Response to Zhao and Huang’s Commentary Letter, “Conversion of Human Cardiac Progenitor Cells using Reprogramming Factors into Heterogeneous Cardiac Pacemaker-like Cells”, regarding our Manuscript: “Conversion of Human Cardiac Progenitor Cells into Cardiac Pacemaker-like Cells”

We wish to comment on Zhao and Huang’s Commentary Letter to the Editor regarding our research manuscript [1] entitled, “Conversion of Human Cardiac Progenitor Cells into Cardiac Pacemaker-like Cells”, where we showed that the SHOX2, HCN2, and TBX5 (SHT5) combination of transcription factors and channel protein can be used to reprogram human cardiac progenitor cells (CPCs) into Pacemaker-like cells as a potential stem cell therapy for sick sinus syndrome (SSS).

As described in our Raghunathan et al. [1] manuscript, we used a screening strategy to test for reprogramming factors for the conversion of human CPCs into Pacemaker-like cells. Specifically, human transcription factors SHOX2, TBX3, TBX5, TBX18, and the channel protein HCN2, were transiently induced as single factors and in trio combinations into CPCs, first being transduced with the connexin 30.2 (CX30.2) mCherry reporter. As pointed out by Zhao and Huang, these same single factors (SHOX2, TBX3, TBX5, TBX18, and HCN2) and combination of factors (SHT3, SHT5, and SHT18) were also used to directly reprogram human adipogenic mesenchymal stem cells (hADMSCs) into pacemaker cells and Purkinje cells, as reported in the US Patent by Alt and Karimi [2]. In both, identical reprogramming factors were used since all the viral vectors and technology in these reports were developed in collaboration in the Schwartz laboratory. However, a key difference between the methodology of these studies is that in Raghunathan et al. [1], the hADMSCs were first reprogrammed using the human transcription factors ETS2/MESP1 for the differentiation into CPCs [3]. In fact, we found at least an order of magnitude higher conversion rates when using CPCs, rather than hADMSCs, as the starting cells to test these reprogramming factors for the conversion into Pacemaker-like cells.

In addition, as reported in Raghunathan et al. [1], we performed RTPCR and RNA sequencing of SHOX2, HCN2, and TBX5 (SHT5) mCherry + and did not observe the conversion of our CPCs into Purkinje cells, as suggested by Zhao and Huang. Instead, SHT5 factors upregulated pacemaker specific gene and transcriptome expression, attributing to the pacemaker phenotype of these cells. Specifically, following screens for reporter CX30.2 mCherry gene activation and FACS enrichment, we observed the definitive expression of many pacemaker specific genes; including, CX30.2, KCNN4, HCN4, HCN3, HCN1, and SCN3b [4–9]. These findings suggest that the SHT5 combination of transcription factors is a much better candidate in driving the CPCs into Pacemaker-like cells than other combinations of transcription factors or individual factors by itself. Therefore, the schematic diagram by Zhao and Huang’s showing the conversion of CPC into Purkinje cells is not supported by Raghunathan et al. [1] The findings of our study [1] nicely show that the SHT5 combination of transcription factors and channel proteins can be used to reprogram CPCs into Pacemaker-like cells towards the construction of biological pacemakers.

Furthermore, we perform single-cell RNA sequencing on the SHT5 activated cells to better understand the transcriptome of the individual cells within the total cell population. Zhao and Huang did not fully appreciate that over 500 cells SHT5 mCherry + cells were single-cell sequenced which revealed cellular enrichment of pacemaker specific markers including SHOX2, GJD3 (CX30.2), TBX5, TBX3, BMP2, KCNN4 [10,11] the pacemaker specific transcripts for calcium and potassium channels including KCND2, KCNK2, CACNB1, CACNA1A [9] as well as the down-regulation of NKX2.5 [12,13]; where NKX2.5 is a specific ventricular conduction marker and highly enriched in Purkinje fibers [14]. Our Pacemaker-like cells did not display Purkinje signature markers. In fact, cells transfected with fewer than three factors did not express the pacemaker gene signature. Raghunathan et al. [1] did not show heterogeneity in the trio converted pacemakers. Thus, Zhao and Huangs argument for the SHT5 activated cells expressing Purkinje cell-specific markers is not supported by Raghunathan et al. [1] However, we did not test the idea that TBX factors are differentially expressed in nodes and their combinations may affect the transcriptome readout. Our plans are to test the combination of TBX factors in future studies.

Finally, we measured the HCN currents, characteristic of pacemaker cells, using patch-clamp recordings of the SHT5 activated cells and thus demonstrating the functional efficiency of these Pacemaker-like cells. Biological pacemaker cells are also sensitive to hormone regulation whereby β-adrenergic receptor (β-AR) stimulated cAMP binds to the HCN channel and thus contributing to the HCN pacemaker currents [15] . In fact, we demonstrated that the SHT5 cells are also sensitive to β-AR stimulation and thus further supporting the functional competence of these cells.

Overall, we demonstrated in Raghunathan et al. [1] that the SHT5 cocktail of transcription factors and channel protein reprogrammed CPCs into Pacemaker-like cells. The SHT5 factors resulted in the up-regulation of pacemaker specific gene expression and transcriptome expression, attributing the pacemaker phenotype to the cells. The SHT5 mCherry + cells also exhibited the funny current via HCN4 channels, attributing the functional characteristic of pacemaker cells. Thus, the findings of this study show that the SHT5 combination of transcription factors and channel protein can be used to reprogram CPCs into Pacemaker-like cells thus providing a strategy for the construction of a biological pacemaker to restore function of the failing sinoatrial node (SAN) and treat sick sinus syndrome (SSS) as well as for other cardiac conduction diseases.