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. 2022 Jul 22;10:908848. doi: 10.3389/fbioe.2022.908848

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Copyright © 2022 Kahn-Krell, Pretorius, Guragain, Lou, Wei, Zhang, Qiao, Nakada, Kamp, Ye and Zhang.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Experimental design. (A) hiPSCs were differentiated into CMs, ECs, SMCs, and CFs via established methods. (B) Spheroids containing all four cell types (C4 spheroids) were produced by combining a 4:2:1:1 ratio of CMs, ECs, SMCs, and CFs in 96-well U plates and allowing the cells to fuse for 7 days; then, (C) the spheroids were transferred to 6-well plates and (D) cultured with shaking for to 60 days. (E) Procedures for spheroid manufacture and experimental analyses are displayed on a timeline.