ex-vivo-placental-explant-flow-culture-mimicking-dynamic-conditions

<em>Ex Vivo</em> Placental Explant Flow Culture - Mimicking the Dynamic Conditions <em>In Utero</em>

The existing ex vivo placental explant culture models are primarily grounded in static culture systems using well plates. However, these models ...

Medical University of Graz

Progress in the understanding of the biology of perinatal tissues has contributed to the breakthrough revelation of the therapeutic effects of perinatal derivatives (PnD), namely birth-associated tissues, cells, and secreted factors. The significant knowledge acquired in the past two decades, along with the increasing interest in perinatal derivatives, fuels an urgent need for the precise identification of PnD and the establishment of updated consensus criteria policies for their characterization. The aim of this review is not to go into detail on preclinical or clinical trials, but rather we address specific issues that are relevant for the definition/characterization of perinatal cells, starting from an understanding of the development of the human placenta, its structure, and the different cell populations that can be isolated from the different perinatal tissues. We describe where the cells are located within the placenta and their cell morphology and phenotype. We also propose nomenclature for the cell populations and derivatives discussed herein. This review is a joint effort from the COST SPRINT Action (CA17116), which broadly aims at approaching consensus for different aspects of PnD research, such as providing inputs for future standards for the processing and  characterization and clinical application of PnD.

Perinatal Derivatives: Where Do We Stand? A Roadmap of the Human Placenta and Consensus for Tissue and Cell Nomenclature.

Preservation of ultrastructural features in biological samples for electron microscopy (EM) is a challenging task that is routinely accomplished through chemical fixation or high-pressure freezing coupled to automated freeze substitution (AFS) using specialized devices. However, samples from clinical (e.g. "biobanking" of bulk biopsies) and preclinical (e.g. whole mouse tissues) specimens are often not specifically prepared for ultrastructural analyses but simply immersed in liquid nitrogen before long-term cryo-storage. We demonstrate that ultrastructural features of such samples are insufficiently conserved using AFS and developed a simple, rapid, and effective method for thawing that does not require specific instrumentation. This procedure consists of dry ice-cooled pre-trimming of frozen tissue and aldehyde fixation for 3 h at 37 °C followed by standard embedding steps. Herein investigated tissues comprised human term placentae, clinical lung samples, as well as mouse tissues of different composition (brown adipose tissue, white adipose tissue, cardiac muscle, skeletal muscle, liver). For all these tissues, we compared electron micrographs prepared from cryo-stored material with our method to images derived from directly prepared fresh tissues with standard chemical fixation. Our protocol yielded highly conserved ultrastructural features and tissue-specific details, largely matching the quality of fresh tissue samples. Furthermore, morphometric analysis of lipid droplets and mitochondria in livers of fasted mice demonstrated that statistically valid quantifications can be derived from samples prepared with our method. Overall, we provide a simple and effective protocol for accurate ultrastructural and morphometric analyses of cryo-stored bulk tissue samples.

Simple method of thawing cryo-stored samples preserves ultrastructural features in electron microscopy.

In early human gestation, maternal arterial blood flow into the intervillous space of the developing placenta is obstructed by invaded trophoblasts, which form cellular plugs in uterine spiral arteries. These trophoblast plugs have recently been described to be loosely cohesive with clear capillary-sized channels into the intervillous space by 7 weeks of gestation. Here, we analysed localisation of maternal platelets at the maternal-foetal interface of human first trimester pregnancy, and tested the hypothesis whether HLA-G, which is primarily expressed by extravillous trophoblasts, affects aggregation and adhesion of isolated platelets. Immunohistochemistry of first trimester placental sections localised maternal platelets in vessel-like channels and adjacent intercellular gaps of extravillous trophoblasts in distal parts of columns. Furthermore, this localisation was confirmed by transmission electron microscopy. Neither co-incubation of HLA-G overexpressing JAR cells with isolated platelets, nor incubation with cell-derived soluble HLA-G or recombinant HLA-G affected platelet adhesion and aggregation. Our study suggests that maternal platelets flow through vessel-like channels of distal trophoblast columns and spread into adjacent lateral intercellular gaps, where platelet-derived factors could contribute to trophoblast differentiation into the invasive phenotype.

Maternal platelets pass interstices of trophoblast columns and are not activated by HLA-G in early human pregnancy.

During pregnancy, there is an intense crosstalk between mother and placenta. During the entire time of pregnancy, the maternal system deals with a huge amount of foreign (fetal) material released from the placenta, which can be referred to as placental exposome. Besides the release of hormones and growth factors, the placenta releases a variety of extracellular vesicles into maternal blood. These vesicles contain specific molecules including proteins, lipids, DNA as well as miRNA, all of which may have specific sites and modes of action on maternal cells. During normal pregnancy, the fine-tuning of factors and vesicles helps maintaining a viable and healthy pregnancy. However, in pregnancy pathologies such as preeclampsia, quantity and quality of the placenta-derived vesicles are altered leading to a deleterious effect on the maternal vascular system. This review focuses on the different types of placenta-derived extracellular vesicles in pregnancy with special emphasis on the interplay between these placental vesicles and the maternal system. Additionally, it displays new techniques and ideas for the analysis of the placental exposome with placental extracellular vesicles as a key aspect.

The endogenous exposome of the pregnant mother: Placental extracellular vesicles and their effect on the maternal system.

During pregnancy, freely floating placental villi are adapted to fluid shear stress due to placental perfusion with maternal plasma and blood. In vitro culture of placental villous explants is widely performed under static conditions, hoping the conditions may represent the in utero environment. However, static placental villous explant culture dramatically differs from the in vivo situation. Thus, we established a flow culture system for placental villous explants and compared commonly used static cultured tissue to flow cultured tissue using transmission and scanning electron microscopy, immunohistochemistry, and lactate dehydrogenase (LDH) and human chorionic gonadotropin (hCG) measurements. The data revealed a better structural and biochemical integrity of flow cultured tissue compared to static cultured tissue. Thus, this new flow system can be used to simulate the blood flow from the mother to the placenta and back in the most native-like in vitro system so far and thus can enable novel study designs.

Placental Villous Explant Culture 2.0: Flow Culture Allows Studies Closer to the In Vivo Situation.

Cancer cells voraciously consume nutrients to support their growth, exposing metabolic vulnerabilities that can be therapeutically exploited. Here, we show in hepatocellular carcinoma (HCC) cells, xenografts, and patient-derived organoids that fasting improves sorafenib efficacy and acts synergistically to sensitize sorafenib-resistant HCC. Mechanistically, sorafenib acts noncanonically as an inhibitor of mitochondrial respiration, causing resistant cells to depend on glycolysis for survival. Fasting, through reduction in glucose and impeded AKT/mTOR signaling, prevents this Warburg shift. Regulating glucose transporter and proapoptotic protein expression, p53 is necessary and sufficient for the sorafenib-sensitizing effect of fasting. p53 is also crucial for fasting-mediated improvement of sorafenib efficacy in an orthotopic HCC mouse model. Together, our data suggest fasting and sorafenib as rational combination therapy for HCC with intact p53 signaling. As HCC therapy is currently severely limited by resistance, these results should instigate clinical studies aimed at improving therapy response in advanced-stage HCC.

Fasting improves therapeutic response in hepatocellular carcinoma through p53-dependent metabolic synergism.

Signaling trough p53is a major cellular stress response mechanism and increases upon nutrient stresses such as starvation. Here, we show in a human hepatoma cell line that starvation leads to robust nuclear p53 stabilization. Using BioID, we determine the cytoplasmic p53 interaction network within the immediate-early starvation response and show that p53 is dissociated from several metabolic enzymes and the kinase PAK2 for which direct binding with the p53 DNA-binding domain was confirmed with NMR studies. Furthermore, proteomics after p53 immunoprecipitation (RIME) uncovered the nuclear interactome under prolonged starvation, where we confirmed the novel p53 interactors SORBS1 (insulin receptor signaling) and UGP2 (glycogen synthesis). Finally, transcriptomics after p53 re-expression revealed a distinct starvation-specific transcriptome response and suggested previously unknown nutrient-dependent p53 target genes. Together, our complementary approaches delineate several nodes of the p53 signaling cascade upon starvation, shedding new light on the mechanisms of p53 as nutrient stress sensor. Given the central role of p53 in cancer biology and the beneficial effects of fasting in cancer treatment, the identified interaction partners and networks could pinpoint novel pharmacologic targets to fine-tune p53 activity.

Complementary omics strategies to dissect p53 signaling networks under nutrient stress.

Human pregnancy depends on the proper development of the embryo prior to implantation and the implantation of the embryo into the uterine wall. During the pre-implantation phase, formation of the morula is followed by internalization of blastomeres that differentiate into the pluripotent inner cell mass lineage, while the cells on the surface undergo polarization and differentiate into the trophectoderm of the blastocyst. The trophectoderm mediates apposition and adhesion of the blastocyst to the uterine epithelium. These processes lead to a stable contact between embryonic and maternal tissues, resulting in the formation of a new organ, the placenta. During implantation, the trophectoderm cells start to differentiate and form the basis for multiple specialized trophoblast subpopulations, all of which fulfilling specific key functions in placentation. They either differentiate into polar cells serving typical epithelial functions, or into apolar invasive cells that adapt the uterine wall to progressing pregnancy. The composition of these trophoblast subpopulations is crucial for human placenta development and alterations are suggested to result in placenta-associated pregnancy pathologies. This review article focuses on what is known about very early processes in human reproduction and emphasizes on morphological and functional aspects of early trophoblast differentiation and subpopulations.

Article	Year
Perinatal Derivatives: Where Do We Stand? A Roadmap of the Human Placenta and Consensus for Tissue and Cell Nomenclature. Frontiers in bioengineering and biotechnology\| PubMed ID: 33392174	2020
Simple method of thawing cryo-stored samples preserves ultrastructural features in electron microscopy. Histochemistry and cell biology\| PubMed ID: 33404705	2021
Maternal platelets pass interstices of trophoblast columns and are not activated by HLA-G in early human pregnancy. Journal of reproductive immunology\| PubMed ID: 33530024	2021
The endogenous exposome of the pregnant mother: Placental extracellular vesicles and their effect on the maternal system. Molecular aspects of medicine\| PubMed ID: 33612320	2022
Placental Villous Explant Culture 2.0: Flow Culture Allows Studies Closer to the In Vivo Situation. International journal of molecular sciences\| PubMed ID: 34299084	2021
Fasting improves therapeutic response in hepatocellular carcinoma through p53-dependent metabolic synergism. Science advances\| PubMed ID: 35061544	2022
Complementary omics strategies to dissect p53 signaling networks under nutrient stress. Cellular and molecular life sciences : CMLS\| PubMed ID: 35635656	2022
Early human trophoblast development: from morphology to function. Cellular and molecular life sciences : CMLS\| PubMed ID: 35661923	2022

Nadja Kupper

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