Chemically defined generation of human cardiomyocytes

Abstract

Existing methods for human induced pluripotent stem cell (hiPSC) cardiac differentiation are efficient but require complex, undefined medium constituents that hinder further elucidation of the molecular mechanisms of cardiomyogenesis. Using hiPSCs derived under chemically defined conditions on synthetic matrices, we systematically developed an optimized cardiac differentiation strategy, using a chemically defined medium consisting of just three components: the basal medium RPMI 1640, L-ascorbic acid 2-phosphate and rice-derived recombinant human albumin. Along with small molecule-based induction of differentiation, this protocol produced contractile sheets of up to 95% TNNT2(+) cardiomyocytes at a yield of up to 100 cardiomyocytes for every input pluripotent cell and was effective in 11 hiPSC lines tested. This chemically defined platform for cardiac specification of hiPSCs will allow the elucidation of cardiomyocyte macromolecular and metabolic requirements and will provide a minimal system for the study of maturation and subtype specification.

Figure 1

Chemically defined differentiation protocol for efficient cardiac differentiation of hiPSCs. a) Schematic of optimized chemically defined cardiac differentiation protocol. E8, chemically defined pluripotency medium; EDTA, ethylenediaminetetraacetic acid used for clump cell passaging; TZV, thiazovivin, a Rho kinase inhibitor; VTN, vitronectin; CDM3, chemically defined medium 3 components; Δ, medium change. b) Simple three-component formula of CDM3. c) Comparison of total live cell yield from differentiations in RPMI+B27-ins and CDM3, measured by flow cytometry for cardiac troponin T (TNNT2) on day 15 cells (hiPSC line 59FSDNC3 shown), n = 4. d) Comparison of cardiac differentiation efficiency from differentiations in RPMI+B27-ins and CDM3, n = 4. e) Typical TNNT2⁺ populations in cells produced from differentiations in RPMI+B27-ins medium and CDM3. Left peak represents isotype control. f) Effect of inhibition of signaling pathways during early mesoderm differentiation. Small molecules were added at designated time points (day 0–2 or day 1–2), at 5 µM for all except Wnt-C59, which was used at 2 µM. Normal doses of CHIR99021, Wnt-C59, and medium change timings were maintained, n = 3. g) Effect of inhibition of signaling pathways post mesoderm induction. The same as f) but assessing cardiac induction time points (day 2–4, day 3–4, or day 4–6). Slight reduction of cardiac differentiation efficiency with Wnt-C59 on day 2 to day 4 was likely due to the combined total dose (4 µM) being suboptimal (Supplementary Fig. 4i), n = 3. All error bars represent S.E.M.

Figure 2

Characterization and purification of cardiomyocytes produced by chemically defined differentiation. a) Immunofluorescence staining for TNNT2 and α-actinin (cardiomyocyte structural markers), α-smooth muscle actin (primitive cardiomyocytes), vWF (endothelial cells), P4HB (fibroblasts), and Ki67 (proliferating cells). Scale bar, 12.5 µm. b) Efficiency of chemically defined cardiac differentiation measured by flow cytometry for TNNT2⁺ on day 15 cells in a range of peripheral blood or fibroblast-derived hiPSC generated with Sendai virus, ‘Yamanaka’ episomal plasmids, or a mini intronic plasmid (CoMiP). All hiPSC and hESC lines were cultured under chemically defined conditions and differentiated in CDM3, n ≥ 3. Error bars represent S.E.M. c) Schematic of chemically defined cardiac differentiation including purification by metabolic selection. CDM3L: CDM3 without D-glucose and supplemented with 5 mM sodium DL-lactate. d) Effect of chemically defined metabolic purification measured by flow cytometry for TNNT2 on day 20 cells. Showing cells with no lactate treatment, 6 days of lactate treatment (day 10–16), and 10 days lactate treatment (day 10–20). e) Assessment of gene expression heterogeneity using single cell real time RT-PCR of day 20 cardiomyocytes differentiated in CDM3 without metabolic purification.

Figure 3

Characterization of atrial vs. ventricular profile of cardiomyocytes produced under chemically defined conditions. a) Flow cytometry assessment of expression of cardiac troponin T (TNNT2), atrial myosin light chain 2 (MLC2A), and ventricular myosin light chain (MLC2V) in cardiomyocytes derived and maintained in CDM3 at differentiation from day 10 through day 60, n = 3. Error bars represent S.E.M. Additional data is provided in Supplementary Figs. 11 and 12. b) Immunofluorescence staining of day 20 cardiomyocytes with the same antibodies used for flow cytometry to demonstrate specificity. Scale bar, 12.5 µm.

Figure 4

Electrophysiological characterization of cardiomyocytes produced under chemically defined conditions. a) Representative action potential (AP) recordings using whole cell patch of three cardiomyocyte subtypes produced from day 15–20 cells and day 30–35 cells. Cells exhibit AP morphologies that can be categorized as atrial-, nodal-, or ventricular-like. b) Proportions of cardiomyocyte subtypes at day 15–20, n = 21 and day 30–35, n = 13. c) Patch clamp recordings of differentiation day 15–20 and day 30–35 cells, demonstrating MDP, maximum diastolic potential; peak voltage; APA, action potential amplitude; AP duration at different levels of repolarization (i.e., 90 or 50%); and dV/dt_max (maximal rate of depolarization). d) Assessment of cells differentiated in CDM3 at day 30 of differentiation using MEA-based nanopillar, representative trace of ventricular-like and atrial-like subtypes. e) Percentages of ventricular-like and atrial-like cells, n = 20. f) Depolarization time; APD, action potential duration; AP duration at different levels of repolarization (i.e., 50 or 90%) and AP morphology used to classify cardiomyocyte subtype. Error bars represent S.E.M.