Direct Production of Human Cardiac Tissues by Pluripotent Stem Cell Encapsulation in Gelatin Methacryloyl

Direct stem cell encapsulation and cardiac differentiation within supporting biomaterial scaffolds are critical for reproducible and scalable production of the functional human tissues needed in regenerative medicine and drug-testing applications. Producing cardiac tissues directly from pluripotent stem cells rather than assembling tissues using pre-differentiated cells can eliminate multiple cell-handling steps that otherwise limit the potential for process automation and production scale-up. Here we asked whether our process for forming 3D developing human engineered cardiac tissues using poly­(ethylene glycol)-fibrinogen hydrogels can be extended to widely used and printable gelatin methacryloyl (GelMA) hydrogels. We demonstrate that low-density GelMA hydrogels can be formed rapidly using visible light (<1 min) and successfully employed to encapsulate human induced pluripotent stem cells while maintaining high cell viability. Resulting constructs had an initial stiffness of approximately 220 Pa, supported tissue growth and dynamic remodeling, and facilitated high-efficiency cardiac differentiation (>70%) to produce spontaneously contracting GelMA human engineered cardiac tissues (GEhECTs). GEhECTs initiated spontaneous contractions on day 8 of differentiation, with synchronicity, frequency, and velocity of contraction increasing over time, and displayed developmentally appropriate temporal changes in cardiac gene expression. GEhECT-dissociated cardiomyocytes displayed well-defined and aligned sarcomeres spaced at 1.85 ± 0.1 μm and responded appropriately to drug treatments, including the β-adrenergic agonist isoproterenol and antagonist propranolol, as well as to outside pacing up to 3.0 Hz. Overall results demonstrate that GelMA is a suitable biomaterial for the production of developing cardiac tissues and has the potential to be employed in scale-up production and bioprinting of GEhECTs

Conserved effects of the Pro552Arg mutation across GluN2 subunits.

<p><b>A</b>,<b>B</b>, Composite concentration-response curves of glutamate in the presence of 100 μM glycine (<b>A</b>) and glycine in the presence of 100 μM glutamate (<b>B</b>) for human GluN1-P557R/GluN2A, GluN1-P557R/GluN2B, GluN1/GluN2B-P553R, and rat GluN1/GluN2C-P550R, and GluN1/GluN2D-P577R. The graph legends refer to GluN1 as N1 and GluN2 as N2. Fitted EC₅₀ values are summarized in Tables <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006536#pgen.1006536.t003" target="_blank">3</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006536#pgen.1006536.t005" target="_blank">5</a>. <b>C</b>,<b>D</b>, human GluN1-P557R/GluN2A significantly prolongs deactivation time course after removal of glutamate (<b>C</b>) or removal of glycine (<b>D</b>) on transfected HEK293 cells, but does not slow the rise time when the receptors were activated by the agonists. <b>E</b>, GluN1/GluN2B-P553R significantly slows rise time and prolongs deactivation time course. Fitted parameters describing the response time course are given in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006536#pgen.1006536.t006" target="_blank">Table 6</a>.</p

Patients’ and mutations’ information.

<p>Patients’ and mutations’ information.</p

Agonist potency of GluN2A-P552R equivalent mutations in GluN2B, GluN2C, and GluN2D.

<p>Agonist potency of GluN2A-P552R equivalent mutations in GluN2B, GluN2C, and GluN2D.</p

Potential interaction between the pre-M1 and M3 helices.

<p><b>A</b>,<b>B</b>, Ribbon structures of the GluN1/GluN2A (<b><i>A</i></b>) and GluN1/GluN2B (<b><i>B</i></b>) receptors without the amino terminal domain is shown. GluN1 is tan and GluN2 is light blue; regions with an OE-ratio below the 5^th percentile are colored purple, and indicate the regions under the strongest purifying selection. <b>C</b>, Side and top down view of the pore forming elements M1, M3, M4 in GluN1/GluN2A receptors colored as in (<b><i>A</i></b>), with regions of purifying selection shown in purple. <b>D</b>, Expanded view of the pre-M1 helix for GluN1 (<i>left panel</i>) and for GluN2A (<i>right panel</i>).</p

Assessment of alternative Pro552 substitutions in GluN2A.

<p><b>A,B</b>, the composite glutamate (in the presence of 100 μM glycine) and glycine (in the presence of 100 μM glutamate) concentration-response curves of GluN2A- P552A, P552G, P552I, P552K, P552Q, P552L constructs. Error bars are SEM and shown when larger than symbol. <b>C,D,E</b>, The response time courses are shown of GluN1/GluN2A(P552K), GluN1/GluN2A(P552G), and GluN1/GluN2A(P552L) receptors activated by rapid application of 100 μM glutamate; 100 μM glycine was present in all solutions. For panel <b>D</b> the rise time is expanded as an inset. The data (mean ± SEM) are given in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006536#pgen.1006536.t007" target="_blank">Table 7</a>.</p

Analysis of genetic variation on S1-M1 linker in <i>GRIN1</i>, <i>GRIN2A</i>, <i>and GRIN2B</i>.

<p>Analysis of genetic variation on S1-M1 linker in <i>GRIN1</i>, <i>GRIN2A</i>, <i>and GRIN2B</i>.</p

Summary of pharmacological and functional properties of pre-M1 mutations.

<p>Summary of pharmacological and functional properties of pre-M1 mutations.</p

Pharmacology and response time course for substitution of GluN2A-ProP552 with Ala, Gly, Lys, Gln, Ile, or Leu.

<p>Pharmacology and response time course for substitution of GluN2A-ProP552 with Ala, Gly, Lys, Gln, Ile, or Leu.</p

Neurotoxic consequences of GluN2A-P552R expression and rescue pharmacology.

<p><b>A</b>, Schematic of experimental timeline indicates the relative dates of neuronal cell culture from embryonic day 16/17 (E16/17), transfection along with memantine/vehicle treatment, and toxicity studies (luciferase assays, cell counts, and confocal imaging). <b>B</b>, Confocal images of cortical neurons transfected with a GFP-expressing construct, with various concentrations of the human GluN2A-P552R-expressing plasmid illustrate the cell morphology. Images were acquired 24 h post-transfection at 20× magnification (scale bars 10 μm). <b>C</b>, Confocal images of cortical neurons transfected with GFP-expressing construct with either 0.6 μg cDNA per well (40% of total transfection cDNA) of WT GluN2A or GluN2A-P552R, the latter in the absence or presence of memantine (50 μM). Images were acquired 24 h post-transfection at 20× magnification, with the exception of the bottom left panel (40×), which highlights GluN2A-P552R-induced dendritic swelling and blebs (scale bar 10 μm). <b>D</b>, The mean cell viability values are shown as a percent of control. Luciferase assays: neuronal cultures were transfected with GFP-N1 plasmid (0.525 μg or 0.825 μg per well) luciferase cDNA (0.375 μg/well) for cell viability assaying, with varied concentrations (0.3 μg or 0.6 μg per well) of pCIneo-vector, WT GluN2A, or GluN2A-P552R cDNA (1.5 μg total DNA per well). Each transfection condition was performed in pairs, either supplemented with vehicle (–) or memantine (20 μM for 0.3 μg; 50 μM for 0.6 μg) treatment (+). Luciferase assays were performed 48 h following transfection and treatment. Experiments were performed in quadruplicate, and independent experiments were repeated (0.3 μg cDNA, n = 7; 0.6 μg cDNA, n = 8). Each condition was normalized to its relevant vector-transfected group to obtain relative viability values, expressed as % control. Data are mean ± SEM of viability (% control) for each condition (ANOVA/Bonferroni; *p <0.05, **p < 0.01, ***p < 0.001). Cell counts: Neuronal cultures were transfected with GFP-N1 plasmid for cell visualization, with either 0.6 μg pCIneo including vector, WT GluN2A, or GluN2A-P552R cDNA (40% of total transfection cDNA). Each transfection condition was performed in duplicate. Cell counts were performed 48 h post-transfection (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006536#sec013" target="_blank">Methods</a>). Data are mean ± SEM of viability (% control) for each condition in 6 independent experiments. ANOVA/Bonferroni (**p < 0.01). See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006536#pgen.1006536.s017" target="_blank">S9 Table</a> for statistics.</p