The MET-FLAM Faculty

Christian WADSACK

Scientific Interests:

In our research, we investigate pregnancy associated factors, placental physiology, and placental function in healthy and compromised pregnancies. Therefore, we have established a panel of cell and tissue culture models and use functional assays, in combination with innovative molecular analyses to address our research questions.

Proposed Dissertation Topic:

Background: Pregnancy represents a unique challenge to the maternal-fetal immune interface. It requires a balance between immunosuppression, which is essential to maintain a semi-allogeneic fetus, and proinflammatory host defenses to protect the maternal-fetal interface from invading microorganisms. Exaggerated inflammatory responses to metabolic derangements in the mother may be critical for an adequate fetal development as the placenta transmits such adverse signals. Extracellular vesicles (EVs) are membrane-bound complexes released by cells and are potent signaling modulators of the immune system under both physiological and pathological conditions [1]. The placenta and the maternal immune system communicate via the release of soluble factors such as cytokines and through components carried by EVs [2]; such released EVs have immunomodulatory roles during pregnancy [3]. However, how the placenta communicates with the fetal circulation and whether EVs act as carriers is largely unknown. We have previously shown that maternal lipoproteins differ from fetal particles in their composition, function, and interaction with the placental barrier [4]. In this context, we suggest that placental EVs derived from endothelial cells (ECs) may play distinct roles in modulating fetal metabolism.

Hypothesis and objectives: Preeclampsia (PE), a severe and frequent disorder in pregnancy, is often accompanied by a reduced tolerance of the semi-allogenic fetus and may lead to unfavorable outcomes. We hypothesize that PE placenta-derived ECEVs cause an exacerbated inflammatory response in fetal immune cells compared to normal ECEVs, thereby inducing a myriad of cellular and metabolic alterations in the unborn. In particular, the student will examine (i) whether there are differences in the ECEVs protein / lipid / mRNA cargo between normal and PE-derived placentae; (ii) whether cord-blood-derived CD34⁺ immune cells interact differently with ECEVs, and (iii) whether ECEVs isolated from PE placentae might trigger an altered inflammatory response in fetal immune cells compared to those derived from normal placentae.

Methods and approaches: In the 1st year, the PhD candidate will be instructed in the isolation and characterization of primary human ECs from placental tissue using proteolytic enzymes and by immuno­fluorescence analyses, respectively. The student will be trained to isolate ECEVs from supernatants of in vitro cultured ECs and from samples after tissue perfusion. The student will also perform proteomic and lipid analyses to reveal compositional differences between ECEVs from gestational age matched healthy and PE placentae. Moreover, the PhD candidate will be trained in the isolation and phenotyping of different immune cell populations from cord blood by multi-color flow cytometry (2nd and 3rd year). In vitro cell culture experiments with different types of immune cells and ECEVs, using ELISAs, western blotting and live-cell imaging microscopy, will determine cell–EV interaction profiles. Finally, the student will be taught to perform placental perfusion experiments to study secreted immunomodulatory factors from whole tissue (3nd and 4th year).

Pitfalls and alternative approaches: The followings are the estimated risk of the proposed project and the contingency plans for them:
 

Risk description:	Factor:	Affected activity:	Proposed strategy:
Characterization of isolated ECEVs by FACS	High background, particle noise	Characterization and purity of ECEVs	Use ultra-filtration to reduce background Test antibodies, reduce flow Change approach: Nanoparticle tracking, immunoblotting
Labelling of ECEVs	Characteristics of used dye (e. g. BODIPY)	Uptake studies of labelled ECEVs on CD34+ cord-blood-derived cells	Use of amphiphilic dyes Test intercalation into membranes Test labelling and uptake with an immortalized cell line (e. g. Hep-2) Include appropriate controls e. g., supernatants of conjugated ECEVs, dye alone…
CD34+ cell uptake studies of ECEVs between normal and PE reveals no differentiation differences	Differentiation conditions, colony forming, inflammatory conditions in vitro	To investigate differentiation potential of CD34+ immune cells between normal and pathophysiological conditions	Test uptake mechanisms, inhibition of endocytosis Subcellular signalling: to test activation of lysosomes and early endosomes Mimic PE conditions by adding pro-inflammatory cocktails to the assay
Potential ethical, safety-related, or regulatory aspects of the submitted project	Access to human placental tissue samples and respective plasma samples	All experiments with primary placental cells and ECEVs isolation / source	Human placenta samples will be used for the project. These will be collected at the University hospital in Graz. This is covered by an ongoing vote (29-319 ex 16/17) by the ethics committee of the Medical University of Graz and allows for collection of normal and PE compromised placentae after birth including maternal and fetal plasma and subsequent use for research after obtaining written consent of the mother.
Gender relevant aspects	Our study specifically and exclusively focuses on (patho)physiological mechanisms during pregnancy. Hence, all human tissues are obtained from female donors.	There is an increasing body of evidence regarding sex-differences during fetal and placental development, especially regarding the handling of stressors in PE.	We will ensure to include considerations of sex-specific differences regarding fetal outcome and placental development in the planned experiments, where appropriate, and divide outcome measures by fetal sex, where relevant.

Involved Faculty members: Christian Wadsack (PI), Gunther Marsche and Eva Sturm (activation assay for neutrophils / monocytes, eosinophils and basophils), Ákos Heinemann (inflammatory lipid mediator profiling), Stefano Angiari (T-cell function), Julia Kargl (live EV imaging, immune-cell characterization).

Facilities: The Wadsack lab is an interdisciplinary team of two post docs, four PhD candidates, two study nurses and one technician. Our lab has access to maternal / fetal blood and placental samples including all clinical information of more than 3,500 normal and compromised deliveries. The wet lab is equipped with facilities for cell culture, immuno­fluorescence, western blot, real-time qPCR that enables isolating, characterizing and culturing all types of primary cells in the placenta. Furthermore, we have established the ex vivo dual placental perfusion model which allows us to perform maternal to fetal transfer studies and to isolate placental extracellular vesicles distinctly. The lab is equipped with ECIS system including a versatile pump system (Ibidi) to monitor barrier function of cells and for the stimulation of physiological systems with defined shear stress, mimicking blood vessels, endothelial cells under flow, rolling and adhesion assays perfusion of cells, spheroids and organoids for optimal nutrition. OMICS approaches are performed in a long-standing cooperation with core facilities of the Med Uni Graz and with Karl Kashofer, head of the Molecular Pathology lab at our University.

Preparatory Findings:

Figure 1. Characterization of sEVs by size and bioactive proteins.
(A, B, C) Size distribution of isolated fetoplacental sEVs from healthy (A, CTRL) and preeclamptic (B, PE) endothelial cells, determined by Nanoparticle Tracking Analysis-Nanosight. (D) sEVs are characterized by endosomal adapter proteins Syntenin and Alix and by the placental and endothelial markers CD31 and Siglec 6 respectively. Apolipoproteins (ApoB and ApoA1) served as negative control.

Figure 2. Glycan profiling of CTRL vs PE sEVs by LecChipTM and predicted cell-to-cell communication via sEVS.
 

1. Théry C, Ostrowski M, Segura E: Membrane Vesicles as Conveyors of Immune Responses. Nat Rev Immunol, 2009; 9(8):​581–589.
   
2. Tannetta D, Masliukaite I, Vatish M, Redman C, Sargent I: Update of Syncytiotrophoblast Derived Extracellular Vesicles in Normal Pregnancy and Preeclampsia. J Reprod Immunol, 2017; 119:​98–106.
   
3. Tong M, Abrahams VM, Chamley LW: Immunological Effects of Placental Extracellular Vesicles. Immunol Cell  Biol, 2018; 96(7):​714–722.
   
4. Stadler JT, Wadsack C, Marsche G: Current Knowledge on Particle Metabolism, Composition and Function in Health and Disease. Biomedicines, 2021; 9(4):349.