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Proteomics of a microphysiological model of human trophoblast invasion during early embryo implantation

ABSTRACT: We have designed an implantation-on-a-chip device to model the invasion of specialized fetal extravillous trophoblasts (EVTs) from an implanting embryo into the maternal decidua. We profiled the proteome of the external factors secreted in the model, containing different cell population compositions: 1) Extravillous trophoblast monoculture - EVTs monoculture, 2) Endothelial cells monoculture - ECs monoculture, 3) EVTs + ECs coculture, and 4) EVTs, ECs, and decidualized stromal cells (DSCs) - triculture. The proteins were extracted using the MPLEx extraction method (Nakayasu et. al. PMCID: PMC5069757), digested with 1:50 (trypsin:protein, w:w) for 3 hr at 37 C. Solid Phase Extraction (SPE) was performed on the samples using 1 mL/50 mg columns from Phenomenex as previously described (PMCID: PMC7192326). Peptidic concentration was assayed using a BCA assay and 5 ul at 0.1 ug/ul were injected on the LC-MS/MS system. Data was searched with MaxQuant.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Dan Dongeun Huh Geremy Clair

PROVIDER: MSV000086888 | MassIVE | Tue Feb 16 12:26:00 GMT 2021

REPOSITORIES: MassIVE

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A microphysiological model of human trophoblast invasion during implantation.

Park Ju Young JY Mani Sneha S Clair Geremy G Olson Heather M HM Paurus Vanessa L VL Ansong Charles K CK Blundell Cassidy C Young Rachel R Kanter Jessica J Gordon Scott S Yi Alex Y AY Mainigi Monica M Huh Dan Dongeun DD

Nature communications 20220315 1

Successful establishment of pregnancy requires adhesion of an embryo to the endometrium and subsequent invasion into the maternal tissue. Abnormalities in this critical process of implantation and placentation lead to many pregnancy complications. Here we present a microenigneered system to model a complex sequence of orchestrated multicellular events that plays an essential role in early pregnancy. Our implantation-on-a-chip is capable of reconstructing the three-dimensional structural organiza ...[more]

PMID: 35292627

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Project description:Background: Experimental studies have suggested that stem cells (SC) may exert their beneficial effects on the ischemic heart by paracrine activation of antiapoptotic pathways. In order to identify potential cardioprotective mediators, we performed a systematic analysis of the differential gene expression of lin-/c-kit+ SC after coculture with cardiomyocytes (CM). Methods: SC were separated from murine bone marrow and labeled with the green fluorescent CFDA. SC were then cocultivated with neonatal rat ventricular CM (NRVCM). After 48 h, we performed two cell sorting steps to generate a highly purified population of conditioned SC (>99%) and isolated the RNA. Next, we performed a genome-wide microarray analysis of cocultured vs. monocultured SC with Illumina Chips (Coculture n=3; Monoculture n=3). Our systematic analysis of differentially expressed genes focused on products that are secretable, membrane-bound and potentially involved in antiapoptotic signal transduction. Results: Our analysis resulted in 3 genes (out of more than 1000 differentially regulated genes) that met the criteria mentioned above and which could also be confirmed by RT-PCR. We found CCL-12 (12x, p<0.05), MIF (2x, p<0.05) and connexin 40 (4.5x; p<0.05) upregulated in our coculture model. An ELISA of cell culture supernatants was performed to proof secretability of candidate genes and showed that supernatants of coculture experiments have higher CCL-12 concentrations than monoculture experiments (20 pg/ml vs. 4 pg/ml; p<0.01). Next, we stimulated NRVCM with concentrated coculture supernatants which resulted in a significantly higher phosphorylation of AKT (p<0.01). Finally, NRVCM were labeled with Annexin-V and apoptosis of NRVCM in a co- and monoculture was measured by FACS. Thereby, we observed a reduction in apoptotic NRVCM in the coculture model (12% vs. 25%; p<0.05). Conclusion: Our results provide evidence that coculture of SC with NRVCM leads to an upregulation of antiapoptotic genes and a paracrine-mediated increase in the phosphorylation of AKT. This results in less apoptotis in cocultured NRVCM. We therefore conclude, that cell-cell interactions lead to a modified gene expression in SC that may in part explain the cardioprotective effects of stem cell therapy. We analysed six RNA samples extracted from three cocultures and three monocultures. Every sample represents an independant biological experiment, derived from individual preparations of cardiac myocytes and bone marrow stem cells.

Dataset Information

Proteomics of a microphysiological model of human trophoblast invasion during early embryo implantation

Publications

A microphysiological model of human trophoblast invasion during implantation.

Similar Datasets

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