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Current protocols for the differentiation of human-induced pluripotent stem cells (hiPSC) into cardiomyocytes only generate a small amount of cardiac pacemaker cells. In previous work, we reported the generation of high amounts of cardiac pacemaker cells by co-culturing hiPSC with mouse visceral endoderm-like (END2) cells. However, potential medical applications of cardiac pacemaker cells generated according to this protocol, comprise an incalculable xenogeneic risk. We thus aimed to establish novel protocols maintaining the differentiation efficiency of the END2 cell-based protocol, yet eliminating the use of END2 cells. Three protocols were based on the activation and inhibition of the Wingless/Integrated (Wnt) signaling pathway, supplemented either with retinoic acid and the Wnt activator CHIR99021 (protocol B) or with the NODAL inhibitor SB431542 (protocol C) or with a combination of all three components (protocol D). An additional fourth protocol (protocol E) was used, which was originally developed by the manufacturer STEMCELL Technologies for the differentiation of hiPSC or hESC into atrial cardiomyocytes. All protocols (B, C, D, E) were compared to the END2 cell-based protocol A, serving as reference, in terms of their ability to differentiate hiPSC into cardiac pacemaker cells. Our analysis revealed that protocol E induced upregulation of 12 out of 15 cardiac pacemaker-specific genes. For comparison, reference protocol A upregulated 11, while protocols B, C and D upregulated 9, 10 and 8 cardiac pacemaker-specific genes, respectively. Cells differentiated according to protocol E displayed intense fluorescence signals of cardiac pacemaker-specific markers and showed excellent rate responsiveness to adrenergic and cholinergic stimulation. In conclusion, we characterized four novel and END2 cell-independent protocols for the differentiation of hiPSC into cardiac pacemaker cells, of which protocol E was the most efficient.

Lymphatic muscle cells orchestrate the contraction of collecting lymphatic vessels in mice.

Human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes raise the possibility of generating pluripotent stem cells from a wide range of human diseases. In the cardiology field, hiPSCs have been used to address the mechanistic bases of primary arrhythmias and in investigations of drug safety. These studies have been focused primarily on atrial and ventricular pathologies. Consequently, many hiPSC-based cardiac differentiation protocols have been developed to differentiate between atrial- or ventricular-like cardiomyocytes. Few protocols have successfully proposed ways to obtain hiPSC-derived cardiac pacemaker cells, despite the very limited availability of human tissues from the sinoatrial node. Providing an in vitro source of pacemaker-like cells would be of paramount importance in terms of furthering our understanding of the mechanisms underlying sinoatrial node pathophysiology and testing innovative clinical strategies against sinoatrial node dysfunction (i.e., biological pacemakers and genetic- and pharmacological- based therapy). Here, we summarize and detail the currently available protocols used to obtain patient-derived pacemaker-like cells.

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 (SANCMs) 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.

Background Glioblastoma is the most aggressive primary brain tumour in adults with a dismal prognosis and median overall survival of 12–14 months despite standard therapy. Novel discoveries in Cancer Neuroscience have revealed excitatory neuron-to-tumour synapses that drive glioma proliferation and invasion via AMPA-receptor activation, and vital tumour-autonomous calcium signalling mediated by KCa3.1 channels that sustain cancer cell network- and therapy resistance. Both mechanisms have been effectively targeted in preclinical studies using perampanel, a non-competitive AMPA-receptor antagonist, and senicapoc, a selective KCa3.1 blocker, suggesting promising avenues for treatment. This trial was designed to test the safety, pharmacokinetics, and biological effects of these drugs, as mono- and combination therapy, when added to standard-of-care treatment in newly diagnosed glioblastoma. Methods SENIPERA is a two-stage, phase 0/1, prospective, open-label, randomised clinical trial with an exploratory window-of-opportunity design. A total of 27–36 adult patients with newly diagnosed glioblastoma will be enrolled. Trial Part A will enrol 9–18 patients in a 3 + 3 dose-escalation design to determine the maximum tolerable dose (MTD) of senicapoc. In Part B, 18 patients will be randomised 1:1 to receive perampanel monotherapy or combination therapy with senicapoc at the established MTD. Study treatment begins upon enrolment 7–14 days before surgery and continues until 30 days after adjuvant radiochemotherapy. Primary endpoints are the MTD of senicapoc (mg) (Part A) and the proportion of patients discontinuing perampanel due to intolerance at the lowest dose (Part B). Secondary endpoints include safety parameters, overall and progression-free survival, objective response rate, and tumour-volume changes. Exploratory analyses will characterise pharmacokinetics, tumour drug penetration, and molecular- genetic tumour profiles. Discussion SENIPERA introduces a dual network-targeting strategy that simultaneously addresses neuronal-driven invasion and tumour network resilience in glioblastoma. By integrating senicapoc and perampanel with standard therapy in an early-phase, window-of-opportunity design, this study will establish the preliminary safety and pharmacological foundation for future trials evaluating safety and efficacy. The comprehensive translational analyses of tissue, cerebrospinal fluid, and blood will provide detailed insight into drug activity within the tumour microenvironment, informing the development of potential biologically guided treatment strategies for glioblastoma. Trial registration EU-CT: 2025–522605-37–00. Trial sponsor. Aarhus University Hospital. Department of Neurosurgery, Palle Juul-Jensens Boulevard 165, J617. Aarhus, Denmark. Protocol version and date. Version 1.0 November 26, 2025.

The embryonic development of sinus nodes (SAN) is co-regulated by multiple signaling pathways. Among these, the bone morphogenetic protein (BMP) and Wnt signaling pathways are involved in the development of SAN. In this study, the effects of BMP and Wnt signaling on the differentiation of SAN-like pacemaker cells (SANLPCs) were investigated. Human induced pluripotent stem cells (hiPSCs) were divided into four groups: control, BMP4, CHIR—3, and BMP4 + CHIR (CHIR: a Wnt signaling activator). The samples were tested at day (D) 15 of differentiation. The final protocol for the activation of BMP signaling at D0–D3 and reactivation of Wnt signaling at D5–D7 in the differentiation of hiPSCs were determined. The results showed that the mRNA levels of pacemaker markers (TBX18, SHOX2, TBX3, HCN4, and HCN1) were higher in the BMP4 + CHIR group than in the control group, and working myocardial genes were downregulated. The immunofluorescence assay revealed that the expression of SHOX2 and HCN4 increased in the BMP4 + CHIR group compared to that in the other groups. In addition, the results of patch clamps revealed that a funny current of higher density and typical SAN action potentials were recorded, except in the control group, in which the L-type calcium current was higher in the BMP4 + CHIR group than in the other groups. Finally, the proportion of SANLPCs (cTnT + NKX2.5 − ) was further enhanced by the combination of BMP4 and CHIR treatment. In summary, the combination of BMP and Wnt signaling promotes the differentiation of SANLPCs from hiPSCs.

Summary We aimed to determine how age-associated changes in mechanisms extrinsic and intrinsic to pacemaker cells relate to basal beating interval variability (BIV) reduction in vivo. Beating intervals (BIs) were measured in aged (23-25 months) and adult (3-4 months) C57BL/6 male mice (i) via ECG in vivo during light anesthesia in the basal state, or in the presence of 0.5 mg mL-1 atropine + 1 mg mL-1 propranolol (in vivo intrinsic conditions), and (ii) via a surface electrogram, in intact isolated pacemaker tissue. BIV was quantified in both time and frequency domains using linear and nonlinear indices. Although the average basal BI did not significantly change with age under intrinsic conditions in vivo and in the intact isolated pacemaker tissue, the average BI was prolonged in advanced age. In vivo basal BIV indices were found to be reduced with age, but this reduction diminished in the intrinsic state. However, in pacemaker tissue BIV indices increased in advanced age vs. adults. In the isolated pacemaker tissue, the sensitivity of the average BI and BIV in response to autonomic receptor stimulation or activation of mechanisms intrinsic to pacemaker cells by broad-spectrum phosphodiesterase inhibition declined in advanced age. Thus, changes in mechanisms intrinsic to pacemaker cells increase the average BIs and BIV in the mice of advanced age. Autonomic neural input to pacemaker tissue compensates for failure of molecular intrinsic mechanisms to preserve average BI. But this compensation reduces the BIV due to both the imbalance of autonomic neural input to the pacemaker cells and altered pacemaker cell responses to neural input.

Sinoatrial node dysfunction can manifest as bradycardia, leading to symptoms of syncope and sudden cardiac death. Electronic pacemakers are the current standard of care but are limited due to a lack of biological chronotropic control, cost of revision surgeries, and risk of lead- and device-related complications. We therefore aimed to develop a biological alternative to electronic devices by using a clinically relevant gene therapy vector to demonstrate conversion of cardiomyocytes into sinoatrial node-like cells in an in vitro context. Neonatal rat ventricular myocytes were transduced with recombinant adeno-associated virus vector 6 encoding either hTBX18 or green fluorescent protein and maintained for 3 weeks. At the endpoint, qPCR, Western blot analysis and immunocytochemistry were used to assess for reprogramming into pacemaker cells. Cell morphology and Arclight action potentials were imaged via confocal microscopy. Compared to GFP, hTBX18-transduced cells showed that hTBX18, HCN4 and Cx45 were upregulated. Cx43 was significantly downregulated, while sarcomeric α-actinin remained unchanged. Cardiomyocytes transduced with hTBX18 acquired the tapering morphology of native pacemaker cells, as compared to the block-like, striated appearance of ventricular cardiomyocytes. Analysis of the action potentials showed phase 4 depolarization and a significant decrease in the APD50 of the hTBX18-transduced cells. We have demonstrated that rAAV-hTBX18 gene transfer to ventricular myocytes results in morphological, molecular, physiological, and functional changes, recapitulating the pacemaker phenotype in an in vitro setting. The generation of these induced pacemaker-like cells using a clinically relevant vector opens new prospects for biological pacemaker development.

The sinoatrial node (SAN), the primary cardiac pacemaker, governs rhythmic heartbeats through spontaneous electrical impulses. While the classical “coupled‐clock” theory, integrating the membrane voltage clock (driven by cyclic ion channel activity) and the calcium clock (orchestrated by rhythmic sarcoplasmic reticulum Ca 2+ release), remains central to understanding pacemaker automaticity, recent research has unveiled multifaceted regulatory mechanisms that may complement this core model. This review synthesises current evidence on the critical roles of pacemaker cell‐microenvironment interaction, glutamatergic signalling via mitochondrial reactive oxygen species (ROS)‐Ca 2+ coupling, and novel molecular modulators such as CIRP, SGO1, and GLP‐1. These insights reveal a highly integrated and dynamic regulatory network that potentially modulates SAN automaticity under physiological and pathological conditions. Elucidating these mechanisms not only deepens our understanding of cardiac pacemaking but also identifies potential therapeutic targets for SAN dysfunction and associated arrhythmias.