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• W4283582110 abstract "Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Research and therapeutic applications using human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) require robust differentiation strategies. Efforts to improve hPSC-CM differentiation have largely overlooked the role of extracellular matrix (ECM). The present study investigates the ability of defined ECM proteins to promote hPSC cardiac differentiation. Fibronectin (FN), laminin-111, and laminin-521 enabled hPSCs to attach and expand. However, only addition of FN promoted cardiac differentiation in response to growth factors Activin A, BMP4, and bFGF in contrast to the inhibition produced by laminin-111 or laminin-521. hPSCs in culture produced endogenous FN which accumulated in the ECM to a critical level necessary for effective cardiac differentiation. Inducible shRNA knockdown of FN prevented Brachyury+ mesoderm formation and subsequent hPSC-CM generation. Antibodies blocking FN binding integrins α4β1 or αVβ1, but not α5β1, inhibited cardiac differentiation. Furthermore, inhibition of integrin-linked kinase led to a decrease in phosphorylated AKT, which was associated with increased apoptosis and inhibition of cardiac differentiation. These results provide new insights into defined matrices for culture of hPSCs that enable production of FN-enriched ECM which is essential for mesoderm formation and efficient cardiac differentiation. Editor's evaluation We found this study important for advancing derivation of cardiac cells from human pluripotent stem cells, as it convincingly supports the critical role of fibronectin in the formation of precardiac mesoderm. We believe that the work will be of interest to developmental biologists, stem cell biologists, and engineers as they work to optimize substrates used for preparation of cardiomyocytes and supporting cardiac cells. https://doi.org/10.7554/eLife.69028.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Cardiomyocytes derived from human pluripotent stem cells (hPSC-CMs) are increasingly used in basic research, drug development, toxicity testing, precision medicine applications, and emerging clinical strategies for cardiac repair and regeneration. Methods to differentiate hPSC-CMs have advanced significantly over the past 15 years (Burridge et al., 2014; Kattman et al., 2011; Lian et al., 2012; Mummery et al., 2012; Zhang et al., 2012). Most cardiac differentiation protocols have focused on the optimal application of soluble molecules including growth factors and small molecules to promote generation of stage-specific cardiac progenitors and ultimately hPSC-CMs. These protocols also require extracellular matrix (ECM) proteins, either endogenously produced or exogenously added as substrates as well as signaling molecules to enable hPSC attachment, survival, proliferation and differentiation. However, the ECM proteins involved in the cardiac differentiation of hPSCs and ECM-activated signaling pathways have been far less investigated and elucidated. Our previous study showed that hPSCs cultured on the commercially available ECM preparation, Matrigel, more efficiently and reproducibly differentiate to hPSC-CMs in response to Activin A/BMP4/bFGF signaling if they concurrently received overlays of Matrigel during the initiation of differentiation – the matrix sandwich protocol (Zhang et al., 2012). The Matrigel overlays promote the initial stage of differentiation, the epithelial-to-mesenchymal transition (EMT) to form Brachyury+ mesoderm, mimicking the primitive streak in development (Nieto et al., 2016). However, Matrigel is a complex mixture of ECM proteins produced from Engelbreth-Holm-Swarm mouse sarcoma cells, is not fully defined, and exhibits batch-to-batch variability. The essential ECM components responsible for promoting the initial stages of cardiogenesis in the matrix sandwich protocol as well as the optimal ECM environment to promote cardiogenesis in general remain to be determined. Complex mixtures of ECM proteins such as Matrigel allow for the attachment and self-renewal of hPSCs in appropriate media. More recently, recombinant ECM proteins and synthetic substrates have been identified that can support long-term culture of hPSCs (Lambshead et al., 2013). These defined substrates mimic the ECM components present in the earliest embryo including laminins, collagens, fibronectin (FN), vitronectin, and proteoglycans. The hPSCs interact with the substrates via transmembrane receptors called integrins and other cell adhesion molecules, such as cadherins. However, for cardiac differentiation protocols a substrate that both allows attachment of the hPSCs and also supports proliferation and subsequent differentiation is needed. Strong signals to maintain self-renewal and pluripotency provided by the ECM will impede the differentiation processes, so a composition of ECM that is dynamic and supports hPSC proliferation, as well as differentiation, is theoretically optimal. Yap and colleagues utilized a combination of recombinant laminins, laminin-521 (LN521) to enable self-renewal of hPSCs (Rodin et al., 2014) and laminin-221 (LN221) to enable differentiation to cardiac progenitors (Yap et al., 2019). Others using a design of experiment statistical approach found a combination of three ECM proteins optimal for cardiac differentiation of hPSCs, collagen type I, laminin-111 (LN111) and FN (Jung et al., 2015; Kupfer et al., 2020). Burridge and colleagues systematically tested a range of different substrates in a defined small molecule-based cardiac differentiation protocol and found a variety of substrates including a synthetic vitronectin-derived peptide, recombinant E-cadherin, recombinant human vitronectin, recombinant human LN521, truncated LN511 and human FN, and a FN mimetic enabled hPSC-CM differentiation (Burridge et al., 2014). However, these studies have not examined the impact of dynamic manipulation of defined ECM proteins or ECM signaling pathways in cardiac differentiation, nor characterized the changes in endogenous ECM proteins that ultimately contribute to the cellular transitions. In the present study, we tested a variety of human recombinant and defined ECM proteins for both attachment and overlay of hPSC cultures in the matrix sandwich protocol and investigated the potential ECM-activated signaling pathways. We chose to test LN111 (the dominant laminin isoform in Matrigel), LN521 (demonstrated adherence and culture of hPSCs [Rodin et al., 2014]), FN (implicated in embryonic developmental studies [Cheng et al., 2013; George et al., 1993; Trinh and Stainier, 2004]), and collagen (common ECM protein used for in vitro cell adherence). We found that of the tested ECM proteins, only FN overlays promoted cardiac differentiation comparable to Matrigel overlays while LN111, LN521 and collagen IV (COL4) overlays inhibited cardiogenesis. Furthermore, hPSCs differentiated efficiently to hPSC-CMs without overlay when grown on FN, LN111, and LN521. Regardless of the ECM preparation used as the attachment substrate, we identified an essential role of FN in promoting the initial stages of hPSC cardiac differentiation acting via transmembrane integrin α4β1 and αVβ1 receptors to activate downstream integrin-linked kinase (ILK) signaling cascades. Results Defined ECM proteins support hPSC adhesion, growth, and cardiac differentiation We tested whether defined human ECM proteins could replace Matrigel in the matrix sandwich cardiac differentiation protocol. The matrix sandwich protocol uses Matrigel coating for hPSC adhesion and expansion followed by overlaying the proliferating hPSCs with Matrigel at day –2 and day 0 of differentiation with the addition of growth factors Activin A at day 0–1, followed by addition of BMP4 and bFGF at day 1–5 (Figure 1A). Therefore, we first tested the ability of defined ECM proteins to support hPSC adhesion and expansion. DF19-19-11T iPSCs or H1 ESCs were seeded on human LN111, LN521, COL4, and FN coated surfaces and cultured in mTeSR1 medium. The hPSCs grew as a monolayer on LN111, LN521, and FN and exhibited similar morphology and expression of the pluripotency markers OCT4 and SSEA4 as hPSCs grown on Matrigel (Figure 1B, Figure 1—figure supplement 1). However, the hPSCs seeded on COL4 did not grow as a confluent monolayer (Figure 1, Figure 1—figure supplement 2), so this matrix coating was not tested further. Figure 1 with 3 supplements see all Download asset Open asset Defined ECM proteins support hPSC adhesion, growth and cardiac differentiation using the matrix sandwich protocol. (A) Schematic method of the matrix sandwich protocol. Defined ECM proteins were tested as coating matrix (blue) and overlay matrix (red). (B) Fluorescence images of DF19-9-11T iPSCs growing on the ECM of Matrigel, LN111, LN521 and FN as confluent monolayer and immuno-labeled with antibodies against OCT4 and SSEA4. Scale bar is 100 µm. (C) cTnT+ cells measured by flow cytometry at 15 days of differentiation of DF19-9-11T iPSCs on different ECM proteins as substrate (bottom) and overlay (overlay). N≥3 biological replicates. Error bars represent SEM. *p<0.05, one-way ANOVA with post-hoc Bonferroni test. For those matrix substrates that supported monolayer growth of hPSCs, including LN111, LN521 and FN, we tested overlay of defined ECM proteins in the matrix sandwich protocol using DF19-9-11T iPSCs and H1 ESCs. Cardiac differentiation was measured by flow cytometry for cTnT+ cells at 15 days of differentiation (Figure 1C, Figure 1—figure supplement 3). hPSCs seeded on Matrigel showed poor cardiac differentiation in response to the growth factors without matrix overlay, but with Matrigel overlays the percentage of cTnT+ cells was significantly increased as we previously reported (Zhang et al., 2012). Interestingly, FN overlays were as effective as Matrigel overlays in promoting cardiac differentiation of the hPSCs growing on Matrigel. However, if cells were seeded on LN111, LN521, or FN coated surfaces, the overlay of Matrigel or FN did not further increase the efficiency of hPSC-CM generation, and overlays of LN111, LN521, and COL4 strongly inhibited cardiac differentiation. These results demonstrated that the defined ECM proteins of LN111, FN and to a lesser extent LN521 support hPSC adhesion, growth and cardiac differentiation in monolayer hPSC culture and do not require a matrix overlay for efficient cardiac differentiation using the Activin A/BMP4/bFGF growth-factor-directed protocol. hPSC monolayer culture on LN111 substrate promote endogenous FN production Since FN promoted cardiac differentiation as both a culture matrix and an overlay, and also because FN plays key roles in EMT during gastrulation and cardiogenesis (Boucaut et al., 1996; Boucaut and Darribere, 1983; Boucaut et al., 1984; Darribère et al., 1988; Johnson et al., 1993; Lee et al., 1984; Lim and Thiery, 2012; Linask and Lash, 1986; Linask and Lash, 1988a, Linask and Lash, 1988b; Nieto et al., 2016; Suzuki et al., 1995; Thiery and Sleeman, 2006), we next examined for the presence of FN ECM in hPSC culture. DF19-9-11T iPSCs plated on Matrigel-coated surfaces and cultured in mTeSR1 per the matrix sandwich protocol were immunolabeled with a FN antibody on days −3,–2, –1 and 0 without permeabilizing the cells to examine extracellular FN protein (Figure 2A). On days –3 and –2, minimal immunolabeled FN in ECM was observed. However, after 4 days of culture (day 0) immunolabeled fibrillar FN ECM was abundant in the matrix sandwich culture (Figure 2A and B). In contrast, the monolayer culture control in the absence of Matrigel overlay had significantly less FN ECM present by day 0 (Figure 2A and B). This suggests that the Matrigel overlay promotes the production of endogenous FN or remodeling of FN ECM relative to the monolayer culture control without Matrigel overlay. Because the hPSCs cultured on LN111 coated surface without matrix overlay enabled efficient cardiac differentiation (Figure 1C, Figure 1—figure supplement 3), we examined the endogenous FN production in the hPSC culture on LN111 coated surface in which no exogenous FN was added. The hPSCs grown on LN111 coated surface without any matrix overlay were immunolabeled with the FN antibody without permeabilizing the cells. Confocal z-scan of the cell culture showed no detectable FN ECM at days –3 and –2, similar to the Matrigel/Matrigel sandwich culture; however, by day 0, dense fibrillary FN ECM was present in the cell culture on LN111 coated surface (Figure 2C, DF19-9-11T iPSCs; Figure 2—figure supplement 1, H1 ESCs). Similar to our previous study of the Matrigel/Matrigel sandwich culture (Zhang et al., 2012), the hPSCs growing on the LN111 coated surface without any matrix overlay formed multilayer cultures as shown in the side view of the confocal z-scan as did FN/FN matrix sandwich culture (Figure 2D). To determine if the hPSC culture results in accumulation of endogenously produced laminin ECM as well, DF19-9-11T iPSCs cultured on FN, LN111, and LN521 coated surfaces were immunolabeled with an antibody detecting laminins without permeabilizing the cells, and did not show measurable laminin ECM after 4 days of growth on these defined matrices (Figure 2—figure supplement 1). These results together with the above cardiac differentiation results supported the potential role of FN in promoting ActivinA/BMP4/bFGF-directed hPSC cardiac differentiation. Figure 2 with 1 supplement see all Download asset Open asset Production of endogenous FN in the hPSC-matrix sandwich culture and LN111 culture. (A) Phase contrast and fluorescence images of the matrix sandwich culture of DF19-9-11T iPSCs grown for 4 days and immunolabeled using anti-FN antibody, compared with the monolayer culture. Scale bar is 200 µm. (B) Quantitative analysis of the FN fluorescence in A by Image J. N≥3 replicates. The box plots summarize the biological replicates with the box enclosing from first to third quartile and middle square indicating mean and line in box indicating median. *p<0.05, one-way ANOVA with post-hoc Bonferroni test. (C) Maximum projection view of the confocal z-scan of DF19-9-11T iPSCs growing on LN111 coated surface immunolabeled with FN antibody at day -3, -2 and -1. Scale bar is 25 µm. (D) The maximum projection view (upper panel) and side view (lower panel) of the confocal z-scan of DF19-9-11T iPSCs grown for 4 days (at day 0) on LN111 coated surface without matrix overlay immunolabeled with the anti-FN antibody. The multilayer growth and FN production are similar to Matrigel/Matrigel and FN/FN matrix sandwich cultures in parallel at the same time (day 0). Scale bar is 25 µm. Figure 2—source data 1 Images and quantitative analysis for Figure 2B. https://cdn.elifesciences.org/articles/69028/elife-69028-fig2-data1-v1.zip Download elife-69028-fig2-data1-v1.zip Differentiation of hPSCs on LN111 substrate undergo EMT and generate mesoderm in the FN-rich ECM To characterize the early stages of cardiac differentiation of hPSCs cultured on LN111 and treated with Activin A/BMP4/bFGF growth factors, we examined markers of EMT, mesoderm and cardiac mesoderm. Gene expression was assessed by quantitative RT-PCR, and upon the addition of Activin A, BMP4 and bFGF, there was significant upregulation of transcription factors associated with EMT including SNAI1 (Leptin and Grunewald, 1990), SNAI2 (Nieto et al., 1994), and TWIST (Thiery et al., 2009; Figure 3A). The mesenchymal cell markers of vimentin (VIM), FN (FN1) and N-cadherin (CDH2) were also greatly upregulated by day 3 (Figure 3A). In contrast, E-cadherin expression (CDH1), an epithelial cadherin, was greatly downregulated by day 3 of differentiation. The mesendoderm/mesoderm transcription factors GSC, MIXL1, SOX17 and TBXT were transiently upregulated followed by expression of cardiac transcription factors of MESP1, ISL1, NKX2-5, and GATA4 at days 3–5 (Figure 3B). Figure 3 with 1 supplement see all Download asset Open asset Expression of EMT, mesendoderm/mesoderm markers and cardiac transcription factors in the cardiac differentiation of hPSCs cultured on LN111 substrate by Activin A/BMP4/bFGF signaling. (A) qRT-PCR for gene expression of EMT markers at days 0–3 of cardiac differentiation. (B) qRT-PCR for gene expression of mesendoderm/mesoderm and cardiac transcription factors at days 0–5 of cardiac differentiation. N=3 technical replicates for each point. (C) Maximum projection view of the confocal z-scan of DF19-9-11T iPSCs at days 0–3 of the cardiac differentiation co-labeled with antibodies against Brachyury (BRY) and FN. Scale bar is 25 µm. Error bars represent SEM. To determine if FN ECM persisted or remodeled during the early stages of cardiac differentiation on LN111 substrate, immunolabeling of the early differentiated DF19-9-11T iPSCs on days 0–3 for FN and Brachyury was performed. Confocal z-scan imaging showed abundant FN ECM at each day, and Brachyury+ cells were associated with the dense network of FN ECM (Figure 3C), suggesting that the Brachyury+ cells interact with FN. Similar Brachyury+ cells and the FN ECM network were also observed in the Matrigel/Matrigel and FN/FN matrix sandwich cultures (Figure 3—figure supplement 1). Together, these results show that hPSCs grown on LN111, undergo the early stages of cardiac differentiation with transitions to mesoderm and cardiac mesoderm progenitors occurring in an endogenously generated FN-rich ECM, similarly as in the Matrigel/Matrigel and FN/FN matrix sandwich cultures. FN is essential for cardiac differentiation of hPSCs To determine if FN is essential for mesoderm formation in our protocol and to investigate the stage-specific roles of FN during cardiac differentiation of hPSCs, we generated a doxycycline (dox) inducible FN knockdown system using FN1 shRNA (Figure 4—figure supplement 1). The two vectors shown in Figure 4A were incorporated into lentivirus and transduced into hPSCs. Clones were selected by neomycin resistance from both hESC line H1 and hiPSC line DF19-9-11T. To confirm the dox inducibility of the FN1 shRNA in the cell lines, we first assessed dox-induced bicistronic mCherry expression (Figure 4—figure supplement 2A). Inducible FN knockdown was demonstrated by immunolabeling with FN antibody (Figure 4—figure supplement 2B) and quantitative western blot for FN expression (Figure 4—figure supplement 2C). Figure 4 with 5 supplements see all Download asset Open asset FN is essential at the initiation of cardiac differentiation of hPSCs. (A) Schematic of the inducible shRNA construct for FN1 knockdown. (B) Schematic method of FN knockdown at differentiation stages of days 0–1, 1–5, and 5–7 in the cardiac differentiation protocol. (C) cTnT+ cells measured by flow cytometry at 15 days of differentiation of the H1 FN1 knockdown clone 34 using the protocol in (B). Dox concentration is 2 µg/ml, N≥3 biological replicates. Error bars represent SEM. *p<0.05, one-way ANOVA with post-hoc Bonferroni test. FNKD indicates FN knockdown by dox induction. +FN indicates exogenous FN added. Cardiac differentiation was performed using the monolayer based protocol with the H1 inducible FN knockdown clones growing on LN111 coated surface and treated with the growth factors Activin A, BMP4 and bFGF as shown in Figure 4B. To probe the stage-specific effect of FN knockdown during cardiac differentiation, dox was added at different time points: day 0–1, day 1–5, and day 5–7, and cardiac differentiation was measured by flow cytometry for cTnT+ cells at 15 days of differentiation. Cardiac differentiation was significantly inhibited when FN was knocked down at day 0–1, whereas FN knockdown at days 1–5, or days 5–7, did not have significant impact on the percentage of cTnT+ cells when compared to the no dox control (Figure 4C). Because FN knockdown at day 0–1 significantly inhibited cardiac differentiation, we next tested if exogenous FN at this stage can rescue cardiac differentiation. Using the same protocol as shown in Figure 4B with dox induction of FN knockdown at day 0–1, we added soluble human FN (3 µg/cm2) in the cell culture on day 0–1. Cells were differentiated for 15 days, and cardiac differentiation was measured by flow cytometry for cTnT+ cells as above. Adding exogenous FN fully rescued the hPSC-CM differentiation, giving rise to a similar percentage of cTnT+ cells compared to the no FN knockdown control (Figure 4C). The effect of dox-induced FN knockdown on day 0–1 was concentration-dependent (Figure 4—figure supplement 3A), but at the highest concentration of dox tested (8 µg/ml), there was evidence for dox toxicity based on the loss of viability of nontransgenic H1 cells undergoing the differentiation protocol (Figure 4—figure supplement 3B). Adding exogenous FN (3 µg/cm2) along with dox at days 1–5 or days 5–7 did not significantly increase the percentage of cTnT+ cells when compared to the no dox control at days 1–5 and days 5–7, respectively (Figure 4C). FN is required for formation of Brachyury+ cells As FN knockdown at day 0–1 dramatically inhibited cardiac differentiation which could be rescued by the addition of exogenous FN, we first evaluated the expression of EMT genes that mark the initial transition of hPSCs to mesoderm over the first two days of differentiation. Quantitative RT-PCR was performed on the same H1 inducible FN knockdown clones at 0, 24, 36, and 48 hr after hPSCs differentiation was initiated (Figure 5A). The effect of dox-induced knockdown of FN1 transcripts was confirmed by the greater than 50% reduction in mRNA levels in the FN knockdown and FN knockdown +exogenous FN cell samples at 24 hr relative to the no dox control (Figure 5A). By 36 hr, FN1 transcripts recovered to the control level for FN knockdown condition or were significantly increased as in the FN knockdown +exogenous FN samples after dox was removed at 24 hr. By 48 hr, there were similar levels of FN1 transcripts in both the control and the FN rescue samples, but no cells survived in the FN knockdown group (Figure 5A). The key EMT transcription factors upregulated during gastrulation (Barrallo-Gimeno and Nieto, 2005; Nieto, 2002; Nieto et al., 2016; Thiery and Sleeman, 2006), SNAI1 and SNAI2, were examined. Quantitative RT-PCR showed SNAI1 expression increased significantly in both the FN knockdown and FN knockdown +exogenous FN samples compared to the control at 24 hr, and its expression continuously upregulated in the FN knockdown sample at 36 hr. By 48 hr, there were similar level of SNAI1 expression in both the control and the FN rescue samples; whereas, SNAI2 expression was not significantly different between the groups at 24 and 36 hr but a general increase in expression over this time window was observed (Figure 5A). VIM expression, similar to SNAI1 expression, was increased significantly in the FN knockdown cells by 36 hr compared to the control (Figure 5A), which is consistent with this mesenchymal marker and known target of SNAI1. Figure 5 with 2 supplements see all Download asset Open asset shRNA knockdown of FN results in loss of Brachyury+ cells. (A) qRT-PCR for gene expression of EMT markers for the H1 FN knockdown clone in the cardiac differentiation time course of 0–48 hr at the no dox control, dox induction at day 0–1 and dox induction at day 0–1 with adding exogenous FN conditons. (B) Total cell number of the H1 FN knockdown clones in the time course of days 0–5 in the cardiac differentiation at the no dox control, dox induction at day 0–1 and dox induction at day 0–1 with adding exogenous FN conditions. (C) Flow cytometry of co-labeling the cells shown in B with Brachyury and Sox17 antibodies. The cardiac differentiation protocol is shown in Figure 4B. Error bars represent SEM. *p<0.05, one-way ANOVA with post-hoc Bonferroni test. We next examined the mesendodermal/mesodermal progenitors generated in the initial differentiation stage of the H1 inducible FN knockdown clones by flow cytometry. The cell counts of the attached cells on days 0–5 of differentiation showed a great reduction of cell number at day 1 in all three groups (Figure 5B); however, the cells in the control and the FN rescue groups rapidly proliferated after day 1. In contrast, no cells survived in the FN knockdown group after day 2 (Figure 5B). Because Brachyury and Sox17 are both expressed in mesendodermal progenitors, we co-labeled the cells with Brachyury and Sox17 antibodies on days 0–5 and analyzed by flow cytometry. Brachyury+ cells started to emerge at day 1 in all three groups. By day 2, 97–98% of the cells were Brachyury+ in both the control and FN rescue groups, but there were no surviving cells in the FN knockdown samples (Figure 5C). The fraction of cells that were Brachyury+ rapidly decreased after day 2 in both the control and FN rescue groups, and by day 5 there were minimal Brachyury+ or Sox17+ cells present in both groups (Figure 5C). We performed the same experiment using the DF19-9-11T inducible FN knockdown clones and observed similar results (Figure 5—figure supplement 1). In contrast to the day 0–1 treatment with dox, a later timed dox pulse from days 1 to 5 did not alter the abundance of Brachyury+ cells in all three groups over the same time course of differentiation (Figure 5—figure supplement 2). Together these results suggest that knockdown of FN at day 0–1 does not stop the initiation of EMT upon addition of Activin A at d0 (Nieto et al., 2016; Thiery and Sleeman, 2006), but it prevents the generation and/or survival of Brachyury+ mesodermal progenitors. In contrast, shRNA knockdown of FN at later time points in the protocol did not impact the fate of the differentiating cells. Addition of blocking antibodies to integrin β1, α4 or αV subunits at mesoderm formation inhibits hPSC-CM differentiation To investigate the mechanisms underlying FN’s essential role in the formation of Brachyury+ mesoderm, we evaluated integrins expressed in undifferentiated hPSCs and known to bind FN. Integrins are a family of heterodimeric transmembrane proteins composed of α and β subunits that interact with ECM. Of the 24 known heterodimeric integrin receptors, 13 have been shown to bind FN (Bachmann et al., 2019; Bharadwaj et al., 2017; Hynes, 2002; Ruoslahti, 1991; Wu et al., 1995). Review of RNA-seq data from undifferentiated hiPSCs shows expression of integrin subunits associated with FN binding including integrin α3, α4, α5, αV, β1, β5, and β8 (Zhang et al., 2019). Of these integrin subunits, knockout studies have implicated only α4, α5, αV, and β1 with various developmental defects impacting the heart (Hynes, 2002), so we focused our studies on these integrins. Integrin β1 shows the highest level of expression of all integrin subunits in hPSCs, and so we first tested blocking integrin β1 with a monoclonal antibody, P5D2, during differentiation. We added P5D2 at day –2 through day –1 (pluripotent stage) or day 0 through day 1 (mesoderm formation) in the matrix sandwich protocol when Matrigel overlays were applied (Figure 6A). Adding P5D2 at day –2 did not block cardiac differentiation; however, adding P5D2 at day 0 significantly inhibited cardiac differentiation as measured by flow cytometry of the cTnT+ cells using DF19-9-11T iPSCs and H1 ESCs (Figure 6B, Figure 6—figure supplement 1A). Furthermore, the P5D2 antibody showed concentration-dependent inhibition of hPSC-CM generation in DF19-9-11T iPSCs and H1 ESCs (Figure 6C, Figure 6—figure supplement 1B). We next tested antibody blocking (3 µg/ml) of relevant integrin α subunits including α5, αV, and α4. Adding the monoclonal antibodies P1D6 (anti-integrin α5), P3G8 (anti-integrin αV), or P4G9 (anti-integrin α4) at day –2 as shown in Figure 6A did not significantly impact hPSC-CMs differentiation as measured by flow cytometry of the cTnT+ cells, similar to the results blocking integrin β1 at day –2 (Figure 6D, Figure 6—figure supplement 1C). When the integrin α blocking antibodies were added on day 0, block of integrin α5 did not show inhibition of hPSC-CMs differentiation for both DF19-9-11T and H1 lines. Whereas block of integrin αV showed borderline inhibition and block of integrin α4 showed significant inhibition of hPSC cardiac differentiation in both DF19-9-11T and H1 lines (Figure 6D, Figure 6—figure supplement 1C). To probe the impact of blocking integrin α4 and integrin αV further, we tested a range of blocking antibody concentrations added at day 0 and found concentration-dependent inhibition of cardiac differentiation by P4G9 (anti-integrin α4), as well as significant inhibition by the highest concentration (5 µg/ml) of P3G8 (anti-integrin αV) (Figure 6E and F). Taken together, these results showed antibody blocking FN integrin receptors of β1, α5, αV, or α4 at the pluripotent stage of hPSCs (day –2) in the matrix sandwich protocol did not inhibit cardiac differentiation; however, blocking integrin β1, α4, or αV at day 0–1, when mesodermal progenitors are generated resulted in significant inhibition of hPSC-CM differentiation compared to the control for multiple hPSC lines. Thus, integrin α4β1 and αVβ1 heterodimers are likely key mediators of the FN effect on early differentiation stages. Figure 6 with 1 supplement see all Download asset Open asset Cardiac differentiation is blocked by anti-integrin β1, α4 or αV antibodies when added at mesoderm formation in the matrix sandwich protocol. (A) Schematic for testing monoclonal antibodies to block integrin β1 (P5D2), α5 (P1D6), αV (P3G8), and α4 (P4G9). (B) cTnT+ cells meas" @default.
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• W4283582110 title "Author response: Cardiac differentiation of human pluripotent stem cells using defined extracellular matrix proteins reveals essential role of fibronectin" @default.
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• W4283582110 workType "peer-review" @default.