Suspended Tissue Open Microfluidic Patterning (STOMP).

Cell-laden hydrogel constructs suspended between pillars are powerful tools for modeling tissue structure and physiology, though current fabrication techniques often limit them to uniform compositions. In contrast, tissues are complex in nature with spatial arrangements of cell types and extracellular matrices. Thus, we present Suspended Tissue Open Microfluidic Patterning (STOMP), which utilizes a removable, open microfluidic patterning channel to pattern multiple spatial regions across a single suspended tissue.

Engineered Heart Tissues for Standard 96-Well Tissue Culture Plates.

Engineered heart tissues (EHTs) have been shown to be a valuable platform for disease investigation and therapeutic testing by increasing human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) maturity and better recreating the native cardiac environment. The protocol detailed in this chapter describes the generation of miniaturized EHTs (mEHTs) incorporating hiPSC-CMs and human stromal cells in a fibrin hydrogel. This platform utilizes an array of silicone posts designed to fit in a standard 96-well tissue culture plate.

Targeted CRISPR activation is functional in engineered human pluripotent stem cells but undergoes silencing after differentiation into cardiomyocytes and endothelium.

Human induced pluripotent stem cells (hiPSCs) offer opportunities to study human biology where primary cell types are limited. CRISPR technology allows forward genetic screens using engineered Cas9-expressing cells. Here, we sought to generate a CRISPR activation (CRISPRa) hiPSC line to activate endogenous genes during pluripotency and differentiation.

Full-length dystrophin deficiency leads to contractile and calcium transient defects in human engineered heart tissues.

Cardiomyopathy is currently the leading cause of death for patients with Duchenne muscular dystrophy (DMD), a severe neuromuscular disorder affecting young boys. Animal models have provided insight into the mechanisms by which dystrophin protein deficiency causes cardiomyopathy, but there remains a need to develop human models of DMD to validate pathogenic mechanisms and identify therapeutic targets. Here, we have developed human engineered heart tissues (EHTs) from CRISPR-edited, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) expressing a truncated dystrophin protein lacking part of the actin-binding domain.