Cardiac differentiation of human pluripotent stem cells using defined extracellular matrix proteins reveals essential role of fibronectin

Figure 1.. 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.

Figure 1—figure supplement 1.. Fluorescence images of H1 ESCs growing on Matrigel, LN111, LN521 and FN coated surface as confluent monolayer immunolabeled with antibodies against OCT4 and SSEA4. Scale bar is 100 µm.

Figure 1—figure supplement 2.. Morphology of DF19-9-11 iPSCs growing on human collagen IV coated surface. The iPSCs were grown for 5 days on collagen IV coated surface and did not form confluent monolayer.

Figure 1—figure supplement 3.. cTnT+ cells measured by flow cytometry at 15 days of differentiation of H1 ESCs on different ECM proteins as substrate (bottom) and overlay (overlay) using the matrix sandwich protocol. N≥3 biological replicates. *p<0.05, one-way ANOVA with post-hoc Bonferroni and test.

Figure 2.. 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…

Figure 2—figure supplement 1.. Fluorescence images of H1 ESCs growing on FN, LN111 and LN521 coated surface immunolabeled with antibodies against FN and laminin. Scale bar is 100 µm.

Figure 3.. 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.

Figure 3—figure supplement 1.. Maximum projection view of the confocal z-scan of DF19-9-11T iPSCs at days 0–3 of the cardiac differentiation using the Matrigel/Matrigel (Mat/Mat) and FN/FN matrix sandwich protocol. The cells were co-labeled with antibodies against Brachyury (BRY) and FN. Scale bar is 25 µm.

Figure 4.. 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.

Figure 4—figure supplement 1.. The FN1 shRNA oligos. Yellow, CDS where shRNA binds; Green, palindrome. Oligo-1 binds in exon 8; Oligo-2 binds in 3’ UTR.

Figure 4—figure supplement 2.. Characterization of hPSC FN1 knockdown clones. (A) Doxycycline-induced mCherry expression in the hPSC culture of H1 FN1 knockdown clone. Scale bar is 200 µm. (B) Phase contrast and immunofluorescence images of FN expression in doxycycline hPSC culture of H1 FN1 knockdown clone. Scale bar is 200 µm. (C) Quantatitave western blots of FN from control of H1 and DF19-9-11T hPSCs and associated FN1 knockdown hPSC clones in the absence and presence of Dox (8 µg/ml). N=3 biological replicates. *p<0.05, one-way ANOVA with post-hoc Bonferroni test.

Figure 4—figure supplement 3.. Effect of dox concentration-dependent FN knockdown at days 0–1 on cardiac differentiation using the protocol as shown in Figure 4A. (A) cTnT+ cells measured by flow cytometry at 15 days of differentiation of the FN1 knockdown clones (H1c8 and H1c34) showing the effect of FN knockdown at days 0–1 at different concentrations of dox (1–8 μg/ml) and the effect of adding exogenous FN (3 μg/cm2) at each concentration of dox. Dox added at 2 μg/ml inhibited generation of cTnT+ cells which was rescued by added exogenous FN. Dox added at 4 µg/ml or higher inhibited cTnT+ cells and could not be rescued by exogenous FN under the conditions tested. (B) Total cells in culture for the unmodified control H1 hESC line undergoing cardiac differentiation measured at days 0–3 when dox was added at days 0–1. Dox concentrations of 0 (no dox ctr), 1 (dox1), 2 (dox2), 4 (dox4), and 8 (dox8) μg/ml were tested. At the highest concentration of dox tested (8 μg/ml) no cells survived.