An investigation of the effects of retinoids and mesenchymal stem/stromal cell secretomes on embryonic stem cells

Abstract

Gastrulation is a landmark event in early embryonic development that marks the formation of the three germ layers. Spatiotemporal activation of retinoic acid receptors (RAR) α, β, and γ is crucial for proper embryonic development. In addition, mesenchymal stem/stromal cell (MSC) secretomes encompass trophic biological factors that provide global signalling pathways capable of regulating immature cell types via paracrine activities. The overall aim of this study was to explore (i) the influence of ATRA and highly selective agonists and antagonists for RARα and RARγ in the regulation of gastrulation and (ii) how the paracrine activity of MSCs regulates immature cell types during development. Transcriptome analysis conducted on a pre-existing mouse ES cell gastruloid dataset (Rossi et al., 2021) revealed the level of expression of RARα and RARγ in developing gastruloids identified heterogeneous cell types. UMAPs showed that mRNA RARα expression was ubiquitous. In contrast, RARγ mRNA expression was restricted to primitive cell types and strongly associated with the expression of stem cell markers, namely Pou5f1 (Oct4), Nanog, Sox2, Sox1, and Tbxt (Brachyury) at days 4, 5, and 6 of gastruloid development. Immunofluorescence (IF) revealed presence of RARα and RARγ in mouse ES cells, both primarily localised within nucleus, with some detected in the cytoplasm. Treatment of mouse ES cells with RARγ agonist significantly reduced ES cell proliferation in a concentration-dependent manner, whereas RARα agonism mirrors control effect. Additionally, WNT-induced gastruloid axial elongation was blocked by ATRA and RARγ agonist treatment. Conversely, 10 nM RARα agonist treatment slightly enhanced elongation, though not statistically significant. Surprisingly, 100 nM RARα agonist disrupted gastruloid elongation. Co-addition of RARγ antagonist to override the effect of ATRA blockage showed marginal effect. Serum-supplemented and serum-free (sf) MSC conditioned medium (CM) bound to the culture substratum had no significant impact on ES cell proliferation but was marginally better than control. 24 h serum or serum-free MSC CM had a comparable effect on ES cell proliferation to their respective control conditions, whereas late harvested MSC CM (48 h and 72 h) inhibited ES cell proliferation. Proteomic analysis of sfMSC CM identified 38 proteins consistently secreted across 48 h sfMSC CM triplicate samples. GO revealed enrichment in excreted factors and extracellular factors, while KEGG pathways identified 12 associated biological processes. STRING analysis revealed a complex, functionally interconnected protein-protein interaction (PPI), highlighting sfMSC secretome coordinated functional roles. Investigation of the paracrine activity of 48 h sfMSC secretomes on mouse ES gastruloids revealed induction of gastruloid elongation structure, albeit at ~58% frequency. Overall, the studies presented in this thesis have made use of a highly tractable gastruloid system to show that signals provided by RARα and RARγ, WNT/β-catenin, and extracellular signalling as provided by paracrine activities of MSC secretomes are integrated to mediate events during gastruloid development.