E2A ablation enhances proportion of nodal-like cardiomyocytes in cardiac-specific differentiation of human embryonic stem cells

Abstract

Background: Human sinoatrial cardiomyocytes are essential building blocks for cell therapies of conduction system disorders. However, current differentiation protocols for deriving nodal cardiomyocytes from human pluripotent stem cells (hPSCs) are very inefficient. Methods: By employing the hPSCs to cardiomyocyte (CM) in vitro differentiation system and generating E2Aknockout hESCs using CRISPR/Cas9 gene editing technology, we analyze the functions of E2A in CM differentiation. Findings: We found that knockout of the transcription factor E2A substantially increased the proportion of nodal-like cells in hESC-derived CMs. The E2A ablated CMs displayed smaller cell size, increased beating rates, weaker contractile force, and other functional characteristics similar to sinoatrial node (SAN) cells. Transcriptomic analyses indicated that ion channel-encoding genes were up-regulated in E2A ablated CMs. E2A directly bounded to the promoters of genes key to SAN development via conserved E-box motif, and promoted their expression. Unexpect enhanced activity of NOTCH pathway after E2A ablation could also facilate to induct ventricle workingtype CMs reprogramming into SAN-like cells. Interpretation: Our study revealed a new role for E2A during directed cardiac differentiation of hESCs and may provide new clues for enhancing induction efficiency of SAN-like cardiomyocytes from hPSCs in the future.

Cadherin‐5 facilitated the differentiation of human induced pluripotent stem cells into sinoatrial node‐like pacemaker cells by regulating β‐catenin

“…Furthermore, immunofluorescence staining showed a high expression level of cTNT in hiPSC‐CMs (Figure 1f). The expression profiles of pluripotency marker genes, including SOX2 , OCT4 , and NANOG , were assessed alongside mesoderm‐associated markers such as Brachyury T and ECOMES , cardiac mesoderm marker MESP1 , cardiac progenitor cell markers KDR , NKX2.5 , and ISL1 , and cardiomyocyte markers TNNT2 , MYH6 , and MYL7 during the differentiation of hiPSCs into cardiomyocytes (Figure 1g), which were consistent with previously reported observations (Li et al, 2021; Zhao, Wang, et al, 2020). These results indicated that our cardiac differentiation protocol was feasible.…”

“…The differentiation of human induced pluripotent stem cells (hiPSCs) has made it feasible to generate cardiomyocytes with characteristics similar to cardiac pacemaker cells. Although the yield of SAN‐like pacemaker cells (SANLPCs) derived from current 2D cardiac‐specific differentiation protocols has shown significant improvements, it is important to acknowledge that these studies have not yet validated the biological pacemaking function of the obtained SANLPCs in animal models (Li et al, 2021; Liang et al, 2020; Liu et al, 2020; Ren et al, 2019; Yechikov et al, 2020). A previous study made a significant advancement in the effectiveness of generating SANLPCs from human pluripotent stem cells through the implementation of 3D differentiation protocol and flow cytometry sorting techniques.…”

Protocatechuic aldehyde alleviates D-galactose-induced cardiomyocyte senescence by regulating the TCF3 / ATG5 axis

“…TCF3, as transcription factor, plays an essential role in treating acute lymphoblastic leukemia, breast cancer, colorectal cancer, etc., and especially heart diseases. 15,[36][37][38] As the prediction of bioinformation analysis, TCF3 was suspected as a transcription factor of ATG5. ATG5 was essential in the early stage of autophagosome formation and development, cell differentiation, and maintenance of homeostasis.…”

“…14 Besides, TCF3 was proved to play an essential role in heart development and cardiac-specific differentiation of human pluripotent stem cells. 15 Moreover, the overexpression of TCF3 in cardiomyocytes caused the disassembly of sarcomere, cell connection interruption, upregulation of immature markers, and a phenotype related to dedifferentiation of cardiomyocytes. 16 Meanwhile, TCF3 played an important role in heart formation.…”

Cardiac stem cells: Current knowledge and future prospects

“…Nodal signaling is vital for the formation of sinoatrial node. Nodal inhibition during the cardiac mesoderm differentiation stage downregulates PITX2c, a transcription factor recognized to inhibit the formation of the sinoatrial in the left atrium during cardiac development[ 19 ]. Moreover, nodal signaling is dispensable for initiation of heart looping; however, it regulates asymmetries that result in a helical shape at the heart tube poles[ 20 ].…”