Generation, functional analysis and applications of isogenic three-dimensional self-aggregating cardiac microtissues from human pluripotent stem cells

Abstract

Tissue-like structures from human pluripotent stem cells containing multiple cell types are transforming our ability to model and understand human development and disease. Here we describe a protocol to generate cardiomyocytes (CMs), cardiac fibroblasts (CFs) and cardiac endothelial cells (ECs), the three principal cell types in the heart, from human induced pluripotent stem cells (hiPSCs) and combine them in three-dimensional (3D) cardiac microtissues (MTs). We include details of how to differentiate, isolate, cryopreserve and thaw the component cells and how to construct and analyze the MTs. The protocol supports hiPSC-CM maturation and allows replacement of one or more of the three heart cell types in the MTs with isogenic variants bearing disease mutations. Differentiation of each cell type takes ~30 d, while MT formation and maturation requires another 20 d. No specialist equipment is needed and the method is inexpensive, requiring just 5,000 cells per MT.

Fig. 1. Outline of in vitro differentiation protocol from hiPSCs to ECs, CFs and CMs.

Schematic of the protocol for differentiation of the three cell types used for MT production with indication of the media and supplements used.

Fig. 2. Outline of in vitro 3D cardiac microtissues (MTs) formation and analysis.

Graphical illustration describing MT generation from frozen stocks of ECs, CFs and CMs and characterization of structural and functional properties using: immunofluorescence, contraction and calcium transient analysis, Seahorse analysis, sharp electrode electrophysiology and single-cell dissociation.

Fig. 3. Morphology of undifferentiated hiPSCs.

Representative brightfield images of hiPSCs cultured in E8/VTN (a) or StemFlex/Laminin (b). Scale bars, 200 μm.

Fig. 4. hiPSC-EC characterization.

(a) Representative brightfield image of CD34⁺ hiPSC-ECs 6 d postisolation and before freezing. Scale bar, 200 μm. (b) Representative brightfield image of CD34⁺ hiPSC-ECs after thawing at higher magnification. Scale bar, 200 μm. (c) Flow cytometry analysis of CD34⁺ hiPSC-ECs showing a representative histogram for the expression of the EC marker CD31. Isotype negative control (grey) and hiPSC-ECs are stained with CD31 (red). (d) Representative immunofluorescence images of the endothelial marker CD31 in hiPSC-ECs (day 12). Nuclei are stained with DAPI (blue). Scale bar, 50 μm.

Fig. 5. hiPSC-EPI and hiPSC-CF characterization.

(a) Brightfield images at different time points showing the epithelial mesenchymal transition of hiPSC-EPI (day 12) toward hiPSC-CFs (day 28) during cardiac fibroblast differentiation. Scale bar, 200 μm. (b) Representative immunofluorescence images of the epicardial markers WT1, TBX18, CX43 (green) and ZO-1 (red) in hiPSC-EPI (day 12). Nuclei are stained with DAPI (blue). Scale bar, 50 μm. (c) Representative immunofluorescence images of the fibroblast markers VIM, COL1A1, GATA4 (red) and CX43 (green) in hiPSC-CFs (day 28). Nuclei are stained with DAPI (blue). Scale bar, 50 μm.

Fig. 6. hiPSC-CM characterization.

(a) Brightfield images of hiPSC-CMs after 21 d of differentiation using LI-BPEL protocol with lactate purification (left panel) or mBEL protocol (right panel). Scale bars, 200 μm. (b) Expression of cardiac Troponin T (cTnT) by hiPSC-CMs. Representative flow cytometry analysis of hiPSC-CMs generated using either the LI-BPEL with lactate purification (left panel) or mBEL (right panel). Histograms show hiPSC-CMs stained with isotype control (LI-BPEL with lactate purification) or unstained (mBEL) (grey), and stained with cTnT (red).

Fig. 7. 4: Examples of optimal and sub-optimal cardiac MTs after 24h after formation.

Representative images of compact and round-shaped MT (a) and not properly formed MT (b). Scale bar 200 μm.

Fig. 8. Overview of correct Seahorse plating conditions.

(a) Bent and stubbed Pasteur pipette used for MT positioning. Scale in cm. (b) MTs directly after plating at an incorrect position in the plate. Scale bar, 400 μm. (c) Correct positioning of MTs in the middle of the Seahorse well. Scale bar, 400 μm.

Fig. 9. Characterization of 3D MTs after 21 d in culture.

(a) Immunofluorescence analysis of cardiac-(TNNI, green), fibroblast-(COL1A1, red) and endothelial-(CD31, grey) markers in d21-MTs. Nuclei are stained in blue with DAPI. Scale bar, 50 μm. (b) Representative immunofluorescence images for cardiac sarcomeric proteins ACTN2 (red) and TNNI (green), showing the organization of sarcomere structures in 3D MTs. Nuclei are stained in blue with DAPI. Scale bar, 20 μm. (c) Representative contraction traces from MTs stimulated at 1Hz, 2Hz and 3Hz, as indicated. (d) Representative traces of mitochondrial stress test (oxygen consumption rate, OCR, upper panel) and glycolytic stress test (extracellular acidification rate, ECAR, lower panel) from Seahorse measurements of MTs. N = 16 MTs/4 wells. Data are shown as mean ± SEM of 4 wells for the line and mean for each well of 4 MTs for the scatter. (e) Representative action potential (AP) traces recorded from hiPSC-CMs from standard 2D culture (left panel) and dissociated from MTs (right panel). The cells were not artificially hyperpolarized and APs were recorded using the perforated patch clamp technique in current clamp mode.