Method for producing sinoatrial node cells (pacemaker cells) from stem cells, and use of the produced sinoatrial node cells

Abstract

The electrical pacemakers currently being used for the therapeutic approaches for treatment of “sick sinus syndrome” are not hormonally regulatable and entail risks through infections or premature battery discharge. These problems could be overcome by means of “biological cardiac pacemakers” obtained from pluripotent stem cells (PSCs). It has been shown that the controlled differentiation of stem cells with TBX, inductors of sinoatrial node cells, and an additional Myh6 promoter-specific antibiotic selection can give cardiomyocyte aggregates consisting to an extent of more than 80% of physiologically functional pacemaker cells. These induced sinoatrial bodies (“iSABs”) for the first time exhibited very high beat frequencies (300-400 bpm), similar to those in a murine heart, and were able to stably rhythmically stimulate heart muscle cells ex vivo. In the iSAB transcriptome decoded by means of RNA-seq, it was possible to assign almost all the genes to the ontologies of heart function/heart development and the structures of contractile cells. Overall, this is the first example of a high-purity functional sinoatrial tissue derived from stem cells, which means that a crucial step for future cell therapy and the testing of medicaments in vitro is being implemented.

Claims

1. A method of producing sinoatrial node cells from human stem cells, the method comprising the steps of: (A) introducing a nucleic acid into the stem cells, wherein the nucleic acid encodes a TBX3 transcription factor, or introducing a TBX3 protein into the stem cells to generate TBX3-overexpressing stem cells; (B) introducing a nucleic acid into the TBX3-overexpressing stem cells, wherein the nucleic acid encodes an antibiotic resistance gene under the control of an alpha-MHC (MYH6) promoter; (C) after introducing the nucleic acid encoding an antibiotic resistance gene under the control of an MYH6 promoter in step (B), culturing the TBX3-overexpressing stem cells under conditions in which the antibiotic resistance gene is expressed; (D) culturing the cells of step (C) with an antibiotic to which the cells expressing the antibiotic resistance gene are resistant; (E) selecting antibiotic-resistant Myh6-TBX3 cells; and (F) differentiating the antibiotic-resistant Myh6-TBX3 cells from step (E) into sinoatrial node cells having 50 to 90 action potentials per minute.

2. The method of producing sinoatrial node cells from stem cells according to claim 1, wherein the sinoatrial node cells are comprised in cell aggregates consisting of spontaneous beating cardiomyocytes (KM cells) and more than 60% of the KM cells are sinoatrial node cells.

3. The method of producing sinoatrial node cells from stem cells according to claim 1, wherein multipotent or pluripotent stem cells are used.

4. The method of producing sinoatrial node cells from stem cells according to claim 1, wherein human embryonic stem cells, human induced stem cells, human parthenogenetic stem cell, or human spermatogonial stem cells are used.

5. The method of producing sinoatrial node cells from stem cells according to claim 1, wherein the nucleic acid is TBX3 DNA.

6. The method of producing sinoatrial node cells from stem cells according to claim 1, wherein the introduction of the TBX3 nucleic acid is effected by means of a vector.

7. The method of producing sinoatrial node cells from stem cells according to claim 1, wherein the antibiotic resistance gene is an aminoglycoside antibiotic resistance gene and the antibiotic is an aminoglycoside antibiotic.

8. A method of producing sinoatrial node cells from murine stem cells, the method comprising the steps of: (A) introducing a nucleic acid into the stem cells, wherein the nucleic acid encodes a TBX3 transcription factor, or introducing a TBX3 protein into the stem cells to generate TBX3-overexpressing stem cells; (B) introducing a nucleic acid into the TBX3-overexpressing stem cells, wherein the nucleic acid encodes an antibiotic resistance gene under the control of an alpha-MHC (MYH6) promoter; (C) after introducing the nucleic acid encoding an antibiotic resistance gene under the control of an MYH6 promoter in step (B), culturing the TBX3-overexpressing stem cells under conditions in which the antibiotic resistance gene is expressed; (D) culturing the cells of step (C) with an antibiotic to which the cells expressing the antibiotic resistance gene are resistant; (E) selecting antibiotic-resistant Myh6-TBX3 cells; and (F) differentiating the antibiotic-resistant Myh6-TBX3 cells from step (E) into sinoatrial node cells having 300 to 700 action potentials per minute.

9. The method of producing sinoatrial node cells from stem cells according to claim 8, wherein the sinoatrial node cells are comprised in cell aggregates consisting of spontaneous beating cardiomyocytes (KM cells) and more than 60% of the KM cells are sinoatrial node cells.

10. The method of producing sinoatrial node cells from stem cells according to claim 8, wherein multipotent or pluripotent stem cells are used.

11. The method of producing sinoatrial node cells from stem cells according to claim 8, wherein the nucleic acid is TBX3 DNA.

12. The method of producing sinoatrial node cells from stem cells according to claim 8, wherein the introduction of the TBX3 nucleic acid is effected by means of a vector.

13. The method of producing sinoatrial node cells from stem cells according to claim 8, wherein the antibiotic resistance gene is an aminoglycoside antibiotic resistance gene and the antibiotic is an aminoglycoside antibiotic.

14. The method of producing sinoatrial node cells from stem cells according to claim 8, wherein murine embryonic stem cells, murine induced stem cells, murine parthenogenetic stem cell, or murine spermatogonial stem cells are used.

Description

DESCRIPTION OF FIGURES

(1) FIG. 1: Functionality of the TBX3 Overexpression Constructs in ES Cells

(2) (A) 20 independent cell clones stably transfected with the overexpression construct containing human TBX3 cDNA. The overexpression levels were analyzed by means of qRT-PCR. For further analysis, four representative clones were selected (data are reported as mean values±SD; n=2). (B) The immune staining of overexpressed TBX3 and actin in the four selected clones confirms the overexpression level of TBX3. (C) FACS analyses of Oct-4/Pou5f1 and Sox2 did not show any influence of TBX3 overexpression on pluripotency with the addition of LIF (data are reported as mean values±SEM; n=5).

(3) FIG. 2: Predominant Occurrence of Pacemaker-Type Cardiomyocytes in Tbx3-Differentiated ES Cells

(4) (A) Increase in spontaneous bead activity in independent TBX3 clones and in ES control cells (GSES) (data are reported as mean values±SEM, n>100). The control and the four clones clone #3, clone #7, clone #15 and clone #19 are each shown in this sequence from right to left, beginning in each case with the control shown in white. (B) Confocal analysis of Myh6 expression in control and TBX3-overexpressing cardiomyocytes. Counter-staining of actin and cell nuclei. Scale: 10 μm. (C) Distribution of the cardiomyocyte subtypes and 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 have significantly increased TBX3 clones on day 18 (data are reported as mean values±SD; n=5).

(5) FIG. 3: Physiological Parameters of the Pacemaker-Type Cardiomyocytes Obtained by Combined Tbx3 Differentiation and Myh6 Promoter Selection

(6) (A) Beat frequencies against time after different treatment regimes of Myh6-neomycin controls and Myh6-TBX3 cells. The highest beat frequencies (300-400 bpm) are achieved by a combination of selective TBX3 programming, antibiotic selection and an additional dissociation step (data are reported as mean values±SD; 5). (B) Expression of HCN4 (left-hand region), Cx45 (middle region) and Cx30.2 (right-hand region) in iSABs. Counter-staining of actin and cell nuclei. Scale: 10 μm. (C) Typical elongated cell form of sinoatrial node cells in the synchronized cell layers obtained by plating out iSAB. Scale: 10 μm. (D) Distribution of the pacemaker cells as apparent from single-cell patch-clamp and funny channel measurement: more than 81% pacemaker-type cells were obtained. Of these, 19% were immature pacemaker cells, while the others were mature pacemaker cells; n=65, (E) Frequencies of spontaneous Ca.sup.2+ transients in Myh6-TBX3 cells decrease significantly after inhibition of the HCN channels by ZD7288 (data are reported as mean values±SEM; n 7). (F) The frequencies of spontaneous Ca.sup.2+ transients in Myh6-TBX3 cells decrease after inhibition of T-type and L-type Ca.sup.2+ channels by mibefradil and nifedipine (data are reported as mean values±SEM; n>12). (G) Spontaneous Ca.sup.2+ transients before and in the presence of 10 mM caffeine in cells derived from iSABs and aCaBs. The amplitude of the caffeine-induced peak in the former cells is comparable to the maximum of the spontaneous Ca.sup.2+ transients. n 8. (H) Blockage of the Ca.sup.2+ uptake into the SR by thapsigargin leads, by contrast to the cells derived from aCaBs, to increased diastolic Ca.sup.2+ levels in the cells derived from iSABs. n≥11. (I) Ca.sup.2+ transients before and after blockage of the Na.sup.+/Ca.sup.2+ exchanger and of the sarcolemmal Ca.sup.2+ channels: in cells derived from iSABs, by contrast with the control cells derived from EBs, a large caffeine peak is observed. n>16. (J) Ca.sup.2+ transients before and after inhibition of Ca.sup.2+ uptake: inhibition of Na.sup.+/Ca.sup.2+ exchangers plus SERCA inhibition causes, by contrast with the cells derived from aCaBs, a rapid rise in intracellular Ca.sup.2+ in cells derived from iSABs. n>24. In addition, in both cases, a small caffeine peak was observed. (K) The analysis of the SR Ca.sup.2+ outflow, which is characteristic of cells active as pacemakers, shows an increased rate of accumulation of intracellular Ca.sup.2+ in cells derived from iSABs compared to aCaBs. Blockage of the Na.sup.+/Ca.sup.2+ exchanger plus inhibition of SERCA brought about intracellular Ca.sup.2+ accumulation comparable to the decrease in the caffeine peak in cells derived from iSABs. No difference in the rate of accumulation of systolic Ca.sup.2+ and amplitude of the caffeine peak in cells derived from aCaBs. Mean value±SEM; #p<0.05 vs. no SERCA inhibition; *p<0.05 vs. control is.

(7) FIG. 4: Functionality of iSABs in the Ex-Vivo Model of Cultivated Murine Ventricle Slices

(8) (A) Ventricle slices cultivated for five days (left-hand region) and MTT stain for checking of vitality (right-hand region). (B) Identification of the Dil-labeled iSABs sown on a slice (right-hand region: overlay). (C) Percentage distribution of iSAB-sown, aCaB-sown and unsown slices containing at least one contracting region over time. The spontaneous activity decreases after day 2, while the activity in the slices sown with iSAB is maintained to a high degree (n≥15). (D) Average number of active regions per iSAB-sown, aCaB-sown and unsown slice over time (data are reported as mean values±SEM (MW SEM); n≥15). (E) Increase in the beat frequencies of slices sown with iSABs from day 1 to day 4 (data are reported as mean values±SEM ((MW SEM); n≥16). (F) Transfer of calcein dye from an iSAB to the recipient slice over time [scale: 200 μm; “h”: hour(s); “d”: day(s)]. (G) The stimulation of slice regions (arrows) in the immediate environment of an iSAB (arrow) is accompanied by highly synchronous Ca.sup.2+ transients.

(9) FIG. 5: Transcriptome Status of iSABs Determined by RNA-seq

(10) Ontology descriptions that are familiar to the person skilled in the art only in the English language have been left here in English.

(11) (A) 82 gene ontologies which describe biological processes comprise 220 upregulated genes in iSABs vs. controls. (B) Cluster of ontology groups relating to heart/muscle function and heart development are dominant. (C) Consolidated network of the gene ontologies which relate to biological processes. (D) 34 gene ontologies which describe the cellular components comprise the 220 upregulated genes in iSABs vs. controls. (E) of ontology groups relating to structures typical of contracting cells are dominant. (F) Consolidated network of the gene ontologies which relate to cellular components.

(12) FIG. 6: Representative Action Potentials Obtained From Tbx3-Programmed Cells

(13) (A, B) Myocardial 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 which are referred to as intermediate-type cells of the murine embryonic heart. Horizontal time axis: 100 ms.

(14) FIG. 7: Representative Action Potentials Obtained From iSAB Cardiomyocytes.

(15) (A) Pacemaker-type action potentials similar to action potentials which have been generated from fully grown murine sinoatrial node cells. (B) Action potentials generated by slightly immature pacemaker-type cells. Horizontal time axis: 100 ms.

(16) FIG. 8: I.sub.f Plots From Isolated iSAB Cells.

(17) (A) Patch-clamp protocol; voltage applied to cause the current activated by hyperpolarization. (B) Example of the I.sub.f current, recorded from an isolated cell originating from an iSAB. (C) Current density and (D) time constant of the activation at −130 mV, showing a robust I.sub.f expression with slow activation kinetics typical of the HCN4 channel subtype and for mature sinoatrial nodes I.sub.f. n=17; error bar: mean value±SD.

(18) FIG. 9: Modulation of Action Potential (AP) Rates of Isolated Tbx3-Programmed Cells and Cells Originating From iSAB.

(19) Reaction to β-adrenergic (isoprotenerol) and muscarinic (carbachol) stimulation leads to typical accelerated vs. slowed AP rates. Cells originating from iSAB show a clearer response to isoprotenerol with beat frequencies which range up to 560 bpm. iSAB/iso: n=9; iSAB/carb: n=5; error bar: mean value±SD.

(20) FIG. 10: Table 1:Action potential parameters of the cardiomyocytes obtained from action of Tbx3-programmed cells.

(21) The data shown in the table are the mean value ± SD (standard deviation). Commas (,) in the numbers are represented by points (.).

(22) FIG. 11: Table 2: Action potential parameters of cardiomyocytes which have been obtained from iSABs.

(23) The data shown in the table are the mean value ± SD. Commas (,) in the numbers are represented by points(.).

LITERATURE

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