Methods for producing sinoatrial node cells (pacemaker cells) from stem cells and uses of the produced sinoatrial node cells

Abstract

Currently used electrical pacemakers in therapeutic regimens for the treatment of "sick sinus syndrome" are not adjustable in terms of hormones and have the risk of infection or premature battery discharge. These problems can be overcome with the aid of "bio pacemakers" from pluripotent stem cells (PSCs). It has been shown that TBX (an inducer of sinoatrial node cells) for the controlled differentiation of stem cells and additional Myh6 promoter-specific antibiotic selection can produce myocardial cell aggregates consisting of more than 80% physiologically functional pacemaker cells. These induced sinoatrial bodies ("iSABs") exhibit very high beating frequencies (300-400 bpm) for the first time, similar to the beating frequencies in murine hearts, and are able to stimulate isolated myocardial cells rhythmically and stably. In the iSAB transcriptome decoded with the aid of RNA-seq, it is possible to assign almost all genes to the ontology of cardiac function / cardiac development and the structure of contractile cells. In summary, this is the first example of high-purity functional sinoatrial tissue derived from stem cells, which means that a key step is being taken towards future cell therapies and in vitro drug testing.

Description

[0001] The term "sick sinus syndrome" is used as a collective term to describe a series of disorders caused by impaired function of the sinoatrial node (the pacemaker of the heart). Structurally, it is formed from specialized myocardial cells innervated by the autonomic nervous system. The disorders include pathologically symptomatic sinus bradycardia, SA block (SA = sinoatrial), sinus arrest, and tachycardia-bradycardia syndrome.

[0002] Sick sinus syndrome is often accompanied by general cardiac disorders, such as ischemic heart disease, cardiomyopathy or myocarditis. These either lead to the formation of damaged action potentials in the sinoatrial node, or lead to damaged conduction of electrical impulses from the sinoatrial node to the atrium (the latter is called "sinoatrial conduction abnormality"). At present, the therapeutic measures of "sick sinus syndrome" are based on the implantation of artificial cardiac pacemakers that are very expensive and not sensitive to hormonal stimulation. Another factor is that the risks caused by infection and premature battery discharge lead to serious limitations. Thus, patients with implanted pacemakers are usually at high risk of serious complications for the rest of their lives.

[0003] These defects can be avoided by the availability of functional SA node cells (synonyms: sinoatrial node cells, node cells, cardiac pacemaker cells) for transplantation or by their de novo generation in vivo. Node cells are characterized by their low membrane potential, diastolic depolarization, and low ascending branch velocity. Several different ionic currents participate in diastolic depolarization and the action potential in the SA node, including the pacemaker current I f This current is carried by HCN channels, which are regulated by cyclic nucleotides. The cAMP binding site in the HCN channel is able to regulate the activation achieved by catecholamines, and this property may control the autonomic regulation of the pacemaker mechanism [1]. The isoforms of the channel (called HCN1 to HCN4) are encoded by four genes. The most prevalent isoform in the SA node is HCN4 [2][3].

[0004] To obtain biological pacemaker cells for future therapy, two approaches have been proposed:

[0005] On the one hand, the goal is to convert beating cardiomyocytes in situ by means of genetic manipulation of cardiac pacemaker cells (“direct reprogramming”). In this regard, TBX3 (an essential early transcription factor) leads to cells with incomplete pacemaker properties [4]. In recent years, it has been reported that viral overexpression of Tbx18 (a member of the same transcription factor family) can reprogram ventricular myocardium into sinoatrial node cells [5]. However, this operation requires a viral vector, which prevents the spatial and temporal regulation of the expression of this reprogramming factor for simulating the in vivo situation of the developing embryo. In addition, the efficiency is very low [5]. In addition, it has been shown that Tbx18 is only transiently expressed in the head of the evolving sinoatrial node, while the Tbx3 factor used here (i.e. in the invented scheme) for controlled differentiation (compared to direct reprogramming) is permanently expressed in vivo in the intact sinoatrial node [6].

[0006] Another approach is based on the transplantation of "biological pacemakers" produced in vitro, which have been obtained from pluripotent stem cells (stem cells = S cells or SC) such as embryonic stem cells (ESC or ES cells) or induced pluripotent stem cells (iPSC) [7][8]. In this regard, it has been assumed that the low molecular weight compound EBIO would to some extent enhance the formation of nodal cells from mouse ES cells [9]. However, the publication does not address the actual ability of the cells to stimulate ventricular cardiomyocytes, and the beating frequency of the cells is also (too) low. In addition, at the electrophysiological level, no distinction is made between relatively mature pacemaker cells and early / intermediate cell types that also contract spontaneously [9]

[10]

[11] .

[0007] It has recently been reported that sinoatrial node cells can be generated from cell populations purified by detection of Alcam expression (CD166). However, the applicability to human systems is unclear here, as the specificity of surface markers is often not conserved between species

[12] .

[0008] It is therefore an object of the present invention to provide cardiac pacemaker cells (sinoatrial node cells) with improved pacing properties, which have been obtained in vitro from stem cells in high yield.

[0009] This object is achieved by the method as claimed in claim 1. Further preferred embodiments are disclosed in the dependent claims. In other words, the object is achieved by a method for the production of sinoatrial node cells ("cardiac pacemaker cells") from stem cells, wherein either a nucleic acid is introduced into the stem cells as a result of which these express a TBX transcription factor, or a TBX protein is introduced into the stem cells, wherein the method is characterized in that additionally a construct for the expression of an antibiotic resistance gene is introduced under the control of the alpha-MHC (MYH6) promoter and the resulting stem cells are differentiated in the presence of the antibiotic. By "resulting stem cells" is meant stem cells which comprise the TBX (nucleic acid or protein) and the construct.

[0010] This constitutes a further development of the alternative type of controlled differentiation of ES cells which was recently introduced by the inventors for the production of specific cardiomyocyte subtypes by various transcription factors

[13]

[14]

[11] . It is an "alternative type" because the focus has been on other subtypes so far.

[0011] By applying the described procedure (i.e. by controlled differentiation by various transcription factors (TBX, in particular TBX3)), it has been demonstrated that this has led to a multiplication of functional pacemaker cells. However, this was still not sufficient to obtain a pure population of these cells. This is related to the fact that TBX3 as a single factor cannot accomplish the functional reprogramming [4].

[0012] Therefore, according to the present application, the method is combined with an antibiotic selection based on the Myh6 promoter

[15] . This provides cell aggregates which exclusively consist of spontaneously beating cardiomyocytes (KM cells) and whose beating frequency for the first time approximates the beating frequency of a (murine) heart.

[0013] In these cells, more than 60%, in particular more than 70%, most preferably more than 80% exhibit the desired pacemaker cell type (based on the cardiomyocyte subtype produced), which is characterized by protein expression, electrophysiological parameters and Ca 2+ The level of transient as well as its efficacy for stable ex vivo stimulation of cardiomyocyte cultures, in particular murine cardiomyocyte cultures, has full functionality. Some literature has mentioned a 60% yield, but without making a distinction between early / intermediate cells and differentiated pacemaker cells. The former also beat spontaneously (like cardiac pacemaker cells), but have in other respects different properties which make them unsuitable as cardiac pacemaker cells (i.e. they are at the beginning of the differentiation phase and can / will still produce all subtypes, i.e. not only pacemaker cells, but also atrial and ventricular cells).

[0014] The results were confirmed by RNA-seq analysis of the cells or cell aggregates obtained according to the invention, also known as "induced sinoatrial bodies" (iSABs), with regard to their overall mRNA expression profile. Thus, the present invention has generated a highly enriched population of pacemaker cells derived from stem cells, which possess all the properties characteristic of this cell type and which will be of great importance for the in vitro evaluation of future cell therapies and drugs.

[0015] If a nucleic acid is introduced into stem cells for the expression of a TBX transcription factor, this is preferably selected from TBX DNA, in particular TBX cDNA; or TBX RNA, in particular TBX mRNA. In the context of RNA, TBX mRNA can be transfected into stem cells, although this does not result in stable genetic modification. Alternatively, it is possible to introduce micro-RNA, which leads to the expression of endogenous TBX. In a preferred embodiment of the nucleic acid introduction, TBX DNA, in particular TBX cDNA, is introduced with the aid of a vector, in particular with the aid of an (over)expression vector. TBX is preferably selected from TBX3 or TBX-18, TBX3 being particularly preferred, and TBX3 cDNA being most preferred. In other words, the most preferred variant is the introduction of TBX3 cDNA using an overexpression vector. With respect to TBX proteins, which likewise do not result in (stable) genetic modification, TBX3 is preferred.

[0016] Human or non-human nucleic acids or proteins are used, preferably those of human origin.

[0017] About the stem cell used, use multipotent (multipotent) or multipotent (pluripotent), preferably multipotent (pluripotent) stem cell.Described stem cell can be selected from people or non-human embryonic stem cell, or people or non-human induced stem cell, or people induced stem cell, or parthenogenetic stem cell, or spermatogonial stem cell.They are preferably non-human embryonic stem cell, or non-human induced stem cell, or people induced stem cell, or parthenogenetic stem cell, or spermatogonial stem cell, more preferably non-human embryonic stem cell, or non-human induced stem cell, or people induced stem cell.In preferred and particularly preferred variant, clearly get rid of human embryonic stem cell.

[0018] The antibiotic selection of the present invention based on the Myh6 promoter preferably utilizes an antibiotic resistance gene selected from aminoglycoside antibiotic resistance genes, more preferably selected from neomycin and puromycin resistance genes, and most preferably a neomycin resistance gene. The antibiotic used for selection is correspondingly selected from aminoglycoside antibiotics, in particular neomycin and puromycin. "Correspondingly selected" means that the antibiotic corresponding to the resistance gene is always used; for example, in the case of a neomycin resistance gene, neomycin is subsequently used for selection.

[0019] In each case, cardiac pacemaker cells (human or non-human) are produced, preferably human cardiac pacemaker cells. For this purpose, human stem cells are combined with preferably human proteins or human nucleic acids. Cross-combinations, such as the introduction of human proteins or human nucleic acids into non-human (e.g., murine) stem cells, are also possible, as are pure combinations of non-human representatives for the production of non-human cardiac pacemaker cells.

[0020] As will be explained in detail below, the sinoatrial node cells of the present invention, which have been produced in vitro from stem cells (hereinafter also synonymously referred to as the sinoatrial node cells of the present invention or sinoatrial node cells produced from stem cells according to the present invention), exhibit the expected behavior when exposed to drugs: for example, the HCN channel blocker ZD-7288 and the muscarinic receptor antagonist carbachol cause a significant decrease in the beat frequency. In contrast, administration of the β-adrenergic receptor agonist isoproterenol leads to an increase in the beat frequency. Because the sinoatrial node cells of the present invention, which have been produced from stem cells, react to administered drugs in exactly the same way as sinoatrial node cells in vivo, they are suitable as models for normal sinoatrial node cells. Sinoatrial node cells produced in vitro from stem cells, in particular those produced in vitro from stem cells according to the present invention, are therefore particularly useful for the in vitro evaluation of drugs. This is to be understood as being synonymous with the investigation of active ingredients (i.e., potential drugs) for their practical suitability ("in vitro drug testing"). Potential candidates for novel drugs must be tested for their quality, safety and efficacy in mandatory preclinical and clinical studies before they are approved for marketing by drug regulatory agencies. Since preliminary biochemical and chemoinformatic studies have not yet given any final conclusions about how novel active ingredients work in vivo, novel active ingredients must be tested in preclinical studies, which has so far made large-scale animal experiments indispensable. The use of sinoatrial node cells produced in vitro from stem cells, in particular the sinoatrial node cells of the present invention, can at least help to reduce the scope of animal experiments. It is also possible to use sinoatrial node cells produced in vitro from stem cells, in particular the sinoatrial node cells of the present invention, in the early preclinical stages, because cell-based in vitro assays can often also be used in the study of active ingredient candidates and in studying toxicity, due to the fact that these basic aspects can be reflected in the pharmacology and toxicology in vivo.

[0021] Sinoatrial node cells produced in vitro from stem cells, in particular those produced according to the present invention, can be used to construct and / or culture cardiac tissue, in particular for therapeutic purposes, and also for de novo production in vivo. More specifically, the focus here is on the production of cell-based biological pacemakers.

[0022] The research underlying the present invention is presented in detail below:

[0023] Generation of a stable ES cell line overexpressing TBX3

[0024] Based on the high conservation of TBX3 protein in vertebrates, human TBX3 was inserted into mouse ES cells because its specific detectability is high there. For overexpression in ES cells, human TBX3-cDNA was inserted into pEF-DEST51 ​​(Invitrogen). From 20 independent clones, based on the results obtained from qRT-PCR ( Figure 1A ) Four clones were selected that represented the entire range of TBX3 mRNA overexpression levels from low to high. This overexpression level was confirmed at the protein level ( Figure 1B In FACS analysis, no effect of TBX3 overexpression on the percentage of pluripotency markers Oct-4 / Pou5f1-positive and Sox2-positive cells was found ( Figure 1C This corresponds to normal undifferentiated colony growth in LIF-containing medium ( Figure 1B ), and is consistent with previous results obtained with overexpression of MESP1 and NKX2-5

[13]

[14]

[11] . It was concluded that, similar to these factors, TBX3 alone does not have the ability to induce ES cell differentiation.

[0025] Effects of TBX3 overexpression on the yield and subtypes of spontaneously beating cardiomyocytes derived from ES cells

[0026] ES cell clones with high and medium TBX3 overexpression began to shrink early in differentiation and showed approximately 5-10 times the shrinkage area ( Figure 2A This increased cardiac differentiation is similar to the effects of MESP1 and NKX2-5 overexpression recently described by the inventors

[13]

[14]

[11] . In accordance with their enhanced beating activity, cardiomyocytes overexpressing TBX3 exhibited a normal expression profile of the sarcomere marker Myh6 ( Figure 2B ).

[0027] To further verify functionality and determine the proportions of cardiomyocyte subtypes, electrophysiological properties were analyzed by single-cell patch-clamp techniques as described

[13]

[14]

[16] , and the density of HCN (or fungal) channels was measured at day 18 of differentiation.

[0028] In general, all described subtypes of isolated beating cardiomyocytes obtained during EB development were present in preparations of TBX3 cell clones (i.e., ventricular, atrial, and SA / AV (pacemaker-type) cells as well as early / intermediate cells) ( Figure 2C; Additional Table 1). Action potentials generated by the different cell types did not differ significantly between TBX3 cells and control cells in terms of their individual parameters (e.g., MDP, DDR, rate of the ascending limb, and duration of the AP plateau phase) or in terms of their responses to β-adrenergic (isoproterenol) or muscarinic (carbachol) stimulation, supporting the hypothesis of correct development of cardiomyocytes ( Figure 2C ; Additional Table 1). However, a very high proportion of pacemaker-type subtypes was found in cells overexpressing TBX3, accounting for 38.5% of all cardiomyocytes. These cells did not have a plateau phase and had a high density of interesting channels (I f ) and a positive MDP > -60 mV, and exhibited the highest DDR values, which generally exceeded 60 mV / s. Conversely, they had the slowest rising limb rate and the fastest depolarization rate (<5 V / s) with the smallest positive overshoot (maximum of +10 mV). Furthermore, SA / AV cells responded to isoproterenol as expected, resulting in an acceleration of AP rate. In accordance with the high proportion of pacemaker cells in TBX3-selective cardiomyocytes, the number of cells expressing HCN4 channels (corresponding to the "interesting channel") was increased (Figure 2D).

[0029] To further increase the yield of SA / AV subtypes obtained by controlled differentiation with the help of TBX3, attempts were made to combine this approach with antibiotic selection based on the Myh6 promoter

[15] , since it was recently demonstrated that the latter approach specifically enriches for pacemaker cells

[17]

[18] , but with very unsatisfactory yields of no more than 40%.

[0030] For this purpose, a plasmid containing the Myh6-neomycin cassette

[15] was additionally inserted into the TBX3 clone. As expected, the administration of antibiotics (neomycin) during differentiation resulted in an enrichment of beating tissue in control cells obtained by antibiotic selection assisted by the Myh6 promoter (but without Tbx3), which was termed antibiotic-selected cardiac bodies ("aCaB"). In the same way, the double transgenic Myh6-TBX3 clones were enriched in spontaneously contracting areas. However, in the latter case, the beating rate of the corresponding beating cells was essentially doubled ( Figure 3A After introducing an additional dissociation step, further increases in beating rate were obtained in antibiotic-selected Myh6 promoter control cells and in double-transgenic Myh6-TBX3 cell clones. In the case of the latter cells, cell aggregates contracting at 300-400 bpm were obtained for the first time, which is close to the beating rate of the mouse heart (about 500 bpm) and suggests the formation of "induced sinoatrial bodies" (iSABs). Figure 3A ). Cells within the iSAB were positive for HCN4 and the connexins Cx45 and Cx30.2 ( Figure 3B ), a characteristic of pacemaker cells [19, 20].

[0031] The iSAB were further cultured for 3 weeks in gelatin-based cell culture dishes, resulting in an accumulated highly synchronized cell layer beating at > 350 bpm. The cells had the typical elongated form of sinoatrial cells auf Figure 3C ).

[0032] To investigate the electrophysiological parameters of cardiomyocytes derived from iSAB, the single-cell patch-clamp technique was used again. Of the 65 cells analyzed, 53 cells (81.5%) now corresponded to the pacemaker cell subtype that regularly and rapidly beats, and only 12 cells (18.5%) represented the cardiomyocyte subtype that irregularly and slowly beats. In addition, 43 of the 53 cells classified as pacemaker cell subtype exhibited the parameters of mature pacemaker cells, whereas 10 of these regularly and spontaneously beating cells were still immature Figure 3D , Table 2, additional). Although they had a short plateau phase and a more negative MDP, which distinguished them from mature pacemaker cells, they did exhibit typical characteristics of pacemaker cells such as the generation of regular spontaneous action potentials with a fast DDR and the typical pacemaker current I f (see, e.g., Figure 3B ).

[0033] Further evidence for their agreement with pacemaker cells, the Ca 2+ currents from the extracellular space and the intracellular Ca 2+ stores were characterized, which are generated by Ca 2+ channels in the sarcolemma or by the release of Ca 2+ from stores. The physiological functionality of the channels within the sarcolemma also determines the frequency of Ca 2+ transients. As a feature of pacemaker cells, the cells were examined for HCN channels, which modulate the frequency of Ca 2+ transients. The application of the HCN channel blocker ZD 7288 reduced the frequency of Ca 2+ transients in iSAB as a function of time Figure 3E ). In addition, the frequency of Ca 2+ transients is likewise based on the activity of voltage-dependent T- and L-type Ca 2+ channels. Accordingly, after inhibition of the L-type Ca 2+ channels with nifedipine, the spontaneous frequency of Ca 2+ transients in cells derived from iSAB dropped sharply, and the inhibition of T-type Ca 2+ channels by mibefradil led to a moderate decrease Figure 3F ).

[0034] Functional sarcoplasmic reticulin (SR) identifies the mature stage of cardiomyocytes. 2+ It plays an important role in spontaneous activity. 2+ When the complete release of Ca was induced by caffeine or when SERCA was inhibited with thapsigargin, the Ca from SR 2+ Spontaneous Ca 2+ This transient effect became clear. In cells derived from iSAB, caffeine-induced Ca 2+ Release will be with Ca 2+ Peak diastolic Ca increased in a comparable manner 2+ level, but in Ca 2+ There is a recognizable spontaneous Ca spike during the 2+ transient with unchanged systolic Ca 2+ value( Figure 3G A similar caffeine-induced increase in SR-Ca expression was not detected in cells derived from aCaB. 2+ Effect of thapsigargin on the release of Ca 2+ Blockade of Ca reuptake into the SR resulted in Ca upregulation only in cells derived from iSABs. 2+ Increased diastolic levels ( Figure 3H ).

[0035] As Ca 2+ transient basal Ca 2+ The predominant proportion of Ca is derived from the extracellular space. 2+ The exchange of Ca 2+ Transient elimination. When sarcolemmal Ca 2+ Inflow is Na + / Ca 2+ Exchanger and Ca 2+ When the channel blockade is eliminated, only intracellular Ca 2+ In this case, the addition of caffeine induces Ca2+ in cells derived from iSABs. 2+ peak, which was 4 times larger than that of cells derived from aCaB ( Figure 3I , K). Additional inhibition of SERCA under these conditions revealed SR leakage in cells derived from iSABs. Intracellular Ca 2+ The rate of accumulation was increased 4-fold compared to the treatment without SERCA inhibition. 2+ In contrast, in control cells, Ca 2+ The peak remained the same with and without SERCA inhibition ( Figure 3J , K), and it is also impossible to detect any intracellular Ca 2+ accumulation.

[0036] To finally examine the functional pacemaker activity of iSABs, we took advantage of the ex vivo model based on ventricular slices from cultured rat hearts

[21] ( Figure 4A Although these have spontaneous beating activity immediately after preparation, it is possible to induce stable contractions down to about 60 bpm in them, and as such they constitute an ideal test system for the functionality of the generated iSABs. As evident from labeling with Dil, iSABs seeded on the slices are able to attach to and survive on the slices ( Figure 4B To determine the effect of iSAB seeding, spontaneous slice activity was first carefully quantified in naive slices. While a maximum of approximately 70% of slices contained at least one area of ​​spontaneous beating activity on day 2 after slice preparation, this percentage dropped dramatically on days 3 and 4 ( Figure 4C Despite the iSAB seeding, slices already showed an approximately 1.5-fold increase in beating activity on days 1 and 2 compared to those seeded with aCaB, and their activity was exclusively maintained thereafter ( Figure 4C In addition, the slices contained significantly more very active areas per slice at days 3 and 4 compared to unseeded and aCaB-seeded slices ( Figure 4D In addition, the beating frequency of slices seeded with iSABs increased continuously and significantly by more than 4-fold from day 1 to day 4 ( Figure 4E ); this was not observed in the case of unseeded and aCaB-seeded slices (dns). Loading iSABs with calcein before transfer to slices confirmed syncytia formation between iSABs and ventricular cells, as can be seen from the dye versus time plot ( Figure 4F ). In addition, the functional coupling is derived from the synchronized Ca2+ transfer between iSAB and sliced ​​cardiomyocytes. 2+ Therefore, the Ca in the slice 2+ The transient peak is smaller but highly synchronized with spontaneous iSAB activity and can be detected within a radius of approximately 200 μm ( Figure 4G ).

[0037] Finally, RNA sequence analysis was used to determine the overall status of the transcriptome of iSAB with the goal of identifying new factors and / or markers important for pacemaker production. By means of this analysis, 220 genes that were significantly upregulated were identified in iSAB by comparison with the control group. Among these were Myh6 (33-fold upregulated), SERCA2 (20-fold upregulated), Ryr2 (39-fold upregulated), and Kcnj5 (45-fold upregulated). In summary, it was possible to assign the upregulated genes to 82 gene ontologies describing biological processes ( Figure 5A Of these, it is possible to classify the dominant number into the group that is (very) closely associated with heart and muscle function and heart development ( Figure 5B ). The underlying network of gene ontology is shown in Figure 5C In the addressing of cellular components, 220 genes were divided into 34 ontologies ( Figure 5D Likewise, they can be classified into groups that are highly related to the typical structures of contractile cells ( Figure 5E ). The underlying network of gene ontology is shown in Figure 5F Interestingly, nearly 12-fold more genes (>2,500) were significantly downregulated in iSAB, potentially related to the role of TBX3 as a transcriptional repressor. However, these genes were more closely linked to ontologies related to housekeeping processes, such as G protein-coupled receptor signaling cascades and mitochondrial function.

[0038] 4. Discussion

[0039] The ability to generate de novo a population of highly enriched cardiac pacemaker cells derived from stem cells, all of which contain the functional parameters of mature sinoatrial node cells, is of great importance for future cell-based therapies. This could be an enabling factor for restoring proper cardiac rhythm in the form of a "biological pacemaker". In addition, in vitro drug screening would benefit from the availability of such purified sinoatrial node cells. Pluripotent stem cells (PSCs) are a focus of these goals, as it has been demonstrated that any cell type in a mammalian organism can be generated from them, including spontaneously beating cardiomyocytes with the characteristic molecular and functional properties of SA cells / pacemaker cells

[10]

[22]

[23]

[24]

[25]

[11] . However, the cell population in the "embryoid body" is typically very heterogeneous, with the consequent need for reliable selection and isolation strategies - this is particularly relevant for the very rare cardiac sinoatrial node cell type. In this regard, cells derived from mouse ES cells have been described that contain EGFP transcriptionally controlled by the promoter of the HCN4 gene, which co-express EGFP, HCN4 and other cardiac markers in spontaneously beating regions

[26] . However, after purification by flow cytometry, spontaneous action potentials were only rarely observed in the EGFP-positive fraction, and interestingly, most cells were positive for nestin (a marker of neurons)

[26] . This is based on the knowledge that HCN4 is a marker for both spontaneously active cardiomyocytes and neurons

[27] . Therefore, as an alternative to this approach, the search for endogenous surface markers for the purification of these very popular cells has accelerated. In this regard, it has recently been reported that purification based on Alcam (CD166) increases the content of nodal cells. However, as is evident from HCN4 staining, the enrichment of the desired cell type does not even exceed 10% of the resulting cells obtained from ES cell cultures. Regarding the cells, it was reported that they were able to stimulate the contraction of ventricular cardiomyocytes cultured in vitro, but these important results were not presented in publication

[12] .

[0040] Other approaches have attempted to increase the yield of nodal cells from differentiating ESCs by pharmacological administration of low molecular weight compounds. Although some success has been reported (3-6 fold increase), this has never resulted in a sufficiently enriched population of functional sinoatrial node cells

[28] . Similarly, in publications related to the administration of the low molecular weight compound EBIO, which describes an increase in the proportion of nodal cells, a comprehensive analysis at the electrophysiological and functional levels is lacking [9].

[0041] To overcome this problem, a protocol was developed that combined programmed controlled differentiation of ES cells using TBX (particularly TBX3) as an important transcription factor with Myh6-assisted antibiotic selection

[15] . This consistently resulted in the very efficient production of sinoatrial node-like pacemaker cell aggregates characterized by spontaneously contracting cardiomyocytes with a highly synchronized beating frequency of 300-400 bpm, which for the first time approaches that in the mouse heart. The majority (>80%) of these cell clusters clearly represented the desired nodal cell type - a purity approaching this has not been achieved anywhere before [9]

[28]

[12] . Since the produced cells had all other properties of full functionality, i.e., protein expression profile, electrophysiological parameters, and Ca 2+ Signaling parameters have been investigated, leading some to propose that these pacemaker aggregates generated through genetic manipulation be termed "induced sinoatrial bodies" (iSABs). Furthermore, the generated iSABs were functional in their ability to stably stimulate isolated ventricular myocardium. Furthermore, RNA-seq-assisted analysis will provide the first total mRNA expression profile of pacemaker cells derived from stem cells, serving as a foundation for the investigation of novel factors and / or marker combinations as tools for programming and purification of pacemaker cells.

[0042] Remarkably, initial pilot experiments using TBX3-based programming alone resulted in a doubling of the number of functional pacemaker cells in the aggregates, with a significantly lower beating frequency. However, the introduction of an additional Myh6-promoter-assisted antibiotic selection step further dramatically increased the enrichment. The basic idea is based on a recent publication describing the enrichment of ventricular and precious cardiomyocytes by Myh6-assisted selection

[15]

[17]

[18] , although the original intention of this approach was to enrich for unlimited cardiomyocyte subtypes.

[0043] On the other hand, the fact that TBX3 alone does not lead to a pure population of ESC-derived nodal cells is based on its inability to achieve a complete direct reprogramming of the ventricular myocardium to form pacemaker cells [4]. In recent years, it has been enthusiastically reported that Tbx18, another T-box transcription factor, is able to convert working myocardium directly into sinoatrial node cells (“iSAN”). Although Tbx18 does appear more promising than Tbx3 in this specific respect, there are concerns such as very low efficacy and a relatively slow beating rate. In addition, this viral regimen is only controllable in vivo with great difficulty [5]. The new paper from this group [5a] accurately shows that the viral vector is also present in the lungs and spleen, and there is no clear area of ​​concentration even in the heart. In addition, in large animal in vivo models, pacemaker cell function could be maintained for only 2 weeks, after which it was lost again. In addition, it is still unclear whether adenoviral infection in the heart leads to inflammation and / or arrhythmias.

[0044] The present approach also differs from previous publications that mimicked biological pacemakers by merely manipulating end-effector molecules that form the basis of sarcolemmal electrophysiology, rather than generating fully functional nodal cells from scratch

[29]

[30] . In contrast, the present approach results in cells with not only electrical oscillations but also refined electrophysiology and Ca 2+ The present invention provides a novel method for the generation of cells that have the signaling properties and the distinct morphological characteristics of natural pacemaker cells. Thus, the method of the present invention represents the basis for a future alternative to electronic pacemakers.

[0045] Although the pacemaker cells generated here form synchronized and rapidly beating syncytia and are able to form electrical connections with isolated ventricular slice cultures and to stimulate contraction therein, their potential to become anchored within the cardia and stimulate contraction at physiological beating rates still needs to be analyzed. The applicability of the described protocol to human pluripotent stem cells (i.e., hESC, hiPSC)

[31]

[32]

[33]

[34]

[35]

[36] is directly possible. This may ultimately be a smooth road to the applicability of cell-based biological pacemakers, which are very important for clinical applications and for in vitro drug screening.

[0046] Materials and methods

[0047] qRT-PCR

[0048] Quantitative real-time PCR was performed using RNA isolated using the RNeasy kit (Qiagen). First-strand cDNA was obtained from 2 μg of RNA at 37°C using AMV reverse transcriptase (Amersham) and random hexamer primers. Real-time PCR was performed using the IQ Syber Green Super Mix Kit (Biorad) with an iCycler and MyiQ detection system (Biorad). Primers were constructed using DNA-Star software and the specificity of each primer pair was confirmed by agarose gel electrophoresis. For all primer pairs, the annealing temperature was 57°C, and the amplified mouse cDNA fragment corresponded to base pairs 812-934 of mGAPDH and base pairs 287-429 of human TBX3 (hTbx3). All samples were analyzed in duplicate, and total RNA from a combination of undifferentiated and differentiated ES cells and from mouse heart was used as a control, and a standard curve for the markers analyzed was drawn. Total RNA from each sample without reverse transcriptase was used as a negative control. In the absence of reverse transcriptase, no signal was obtained after 40 PCR cycles, indicating that all samples did not contain contaminating DNA. In addition, when reverse transcriptase was added in the absence of RNA template, no signal was obtained, indicating that there was no pollution of exogenous RNA or DNA. The standard curve of all genes showed an increase from the threshold cycle for each half of the template concentration. The evaluation of relative gene expression intensity was carried out based on the Δ CT method. Using GAPDH as a reference gene, the change multiple of the relative intensity of mRNA expression was calculated, and the expression value in the control cells was defined as 1.

[0049] Diaphragm clamp technology

[0050] Spontaneous action potentials and currents of contracting cardiomyocytes were recorded in a perforated patch configuration at 37°C using a MultiClamp 700B amplifier and pClamp10 software (Molecular Devices, Union City, USA). Offline data analysis was performed using Clampfit software (Molecular Devices, Union City, USA) or with the aid of Origin 6.0 software (Microcal, Northampton, USA). Patch pipettes were made from borosilicate glass and heat-polished and had a resistance of 2-5 MΩ after filling with intracellular solution containing 10 mM NaCl, 130 mM potassium aspartate, 0.04 mM CaCl2, 3 mM Mg-ATP, 10 mM HEPES, and 200 μg / ml amphotericin B, with pH adjusted to 7.2 with KOH. The extracellular (bath) solution contained: 140 mM NaCl, 5.4 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 5 mM HEPES, 5.5 mM glucose; the pH was adjusted to 7.4 with NaOH. f In some cases, 2 mM BaCl2 and 0.3 mM CdCl2 were added to the bath solution to block I KI and I Ca The I was measured by stepping from a holding potential of -40 mV to a test potential between -130 mV and +20 mV. f The current amplitude after 3 seconds during the -130 mV pulse was divided by the cell capacitance to determine the current (I f ) density. In order to determine I f The activation kinetics were obtained by fitting the current trace at a −130 mV step after the initial delay using the sum of two exponential functions. Where τ1 and τ2 are the fast and slow time constants of activation; τ1 is correspondingly referred to as τ because the slow component (A2) of HCN channel activation is typically <10% of the current amplitude. Isoproterenol or carbachol (Sigma, Taufkirchen, Germany) was dissolved directly in the bath solution on the day of the experiment and applied to the cells with the aid of a rapid exchange surface perfusion system. APs were recorded at a sampling rate of 10 kHz. Analysis was performed on the raw traces. The slope of the linear fit of the distance from the MDP to the potential threshold is the DDR; the AP duration is the time from the threshold potential to the MDP.

[0051] Ca 2+ Imaging

[0052] All Ca 2+Transients were measured by fluorescence imaging microscopy (Visitron Systems) and The images were analyzed using imaging software. A cooled CCD digital camera was used in 4*4 frame mode to record Ca2+ at an excitation wavelength of 470 / 40 nm (50 ms) and an emission wavelength of 525 / 50 nm. 2+ iSAB and aCaB were loaded with 2.5 μM Fluo-4 / AM in differentiation medium and kept at 37°C for 30 min. After the second medium change, the measurement was performed at 28°C. The peak fluorescence intensity (F) of the Ca in ESCs was used as the 2+ Transients were normalized to the minimum fluorescence intensity (Fo) during the time of analysis.

[0053] The HCN channels were inhibited by 5 μM ZD 7288 (Sigma-Aldrich), the voltage-dependent T-type Ca channels were inhibited by 1 μM mibefradil (Sigma-Aldrich), and the 2+ channels and inhibited the voltage-dependent L-type Ca 2+ channels with 1 μM nifedipine (Sigma-Aldrich). 2+ channels, affecting spontaneous Ca 2+ Transient. For sarcolemmal Ca 2+ For experiments related to transport blockade, the culture medium was replaced with Tyrode's solution with the following composition (in mM): LiCl 140, KCl 6, MgCl 1, glucose 10, EGTA 1, HEPES 5, pH 7.4 (adjusted with KOH). Caffeine (10 mM, Sigma-Aldrich) was added (to open the ryanodine-coupled SR-Ca 2+ channels) and thapsigargin (2 μM, Sigma-Aldrich) (to inhibit SERCA), the Ca 2+ Ca storage 2+ Transient Effects: ZD 7288, nifedipine, and Fluo-4 / AM were dissolved in DMSO (final concentration <0.1%), and thapsigargin was dissolved in ethanol (final concentration 0.2%). Mibefradil and caffeine were dissolved in H2O.

[0054] Slice preparation and culture

[0055] Animal experiments were performed according to the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, Publication No. 85-23, revised 1996) and approved by the Bavarian government. Hearts were rapidly removed from adult mice of both sexes and transferred to ice-cold modified Tyrode's solution (composition in mM): NaCl 136, KCl 5.4, MgCl2 1, CaCl2 0.9, NaH2PO4 0.33, glucose 10, 2,3-butanedione monoxime 30, HEPES 5, pH 7.4 (adjusted with NaOH). The ventricles were freed of arteries, flaps, and blood vessels and embedded in 4% low-melting-point agarose gel, which was dissolved in modified Tyrode's solution without glucose. The heart containing agarose was adhered to the sample container of a Vibratom (VT1200S, Leica) and quickly covered with ice-cold Tyrode's solution. The heart was cut into 300 μm-thick tissue sections parallel to the plane of the flap using a steel knife (Wilkinson), which produced circular sections of the ventricular myocardium.

[0056] After incubation in ice-cold Tyrode's solution for 30 min, the sections were applied to the Biopore tissue culture inserts (Millicell, Millipore). TM Membrane, for cultivating at air / culture medium interface.Tissue culture insert is put into the culture dish containing culture medium M199, and described culture medium M199 is rich in 1% insulin-transferrin-selenium (Gibco) and 1% penicillin / streptomycin (Sigma).Slice is cultivated in incubator (37 ℃, 5%CO 2) until the co-cultivation stage.For the routine determination of the viability of slice, they are incubated 40min at 37 ℃ with thiazolyl blue-tetrazolium bromide (MTT, 0.5mg / mL, Sigma), and analyzed under optical microscope.

[0057] Co-culture of iSAB and cardiac slices

[0058] Use CalceinRed TM (AAT Bioquest) labeled iSAB to observe living cells. For this purpose, iSAB was incubated with 10 mg / ml Calcein Red in an incubator. TMiSABs were incubated with iSABs / AM in differentiation medium at 37°C for 30 min. After centrifugation (1000 rpm, 5 min) and resuspension in differentiation medium, iSABs were applied to mouse heart slices using a pipette under a microscope. iSABs and heart slices were co-cultured in an incubator (37°C, 5% CO2). The beating frequency of the iSABs and the slice area was recorded daily using a Sony NEX-5N camera. The iSABs were detected by fluorescence imaging (Ex / Em 646 / 659 nm, Visitron Systems) using CalceinRed TM The transfer of iSAB into the slice cells was used to detect the coupling between iSAB and the slice. The contact area between iSAB and the slice was recorded every day and CalceinRed was analyzed visually. TM Distribution of fluorescence.

[0059] Ca in the contact area between iSAB and slice 2+ Imaging

[0060] Slices containing iSAB were loaded with 5 μM Fluo-4 / AM (Invitrogen) in M199 enriched with 1% insulin-transferrin selenium (Gibco) and 1% penicillin / streptomycin (Sigma) for 30 min at 37° C. Ca was recorded by fluorescence imaging microscopy (Visitron Systems) using a cooled CCD digital camera in 4×4 binning mode at an emission wavelength of 525 / 50 nm and an excitation wavelength of 470 / 40 nm (exposure time 30 ms). 2+ Signal. use Imaging software analyzes all Ca 2+ For transient states, the peak value reported is the fluorescence intensity (F), which is normalized to the minimum fluorescence intensity (Fo) in the corresponding region within the analysis time (20 s).

[0061] Cell culture

[0062] ES cell lines derived from the murine cell line GSES

[37] were cultured in DMEM medium containing high concentrations of glucose and stabilized glutamine (GIBCO) in the presence of 1000 U / mL leukemia inhibitory factor (LIF, Millipore), containing 10% FBS Superior (Biochrom), 100 μM non-essential amino acids (GIBCO), 1% penicillin / streptomycin (GIBCO), and 100 μM β-mercaptoethanol (Sigma). Differentiation was performed according to standard protocols in Iscove's basal medium (Biochrom) containing 10% FBS (Biochrom), 100 μM non-essential amino acids (GIBCO), 1% penicillin / streptomycin (GIBCO), and 450 μM 1-thioglycerol

[13] . Cells were passaged using trypsin / EDTA (GIBCO) at 70% confluence, which was usually achieved after 2-3 days. Differentiation was often performed for 3-4 passages after cell thawing. Cells were transformed with 15 μg of plasmid DNA using JetPEI (Peqlab) and subsequently selected with 10 μg / mL blasticidin (Invivogen) or 250 μg / mL hygromycin (Invitrogen) in 10 cm tissue culture dishes. Stable clones were manually selected, cultured, and tested with the aid of qRT-PCR and differentiation assays

[13] . To induce spontaneous formation of aCaBs and iSABs, positive clones as recently described

[15] were treated with antibiotics for enrichment of cardiomyocytes. Single cells required for the following physiological analyses were enzymatically isolated as described

[14]

[16] . To ensure successful production of aCaBs and iSABs, possible mycoplasma contamination was regularly monitored twice a week using the PCR-based MycoSPY kit system (Biontex).

[0063] RNA sequencing

[0064] For the generation of library and for sequencing, the culture medium of the adherent growth cells of cultivation is decanted, and the cells are washed and directly cracked by adding lysis buffer. In this lysate, 1 μ L is used for cDNA synthesis and amplified according to the manufacturer's instructions with SMARTer test kit (Clontech, Mountain View CA, USA). By annealing the primers specific for polyadenylic acid and adding the reverse transcriptase with terminal transferase activity, cDNA synthesis is started. Subsequently, the first chain cDNA of new synthesis is first extended with a homopolymer section by terminal transferase, then extended with a specific application tag by means of template conversion. The double-labeled cDNA obtained is amplified by means of PCR, by ultrasonic treatment (Bioruptor, Diagenode, Liège, Belgium; 25 cycles of 30s on / 30s off) fragmentation, and use NEBnext Ultra DNA library preparation kit (New England Biolabs, Ipswich MA, USA) to be converted into the Illumina sequence library with barcode. After PCR enrichment, the cDNA libraries were purified with AmpureXP magnetic beads (Beckman-Coulter, Brea CA, USA) and quantified in a Bioanalyzer 2100 (Agilent, Santa Clara CA, USA). The cDNA libraries were combined in equimolar amounts and sequenced in an Illumina Genome Analyzer IIx in single-read mode using a read length of 78 nucleotides and a range of 21 to 32 million crude reads per replicate.

[0065] For subsequent mapping and expression analysis, reads from Illumina sequencing were demultiplexed and mapped to the mm9 mouse genome using TOPHAT slicing (v1.4)

[38] and genome annotations (obtained from iGenomes, http: / / cufflinks.cbcb.umd.edu / igenomes.html) to aid in the detection of exon-exon boundaries. The assignment of reads to each gene was obtained using HTSeq (http: / / www-huber.embl.de / users / anders / HTSeq / ) and normalized using the DESeq R package

[39] . Differential expression was examined with the aid of DESeq using pairwise comparisons of each group with three biological replicates and a false discovery rate set to 0.05. All sequence and data analysis steps were performed on a local server on the Galaxy platform

[40] .

[0066] Description of the drawings

[0067] Figure 1: Functionality of TBX3 overexpression constructs in ES cells

[0068] (A) Twenty independent cell clones were stably transfected with an overexpression construct containing human TBX3 cDNA. Overexpression levels were analyzed by qRT-PCR. Four representative clones were selected for further analysis (data are reported as mean ± SD; n = 2). (B) Immunostaining of overexpressed TBX3 and actin in four selected clones confirmed the overexpression level of TBX3. (C) FACS analysis of Oct-4 / Pou5f1 and Sox2 did not show any effect of TBX3 overexpression with the addition of LIF on pluripotency (data are reported as mean ± SEM; n = 5).

[0069] Figure 2: Predominant emergence of pacemaker-type cardiomyocytes in Tbx3-differentiated ES cells

[0070] (A) Increase in spontaneous beating activity in independent TBX3 clones and in ES control cells (GSES) (data are reported as mean ± SEM, n>100). The control and four clones: clone #3, clone #7, clone #15, and clone #19 are each shown in this order from right to left, starting with the control shown in white in each case. (B) Confocal analysis of Myh6 expression in control and TBX3-overexpressing cardiomyocytes. Counterstaining of actin and nuclei. Scale bar: 10 μm. (C) Distribution of cardiomyocyte subtypes, including Ventr. - ventricular (23.1%); Atr. - atrial (8%); Pace. - sinoatrial node-like (38.5%); Interm. - intermediate / early type (38.8%). Horizontal bar: 100 ms; vertical bar: 20 mV. (D) HCN4-expressing cells had significantly increased TBX3 colonies at day 18 (data reported as mean ± SD; n = 5).

[0071] Figure 3: Physiological parameters of pacemaker-type cardiomyocytes derived by combined Tbx3 differentiation and Myh6 promoter selection

[0072] (A) Beating frequency over time after different treatment regimens of Myh6-neomycin control and Myh6-TBX3 cells. The highest beating frequency (300-400 bpm) was achieved by a combination of selective TBX3 programming, antibiotic selection, and an additional dissociation step (data reported as mean ± SD; n ≥ 5). (B) Expression of HCN4 (left-hand panel), Cx45 (middle panel), and Cx30.2 (right-hand panel) in iSAB. Actin and nuclei were counterstained. Scale bar: 10 μm. (C) Typical elongated cell morphology of sinoatrial node cells in synchronized cell layers obtained by plating with iSAB. Scale bar: 10 μm. (D) The distribution of pacemaker cells was evident from single-cell patch clamp and fungal channel measurements: over 81% pacemaker-type cells were obtained. Of these, 19% were immature pacemaker cells, while the others were mature pacemaker cells; n=65. (E) Spontaneous Ca 2+ activity in Myh6-TBX3 cells following inhibition of HCN channels with ZD7288. 2+ The frequency of transient states was significantly decreased (data reported as mean ± SEM; n≥7). (F) In the presence of T-type and L-type Ca2+ expression in the mitochondria and mitochondria, the transient states were significantly decreased (data reported as mean ± SEM; n≥7). 2+ After the passage, spontaneous Ca 2+ The frequency of spontaneous Ca transients decreased (data are reported as mean ± SEM; n>12). (G) Spontaneous Ca transients in cells derived from iSAB and aCaB before and in the presence of 10 mM caffeine. 2+ transient. The amplitude of the caffeine-induced spike in the former cells is related to the spontaneous Ca 2+ The transient maximum values ​​were comparable. n≥8. (H) Unlike cells derived from aCaB, thapsigargin had no effect on Ca 2+ Blockade of uptake into the SR results in increased diastolic Ca in iSAB-derived cells. 2+ Level. n≥11. (I) Blocking Na + / Ca 2+ Exchangers and sarcolemmal Ca 2+ Ca channels before and after 2+ Transient: Unlike control cells derived from EBs, a large caffeine peak was observed in cells derived from iSABs. n>16. (J) Inhibition of Ca 2+ Ca before and after ingestion 2+ Transient: Unlike cells derived from aCaB, in cells derived from iSAB, Na + / Ca 2+ Inhibition of the exchanger + SERCA inhibition results in intracellular Ca 2+ A rapid rise in n>24. In addition, a small caffeine peak was observed in both cases. (K) SRCa2+ Analysis of Ca efflux, a characteristic of cells with pacemaker activity, revealed increased intracellular Ca 2+ in cells derived from iSAB relative to aCaB. 2+ Accumulation rate. + / Ca 2+ Blockade of the exchanger plus inhibition of SERCA resulted in a decrease in intracellular Ca2+ that was comparable to the caffeine peak in cells derived from iSAB. 2+ In cells derived from aCaB, systolic Ca 2+ There was no difference in the accumulation rate or amplitude of the caffeine peak. Mean ± SEM; #p < 0.05 relative to no SERCA inhibition; *p < 0.05 relative to control.

[0073] Figure 4: Functionality of iSABs in an ex vivo model of cultured murine ventricular slices

[0074] (A) Ventricular slices cultured for 5 days (left-hand panel) and MTT staining for viability (right-hand panel). (B) Identification of DiI-labeled iSAB seeded on slices (right-hand panel: overlay). (C) Percentage distribution of iSAB-seeded, aCaB-seeded, and unseeded slices with at least one contraction zone over time. Spontaneous activity decreased after day 2, while activity in iSAB-seeded slices remained high (n ≥ 15). (D) Average number of active zones per iSAB-seeded, aCaB-seeded, and unseeded slice over time (data reported as mean ± SEM (MWSEM); n ≥ 15). (E) Increase in beating frequency in slices seeded with iSABs from day 1 to day 4 (data reported as mean ± SEM ((MW SEM); n ≥ 16). (F) Transfer of calcein dye from iSABs to recipient slices over time [Scale bar: 200 μm; “h”: hours; “d”: days]. (G) Stimulation of a slice region (arrowhead) in the direct environment of an iSAB (arrowhead) is accompanied by highly synchronized Ca 2+ transient.

[0075] Figure 5: Transcriptome status of iSAB determined by RNA-seq

[0076] Ontology descriptions that are familiar to those skilled in the art and are presented only in English are shown here in English.

[0077] (A) 82 gene ontologies describing biological processes that included 220 upregulated genes in iSAB relative to controls. (B) Clusters of ontologies related to cardiac / muscle function and cardiac development were predominant. (C) Consolidated network of gene ontologies related to biological processes. (D) 34 gene ontologies describing cellular components that included 220 upregulated genes in iSAB relative to controls. (E) Clusters of ontologies related to the typical structure of contractile cells were predominant. (F) Consolidated network of gene ontologies related to cellular components.

[0078] 7. Additional Information

[0079] Table 1: Cardiomyocyte action potential parameters obtained from Tbx3-programmed cell actions.

[0080] Data are mean ± SD (standard deviation).

[0081] In the table, a comma (,) in a number is represented by a dot (.).

[0082] Figure 6 : Representative action potentials obtained from Tbx3-programmed cells

[0083] (A, B) Cardiac muscle types with (A) ventricular-type and (B) atrial-type action potentials. (C, D) Pacemaker-type action potentials with (C) mature sinoatrial-type action potentials and (D) slightly immature pacemaker-type cells (called intermediate cells in the mouse embryonic heart). Horizontal time axis: 100 ms.

[0084] Table 2: Cardiomyocyte action potential parameters that have been obtained from iSAB.

[0085] Data are mean ± SD.

[0086] In the table, a comma (,) in a number is represented by a dot (.).

[0087] Figure 7 : Representative action potentials obtained from iSAB cardiomyocytes.

[0088] (A) Pacemaker-type action potential, similar to that generated from a fully grown rat sinoatrial node cell. (B) Action potential generated by a slightly immature pacemaker-type cell. Horizontal time axis: 100 ms.

[0089] Figure 8 : I from isolated iSAB cells f picture.

[0090] (A) Patch clamp scheme; voltage is applied to induce a current activated by hyperpolarization. (B) I fExamples of currents recorded from isolated iSAB-derived cells. (C) Current density and (D) time constant activated at -130 mV, showing robust I with slow activation kinetics typical of HCN4 channel subtypes. f Expression and maturation of sinoatrial node I f n=17; error bars: mean ± SD.

[0091] Figure 9 : Regulation of action potential (AP) rate in isolated Tbx3-programmed cells and iSAB-derived cells.

[0092] Responses to β-adrenergic (isoproterenol) and muscarinic (carbachol) stimulation resulted in typical accelerated versus slowed AP rates. iSAB-derived cells exhibited a more pronounced response to isoproterenol, with beat rates ranging up to 560 bpm. iSAB / iso: n = 9; iSAB / carb: n = 5; Error bars: mean ± SD.

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Claims

1. A method for producing sinoatrial node cells from stem cells, wherein a nucleic acid is introduced into the stem cells, as a result of which these express the TBX3 transcription factor, or a TBX3 protein is introduced into the stem cells, characterized in that A construct for expressing an antibiotic resistance gene controlled by an α-MHC (MYH6) promoter is additionally introduced, and the resulting stem cells are differentiated in the presence of antibiotics; and the method comprises an additional dissociation step.

2. The method for producing sinoatrial node cells from stem cells according to claim 1, wherein The stem cells are pluripotent stem cells.

3. The method for producing sinoatrial node cells from stem cells according to claim 1, wherein Based on the subtype of the obtained cardiomyocytes, the sinoatrial node cells were obtained with a high purity of over 60%.

4. The method for producing sinoatrial node cells from stem cells according to claim 1, wherein Based on the subtype of the obtained cardiomyocytes, the sinoatrial node cells were obtained with a high purity of over 70%.

5. The method for producing sinoatrial node cells from stem cells according to claim 1, wherein Based on the subtype of the obtained cardiomyocytes, the sinoatrial node cells were obtained with a high purity of over 80%.

6. The method for producing sinoatrial node cells from stem cells according to any one of claims 1 to 5, characterized in that: Multipotent stem cells or pluripotent stem cells are used.

7. The method for producing sinoatrial node cells from stem cells according to any one of claims 1 to 5, characterized in that: Non-human embryonic stem cells, non-human induced stem cells, human induced stem cells, parthenogenetic stem cells, or spermatogonial stem cells are used.

8. The method for producing sinoatrial node cells from stem cells according to any one of claims 1 to 5, characterized in that: Non-human embryonic stem cells, non-human induced stem cells, or human induced stem cells are used.

9. The method for producing sinoatrial node cells from stem cells according to any one of claims 1 to 5, characterized in that: The nucleic acid is selected from TBX3 DNA; TBX3 RNA.

10. The method for producing sinoatrial node cells from stem cells according to claim 9, wherein: The TBX3 DNA is TBX3 cDNA.

11. The method for producing sinoatrial node cells from stem cells according to claim 9, wherein: The TBX3 RNA is TBX3 mRNA.

12. The method for producing sinoatrial node cells from stem cells according to any one of claims 1 to 5, characterized in that: The introduction of TBX3 nucleic acid is achieved with the help of a vector.

13. The method for producing sinoatrial node cells from stem cells according to any one of claims 1 to 5, characterized in that: The introduction of TBX3 nucleic acid is achieved with the aid of an expression vector.

14. The method for producing sinoatrial node cells from stem cells according to any one of claims 1 to 5, characterized in that: The introduction of TBX3 nucleic acid was achieved with the aid of an overexpression vector.

15. The method for producing sinoatrial node cells from stem cells according to any one of claims 1 to 5, characterized in that: The antibiotic resistance gene is selected from aminoglycoside antibiotic resistance genes; and the antibiotic is correspondingly selected from aminoglycoside antibiotics.

16. The method for producing sinoatrial node cells from stem cells according to any one of claims 1 to 5, characterized in that: The antibiotic resistance gene is selected from neomycin and puromycin resistance genes; and the antibiotic is correspondingly selected from neomycin and puromycin.

17. The method for producing sinoatrial node cells from stem cells according to any one of claims 1 to 5, characterized in that: The antibiotic resistance gene is a neomycin resistance gene.

18. Use of sinoatrial node cells produced in vitro from stem cells according to the method of one of claims 1 to 17 for in vitro evaluation of drugs.

19. Use of sinoatrial node cells produced in vitro from stem cells according to the method of one of claims 1 to 17 for constructing a cardiac tissue construct and / or culturing cardiac tissue.

20. Use of the sinoatrial node cells produced in vitro from stem cells according to claim 19 for producing a cell-based biological cardiac pacemaker.

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