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Transcriptomic Profiling of JEG-3 cells using human leiomyoma derived matrix.

ABSTRACT: Oxygen tension varies during placental and fetal development. Although hypoxia drives early trophoblast invasion, low placental oxygen levels during pregnancy show association with pregnancy complications including fetal growth restriction and preeclampsia. JEG-3 cells are often used as a trophoblast model. We studied transcriptional changes of JEG-3 cells on a uterine leiomyoma derived matrix Myogel. This might be the closest condition to the real uterine environment that we can get for an in vitro model. We observed that culturing JEG-3 cells on the leiomyoma matrix leads to strong stimulation of ribosomal pathways, energy metabolism, and ATP production. Furthermore, Myogel improved JEG-3 cell adherence in comparison to tissue culture treated plastic. We also included PDMS microchip hypoxia creation, and observed changes in oxidative phosphorylation, oxygen related genes and several hypoxia genes. Our study highlights the effects of Myogel matrix on growing JEG-3 cells, especially on mitochondria, energy metabolism, and protein synthesis.

SUBMITTER: Barmaki S

PROVIDER: S-EPMC9934486 | biostudies-literature | 2022 Aug

REPOSITORIES: biostudies-literature

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Transcriptomic Profiling of JEG-3 cells using human leiomyoma derived matrix.

Barmaki Samineh S Al-Samadi Ahmed A Leskinen Katarzyna K Wahbi Wafa W Jokinen Ville V Vuoristo Sanna S Salo Tuula T Kere Juha J Wedenoja Satu S Saavalainen Päivi P

Biomaterials and biosystems 20220617

Oxygen tension varies during placental and fetal development. Although hypoxia drives early trophoblast invasion, low placental oxygen levels during pregnancy show association with pregnancy complications including fetal growth restriction and preeclampsia. JEG-3 cells are often used as a trophoblast model. We studied transcriptional changes of JEG-3 cells on a uterine leiomyoma derived matrix Myogel. This might be the closest condition to the real uterine environment that we can get for an in v ...[more]

PMID: 36824489

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Project description:BackgroundTendon healing is clinically challenging largely due to its inferior regenerative capacity. We have previously prepared a soluble, DNA-free, urea-extracted bovine tendon-derived extracellular matrix (tECM) that exhibits strong pro-tenogenic bioactivity on human adipose-derived stem cells (hASCs). In this study, we aimed to elucidate the mechanism of tECM bioactivity via characterization of tECM protein composition and comparison of transcriptomic profiles of hASC cultures treated with tECM versus collagen type I (Col1) as a control ECM component.MethodsThe protein composition of tECM was characterized by SDS-PAGE, hydroxyproline assay, and proteomics analysis. To investigate tECM pro-tenogenic bioactivity and mechanism of action, differentiation of tECM-treated hASC cultures was compared to serum control medium or Col1-treated groups, as assessed via immunofluorescence for tenogenic markers and RNA Sequencing (RNA-Seq).ResultsUrea-extracted tECM yielded consistent protein composition, including collagens (20% w/w) and at least 17 non-collagenous proteins (< 100 kDa) based on MS analysis. Compared to current literature, tECM included key tendon ECM components that are functionally involved in tendon regeneration, as well as those that are involved in similar principal Gene Ontology (GO) functions (ECM-receptor interaction and collagen formation) and signaling pathways (ECM-receptor interaction and focal adhesion). When used as a cell culture supplement, tECM enhanced hASC proliferation and tenogenic differentiation compared to the Col1 and FBS treatment groups based on immunostaining of tenogenesis-associated markers. Furthermore, RNA-Seq analysis revealed a total of 584 genes differentially expressed among the three culture groups. Specifically, Col1-treated hASCs predominantly exhibited expression of genes and pathways related to ECM-associated processes, while tECM-treated hASCs expressed a mixture of ECM- and cell activity-associated processes, which may explain in part the enhanced proliferation and tenogenic differentiation of tECM-treated hASCs.ConclusionsOur findings showed that urea-extracted tECM contained 20% w/w collagens and is significantly enriched with other non-collagenous tendon ECM components. Compared to Col1 treatment, tECM supplementation enhanced hASC proliferation and tenogenic differentiation as well as induced distinct gene expression profiles. These findings provide insights into the potential mechanism of the pro-tenogenic bioactivity of tECM and support the development of future tECM-based approaches for tendon repair.

Project description:The zebrafish inner ear organs and lateral line neuromasts are comprised of a variety of cell types, including mechanosensitive hair cells. Zebrafish hair cells are evolutionarily homologous to mammalian hair cells, and have been particularly useful for studying normal hair cell development and function. However, the relative scarcity of hair cells within these complex organs, as well as the difficulty of fine dissection at early developmental time points, makes hair cell-specific gene expression profiling technically challenging. Cell sorting methods, as well as single-cell RNA-Seq, have proved to be very informative in studying hair cell-specific gene expression. However, these methods require that tissues are dissociated, the processing for which can lead to changes in gene expression prior to RNA extraction. To bypass this problem, we have developed a transgenic zebrafish model to evaluate the translatome of the inner ear and lateral line hair cells in their native tissue environment; the Tg(myo6b:RiboTag) zebrafish. This model expresses both GFP and a hemagglutinin (HA) tagged rpl10a gene under control of the myo6b promoter (myo6b:GFP-2A-rpl10a-3xHA), resulting in HA-tagged ribosomes expressed specifically in hair cells. Consequently, intact zebrafish larvae can be used to enrich for actively translated hair cell mRNA via an immunoprecipitation protocol using an antibody for the HA-tag (similar to the RiboTag mice). We demonstrate that this model can be used to reliably enrich for actively translated zebrafish hair cell mRNA. Additionally, we perform a global hair cell translatome analysis using RNA-Seq and show enrichment of known hair cell expressed transcripts and depletion of non-hair cell expressed transcripts in the immunoprecipitated material compared with mRNA extracted from whole fish (input). Our results show that our model can identify novel hair cell expressed genes in intact zebrafish, without inducing changes to gene expression that result from tissue dissociation and delays during cell sorting. Overall, we believe that this model will be highly useful for studying changes in zebrafish hair cell-specific gene expression in response to developmental progression, mutations, as well as hair cell damage by noise or ototoxic drug exposure.

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Transcriptomic Profiling of JEG-3 cells using human leiomyoma derived matrix.

Publications

Transcriptomic Profiling of JEG-3 cells using human leiomyoma derived matrix.

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