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    • The expression of T BRY

    2018-11-02

    The expression of T (BRY), taken as a marker of precursors of all mesodermal tissues, was here induced through the activation of the canonical Wnt signalling which has been identified as an inducer for the primitive streak. A novel GSK3β inhibitor compound, active at a lower concentration than that reported for the commercially available analogue CHIR99021 (Lam et al., 2014; Mae et al., 2013; Takasato et al., 2014), was used in combination with BMP4 to induce robust generation of IM cells as soon as 2days after differentiation. Strong IM commitment was gained within 3days of differentiation, as proven by the co-expression of the specific IM markers OSR1, LHX1 and PAX2. Although expression of these markers is not unique to kidney development (Fujii et al., 1994; Puschel et al., 1992), their co-expression in the same cells identifies a very specific cell population in the developing kidney. Using a combination of RA, BMP7 and FGF9, we could direct the maturation of IM like cells into more mature cells expressing multiple markers of nephron progenitors of the cap mesenchyme (CM), particularly SIX2, PAX2 and WT1. During nephrogenesis, the CM cells, when stimulated, undergo epithelialisation to form podocytes. Studies on zebrafish pronephros formation, a model that mirrors mammalian kidney development, have shown that RA actively triggers WT1 expression (Wingert et al., 2007). RA, by binding to the retinoic tachykinin receptor response elements, modulates the transcription of numerous genes in stem cells, leading them to exit the self-renewing state and promoting their differentiation (Gudas, 2013). In the kidney, the retinoids act on renal progenitors by promoting their differentiation into mature podocytes. Indeed, RA is a stronger inducer of WT1 (Bollig et al., 2009), which is a transcription factor that can physically interact with different co-factors to modulate the transcriptional activity of essential podocyte genes like podocalyxin (O\'Brien et al., 2011) and nephrin (Guo et al., 2004). In our study, we induced podocyte differentiation by exposing renal progenitor cells to the VRAD medium, which contains RA. At day 13 of differentiation, cells showed high expression levels for genes like WT1, SYNPO, NPHS1, ACTN4, CD2AP, and VEGF-A that have been previously identified as essential for defining a cell as a podocyte (Shankland et al., 2007). The expression of the main podocyte markers not only reveals the cellular identity of the generated cells, but also demonstrates the differentiation efficiency of our protocol, with more than 90% of the cells co-expressing WT1 and α-Actinin-4 proteins, and the overall cells expressing synaptopodin and P-cadherin. Notably, the protocol was strongly reproducible, generating comparable results across three hiPSC lines generated with different reprogramming methods (integrative and non-integrative) and from distinct cell sources (dermal fibroblasts and PBMCs). Human podocytes are terminally differentiated cells with the typical feature of bi- and multinucleated cells, both in vitro and in vivo (Pavenstadt et al., 2003). Here, iPSC-derived podocytes share the same characteristics as primary podocytes, as shown by growth arrest and the presence of a fraction of bi- or multinucleated cells. They acquired the typical morphology of primary cells with an arborized appearance, the presence of processes extending from the cell body, and a large cytoplasmic to nuclear volume ratio (Shankland et al., 2007). Most importantly, iPSC-derived podocytes are functional. A recent study from our group demonstrated that Ang II plays an important role in perpetuating glomerular injury in experimental and human diabetic nephropathy by persistently activating Notch1 and Snail signalling in podocytes, eventually down-regulating nephrin expression, the integrity of which is crucial for the glomerular filtration barrier (Gagliardini et al., 2013). Ang II induces actin cytoskeleton rearrangement, which is instrumental to podocyte permselective dysfunction (Macconi et al., 2006). In our in vitro model, hiPSC-derived podocytes exposed to Ang II show a rearrangement of the cytoskeleton, which is also accompanied by a de-differentiation process of the podocytes detected by a marked and significant decrease in the expression of the specific podocyte markers. Notably, as previously demonstrated in conditionally immortalized podocytes (Eyre et al., 2007) and in podocytes generated in organoids from amniotic fluid stem cells (Xinaris et al., 2015), our cells behaved like functional mature podocytes, able to internalize albumin. Furthermore, iPSC-derived podocytes interacted efficiently with mouse renal progenitors to form fine-grained chimeras, and incorporated abundantly into WT1-positive induced metanephric mesenchyme and developing glomeruli, confirming their nephrogenic potential.