FZD4 Marks Lateral Plate Mesoderm and Signals with NORRIN to Increase Cardiomyocyte Induction from Pluripotent Stem Cell-Derived Cardiac Progenitors

Abstract

SummaryThe identification of cell surface proteins on stem cells or stem cell derivatives is a key strategy for the functional characterization, isolation, and understanding of stem cell population dynamics. Here, using an integrated mass spectrometry- and microarray-based approach, we analyzed the surface proteome and transcriptome of cardiac progenitor cells (CPCs) generated from the stage-specific differentiation of mouse and human pluripotent stem cells. Through bioinformatics analysis, we have identified and characterized FZD4 as a marker for lateral plate mesoderm. Additionally, we utilized FZD4, in conjunction with FLK1 and PDGFRA, to further purify CPCs and increase cardiomyocyte (CM) enrichment in both mouse and human systems. Moreover, we have shown that NORRIN presented to FZD4 further increases CM output via proliferation through the canonical WNT pathway. Taken together, these findings demonstrate a role for FZD4 in mammalian cardiac development.

Continuous WNT Control Enables Advanced hPSC Cardiac Processing and Prognostic Surface Marker Identification in Chemically Defined Suspension Culture

“…Patterning into cardiac versus presumptive presomitic mesoderm was confirmed by targeted heatmap analysis for associated GO terms, including osteoblast, skeletal muscle, mesenchyme, somite, and limb development. Remarkably, day-3 analysis of IWP-treated cells showed robust upregulation of key cardiac progenitor markers including ISL1 , GATA4 , MEF2C , NXK2-6 (Biben et al., 1998), FZD4 (Yoon et al., 2018), and FOXC1/2 , as well as TTN and TNNI1 (Figure 1E). Conversely, markers described to inhibit cardiac mesoderm but to promote presomitic specification including CDX1/2 and MSX1/2 (Greber et al., 2010, Mendjan et al., 2014) were significantly upregulated in the absence of early IWP2 treatment (confirmed for CDX2 by FC; Figure S1E).…”

“…Opposing the gene expression patterns specific to cardiac mesoderm (resulting from immediate IWP addition on day 1) versus presomitic mesoderm (achieved by the lack of IWP before day 3) allowed us to evaluate several known cardiac/mesodermal progenitor markers including KDR, PDGFRα, SSEA1, NCAM, ROR2, and CD13 (Ardehali et al., 2013, Evseenko et al., 2010, Kattman et al., 2011, Lee et al., 2017, Nelson et al., 2008, Yang et al., 2008, Yoon et al., 2018) regarding their suitability for early process monitoring and eventually predicting functional CM formation at later stages. Interestingly, none of the established markers was well suited to discriminate early cardiac from presomitic mesoderm.…”

“…However, in line with our data, distinct ROR1 expression was reported in the presumptive cardiac PS region in birds and mammals (Alev et al., 2010, Kinder et al., 1999, Peng et al., 2016) and in embryonic heart tissue as well (Kim et al., 2014). This distinct pattern may hint toward a broader functional role of ROR1 during early heart development, potentially involving non-canonical WNT signaling via FZD4 during lateral plate mesoderm formation (Grumolato et al., 2010, Yoon et al., 2018). In summary, we show that ROR1 may be exploited to identify primed hPSC states and predict lineage commitment, complementing recent findings on cKIT, FZD4, and GARP (Bargaje et al., 2017, Loh et al., 2016, Yoon et al., 2018).…”

Cardiopharyngeal Progenitor Specification: Multiple Roads to the Heart and Head Muscles

“…Expression of Flk1 and Pdgfra together is generally predictive of CM potential in hPSCs (Kattman et al 2011). Several other markers have recently been proposed for the isolation of hPSC-derived cardiac progenitors, such as Ror2 and Cd13 (Drukker et al 2012;Den Hartogh et al 2015;Skelton et al 2016), or Fzd4 (Yoon et al 2018).…”

Automated Hypothesis Generation to Identify Signals Relevant in the Development of Mammalian Cell and Tissue Bioprocesses, With Validation in a Retinal Culture System

“…While pathways that are active in a given type of cell may be inferred from previous developmental or functional studies (Zhou et al, 2015;Fan et al, 2016;Yoon et al, 2018;van der Kant et al, 2019), such research may not yet have been completed for a given tissue of interest. Even if it has, there is no guarantee that all relevant interactions have been identified.…”