Induced pacemaker and Purkinje cells from adult stem cells

Abstract

Adult stem cells are reprogrammed to form pacemaker cells and Purkinje cells through the sequential activation of SHOX2>TBX5>HCN2. These Purkinje cells spontaneously surround and connect with the larger pacemaker cells, thus forming an induced sinoatrial body that produces funny current and can make cardiovascular tissues beat in a manner similar to a natural sinoatrial node.

Claims

1. A method of treating an arrhythmia, comprising a) obtaining adult tissue-derived stem cells from a patient with an arrhythmic heart; b) inducing lineage commitment of said adult tissue-derived stem cells towards Purkinje and pacemaker cardiomyocyte cells through sequential epigenetic reprogramming by sequentially expressing SHOX2 before TBX5 before HCN2 (SHOX2>TBX5>HCN2), and c) introducing said Purkinje cells or said pacemaker cells, or both, into said arrhythmic heart.

2. The method of claim 1, wherein the adult tissue-derived stem cells are uncultured prior to the inducing step b).

3. The method of claim 1, wherein both said pacemaker cells and said Purkinje cells are used together to reconstitute a damaged sinus node of said patient.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1A. ANATOMY OF THE HEART: Diagram of the anatomy of a heart in cross section, with EKG trace (upper right).

(2) FIG. 1B. ELECTROPHYSIOLOGY OF THE HEART: Electrical system of the heart and EKG trace in detail.

(3) FIG. 2. GENE ACTIVATION SEQUENCE: Schematic algorithm representing the critical genes involving in different stages of cardiac pacemaker cell lineage development.

(4) FIG. 3. EXPERIMENTAL OVERVIEW: Schematic diagram, representative of sequence of experimental procedures for generation of cardiac pacemaker cells.

(5) FIG. 4. VECTOR MAP: PNL-TREPiTT-EGFP deltaU3-IRES2-EGFP plasmid used as an expression vector for TBX3, TBX5, TBX18, HCN2, SHOX2 constructs.

(6) FIG. 5. ADSC BEFORE AND AFTER TRANSFECTION: Changes in the morphology of ADSCs after transfection with cardiac pacemaker inducing factors SHOX2, TBX5 and HCN2 from the typical fibroblastic like morphology of ADSCs (A) to the colonies of small network forming cells (B), see arrow, 7 days after initiation under a doxycycline dependent switch.

(7) FIG. 6. CHANGING MORPHOLOGY: Changes in the morphology of ADSCs after transfection with cardiac pacemaker inducing factors SHOX2, TBX5 and HCN2 from the typical fibroblastic like morphology of ADSCs to the colonies of small and large network forming cells within 1 week after initiation of doxycycline switch controlled induction.

(8) FIG. 7. CELL TYPES AND ALIGNMENT: Observation of 2 different cell types in culture plate of ADSCs transfected with SHOX2, TBX5 and HCN2. Right-arrowSmall cell population with spike shape projections. Small cells start to form a special aligned growth pattern and make channel like structures. Left arrowLarger cells with special spider shape morphology.

(9) FIG. 8. CLOSEUP OF LARGE CELLS: Appearance of typical spiderlike morphology of large cells (>50 m) within 14 days after initiation of doxycycline controlled induction of SHOX2, TBX5 and HCN2.

(10) FIG. 9. NETWORKS: Large spider shape cells start to form networks with each other and with small cells within 3 weeks after induction of SHOX2, TBX5 and HCN2.

(11) FIG. 10. DAPI AND CX30.2 IN SMALL CELLS. Expression of CX30.2 (a specific marker of pacemaker cell lineage) in small network forming cells. The blue DAPI stain lights up DNA, indicating the nucleus.

(12) FIG. 11. DAPI and CX30.2 IN LARGE CELLS. Expression of CX30.2 in both small and large spider shaped cells.

(13) FIG. 12. DAPI AND HCN4 (a specific marker of pacemaker cell lineage) in both small and large cells.

(14) FIG. 13. RNA EXPRESSION LEVELS. mRNA expression levels of specific marker genes of cardiac Purkinje cells in ADSCs transfected with different combinations of pacemaker inducing factors, 2 weeks after the initiation of induction.

(15) FIG. 14. RNA EXPRESSION LEVELS CONT. mRNA expression levels of specific marker genes of cardiac pacemaker cells in ADSCs transfected with different combinations of pacemaker inducing factors, 2 weeks after the initiation of SHOX2, TBX5 and HCN2 induction.

(16) FIG. 15. SINGLE CELL VOLTAGE CLAMP EXPERIMENTS, FUNNY CURRENT CONTROL CELLS: Representative funny current (If) recorded from ADSCs (Control 2) cells. The If currents were elicited with voltage steps from 100 mV to 40 mV for 500 mS with 10 mV increment from a holding potential of 40 mV.

(17) FIG. 16. FUNNY CURRENT IN SMALL CELLS: representative funny current (If) recorded from small size fraction (<20 m cell size) of ADSCs transfected with SHOX2, TBX5, and HCN2. The If currents were elicited with voltage steps from 100 mV to 40 mV for 500 mS with 10 mV increment from a holding potential of 40 mV.

(18) FIG. 17. FUNNY CURRENT IN LARGE CELLS. Representative funny current (If) recorded from large size fraction (>50 m cell size) of SHOX2, TBX5, HCN2 transfected ADSCs. The If currents were elicited with voltage steps from 100 mV to 40 mV for 500 mS with 10 mV increment from a holding potential of 40 mV.

DETAILED DESCRIPTION

(19) The invention provides novel methods of making pacemaker cells and Purkinje cells or sinoatrial nodes (SANs); the pacemaker cells, Purkinje cells, and SANs made thereby; and methods of using same, e.g., to replace or supplement damaged pacemakers and Purkinje fibers in the heart of patients, such as human patients. The cells can be surgically delivered directly or by catheter based injection to the place where they are required. For the repair of the sinus node for example, a catheter based injection of a few thousand cells into the damaged and reduced functioning sinus node is sufficient to effect repair.

(20) For the repair of tachycardic arrhythmias, Purkinje cell are injected into the slow conducting zone, such as the border zone of an infarction, in order to accelerate the speed of conduction in this arrhythmogenic substrate and thereby to close the re-entry pathway by accelerating the circulating impulse in such a way that it meets refractory myocardium and the re-entry is interrupted.

(21) Preferably, the cells are made from autologous cells, using any available stem cell source from the patient, such as bone marrow derived stem cells or adipose derived or blood, skin and umbilical cord tissue derived stem cells.

Lentiviral-Based Reprogramming

(22) To induce ADSCs to differentiate into cardiac pacemaker cells, they are sequentially transfected with different combinations of cardiac pacemaker inducing factors including SHOX2, TBX5, TBX3, TBX18 and HCN2 by applying a lentiviral vector system. The lentiviral vector is under the control of a switch, for example a doxycycline switch.

(23) The expression of cardiac pacemaker inducing factors was achieved by lentiviral vectors, applied to the cells in the sequential way in 24-hour intervals (FIG. 3). Cells were cultured in -MEM supplanted with 5% horse serum, 2 mM glutamine, 0.1 mM non-essential amino acids. Cultures were treated daily with 400 ng/ml doxycycline for 3 days, then sustained for 2 weeks in -MEM with above mentioned supplements. Daily microscopic observation of cells was performed to study the morphological changes of cells after the transfection. RNA samples were collected from different experimental groups at day 14 after the initiation of Doxycycline induction.

(24) TABLE-US-00003 TABLE 1 List of experimental groups by controlled expression of single, double and triple combinations of pacemaker inducing factors in ADSCs Experimental Groups' Name Description of Treatments T3 ADSCs transfected with TBX3 only T5 ADSCs transfected with TBX5 only T18 ADSCs transfected with TBX18 only HCN- 2 ADSCs transfected with HCN2 only SHOX2 ADSCs transfected with SHOX2 only SH ADSCs transfected with double combination of SHOX2 and HCN2 SHT- 3 ADSCs transfected with triple combination of SHOX2, TBX3 and HCN2 SHT- 5 ADSCs transfected with triple combination of SHOX2, TBX5 and HCN2 SHT- 18 ADSCs transfected with triple combination of SHOX2, TBX18 and HCN2

(25) Human ADSCs (hADSCs) were obtained from INGENERON (Houston, Tex.). Adipose tissue from donors aged 30-40 years old were obtained with informed consent under a tissue acquisition protocol approved by the Institutional Review Board. The ADSCs were prepared from lipoaspirate acquired from donors undergoing elective lipoplasty. The hASDCs were isolated as described previously.

(26) Briefly, fat tissue was minced and incubated for 30 min at 39 C. with Matrase (INGENERON) at a concentration of 1 unit per gram of fat tissue in Ringer solution in the Transpose RT processing unit (INGENERON). The processed tissue was subsequently filtered through a 100 m filter and centrifuged at 450 g for 10 min. The supernatant containing adipocytes and debris were discarded, and the pelleted cells were washed twice with Hanks' balanced salt solution (CELLGRO) and finally suspended in growth media. The process is described in detail by instructions for use of InGeneron Transpose RT system (INGENERON). Growth media contained alpha-modification of Eagle's medium (CELLGRO), 20% FBS (ATLANTA BIOLOGICALS), 2 mM glutamine, 100 units/ml penicillin with 100 g/ml streptomycin (CELLGRO).

(27) Adherent cells were called hADSCs and grown in culture flasks at 37 C. in a humidified atmosphere containing 5% CO.sub.2 followed by daily washes to remove red blood cells and non-attached cells. On reaching 80% confluence, cells were detached applying Trypsin solution 0.25% and seeded at the density of 3000 cells/cm.sup.2 in fresh cell culture flasks.

(28) To differentiate ADSCs into cardiac pacemaker cells, the ADSCs were transfected with different combinations of cardiac pacemaker inducing factors including SHOX2, TBX3, TBX5, TBX18 and HCN2 using a lentiviral vector system (Table 1). The lentiviral vector used herein includes packing vector psPAX2 and envelope vector pMDS2.G (ADDGENE) (Islas, 2012). In addition, a Doxycycline controlled transactivator (rtTA2) was used as a transcriptional inductive switch of the system. Plasmid PNL-TREPiTT-EGFP delta U3-IRES2-EGFP (FIG. 4) was used as a backbone vector for every single gene. A commercial cell line293FTwas used for viral packaging. The 293FT cell line is a fast-growing, highly transfectable clonal isolate derived from human embryonal kidney cells transformed with the SV40 large T antigen and is available from THERMO FISCHER SCIENTIFIC.

(29) Briefly viral particles were produced in 293 T cells transfected with psPAX2, pMDs2.G vectors and plasmids containing every single gene of interest including SHOX2, TBX3 TBX5, TBX18, HCN2. Reprogramming of about 80% confluent ADSCs into proliferative state were accomplished by infection with different combinations of the viral particles containing SHOX2, TBX3, TBX5, TBX18, HCN2 vectors according to the predefined experimental groups (see Table 1). The 4 and 5 gene combinations also present good preliminary results.

(30) Transfected cells were cultivated in large tissue culture plates by -MEM (INVITROGEN) supplemented with 5% (vol/vol) horse serum, 0.1 mM non-essential amino acids, 2 mM L-glutamate.

(31) The stable integration and mRNA expression of each gene construct were confirmed 72 hours after the transfection using PCR.

(32) To study any changes occurring in the cells after the transfection, delicate microscopic observation of cells were performed every day. Representative data are shown herein, that belonging to the triplet combination of the sequential transfections of SHOX2>TBX5>HCN2.

(33) Starting from week 2 after Doxycycline triggered treatment genetically programmed pacemaker cells from different experimental groups initiated to convert their fibroblastic like morphology and formed new colonies containing a particular network forming cell type with several large spike shape projections. The sequential transfections of SHOX2>TBX5>HCN2 are shown in FIG. 5, 6.

(34) During the differentiation period in various experimental groups, gradually two different types of cells appeared, including a particular large spider shape cell type and small spindle shape cells with long spike like projections. FIG. 7, 8 shows the appearance of the two cell types in the triple sequential transfections of SHOX2>TBX5>HCN2. Subsequently, both populations of spindle and spider shape cells began to form highly interconnected networks with each other (FIG. 9). In addition small spike shape cells started to form particular, highly aligned growth patterns (see linear groove forming in FIG. 7, likely corresponding to Purkinje cell networks in the natural conduction system of heart.

(35) According to the results of previous studies on isolation of pacemaker cells from fetal SAN, spider shape morphology has been defined as a typical morphology of pacemaker cells. Therefore, our ordered transfection experiments showed that the triplet sequential transfections of SHOX2>TBX5>HCN2 made cells with a morphology very much like natural pacemaker cells.

(36) Although not shown herein, the similar observations were made in different groups of ADSCs transfected with various combinations of pacemaker inducing factors. However, more robust morphological changes toward the typical morphology of cardiac conduction cells were observed in ADSC groups transfected with TBX transcription factors (particularly TBX5) in comparison with the groups transfected with individual or double combinations of SHOX2 and HCN2. The most robust morphological changes were observed in ADSCs group transfected with triple combinations of SHOX2, TBX5 and HCN2, and therefore these results are shown in part herein. Similar results were observed in expression of molecular markers of cardiac conductive system by qPCR assay (FIG. 13, 14).

(37) Results of earlier in vivo studies on identifying the main molecular regulators of cardiac conduction system development identified T-box proteins as essential factors for cardiac conduction system morphogenesis as well as the up-regulation of genes encoding the ion channel proteins that contribute to the electrophysiological functionality of cardiac conduction cells.

(38) Hoogaars e al., 2007 indicated that TBX3 controls the sinoatrial node gene program and imposes pacemaker function on the atria. Results of other studies indicating TBX5 is a critical transcription factor regulating developmental networks required for maturation of and functionality of cardiac conduction cells.

(39) Results of genome wide associate studies (GWAS) have identified numerous loci associated with the developmental processes of human cardiac conduction system including TBX5 and ion channels. Expression of ion channels is critical for electrophysiological functionality of the system. Arnold et al., 2012 found that deletion of TBX5 results in severe malfunction in cardiac conduction system including loss of fast conduction, arrhythmias and sudden death.

(40) In addition based on the results of genome wide associate studies (GWAS) a molecular link was identified between TBX5 and SCN5A (UNIPROT Q14524), aka NAV1.5, a key mediator of fast conduction system. Results of this study identified a TBX5-responsive enhancer downstream of SCN5A, which is sufficient for the lineage specification of ventricular conduction system (Arnolds, 2012).

(41) The smaller cells correspond to Purkinje cells, as determined by spindle like morphology and long cellular projections: Two weeks after transfection these small spindle like cells illustrated particularly a highly aligned growth pattern, including formation of multicellular strands and networks through the tight connection of individual cells to each other. These cells illustrated moderate levels of funny currents in single cell patch clump assay. According to the results of previous in vivo studies on isolation and characterization of various cardiac cell types, this particular strand and network forming growth pattern has been defined as the typical characteristics of Purkinje cells. Similar morphological properties were reported in a previous attempt for in vitro production of cardiac nodal cells through the overexpression of TBX3 in ESCs. In addition in agreement with our findings, results of previous studies on characterization of different types of cardiac conductive cells also revealed the lower levels of electrophysiological activity in cardiac Purkinje cells in comparison with the pacemaker cells.

(42) The two cells types associate to form interconnected networks wherein one pacemaker cell is surrounded and connected to several Purkinje cells to form a network. The Purkinje cells have the purpose to act as amplifier and conductors of the initial spontaneous depolarization induced by the pacemaker cells. The formation of these interconnected networks is closely correlated with the expression of specific membrane junctions and ion channels of cardiac conduction cells including CX30.2 and HCN4, essential for the electrophysiological functionality of cells. HCN4, which is a member of hyperpolarization activated cyclic nucleotide-gated sodium channels, is required for If, the specific pacemaker current. CX30.2 is responsible for the cell-cell junctions and formation of networks between spontaneously depolarizing cardiomyocyte cells.

RNA-Based Reprogramming

(43) Another successful way to induce a programmed pacemaker non-contractile cardiomyocyte cell is using a mRNA-based transfection method. This methodology has not yet been fully completed on all different inducing factors, but our current results suggest the method to be equally viable and effective.

(44) Briefly, coding DNA sequence is amplified by PCR using specific primers. PCR products then are purified and the quality of the generated DNA is determined. Using the in vitro transcription (IVT) process, mRNA is generated from the DNA product. Subsequently, the product is purified and treated with phosphatase to remove 5-triphosphates. After the additional purification and quality control of generated mRNA, transfection experiments will be performed.

(45) To obtain, the DNA template for the IVT, all TBX18, TBX3 and HCN2 plasmids are amplified using PCR. Thereby, a poly T-tail of 120 thymidines (T) is added to the insert by using a reverse primer with a T.sub.120 extension. Thus, after IVT, the generated mRNAs obtain a poly A-tail with a defined length. PCR reactions of 100 l will are performed using e.g., HOTSTAR HIFIDELITY POLYMERASE KIT (QIAGEN, Germany) and contained 0.7 M of each forward and reverse primer, 1 Q-solution, 1 HOTSTAR HIFIDELITY PCR buffer, 50 ng plasmid DNA, 2.5 U HOTSTAR HIFIDELITY DNA polymerase.

(46) Amplification is performed using e.g., the following cycling protocol: initial activation step at 95 C. for 5 min, followed by 25 cycles of denaturation at 95 C. for 45 s, annealing at 55 C. for 1 min, extension at 72 C. for 1 min and final extension at 72 C. for 10 min. PCR products will be purified using QIAQUICK PCR PURIFICATION KIT (QIAGEN, Germany) according manufacturer's instructions and the DNA eluted using 20 l nuclease-free water. The quality and purity of the DNA can be assessed by 1% agarose gel electrophoresis.

(47) After the PCR, the genetic information is transcribed from DNA to mRNA in vitro using e.g., MEGASCRIPT T7 Kit (LIFE TECHNOLOGIES, Germany). The mRNA transcript then will be used to induce protein expression in cells. At first, 23 l NTP/cap analog mixture containing 7.5 mM ATP, 1.875 mM GTP (both from MEGASCRIPT T7 Kit), 7.5 mM Me-CTP, 7.5 mM Pseudo-UTP (both from TRILINK BIOTECHNOLOGIES, CA), and 2.5 mM 3-O-Me-m.sup.7G(5)ppp(5)G RNA cap structure analog (NEW ENGLAND BIOLABS, Germany) will be prepared and mixed thoroughly.

(48) The IVT reaction mixture of 40 l then will be assembled by adding 40 U RIBOLOCK RNase inhibitor (THERMO FISHER SCIENTIFIC), 1 g PCR product, 1 reaction buffer and 1 T7 RNA polymerase enzyme mix. The IVT reaction mixture will be incubated at 37 C. for 3 hr in a thermomixer. To remove the template DNA, 1 l TURBO DNase (from MEGASCRIPT T7 Kit) is added to the IVT reaction mixture and incubated for 15 min at 37 C. Then, the reaction mixture is purified using RNEASY Mini Kit (QIAGEN) according to manufacturer's instructions. The modified mRNA will be eluted from the spin column membrane twice with 40 l nuclease-free water.

(49) The generated mRNA will be treated with Antarctic phosphatase (NEW ENGLAND BIOLABS) to remove 5 triphosphates, which can be recognized by RIG-1, and lead to the immune activation. Furthermore, the phosphatase treatment prevents the recircularization in a self-ligation reaction. For this purpose, 9 l of 10 Antarctic phosphatase reaction buffer is added to 79 l of purified mRNA solution. Subsequently, 2 l of Antarctic phosphatase (5 U/l) is added to the reaction mixture and incubated at 37 C. for 30 min.

(50) The treated mRNA can be purified using e.g., RNeasy Mini Kit (QIAGEN) according to manufacturer's instructions. The modified mRNA will be eluted from the spin column membrane twice with 50 l nuclease-free water. The concentration will be measured using SCANDROP spectrophotometer (ANALYTIC JENA, Germany). The concentration of mRNA will be adjusted to 100 ng/l by adding nuclease-free water. The quality and purity of synthesized modified mRNA will be determined by 1% agarose gel electrophoresis. The modified mRNA will be aliquoted and stored at 80 C. and used for transfections.

(51) ADSCs will be cultivated in -MEM media supplemented with 10% FBS (LIFE TECH.), 2 mM L-glutamine (PAA LABORATORIES, Austria), and 1% penicillin/streptomycin (PAA LAB.). Cells will be kept at 37 C. with 5% CO.sub.2 and media will be changed every 3 days. Cells will be passaged using trypsin/EDTA (0.04%/0.03%, PROMOCELL, Germany). For performing of transfection experiments, 1.510.sup.5 cells will be plated per well of 24-well plate. The cells will be incubated overnight at 37 C. in a cell incubator. Next day, transfection experiments can be performed.

(52) Transfection of ADSCs with different mRNA of the invention are performed with LIPOFECTAMINE 2000 (LIFE TECH.). To determine the required amount of LIPOFECTAMINE 2000 for forming of lipoplexes, different amounts of LIPOFECTAMINE 2000, of 1, 2, 4, 6 l, were used to transfect the cells. For transfection of one well of 24-well plate, 250 l Opti-MEM I reduced serum media was prepared containing 2.5 g of each mRNA of interest and respective amount of LIPOFECTAMINE 2000 according to the manufacturer's instruction.

(53) The components are gently mixed by pipetting. The transfection mixture then is incubated at room temperature for 20 min to generate lipoplexes for transfection. Cells will be washed with 250 l DPBS/well, the transfection mixture pipetted into the well. After 4 hr incubation at 37 C. and 5% CO.sub.2, the transfection mixture is replaced by 1 ml complete cell culture medium. Cells will be cultivated for 24 hr in the cell incubator and analyzed using flow cytometry.

(54) To determine the required amount of mRNA for induction of protein expression, firstly different amounts of every eGFP-mRNA, 0, 0.5, 1, 1.5, 2, 2.5 g, are used to perform the transfection of cells. For transfection of one well of 24-well plate, 250 l Opti-MEM I reduced serum media with respective amount of mRNA and 1 l of LIPOFECTAMINE 2000 will be prepared.

(55) The components are gently mixed and incubated for 20 min at room temperature. Cells will be washed with 500 l DPBS/well and the transfection mixture will be added. Cells will be incubated for 4 hr at 37 C. and 5% CO.sub.2. Afterwards, the transfection mixture will be aspirated and 1 ml complete cell culture medium will be added to the cells. Cells will be incubated for 24 hr in the incubator.

(56) Using flow cytometry, the eGFP expression in the cells is verified. After determining the required amount of mRNA for induction of protein expression by eGFP, the same concentration is applied for the respective mRNA according to the invention.

Immunohistochemistry

(57) For further characterization of induced pacemaker cells, immunohistochemistry (IHC) staining for the major maker genes of cardiac pacemaker cell lineages including CX30.2 and HCN4 were performed 2 weeks after the initiation of Doxycycline triggered induction.

(58) IHC staining was performed for two major marker genes respective of non-contractile cardiomyocytes including CX30.2 and HCN4. Results of IHC staining in cells sequentially transformed with the SHOX2>TBX5>HCN2 triplet revealed the expression of CX30.2 and HCN4 in both spindle and spider cell populations (FIG. 10, 11, 12). HCN4 is a member of hyperpolarization activated cyclic nucleotide-gated sodium channels required for If, the specific pacemaker current. CX30.2 is responsible for the cell-cell junctions and formation of networks between respective non-contractile cardiomyocytes.

(59) Similar immunophenotype were also, observed in different groups of ADSCs transfected with other combinations of pacemaker inducing factors. However more robust changes in morphological properties and immunophenotype of transfected ADSCs towards the spontaneously depolarizing, non-contractile cardiomyocytes were observed in ADSC groups transfected with TBX transcription factors (particularly TBX5) in comparison to the groups transfected with individual or double combinations of SHOX2 and HCN2. The most robust changes in morphological properties and immunophenotype of transfected ADSCs toward the sinoatrial node's cells were observed by triple combinations of SHOX2, TBX5 and HCN2. It's also possible that TBX3 and TBX18 can be used in addition to or in replacement of TBX5, and that HCN4 can be used in addition to or in replacement of HCN2.

Expression Levels of Key Genes

(60) To evaluate the effect of various combinations of cardiac pacemaker inducing factors on differentiation of ADSCs towards the different types of cardiac conduction cell lineages including pacemaker and Purkinje cells, mRNA expression level of specific marker genes of both cell lineages were analyzed.

(61) To this end mRNA expression for a panel of downstream late stage markers of cardiac pace maker cells, including HCN1, HCN3, HCN4, SCN3B (UniProt Q9NY72), CX30.2 (properly known as GJC3, UniProt Q8NFK1) as well as Purkinje cells' specific marker genes including IRX3 (UniProt P78415), IRX5 (UniProt P78411), SEMA3B (UniProt Q6PI51), SCN10A (UniProt Q9Y5Y9), SHH (UniProt Q15465) were determined applying qPCR analysis on a mixed cell type population (e.g., cells were not first separated into pacemaker and Purkinje cells, although this experiment is planned).

(62) Briefly total RNA was isolated using the QIAGEN's RNEASY Kit and were reverse transcribed into cDNA using Superscript III (INVITROGEN). Quantitative PCR were performed with the ABI Prism 7000 System Detection Sequence (SDS) and software (APPLIED BIOSYSTEMS) using SYBR Green (APPLIED BIOSYSTEMS) as the detector.

(63) The mRNA expression level for a panel of specific marker genes of cardiac pacemaker marker (Table 3 and FIG. 12) and Purkinje cells (Table 2 and FIG. 13) were analyzed thereby. Based on the results of qPCR analysis expression of marker genes of cardiac Purkinje cell can be observed in all different experimental groups transfected with different combinations of pacemaker inducing factors. However different combinations of cardiac inducing factors play various roles on up-regulation of downstream cardiac pacemaker marker genes including HCN1, HCN3, HCN4, SCN3B (UniProt Q9NY72), CX30.2 (GJC3, UniProt Q8NFK1).

(64) For example the highest mRNA expression level of HCN3B, HCN3 and CX30.2 were observed in ADSCs transfected with triple combination of SHOX2, TBX5 and HCN2, while mRNA expression of HCN1 is more correlated with the up-regulation of TBX18. According to the results of this study, mRNA expression of HCN4 is highly correlated with the up-regulation of SHOX2. In addition moderate expression level of HCN4 was observed in ADSCs transfected with triple combinations of SHOX2, TBX5 and HCN2. All together based on the results of qPCR, transfection of ADSCs with triple combinations of SHOX2, TBX5 and HCN2 leads the most consistent up-regulation of different marker genes of cardiac conductive cells. It can be concluded that the most robust changes in gene expression pattern of transfected ADSCs towards the spontaneously depolarizing, non-contractile cardiomyocytes were observed in ADSC groups transfected with TBX transcription factors (particularly TBX5) in comparison to the groups transfected with individual or double combinations of SHOX2 and HCN2. The most robust and consistent changes in gene expression pattern of transfected ADSCs toward the spontaneously depolarizing, non-contractile cardiomyocytes were observed by triple combinations of SHOX2, TBX5 and HCN2.

(65) Generally it can be concluded that triple combinations of SHOX2, TBX5 and HCN2 can be applied effectively for generation of both cell types of cardiac conductive system including Purkinje and Pacemaker cells.

(66) TABLE-US-00004 TABLE2 Listofforward(F)andreverse(R) primersequencesforspecificmarker genesofcardiacPurkinjecells Primer's Nucleotide Genename name sequenceofprimer Iroquois IRX3F GAGGGAAACGCTTATGGG homebox- (SEQIDNO.1) AGC 5(IRX3) IRX3R CGCCGTCTAAGTTCTCCA (SEQIDNO.2) AATC Iroquois IRX5F TCAGCGACTCGGATTTTA homebox- (SEQIDNO.3) AGGA 5(IRX5) IRX5R GGAGGCGGCGAATGGATA (SEQIDNO.4) A Semaphorine SEMA3BF ACATTGGTACTGAGTGCA (SEMA3B) (SEQIDNO.5) TGAAC SEMA3BR GCCATCCTCTATCCTTCC (SEQIDNO.6) TGG Sodiumchannel SCN10AF TCCCTCGAAACTAACAAC voltage (SEQIDNO.7) TTCCG gated10 SCN10AR TCTGCTCCCTATGCTTCT (SCN10A) (SEQIDNO.8) CTC SonicHedge SHHF CCAAGGCACATATCCACT hog(SHH) (SEQIDNO.9) GCT SHHR GTCTCGATCACGTAGAAG (SEQIDNO.10) ACCT

(67) TABLE-US-00005 TABLE3 Listofforward(F)andreverse(R) primersequencesforspecificmarker genesofcardiacPacemakercells Nucleotide Genename Primer'sname sequenceofprimer Hyperpolarization HCN1F CATGCCACCGCTTTAATC activatedcyclic (SEQIDNO.11) CAG nucleotideion HCN1R ATTGTAGCCACCAGTTTC channel-1(HCN1) (SEQIDNO.12) CGA Hyperpolarization HCN3F AGCAGTGGAAATCGAGCA activatedcyclic (SEQIDNO.13) GG nucleotideion HCN3R GGTCCCAGTAAAACCGGA channel-3(HCN3) (SEQIDNO.14) AGT Hyperpolarization HCN4F GAACAGGAGAGGGTCAAG activatedcyclic (SEQIDNO.15) TCG nucleotideion HCN4R CATTGAAGACAATCCAGG channel-3(HCN3) (SEQIDNO.16) GTGT SodiumChannel, SCN3BF GCCTTCAATAGATTGTTT Voltagegated3b (SEQIDNO.17) CCCCT (SCN3B) SCN3BR CTCGGGCCTGTAGAACCA (SEQIDNO.18) T Connexin30.2 CX30.2F TGGAGTCAGCGGTTTCTG (CX30.2) (SEQIDNO.19) TC CX-30.2R TTGTGTCTTCTGGTGCTC (SEQIDNO.20) TCT

Patch Clamp Assays

(68) To further verify the functionality of genetically programmed pacemaker cells their electrophysiological properties we performed single-cell patch clamp experiments to measure the currents generated by the cells (White, 2005).

(69) Whole cell voltage-clamp experiments were carried out using the standard patch-clamp method. Recording electrodes were made from 1.5-mm thin-walled borosilicate glass (no. 7052, GARNER GLASS, CA) using a Flaming-Brown microelectrode puller (P-97, SUTTER INSTRUMENTS, CA) and heat polished before use. Each of the pipettes have a tip resistance of 2-5 M when filled with internal solution. Recordings were performed using an Axoclamp 2B patch-clamp amplifier (AXON INSTRUMENTS; CA). Data were filtered at 2 kHz, and data acquired using Clampex 8 software (AXON INSTRUMENTS).

(70) Patch pipettes were pulled from borosilicate glass and heat polished. They had a resistance of 2-5 M when filled with intercellular solution.

(71) For current-clamp recordings, the intercellular solution was containing 10 mM NaCl, 130 mM potassium aspartate, 0.04 mM CaCl.sub.2. 3 mM Mg-ATP, 10 mM HEPES. pH was adjusted to 7.2 with KOH. The extracellular (bath) solution contained 140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl.sub.2, 1 mM MgCl.sub.2, 10 mM glucose, and 5 mM HEPES at pH 7.4.

(72) Funny Current (If) density was measured with voltage steps from 100 mV to 40 mV for 500 mS with 10 mV increments from a holding potential of 40 mV. Because an important characteristic of cardiac a pacemaker cell is the expression of an inward funny current (If). According to the results of this study, both populations of small spindle cells as well as large spider shape cells demonstrated a robust If current typical current of HCN channel subtypes (FIG. 15, 16, 17).

(73) The present invention is exemplified with respect to lentiviral vectors. However, this is exemplary only, and the invention can be broadly applied to include any means for activating the requisite genes in stem cells, such as ADSC. The following examples are intended to be illustrative only, and not unduly limit the scope of the appended claims.

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(75) All UniProt cites herein are incorporated by reference herein, including the sequences linked thereto, in their entirety for all purposes.