Transcriptome analysis of non human primate-induced pluripotent stem cell-derived cardiomyocytes in 2D monolayer culture vs. 3D engineered heart tissue

Abstract

Aims: Stem cell therapy has shown promise for treating myocardial infarction via re-muscularization and paracrine signalling in both small and large animals. Non-human primates (NHPs), such as rhesus macaques (Macaca mulatta), are primarily utilized in preclinical trials due to their similarity to humans, both genetically and physiologically. Currently, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) are delivered into the infarcted myocardium by either direct cell injection or an engineered tissue patch. Although both approaches have advantages in terms of sample preparation, cell-host interaction, and engraftment, how the iPSC-CMs respond to ischaemic conditions in the infarcted heart under these two different delivery approaches remains unclear. Here, we aim to gain a better understanding of the effects of hypoxia on iPSC-CMs at the transcriptome level.

Methods and results: NHP iPSC-CMs in both monolayer culture (2D) and engineered heart tissue (EHT) (3D) format were exposed to hypoxic conditions to serve as surrogates of direct cell injection and tissue implantation in vivo, respectively. Outcomes were compared at the transcriptome level. We found the 3D EHT model was more sensitive to ischaemic conditions and similar to the native in vivo myocardium in terms of cell-extracellular matrix/cell-cell interactions, energy metabolism, and paracrine signalling.

Conclusion: By exposing NHP iPSC-CMs to different culture conditions, transcriptome profiling improves our understanding of the mechanism of ischaemic injury.

Keywords: Cardiomyocytes; Engineered heart tissue; Hypoxia; Induced pluripotent stem cells; Non-human primate; Transcriptome.

Figure 1

Diagram of experimental design. Skin fibroblasts were grown from a 1-year-old male rhesus monkey skin biopsy. This was followed by generating iPSCs which were differentiated using a cardiac differentiation protocol to derive the NHP iPSC-CMs for generation of the 2D monolayer and 3D EHT. Both culture conditions (2D, 3D) were then exposed to hypoxic conditions to allow transcriptional analysis by RNA-seq.

Figure 2

Design and fabrication of the master mold, PDMS mold, and NHP EHTs. (A) Master mold designed by AutoCAD and fabricated by 3D printing, and sizes in three dimensions are shown. (B) PDMS mold with flexible posts. (C) EHT formation in the PDMS mold at 30 min and 24 h. (D) Immunofluorescence staining of NHP EHTs at Day 50 with alpha-actinin (green), troponin T (red), and DAPI (blue) showing striated sarcomeres.

Figure 3

Differential transcriptome of NHP iPSC-CMs in 2D and 3D culture models under normoxic or hypoxic conditions by RNA-seq analysis. (A) Heatmap of four groups: 2D-Normoxia, 2D-Hypoxia, 3D-Normoxia, and 3D-Hypoxia, and with four clusters segregated, N = 3 biological samples in each group. (B) PCA for each group: red represents hypoxia, green represents normoxia, a solid circle represents 2D, and solid triangle represents 3D. (C) Overall gene expression trends of four clusters in 2D and 3D culture models under normoxic vs. hypoxic conditions. N = 3 biological samples in each group. The detailed RNA-seq and statistical analysis are in the Methods section.

Figure 4

Enrichment analysis in the groups of 2D-Normoxia, 2D-Hypoxia, 3D-Normoxia, and 3D-Hypoxia. (A) Top 10 pathways in four clusters by GO enrichment analysis. (B) Corresponding genes of the activated pathways with GO numbers in the following four categories: cell–cell/cell–ECM interactions, energy metabolism, hypoxic responses, and paracrine signalling.

Figure 5

Networks of selective genes and pathways. The corresponding four clusters by the RNA-seq enrichment analysis interconnect genes and pathways belonging to the cell–cell/cell–ECM interactions, energy metabolism, hypoxic responses, and paracrine signalling.

Figure 6

Validation of gene and protein expressions in four groups: 2D-Normoxia, 2D-Hypoxia, 3D-Normoxia, and 3D-Hypoxia. (A) Relative VEGF, IL-8, and MCP-1 secretions. N = 4∼6 biological samples in each group, Student’s t-test. (B) Relative VEGFA, CXCL8, and CCL2 expressions. N = 3 biological samples in each group, Student’s t-test. (C) Profile of key genes expressed in 2D and 3D culture models under normoxic or hypoxic conditions to validate the enrichment analysis in the categories: cell–cell/cell–ECM interactions, energy metabolism, hypoxic responses, and paracrine signalling, N = 3 biological samples in each group.