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A single cell transcriptional roadmap of human pacemaker cell differentiation

ABSTRACT: Each heartbeat is triggered by the sinoatrial node (SAN), the primary pacemaker of the heart. Studies in animal models have revealed that pacemaker cells share a common progenitor with the (pro)epicardium, and that the pacemaker cardiomyocytes further diversify into “transitional”, “tail” and “head” subtypes. However, the underlying molecular mechanisms, especially of human pacemaker cell development are poorly understood. Here, we performed single cell RNA sequencing (scRNA-seq) and trajectory inference on human induced pluripotent stem cells (hiPSCs) differentiating to SAN-like cardiomyocytes (SANCM) to construct a roadmap of transcriptional changes and lineage decisions. In differentiated SANCM, we identified distinct clusters that closely resemble different subpopulations of the in vivo SAN. Moreover, the presence of a side population of proepicardial cells suggested their shared ontogeny with SANCM, as also reported in vivo. Our results demonstrate that the divergence of SANCM and proepicardial lineages is determined by WNT signaling. Furthermore, we uncovered roles for TGFβ and WNT signaling in the branching of transitional and head SANCM subtypes, respectively. These findings provide new insights into the molecular processes involved in human pacemaker cell differentiation, opening new avenues for complex disease modeling in vitro and inform approaches for cell-therapy based regeneration of the SAN.

ORGANISM(S): Homo sapiens

PROVIDER: GSE189782 | GEO | 2022/09/01

REPOSITORIES: GEO

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Project description:Background: Ion channels are key determinants for the function of excitable cells but little is known about their role and involvement during cardiac development. Earlier work identified Ca2+-activated potassium channels of small and intermediate conductance (SKCas) as important regulators of neural stem cell fate. Here, we have investigated their impact on the differentiation of pluripotent cells towards the cardiac lineage. Methods and Results: We have applied the SKCa-activator EBIO on embryonic stem cells and identified this particular ion channel family as a new critical target involved in the generation of cardiac pacemaker-like cells: SKCa-activation led to rapid remodeling of the actin cytoskeleton, inhibition of proliferation, induction of differentiation and diminished teratoma formation. Time-restricted SKCa-activation induced cardiac mesoderm and commitment to the cardiac lineage as shown by gene regulation, protein and functional electrophysiological studies. In addition, the differentiation into cardiomyocytes was modulated in a qualitative fashion, resulting in a strong enrichment of pacemaker-like cells. This was accompanied by induction of the sino-atrial gene program and in parallel by a loss of the chamber-specific myocardium. In addition, SKCa activity induced activation of the Ras-Mek-Erk signaling cascade, a signaling pathway involved in the EBIO-induced effects. Conclusions: SKCa-activation drives the fate of pluripotent cells towards the cardiac lineage and preferentially into pacemaker-like cardiomyocytes. This provides a novel strategy for the enrichment of cardiomyocytes and in particular, the generation of a specific subtype of cardiomyocytes, pacemaker-like cells, without genetic modification. Untreated ES cells in three independent experiments: - Untreated control ES cells sample 1 (Con_1) - Untreated control ES cells sample 2 (Con_2) - Untreated control ES cells sample 3 (Con_3) EBIO-treated ES cells in three independent experiments: - EBIO-treated ES cells sample 1 (EBIO_1) - EBIO-treated ES cells sample 2 (EBIO_2) - EBIO-treated ES cells sample 3 (EBIO_3) Untreated differentiated ES cells in two independent experiments: - Untreated control differentiated ES cells sample 1 (Con_day5+10_1) - Untreated control differentiated ES cells sample 2 (Con_day5+10_2) EBIO-treated differentiated ES cells in two independent experiments: - EBIO-treated differentiated ES cells sample 1 (EBIO_day5+10_1) - EBIO-treated differentiated ES cells sample 2 (EBIO_day5+10_2)

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A single cell transcriptional roadmap of human pacemaker cell differentiation

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