Comparative Single-Cell RNAseq Analysis of Cardiac Progenitor Cells from a Holt-Oram Syndrome Patient iPS Line and the CRISPR/Cas9 Corrected Isogenic iPS Line

Background: To investigate the role of TBX5 during human cardiogenesis we used a patient-specific human iPS (hiPS) line from a male Holt-Oram Syndrome (HOS) patient with a TBX5 mutation in the DNA-binding domain (c.920_C>A) leading to an amino acid change (Pro85Thr) and dramatically reduced TBX5 protein activity. Cardiac progenitor cells (CPCs) generated from the patient-specific hiPS line (1460) and the isogenic CRISPR/Cas9 corrected TBX5 hiPS line (1460corr) were analyzed to study TBX5 function in a human patient specific model.

Method: Patient-specific and isogenic CRISPR/Cas9 TBX5 corrected hiPS cells were differentiated in parallel using a directed cardiac differentiation protocol. CPCs (day 8) from both lines were dissociated into single cells (SCs) and processed by droplet-based sc-RNAseq with barcodes for each cell and Unique Molecule Identifiers for each RNA transcript, followed by several amplification and purification steps before sequencing. SC transcriptomes were subjected to downstream bioinformatic analysis including unsupervised clustering and differential expression testing. Identity of clusters was assigned using characteristic cardiac subtype markers.

Results: Sc-RNAseq revealed 13 distinct identities without any fibroblasts or smooth muscle cells and very few endothelial cells (1460 = 0.06%, 1460corr = 0.95% of total cells). Eight of 13 identities were clearly defined as CPCs for both lines (1460corr = 95.3%, 1460 = 89.3% of total cells). Surprisingly, proportion of cells in the CPC identities clearly differed between the lines. CPC identities showing first, and second heart field markers were 19.5 to 31.3% lower in 1460 compared with 1460corr. Common differences for all CPC identities between the lines were seen for heart chamber development (WNT2, TNNI3), maturation (KRT19, PLN) and energy metabolism (ENO1). Furthermore, genes like FAM162A (pro-apoptotic) and the transcription factor GATA4 were differentially expressed. Common upregulated genes in the mutated CPC identities were assigned to biosynthetic/metabolic processes.

Conclusion: Sc-RNAseq of CPCs of isogenic hiPS lines, which only differ in the TBX5 mutation revealed differences in cell composition and gene expression, possibly contributing to the development of HOS. Accordingly, the patient-specific hiPS model is well suitable to define temporary and spatial cardiac lineage specific expression patterns in comparative analyses to define the impact of TBX5 in cardiogenesis.

Publication History

Article published online:
28 January 2023