Fetal Liver Research Articles

Proteomics reveals dynamic metabolic changes in human hematopoietic stem progenitor cells from fetal to adulthood

BackgroundHematopoietic stem progenitor cells (HSPCs) undergo phenotypical and functional changes during their emergence and development. Although the molecular programs governing the development of human hematopoietic stem cells (HSCs) have been investigated broadly, the relationships between dynamic metabolic alterations and their functions remain poorly characterized.MethodsIn this study, we comprehensively described the proteomics of HSPCs in the human fetal liver (FL), umbilical cord blood (UCB), and adult bone marrow (aBM). The metabolic state of human HSPCs was assessed via a Seahorse assay, RT‒PCR, and flow cytometry-based metabolic-related analysis. To investigate whether perturbing glutathione metabolism affects reactive oxygen species (ROS) production, the metabolic state, and the expansion of human HSPCs, HSPCs were treated with buthionine sulfoximine (BSO), an inhibitor of glutathione synthetase, and N-acetyl-L-cysteine (NAC).ResultsWe investigated the metabolomic landscape of human HSPCs from the fetal, perinatal, and adult developmental stages by in-depth quantitative proteomics and predicted a metabolic switch from the oxidative state to the glycolytic state during human HSPC development. Seahorse assays, mitochondrial activity, ROS level, glucose uptake, and protein synthesis rate analysis supported our findings. In addition, immune-related pathways and antigen presentation were upregulated in UCB or aBM HSPCs, indicating their functional maturation upon development. Glutathione-related metabolic perturbations resulted in distinct responses in human HSPCs and progenitors. Furthermore, the molecular and immunophenotypic differences between human HSPCs at different developmental stages were revealed at the protein level for the first time.ConclusionThe metabolic landscape of human HSPCs at three developmental stages (FL, UCB, and aBM), combined with proteomics and functional validations, substantially extends our understanding of HSC metabolic regulation. These findings provide valuable resources for understanding human HSC function and development during fetal and adult life.

Abstract A074: Integrated clinical and single-cell profiling analysis reveals cellular states associated with metastatic organotropism and survival in patients with pancreatic ductal adenocarcinoma

Abstract Some patients with localized pancreatic ductal adenocarcinoma (PDAC) undergo resection with curative intent, but most experience disease recurrence and succumb to their disease. Through examination of 743 PDAC patients in a single center we found that the site of first recurrence is a major predictor for survival. Patients with liver-metastatic recurrence (LIV-MR) had significantly shorter disease free and overall survival compared to those with recurrence to the lung (LUN-MR). We performed single-nucleus RNA-seq (snRNA-seq) and whole-genome sequencing (WGS) of primary PDAC with either LIV-MR or LUN-MR and found that malignant cells adopt transcriptional programs that mimicked that of their ultimate metastatic target organ. We validated this observation in independent data sets of human adult and fetal liver and lung tissues, and two additional patient cohorts who underwent molecular profiling of their actual PDAC liver and lung metastatic lesions. We reconstructed two different trajectories of malignant transformation in PDAC and found that the LIV-MR and LUN-MR are associated with these, suggesting that subsequent metastatic organotropism may be imprinted early in tumorigenesis. Furthermore, primary PDAC tumors recurring with LIV-MR compared to LUN-MR were depleted of infiltrating T cells and enriched with M2-like macrophages, highlighting potential contributions to subsequent outcomes. Taken together, this work provides evidence for pre-existing cellular adaptations that may determine metastatic organotropism in the context of cancer recurrence. These insights may be useful for prognostication and development of therapeutic strategies at the time of diagnosis of resectable PDAC. Citation Format: Morteza Chalabi Hajkarim, Michael May, Amit Dipak Amin, Jacob Jamison, Somnath Tagore, Lindsay Caprio, Zachary Walsh, Parin Shah, Sinan Abuzaid, Alissa Michel, Sun Dajiang, Winston Wong, Neha Shaikh, Priyanka Ramaradj, Basil Bakir, Michael D Kluger, John Chabot, Hanina Hibshoosh, Anil K Rustgi, Alexander Raufi, Gulam A Manji, Benjamin Izar. Integrated clinical and single-cell profiling analysis reveals cellular states associated with metastatic organotropism and survival in patients with pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A074.

PSVII-13 Influence of early maternal gestational body weight gain on liver gene expression across generations in offspring

Abstract Maternal nutrition in the periconceptional period influences gene regulation in the fetal liver across generations, affecting long-term metabolism. This study aimed to examine the effects of the rate of body weight (BW) gain of the dam (F0) during early gestation on the differential gene expression of the liver of first (F1) and second-generation (F2) offspring. Crossbred Angus heifers (F0) were assigned into two groups based on their targeted rate of BW gain during the first 84 d of gestation: low gain (LG; 0.28 kg/d; n = 8) or moderate gain (MG; 0.79 kg/d; n = 8). Following this period, all heifers (F0) were kept on a forage-based diet until the F1 offspring were weaned at 8 mo. The F1 heifers underwent estrus synchronization and were artificially inseminated at 15 mo of age. Liver samples were obtained from the F1 heifers at birth and later when harvested at 84 d of gestation, along with liver samples from their F2 fetuses, for a comprehensive multigenerational RNA-Seq analysis. Differentially expressed genes (DEGs) were identified using the DESeq2 R-package, focusing on significant genes determined by a P-value ≤ 0.05 and a log2 fold change |0.5|. Metabolic pathways and biological processes were analyzed using the WebGestalt tool to understand the implications of the observed gene expression changes. At birth, F1 heifers from MG dams exhibited 281 DEGs, comprising 152 downregulated and 129 upregulated genes compared with the offspring of LG dams. These genes were over-represented in lipid metabolism, cellular homeostasis, and signaling pathways, such as MAPK and chemokine signaling. At harvest, 159 DEGs were identified in F1 heifers, with 67 genes downregulated and 92 genes upregulated in the MG group. These genes were related to cellular morphogenesis and biogenesis processes, with a notable downregulation of metabolic pathways. For the F2 fetuses, 192 DEGs were identified, where 71 genes were downregulated, and 121 genes were upregulated. These genes were involved in biological functions such as peptide secretion to cell proliferation and signaling pathways, including PI3K-Akt and Hippo signaling. The analysis of DEGs shared between F1 heifers and F2 fetuses highlighted the influence of the moderate BW weight gain of the F0 dams. At birth and harvest, F1 heifers shared three genes (NAV2, FAM131C, SH3D21). F1 heifers at birth and F2 fetuses shared five genes (CD3E, H1-12, SLC7A11, PIP4P2, ENSBTAG00000051730). From the harvest at d 84 in F1 heifers and F2 fetuses, five genes were shared (DCDC2, RIN1, ENSBTAG00000008911, ENSBTAG00000040518, ENSBTAG00000048049). No DEGs were shared across all comparisons. These findings emphasize the multigenerational impact of early gestational BW gain of the dam on liver gene expression, influencing metabolic pathways and signaling across generations.

PSV-13 Betaine supplementation and nutrient restriction during gestation: Influences on growth in fetal sheep offspring

Abstract Restricted nutrition during gestation can reduce offspring productivity. Supplementation of betaine, a methyl donor, promotes fetal growth and liver function. We hypothesized that nutrient restriction during gestation impairs growth, including organ weights and muscle mass, while betaine supplementation would alleviate these negative effects. To determine if maternal betaine supplementation during gestation would improve growth of offspring of restricted-fed dams, multiparous Dorset ewes (n = 47) were estrus synchronized and bred to one of three rams. Ewes confirmed pregnant with twins (n = 32) and singletons (n = 15) were randomly assigned to one of four diets [100% NRC requirements (CON), 60% NRC (RES), CON+betaine (CON+BET; 2 g/d), and RES+BET] from d 30 to 130 of gestation. Among the twins, there were 27 males (CON-8, RES-5, CON+BET-5, and RES+BET-9) and 37 females (CON-8, RES-9, CON+BET-11, and RES+BET-9). Within singletons, there were 7 males (CON-2, RES-2, CON+BET-2, and RES+BET-1) and 8 females (CON-3, RES-1, CON+BET-1, and RES+BET-3). Dam body weight (BW) was measured weekly from d 30 to d 130 of gestation, and diet adjusted accordingly. Dams were euthanized at d 130 of gestation and fetal BW and tissue weights were collected. Data were analyzed using RStudio with P ≤ 0.05 considered significant and 0.05 < P ≤ 0.10 as tendency. At d 130, dams carrying twin fetuses in the CON group were 11.3% and 19.1% heavier than RES and RES+BET dams, respectively (P ≤ 0.05), and CON+BET dams were 18.4% and 10.5% heavier than RES and BET+RES dams, respectively (P ≤ 0.07). However, dams with singletons showed no significant difference in BW (P ≥ 0.37). Regarding fetal BW for singletons, RES fetuses (3.80 ± 0.28kg) were lighter than CON (4.60 ± 0.21kg), CON+BET (4.72 ± 0.27kg), and RES+BET (4.59 ± 0.24kg; P ≤ 0.03). Whereas in twins, RES+BET fetuses (3.19 ± 0.11kg) were lighter than CON (3.68 ± 0.12kg), RES (3.73 ± 0.14kg), and CON+BET (3.60 ± 0.13kg; P ≤ 0.02). In all fetuses, the livers of male RES (28.0 ± 1.49g) and male RES+BET (27.7 ± 1.44g) tended to be heavier than those of female RES+BET (24.5 ± 1.19g; P ≤ 0.07). In terms of muscle mass, female CON+BET (9.33 ± 0.43g) fetuses tended to have greater longissimus muscle (LM) mass compared with female CON (8.30 ± 0.35; P = 0.06), whereas male RES (9.06 ± 0.44g) tended to have heavier LM than male CON+BET (8.02 ± 0.43g; P = 0.09). There were no observed effects of diet, fetal sex, or fetal number on the weights of heart, kidney, perirenal fat, pancreas, semitendinosus muscle, and triceps brachii muscle (P ≥ 0.11). These findings suggest that betaine supplementation may have a significant role in improving fetal BW in singleton pregnancies, with variable effects in twins. Additionally, betaine supplementation may have sex-specific effects on muscle development.

169 Vitamin and mineral supplementation to gestating beef heifers: Fetal physiology and metabolic programming

Abstract Vitamins and minerals have key roles in hormone production and action, enzyme activity, tissue synthesis, oxygen transport, and energy production. These micronutrients are efficiently transferred from the dam to the fetus during gestation to be partitioned for metabolic use and stored as postnatal mineral reserves. Maternal nutrient intake is one of the main factors that influence the availability of vitamins and minerals to the fetus. Thus, an inadequate supply of these critical nutrients can have a long-lasting impact on offspring growth and health. Evidence suggests that biological processes regulating normal growth, development, and nutrient utilization are programmed in utero, even during the earliest developmental stages. Therefore, there is a critical need to evaluate the effects of micronutrient supplementation during gestation on fetal physiology and metabolic programming. Our research group has developed research models examining the impact of maternal vitamin and mineral supplementation (VTM, supplemented vs. NoVTM, not supplemented) during the periconceptual period or throughout gestation in beef heifers. A metabolomic analysis revealed that metabolites in the oxidative phosphorylation pathway were more abundant in the liver of fetuses from VTM than NoVTM dams, suggesting that a greater supply of micronutrients during the periconceptual period and first trimester of pregnancy may positively modulate mitochondrial energy metabolism in offspring. These changes in the abundance of metabolites suggested physiological adaptations to meet fetal metabolic needs. Further, high-resolution respirometry analysis revealed greater efficiency of energy utilization in small intestinal samples of neonatal calves from VTM dams. In fact, VTM offspring were heavier from weaning to breeding phase than NoVTM offspring, suggesting that maternal nutrition affects physiological mechanisms in utero that modulate offspring energetics and efficiency of nutrient utilization in the postnatal period. Furthermore, we reported increased concentrations of histidine, aspartate, and 12 out of 14 neutral amino acids in the allantoic fluid of VTM-supplemented dams. Finally, genes involved with amino acid transport in fetal liver were upregulated in response to VTM supplementation, highlighting the intricate relationship between maternal and fetal nutrition. Vitamin and mineral supplementation were also associated with changes in gene expression, biological processes and pathways in placental tissue at d 83 of gestation. We observed an upregulation of genes in the calcium signaling and cyclic guanosine monophosphate (CGMP)-PKG signaling pathways in response to micronutrient supplementation. Calcium-mediated systems may modulate cell proliferation and steroidogenic activity in bovine placentomes, while CGMP-PKG plays a key role in vascular homeostasis. Placental blood vessel vascularity at term was indeed increased in heifers supplemented throughout gestation, emphasizing the importance of micronutrient supplementation beyond early pregnancy. Altogether, our findings suggest that vitamin and mineral supplementation in the periconceptual period and during gestation play a pivotal role in fetal metabolic programming with consequences extending to the postnatal period.

The role of the haematopoietic stem cell niche in development and ageing.

Blood production depends on rare haematopoietic stem cells (HSCs) and haematopoietic stem and progenitor cells (HSPCs) that ultimately take up residence in the bone marrow during development. HSPCs and HSCs are subject to extrinsic regulation by the bone marrow microenvironment, or niche. Studying the interactions between HSCs and their niche is critical for improving ex vivo culturing conditions and genetic manipulation of HSCs, which is pivotal for improving autologous HSC therapies and transplantations. Additionally, understanding how the complex molecular network in the bone marrow is altered during ageing is paramount for developing novel therapeutics for ageing-related haematopoietic disorders. HSCs are unique amongst stem and progenitor cell pools in that they engage with multiple physically distinct niches during their ontogeny. HSCs are specified from haemogenic endothelium in the aorta, migrate to the fetal liver and, ultimately, colonize their final niche in the bone marrow. Recent studies employing single-cell transcriptomics and microscopy have identified novel cellular interactions thatgovern HSC specification and engagement with their niches throughout ontogeny. New lineage-tracing models and microscopy tools have raised questions about the numbers of HSCs specified, as well as the functional consequences of HSCs interacting with each developmental niche. Advances have also been made in understanding how these niches are modified and perturbed during ageing, and the role of these altered interactions in haematopoietic diseases. In this Review, we discuss these new findings and highlight the questions that remain to be explored.

Correlation of Placental and Fetal Liver Morphometry in 2nd and 3rd Trimester Ultrasound Findings

Correlation of Placental and Fetal Liver Morphometry in 2nd and 3rd Trimester Ultrasound Findings

PSVI-2 Multigenerational effects of maternal rate of body weight gain on fetal liver miRNA expression

PSVI-2 Multigenerational effects of maternal rate of body weight gain on fetal liver miRNA expression

Evaluating the impact of Tartrazine on female Wistar rats and their fetuses during gestation

ABSTRACT Tartrazine (TAZ), a synthetic lemon-yellow azo dye, finds uses in food ingredients, pharmaceuticals, and cosmetics. Treatment with TAZ during pregnancy in Wistar rats will be investigated to assess its impact on embryonic development. Eighteen pregnant rats were divided into three groups: the control group and two groups administered TAZ via gavage at doses of 7.5 mg/kg and 15 mg/kg from day 5 to 18. At 19 days of gestation, female rats were euthanised and their foetuses were extracted for assessment. Analysis was conducted to evaluate mortality, growth, morphological, and skeletal abnormalities. Furthermore, the maternal and fetal tissues were subjected to histological analysis and the oxidative state was evaluated. No impact was observed on the corrected body weight of pregnant rats by TAZ. However, it resulted in inhibited fetal development, morphological defects, resorption, and delayed skeletal ossification. Daily administration of TAZ altered the histology of the placenta, maternal and fetal liver, and kidney. Malonaldehyde increased and antioxidant markers such as glutathione, catalase and superoxide dismutase decreased, causing hepatic oxidative stress. Our study indicates that TAZ is embryotoxic in pregnant Wistar rats. Therefore, it should be avoided during the process of organogenesis and the crucial gestational time.

Maternal obesogenic diet during pregnancy and its impact on fetal hepatic function in baboons.

Maternal obesity (MO) increases the risk of later-life liver disease in offspring, especially in males. This may be due to impaired cytochrome P450 (CYP) enzyme activity driven by an altered maternal-fetal hormonal milieu. MO increases fetal cortisol concentrations that may increase CYP activity; however, glucocorticoid receptor (GR)-mediated signaling can be modulated by alternative GR isoform expression. We hypothesized that MO induces sex-specific changes in GR isoform expression and localization that contribute to reduced hepatic CYP activity. Nonpregnant, nulliparous female baboons were assigned to either an ad libitum control diet or a high-fat, high-energy diet (HF-HED) at 9 months pre pregnancy. At 165 days' gestation (term = 180 days), fetal liver samples were collected (n = 6/sex/group). CYP activity was quantified using functional assays, and GR was measured using quantitative RT-PCR and Western blot. CYP3A activity was reduced in the HF-HED group, whereas CYP2B6 activity was reduced in HF-HED males only. Total GR expression was increased in the HF-HED group. Relative nuclear expression of the antagonistic GR isoform GRβ was increased in HF-HED males only. Reduced CYP activity in HF-HED males may be driven in part by dampened hepatic-specific glucocorticoid signaling via altered GR isoform expression. These findings highlight targetable mechanisms that may reduce later-life sex-specific disease risk.

The Effects of Maternal Nutrient Restriction during Mid to Late Gestation with Realimentation on Fetal Metabolic Profiles in the Liver, Skeletal Muscle, and Blood in Sheep.

Poor maternal nutrition during gestation negatively affects offspring growth and metabolism. To evaluate the impact of maternal nutrient restriction and realimentation on metabolism in the fetal liver, skeletal muscle, and circulation, on day 50 of gestation, ewes (n = 48) pregnant with singletons were fed 100% (CON) or 60% (RES) of requirements until day 90 of gestation, when a subset of ewes (n = 7/treatment) were euthanized, and fetal samples were collected. The remaining ewes were maintained on a current diet (CON-CON, n = 6; RES-RES, n = 7) or switched to an alternative diet (CON-RES, RES-CON; n = 7/treatment). On day 130 of gestation, the remaining ewes were euthanized, and fetal samples were collected. Fetal liver, longissimus dorsi (LD), and blood metabolites were analyzed using LC-MS/MS, and pathway enrichment analysis was conducted using MetaboAnalyst. Then, 600, 518, and 524 metabolites were identified in the liver, LD, and blood, respectively, including 345 metabolites that were present in all three. Nutrient restriction was associated with changes in amino acid, carbohydrate, lipid, and transulfuration/methionine metabolic pathways, some of which were alleviated by realimentation. Fetal age also affected metabolite abundance. The differential abundance of metabolites involved in amino acid, methionine, betaine, and bile acid metabolism could impact fetal epigenetic regulation, protein synthesis, lipid metabolism, and signaling associated with glucose and lipid metabolism.

Alterations in one‑carbon metabolism and protein synthesis signals due to methionine supplementation and lipopolysaccharide challenge in Holstein fetal liver explants

One‑carbon metabolism (OCM) fueled by methionine (Met), choline, and folic acid is key for embryo development and fetal growth. We investigated effects of lipopolysaccharide (LPS) to induce inflammation in fetal liver tissue with or without Met on components of OCM and protein synthesis activity. Fetal liver harvested at slaughter from six multiparous pregnant Holstein dairy cows (37 ± 6 kg milk/d, 100 ± 3 d gestation) were incubated (0.2 ± 0.02 g) for 4 h at 37 °C with each of the following: ideal profile of amino acids (control; Lysine:Met 2.9:1), control plus LPS (1 μg/mL), increased Met supply (Met, Lys:Met 2.5:1), and Met+LPS. Data were analyzed as a 2 × 2 factorial (PROC MIXED, SAS 9.4). Ratios of mechanistic target of rapamycin (p-mTOR:mTOR) and eukaryotic elongation factor 2 (p-eEF2:eEF2) protein were lowest (P < 0.0 5) with LPS and highest with Met. Tissue amino acid concentrations were lowest (P < 0.0 5) with Met regardless of LPS suggesting enhanced use via mTOR. The marked increase (P = 0.02) in phosphorylation of S6 ribosomal protein (p-RPS6) with LPS suggested a pro-inflammatory response that was partly alleviated with Met+LPS. No effect (P = 0.4 5) on methionine adenosyl transferase 1 A (MAT1A) protein abundance was detected. Activity of betaine-homocysteine S-methyltransferase (BHMT) was greatest with Met, but Met+LPS dampened this effect (P = 0.0 5). Overall, fetal liver responds to inflammatory challenges and Met supply. The latter can stimulate protein synthesis via mTOR and alter some OCM reactions while having a modest anti-inflammatory effect.

Gestational Exposure to Endocrine Disrupting Chemicals and Fetal Liver Development: Findings from the HPP-3D Study

Gestational Exposure to Endocrine Disrupting Chemicals and Fetal Liver Development: Findings from the HPP-3D Study

Transcriptomic Signatures of the Foetal Liver and Late Prenatal Development in Vitrified Rabbit Embryos.

Assisted reproduction technologies (ARTs) are generally considered safe; however, emerging evidence highlights the need to evaluate potential risks in adulthood to improve safety further. ART procedures like rederivation of embryos by vitrification differ from natural conditions, causing significant disparities between in vitro and in vivo embryos, affecting foetal physiology and postnatal life. This study aims to investigate whether hepatic transcriptome and metabolome changes observed postnatally are already present in foetal livers at the end of gestation. This study compared fresh and vitrified rabbit embryos, finding differences between foetuses obtained by the transfer of fresh and vitrified embryos at 24 days of gestation. Rederived embryos had reduced foetal and liver weights and crown-rump length. However, the offspring of vitrified embryos tended to be born with higher weight, showing compensatory growth in the final week of gestation (59.2 vs. 49.8 g). RNA-Seq analysis revealed 43 differentially expressed genes (DEGs) in the foetal liver of vitrified embryos compared to the fresh group. Notably, downregulated genes included BRAT1, CYP4A7, CYP2B4, RPL23, RPL22L1, PPILAL1, A1BG, IFGGC1, LRRC57, DIPP2, UGT2B14, IRGM1, NUTF2, MPST, and PPP1R1B, while upregulated genes included ACOT8, ERICH3, UBXN2A, METTL9, ALDH3A2, DERPC-like, NR5A2-like, AP-1, COG8, INHBE, and PLA2G4C. Overall, a functional annotation of these DEGs indicated an involvement in lipid metabolism and the stress and inflammatory process or immune response. Thus, our results suggest that vitrification and embryo transfer manipulation induce an adaptive response that can be observed in the liver during the last week of gestation.

Evaluating fetal lung development at various gestational weeks using two-dimensional shear wave elastography.

Noninvasive evaluation of fetal lung development is a critical area of study. Two-dimensional shear-wave elastography (2D-SWE) provides valuable insights into tissue stiffness, potentially correlating with different stages of lung development. This study aims to explore the potential of the 2D-SWE technique for assessing the maturity of fetal lung development. This prospective cohort study included pregnant women undergoing routine antenatal ultrasound examinations at the Second Affiliated Hospital of Fujian Medical University and Quanzhou Women's and Children's Hospital from September 2022 to September 2023. The study consecutively recruited 300 pregnant women with normal pregnancies and 15 who opted for induced labor. Among those with normal pregnancies, the study assessed the differences in fetal pulmonary and hepatic elasticity measurements across different gestational weeks (GW) using one-way analysis of variance (ANOVA). Furthermore, regression analyses using linear, quadratic, and cubic equations were conducted to investigate the relationship between fetal parameters and GW. For those who opted for induced labor, elasticity measurements were taken before induction, and fetal lung tissue specimens were collected for post-induction observation. Fetal lung and liver elasticity values, along with the lung-to-liver elasticity ratio (LLE ratio), showed significant variations across different GW (P<0.05). Specifically, fetal lung elasticity values initially increased and then decreased as GW advanced (R2=0.41). Liver elasticity values continuously increased throughout GW, though the rate of increase diminished during the prenatal period (R2=0.37). The LLE ratio values increased and then decreased over GW, fluctuating overall between 0.8 and 0.9 (R2=0.14). A 71.4% concordance was observed between the predicted stage of lung development, based on lung elasticity values, and the histological stage of lung development in the induced fetuses. 2D-SWE can depict the maturation of fetal lung development at various stages.

Comparative dissection of transcriptional landscapes of human iPSC-NK differentiation and NK cell development

Abstract Clinical and preclinical research has demonstrated that iPSC-derived NK (iNK) cells have a high therapeutic potential, yet poor understanding of the detailed process of their differentiation in vitro and their counterpart cell development in vivo has hindered therapeutic iNK cell production and engineering. Here we dissect the crucial differentiation of both fetal liver NK cells and iNK cells to enable the rational design of advanced iNK production protocols. We use a comparative analysis of single-cell RNA-seq (scRNA-seq) to pinpoint key factors lacking in the induced setting which we hypothesized would hinder iNK differentiation and/ or functionality. By analyzing key transcription factor regulatory networks, we discovered the importance of TBX21, EOMES, and STAT5A in the differentiation timeline. This analysis provides a blueprint for further engineering new iPSC lines to obtain iNK cells with enhanced functions. We validated this approach by creating a new line of STAT5A-iPSCs which can be differentiated to STAT5A-expressing macrophages with both NK cell and macrophage features such as perforin production, phagocytosis, and anti-tumor functions.

Circular RNAs mediate the regulation of maternal placental nutrient transport and fetal development by sugar-sweetened beverages

Epidemiological and experimental studies have demonstrated a strong association between maternal diet and fetal birth weight, obesity, and metabolic syndrome. We investigated the pathways and modes of action of circular RNAs (circRNAs) that mediate the regulation of maternal reproductive performance and fetal development by sugar-sweetened beverages (20 % sucrose water, SSBs) using C57BL/6J mice as a model. Results showed that SSBs significantly increased the reproductive performance (P<0.05), body weight (P<0.01), fetal birth weight (P<0.05), placental weight (P<0.01), and the expression of nutrient transporter genes in the placenta and fetal liver (P<0.05), mainly by accelerating the maternal energy metabolism during pregnancy. However, maternal serum biochemical indices, antioxidant indices, and pathological damage to the liver and placenta predicted that the mother would be at greater health risks during this period. Moreover, transcriptomics results indicated that the differentially expressed (DE) circRNAs in the placenta regulate the maternal multiple metabolic pathways and the placental nutrient transport efficiency by sponging miRNAs and forming growth factors and proteins, ultimately improving the maternal reproductive performance. In addition, we verified the reliability of the sequencing results using reverse transcription polymerase chain reaction and identified the possibility of DE circRNAs binding to nutrient transporter genes using targeting relationship prediction. Finally, we constructed a correlation network that regulates maternal placental nutrient transport based on DE circRNAs, targeted miRNAs and nutrient transport-related genes. This study will provide scientific dietary guidance for pregnant women and new research ideas for preventing and treating pregnancy complications.

HES1 is required for mouse fetal hematopoiesis

BackgroundHematopoiesis in mammal is a complex and highly regulated process in which hematopoietic stem cells (HSCs) give rise to all types of differentiated blood cells. Previous studies have shown that hairy and enhancer of split (HES) repressors are essential regulators of adult HSC development downstream of Notch signaling.MethodsIn this study, we investigated the role of HES1, a member of HES family, in fetal hematopoiesis using an embryonic hematopoietic specific Hes1 conditional knockout mouse model by using phenotypic flow cytometry, histopathology analysis, and functional in vitro colony forming unit (CFU) assay and in vivo bone marrow transplant (BMT) assay.ResultsWe found that loss of Hes1 in early embryonic stage leads to smaller embryos and fetal livers, decreases hematopoietic stem progenitor cell (HSPC) pool, results in defective multi-lineage differentiation. Functionally, fetal hematopoietic cells deficient for Hes1 exhibit reduced in vitro progenitor activity and compromised in vivo repopulation capacity in the transplanted recipients. Further analysis shows that fetal hematopoiesis defects in Hes1fl/flFlt3Cre embryos are resulted from decreased proliferation and elevated apoptosis, associated with de-repressed HES1 targets, p27 and PTEN in Hes1-KO fetal HSPCs. Finally, pharmacological inhibition of p27 or PTEN improves fetal HSPCs function both in vitro and in vivo.ConclusionTogether, our findings reveal a previously unappreciated role for HES1 in regulating fetal hematopoiesis, and provide new insight into the differences between fetal and adult HSC maintenance.

Human iPSC-liver organoid transplantation reduces fibrosis through immunomodulation.

Donor organ shortages for transplantation remain a serious global concern, and alternative treatment is in high demand. Fetal cells and tissues have considerable therapeutic potential as, for example, organoid technology that uses human induced pluripotent stem cells (hiPSCs) to generate unlimited human fetal-like cells and tissues. We previously reported the in vivo vascularization of early fetal liver-like hiPSC-derived liver buds (LBs) and subsquent improved survival of recipient mice with subacute liver failure. Here, we show hiPSC-liver organoids (LOs) that recapitulate midgestational fetal liver promote de novo liver generation when grafted onto the surface of host livers in chemical fibrosis models, thereby recovering liver function. We found that fetal liver, a hematopoietic tissue, highly expressed macrophage-recruiting factors and antifibrotic M2 macrophage polarization factors compared with the adult liver, resulting in fibrosis reduction because of CD163+ M2-macrophage polarization. Next, we created midgestational fetal liver-like hiPSC-LOs by fusion of hiPSC-LBs to induce static cell-cell interactions and found that these contained complex structures such as hepatocytes, vasculature, and bile ducts after transplantation. This fusion allowed the generation of a large human tissue suitable for transplantation into immunodeficient rodent models of liver fibrosis. hiPSC-LOs showed superior liver function compared with hiPSC-LBs and improved survival and liver function upon transplantation. In addition, hiPSC-LO transplantation ameliorated chemically induced liver fibrosis, a symptom of liver cirrhosis that leads to organ dysfunction, through immunomodulatory effects, particularly on CD163+ phagocytic M2-macrophage polarization. Together, our results suggest hiPSC-LO transplantation as a promising therapeutic option for liver fibrosis.

Maternal Vitamin D3 Supplementation in an Oxidized-Oil Diet Protects Fetus from Developmental Impairment and Ameliorates Oxidative Stress in Mouse Placenta and Fetus

BackgroundFried food has increased in popularity worldwide. However, deep frying can increase the production of peroxidative toxins in food, which might be harmful to fetal development. The antioxidative effect of vitamin D3 (VD3) has been reported previously. ObjectivesThis study aimed to explore how maternal VD3 supplementation in an oxidized-oil diet during gestation affects fetal antioxidative ability and development. MethodsPregnant mice were randomly assigned into 3 groups: Control group (diet with fresh soybean oil), OSO group [diet with oxidized soybean oil (OSO)], and OSOV group (diet with OSO and 10,000 IU/Kg VD3). Mice were fed with the corresponding diet during gestation. On day 16.5 of gestation, the placenta and fetus were harvested to analyze antioxidative status. ResultsMaternal oxidized-oil diet during gestation significantly reduced placental vessel abundance, labyrinth zone area, and fetal body weight. However, dietary VD3 supplementation prevented these negative effects of oxidized-oil diet. Maternal intake of oxidized-oil diet increased serum concentrations of malondialdehyde, total-nitric oxide synthase, and inducible nitric oxide synthase, whereas VD3 supplementation showed a protection effect on it. Additionally, maternal VD3 supplementation increased the levels of antioxidative enzymes and the nuclear translocation of nuclear factor erythroid 2-related factor 2 (NRF2), thereby protecting placenta and fetus from apoptosis and oxidative stress caused by an oxidized-oil diet. The gene expression and protein levels of a fatty acid transporter solute carrier family 27 member 1 in the fetal liver were increased by maternal VD3 supplementation under oxidized-oil diet. Notably, NRF2 could be co-immunoprecipitated with the VD receptor in the placenta. ConclusionsMaternal VD3 supplementation could protect fetus from oxidized-oil diet induced developmental impairment by alleviating oxidative stress in the placenta and fetus through the VD receptor/NRF2 pathway, at least partially. Thus, ensuring adequate levels of VD3 through supplementation is often critical during pregnancy.