Use of Adeno-Associated Virus to Enrich Cardiomyocytes Derived from Human Stem Cells

Abstract

Cardiomyocytes derived from human induced pluripotent stem cells (iPSCs) show great promise as autologous donor cells to treat heart disease. A major technical obstacle to this approach is that available induction methods often produce heterogeneous cell population with low percentage of cardiomyocytes. Here we describe a cardiac enrichment approach using nonintegrating adeno-associated virus (AAV). We first examined several AAV serotypes for their ability to selectively transduce iPSC-derived cardiomyocytes. Results showed that AAV1 demonstrated the highest in vitro transduction efficiency among seven widely used serotypes. Next, differentiated iPSC derivatives were transduced with drug-selectable AAV1 expressing neomycin resistance gene. Selection with G418 enriched the cardiac cell fraction from 27% to 57% in 2 weeks. Compared with other enrichment strategies such as integrative genetic selection, mitochondria labeling, or surface marker cell sorting, this simple AAV method described herein bypasses antibody or dye labeling. These findings provide proof of concept for large-scale cardiomyocyte enrichment by exploiting AAV's intrinsic tissue tropism.

Chronic optical pacing conditioning of h-iPSC engineered cardiac tissues

“…We chose ChIEF because this channel does not desensitize with chronic light stimulation, and we chose an AAV1 vector because of its safety and efficient transfection of h-iPSC-CMs. 52,64,65 These three properties exhibited utility as demonstrated by the significant results of C-OP (Figures 1 and 5(b)). Similar CM percentages between D14 control and D14C-OP ECTs supported low AAV toxicity (Figure 5(e)).…”

Modeling reduced contractility and impaired desmosome assembly due to plakophilin-2 deficiency using isogenic iPS cell-derived cardiomyocytes

“…To test proof of concept for gene replacement therapy in human cells, we generated an AAV, containing the N-terminal, FLAG-tagged, full-length human PKP2 sequence driven by the CMV promoter (AAV2 -PKP2 ; Figure 6 A). We selected the AAV2 serotype because AAV2 has been shown to efficiently transduce iPSC-CMs ( Guan et al., 2015 ), and the use of AAV2 led to high transduction efficiency in iPSC-CMs under our experimental conditions ( Kohama et al., 2020 ; Shiba et al., 2021 ) ( Figure S6 A). FLAG-tagged plakophilin-2 protein localized properly at the cellular periphery in iPSC-CMs ( Figure 6 B), and AAV2-mediated delivery of plakophilin-2 restored the localization of desmoglein-2, desmocollin-2, and N-cadherin in the cell-cell junctions of NHEJ-iPSC-CMs ( Figures S6 B and S6C).…”

“…AAV-mediated gene delivery of PKP2 recovered contractility and desmosome assembly in plakophilin-2-deficient iPSC-CMs To test proof of concept for gene replacement therapy in human cells, we generated an AAV, containing the N-terminal, FLAG-tagged, full-length human PKP2 sequence driven by the CMV promoter (AAV2-PKP2; Figure 6A). We selected the AAV2 serotype because AAV2 has been shown to efficiently transduce iPSC-CMs (Guan et al, 2015), and the use of AAV2 led to high transduction efficiency in iPSC-CMs under our experimental conditions (Kohama et al, (E) The images shown in (D) were quantitatively analyzed using high-content imaging. Top: raw immunostained images and captured intensity images detected using high-content imaging.…”

Adeno-associated virus-mediated gene delivery promotes S-phase entry-independent precise targeted integration in cardiomyocytes

“…gRNA was designed in an antisense direction targeting a PAM sequence located 19 bp downstream of the MYL2 stop codon to avoid Cas9-mediated re-cleavage of exon 7. We generated AAV2 encoding a gRNA sequence and repair template, because AAV2 was previously shown to efficiently transduce hiPSC-CMs 26 , and was found to transduce hiPSC-CMs with high efficiency in our experimental conditions (Supplementary Fig. 5 B).…”