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. We first targeted catalytically inactive Cas9 fused to VP64, p65 and Rta activators (dCas9-VPR) regulated by the constitutive CAG promoter to the AAVS1 safe harbor site. These CRISPRa hiPSC lines effectively activate target genes in pluripotency, however the dCas9-VPR transgene expression is silenced after differentiation into cardiomyocytes and endothelial cells. To understand this silencing, we systematically tested different safe harbor sites and different promoters. Targeting to safe harbor sites hROSA26 and CLYBL loci also yielded hiPSCs that expressed dCas9-VPR in pluripotency but silenced during differentiation. Muscle-specific regulatory cassettes, derived from cardiac troponin T or muscle creatine kinase promoters, were also silent after differentiation when dCas9-VPR was introduced. In contrast, in cell lines where the dCas9-VPR sequence was replaced with cDNAs encoding fluorescent proteins, expression persisted during differentiation in all loci and with all promoters. Promoter DNA was hypermethylated in CRISPRa-engineered lines, and demethylation with 5-azacytidine enhanced dCas9-VPR gene expression. In summary, the dCas9-VPR cDNA is readily expressed from multiple loci during pluripotency but induces silencing in a locus- and promoter-independent manner during differentiation to mesoderm derivatives. Researchers intending to use this CRISPRa strategy during stem cell differentiation should pilot their system to ensure it remains active in their population of interest.

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. Stromal cells and hiPSC-CMs are cast in a fibrin matrix suspended between two silicone posts, forming an mEHT that produces synchronous muscle contractions. The platform presented here has the potential to be used for high throughput characterization and screening of disease phenotypes and novel therapeutics through measurements of the myocardial function, including contractile force and calcium handling, and its compatibility with immunostaining.

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. The STOMP platform contains capillary pinning features along the open channel that controls the fluid front, allowing multiple cell and extracellular matrix precursors to be pipetted into one tissue. We have used this technique to pattern suspended tissues with multiple regional components using a variety of native and synthetic extracellular matrices, including fibrin, collagen, and poly(ethylene glycol). Here, we demonstrate that STOMP models a region of fibrosis in a functional heart tissue and a bone-ligament junction in periodontal tissues. Additionally, the STOMP platform can be customized to allow patterning of suspended cores and more spatial configurations, enhancing its utility in complex tissue modeling. STOMP is a versatile technique for generating suspended tissue models with increased control over cell and hydrogel composition to model interfacial tissue regions in a suspended tissue.

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. The 3D EHT platform enables direct measurement of contractile force, simultaneous monitoring of Ca2+ transients, and assessment of myofibril structure. Dystrophin-mutant EHTs produced less contractile force as well as delayed kinetics of force generation and relaxation, as compared to isogenic controls. Contractile dysfunction was accompanied by reduced sarcomere length, increased resting cytosolic Ca2+ levels, delayed Ca2+ release and reuptake, and increased beat rate irregularity. Transcriptomic analysis revealed clear differences between dystrophin-deficient and control EHTs, including downregulation of genes related to Ca2+ homeostasis and extracellular matrix organization, and upregulation of genes related to regulation of membrane potential, cardiac muscle development, and heart contraction. These findings indicate that the EHT platform provides the cues necessary to expose the clinically-relevant, functional phenotype of force production as well as mechanistic insights into the role of Ca2+ handling and transcriptomic dysregulation in dystrophic cardiac function, ultimately providing a powerful platform for further studies in disease modeling and drug discovery.