Comparative expression analysis of Shox2-deficient embryonic stem cell-derived sinoatrial node-like cells

Abstract

The homeodomain transcription factor Shox2 controls the development and function of the native cardiac pacemaker, the sinoatrial node (SAN). Moreover, SHOX2 mutations have been associated with cardiac arrhythmias in humans. For detailed examination of Shox2-dependent developmental mechanisms in SAN cells, we established a murine embryonic stem cell (ESC)-based model using Shox2 as a molecular tool. Shox2 and Shox2 ESC clones were isolated and differentiated according to five different protocols in order to evaluate the most efficient enrichment of SAN-like cells. Expression analysis of cell subtype-specific marker genes revealed most efficient enrichment after CD166-based cell sorting. Comparative cardiac expression profiles of Shox2 and Shox2 ESCs were examined by nCounter technology. Among other genes, we identified Nppb as a novel putative Shox2 target during differentiation in ESCs. Differential expression of Nppb could be confirmed in heart tissue of Shox2 embryos. Taken together, we established an ESC-based cardiac differentiation model and successfully purified Shox2 and Shox2 SAN-like cells. This now provides an excellent basis for the investigation of molecular mechanisms under physiological and pathophysiological conditions for evaluating novel therapeutic approaches.

Subtype-specific differentiation of cardiac pacemaker cell clusters from human induced pluripotent stem cells

“…This is underlined by the fact that culturing in serum-enriched medium leads to upregulation of SHOX2 (Fig. 2c ), a transcription factor known to antagonize the transcriptional output of Nkx2.5 [ 52 ]. Moreover, the role of Nkx2.5 is further supported by a recent study demonstrating that pacemaker-type cells are recruited from Nkx2.5-negative progenitors [ 10 ].…”

“…Notably, these approaches beside the hanging drop method and diverse enrichment strategies usually apply a considerable amount of FBS (10–20%) as an integral component to achieve differentiation of attached cells. To investigate the subtype specification of SAN-like cells during differentiation, comparative expression analysis of SAN-specific marker genes SHOX2 and HCN4 have been established for positive control in these studies [ 52 ], according to our approach.…”

Functional Characterization of Rare Variants in the SHOX2 Gene Identified in Sinus Node Dysfunction and Atrial Fibrillation

“…Recently, our findings were confirmed by the identification of a heterozygous nonsense mutation (p.R194X) in a patient with atrial fibrillation, which was absent in controls and co-segregated with the disease phenotype in a family with multiple affected members with complete penetrance (Li et al, 2018). Shox2 regulatory networks tightly control the pacemaker gene program in the developing sinus node and in nodal-like cells of the pulmonary vein myocardium, as demonstrated in different models (Puskaric et al, 2010; Hoffmann et al, 2013; Ye et al, 2015a; Ye et al, 2015b; Hoffmann et al, 2017). Several genes with pathogenic variants encoding specific ion channels (e.g.…”

Exploring Shared Susceptibility between Two Neural Crest Cells Originating Conditions: Neuroblastoma and Congenital Heart Disease

“…Another relevant gene is SHOX2 (3q25.32), a member of the homeobox family which is one of the major genes involved in the development of the sinoatrial node [50]; its proper function is of crucial relevance for the origin of arrhythmogenic heart disease [51]. Moreover, SHOX2 is implicated in specifying neural systems involved in processing somatosensory information, as well as in face and body structure formation [52,53] and has been reported as involved in Cornelia de Lange syndrome—a condition that implies heart defects [52,54].…”