Gene

Cell type

Cell biology

Induced pluripotent stem cell

Biology

Endothelial stem cell

Human Induced Pluripotent Stem Cells

Cell culture

Stem cell

Tissue engineering

Computational biology

Genetics

Embryonic stem cell

In vitro

Cell

Cellular differentiation

Nature Protocols

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.

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