measurement may improve the specificity of assessment and guide care to alleviate pain. The purpose of this study was fourfold: (a) assess the feasibility of a method of saliva collection in premature infants, (b) assess reliability of a method of measuring salivary cortisol in response to heelstick, (c) identify relationships between salivary cortisol and a measure of pain behavior (using CRIES) following heelstick, and (d) identify peak response times for elevations of salivary cortisol following heelstick in preterm infants. This was a prospective, descriptive pilot study. Serial saliva samples were collected from eight healthy infants 30 to 36 weeks' gestational age in a Newborn Intensive Care Unit. Cortisol levels were determined using enzyme-immune assay. Samples were collected without use of stimulants. Sample means supported peak and trough patterns previously described in the literature. Behavioral measures of pain did not correlate well with peak cortisol levels.