| ObjectiveIn this study, we aim to develop an alternative treatment of complete atrioventricular block (AVB). To do this we use combined cell and gene therapy to construct a biological pacemaker based on porcine adipose derived stem cells (ADSCs) adenovirally transduced with HCN2. Sustained ventricular pacing could indeed be demonstrated in a porcine model of complete heart blockMethods1. Isolation, cultivation and identification of porcine ADSCsAdipose tissue was obtained from the neck of swine and isolated by 0.075%Ⅱcollagenase to obtain ADSCs. ADSCs were cultured in DMEM medium with 10% fetal bovine serum, and the medium was changed every 72 h. The cells were split after they reached 90% confluence. The morphological characteristics of the cultured cells were observed, and the growth curve and cycle of the cells were detected. Flow Cytometry and immunofluorescence detected the cell surface markers CD29,CD34,CD44,CD45,HLA-DR and c-kit. After induced differentiation of ADSCs in adipogenic, osteogenic, chondrogenic and hepatic cell lines, and then the characters of the differentiated cells were identified.2. Construction and examination of Ad.HCN2Previously constructed pShuttleCMV.HCN2 used to generate a first seeding stock (using cationic transduction) of adenovirus. Subsequently, Ad.HCN2 was amplified, purified, and titrated. To measure Ad.HCN2 vector cytotoxicity, Hele-cells were transduced and the minimal multiplicity of infection (MOI) that induces cytotoxicity was detected. Thereafter, a similar experiment was performed to determine cytotoxicity of both Ad.GFP and Ad.HCN2 in ADSCs. To quantify cell cycle activity and cell viability we respectively constructed growth curves and performed flow cytometry.3. Construction of pacemaker cells from Ad.HCN2-transduced ADSCsAd.HCN2-transduced ADSCs were collected and tested for the expression of HCN2 mRNA (RT-PCR), and protein (immunofluorescence and Western-blot). The percentage of HCN2-expressing cells was calculated by flow cytometry. The electrophysiology of Ad.HCN2-transduced ADSCs was detected by patch clamp. To test functionality of the HCN2 loaded ADSCs we performed a coculture experiment with cultured neonatalrat's cardiomyocytes (NRCs). First, we isolated NRCs and characterized the obtained cells using immunohistochemistry. Subsequently, NRCs were cocultured with HCN2 loaded ADSCs and beating frequencies, both at baseline and in the presence of isoproterenol, were counted. In addition, Cx43 immunofluorescence was detected between Ad.HCN2-transduced ADSCs and NRCs.4. Transplantation of autologous pacemaker cells in treatment of complete atrioventricular blockTo establish in vivo function of HCN2 loaded ADSCs, we performed autologous transplantation studies in porcine with complete AVB. First, we implanted a right ventricular endocardial pacemaker and created AVB using His-bundle procedures. Two days later, we exposed the heart through a right lateral thoracotomy and injected pacemaker cells in a single site of the right anterior ventricular wall. After implantation, ECG and 24h Holter was regularly performed. In addition we tested autonomic response using isoproterenol and atropine. After termination, HE staining and immunofluorescence were performed on the injection site.Results1. Isolation, cultivation and identification of porcine ADSCsCell viability of ADSCs after isolation was 93.5%. And they looked round or in round-like shape.3 h later, some cells attached to the plate, and the number of adherent cells dramatically increased after 16 h. Most of the cells got attached after 48 h, and then started to stretch. Media were changed with the removal of red blood cells and some other debris after 72 h. Cells had polygon or spindle-like shape.6-7 d later, after cells got confluent into a single layer, local arrangement had a certain direction, and the cells became elongated, showing a typical fibroblast-like shape.1 h after the passage of ADSCs, they started to adhere to the plates.5-10 h later, the adherence increased.24 hours later, cells completely became adherent.3-4 d later, the cells got fully confluent. There were no significant changes in cell morphology after continuous passage of 9 to 10 generations. Cell growth curves of the 3rd,5th, and 10th generation of ADSCs were similar.1-2 d after passage, cell growth was at a standstill period.3-6 d later, cells entered the logarithmic growth phase.6-7 d later, cell growth stayed in plateau phase. The proliferation rate of the 10th generation was much slower than the 1st and 5th generation. 89.3% of cells were in G0/G1 phase. The expression of CD29 and CD44 were both positive in the 3rd generation of ADSCs shown by flow cytometry and immunofluorescence assay. The expressions of CD34, CD45, c-kit, HLA-DR were weakly positive or not expressed. ADSCs induced in different environments could be differentiated into adipose cells, osteocytes, chondrocytes and hepatocytes.2. Construction and examination of Ad.HCN2Construction of the pShuttleCMV.HCN2 was confirmed by PCR and gene sequencing. Construction of the pAd.HCN2 was confirmed by PCR. By cationic transduction, recombinant adenovirus plasmid pAd.HCN2 was introduced into HEK293 cells to produce recombinant adenovirus. PCR results confirmed that the recombinant gene carries HCN2 gene. The titer of the fourth generation of Ad.HCN2 was 1.5×109 pfu/ml. HeLa cells transduced with Ad.HCN2 at MOI= 50 did not appear cytopathic effect (CPE) after 7 days, since cell numbers at different time points showed no statistical difference between the experimental group and control group.48-72 h after ADSCs were transduced with Ad.GFP, green fluorescent cells were visible under fluorescence microscopy. Different MOI values correspond to different rates of transduction efficiency. With an increase in MOI values, the ratio of GFP-positive cells was also increased, together with a higher green fluorescence under fluorescence microscope. When MOI=50, the transduced rate of ADSCs was over 95%, and meanwhile cells looked much better than cells at the conditions of MOI=100 or 200.72 h after ADSCs were transduced with Ad.GFP, GFP protein expression was observed and the intensity of green fluorescence increased over time. There was no statistical significance of cell viability between transduced group and control groups 3 d and 10 d after ADSCs were transduced by Ad.HCN2. Neither was the cell cycle between the transduction group and the control group, and even among the same group of cells. No statistical significance in proliferation rate was observed between the Ad.HCN2-transduced group, Ad.Null-transduced group and non-transduced ADSCs groups.3. Construction of pacemaker cells from Ad.HCN2-transduced ADSCsAfter RT-PCR, two sharp bands were observed as 28S and 18S bands. The brightness of 28S was twice more than that of the 18S band. There was no difference of 28S and 18S between two different groups after incubation test. The amplified band in the experimental group was the same with the band derived from the recombinant plasmid carrying HCN2 gene. Red fluorescent cells were visible by immunofluorescence detection. Flow cytometry results showed that, the percentage of Ad.HCN2-positive cells was significantly higher in Ad.HCN2-transduced group than that in Ad.Null-transduced group and non-transduced group, with significant statistical differences compared with the blank control and positive control. However, no statistically significant difference was observed between Ad.Null-transduced group and non-transduced group, or between the blank control and positive control, suggesting the high expression of HCN2 channel protein in the membrane of Ad.HCN2-transduced ADSCs. Western blot result indicated that, a specific electrophoretic band with molecular weight of 96 kd was observed in Ad.HCN2-transduced group. By patch-clamp, a hyperpolarization-activated inward current (If) was detected in the experimental group, with the activation potential of about-60 mV, and fully activation potential of-140 mV, which was voltage-dependent. If was almost completely suppressed when the media was added with 4 mmol/L of CsCl2 in experimental group. After elution of CsCl2, If reappeared. When ADSCs were cocultured with the NRCs, after 2 d, synchronized contraction of cells was only observed in the experimental group, but not in the control group.5 d later, some of the NRCs in positive control group showed synchronized contraction, but the contraction frequency was slower than that of the experimental group.21 d later, there was significant statistical difference in cell contraction frequency between the experimental group and the positive control group, while no cell contraction was observed between the negative control and blank control group. The cells in each group were added isoprenaline, cell contraction frequency in both experimental group and the positive control group was increased, with an even higher heart rate in the experimental group compared with the positive control group. Anti-Cx43 immunofluorescence staining could be observed under fluorescence microscope.4. Transplantation of autologous pacemaker cells in treatment of complete atrioventricular blockThe surface ECG showed P wave and QRS wave was totally separated in the animal mode of complete atrioventricular block. The rate of the ventricular, which was under 50 bpm, was slower than the rate of atrium.48 h after implantation, there was no statistical significance of heart rate among each group. After 72 h, the heart rate of experimental group became faster significantly, and the rate was stable at maximum after 7 d. There were significant statistical differences compared with the experimental group and the control groups after 7 d, but no statistically significant difference was observed between the negative control and the blank control. The shape and direction of the QRS was the same between the ECG and pace mapping of the experimental group. The result of 24 h Holter showed that there were significant statistical differences compared with the experimental group and the control groups. The heart rate of the experimental group was decreased from 70.2±2.9 bpm after 2 w to 54.9±3.4 bpm after 6 w. Added isoprenaline, the rate of all animals increased, and there was significantly statistical difference among all groups. And there were significant statistical differences in increased heart rates compared with the experimental group and the control groups. Added atropine, the rate of all animals increased as well. No statistically significant difference of changed heart rate in the experimental group was observed, but there was significant statistical difference of the control groups. And there were significant statistical differences in increased heart rates compared with the experimental group and the control groups. HE staining showed there was no significant fibroplasia in injection region and immunochemistry showed red fluorescent cells were visible.Conclusion1. Isolation, cultivation and identification of porcine ADSCsThe resource of ADSCs is widespread. The residue adipose tissue at donor site still has the ability to amplify and is easy to obtain in a large number, without severe damage. Comparing to other stem cells, ADSCs have higher isolation efficiency and stronger amplification ability, which render them easy to passage and obtain in large quantities. ADSCs express CD29 and CD44, which indicates theirs characteristics of mesenchymal stem cells; ADSCs have no expression of CD34, CD45 and c-kit, which demonstrates that they are non-hematopoietic. MHC II associated protein HLA-DR is rarely expressed in ADSCs, which suggests they have only very few antigen molecules related to immunological rejection and they have the potential of xenograft due to their possible ability of immune escape. Adipogenic, osteogenic, osteogenic and heptogenic differentiation demonstrates that ADSCs are multipotent.2. Construction and examination of Ad.HCN2Recombinant adenovirus carrier Ad.HCN2 was constructed by cloning HCN2 into adenovirus vector. CPE didn't show in infected Hela cells, which demonstrates there is no wild type virus in the recombinant adenovirus and it is safe to infect cells in vitro. In order to get the best cost-effectiveness ratio, MOI=50 is taken as the standard because of the high infection efficiency of Ad.GFP with MOI=50 and good condition of cells. The intensity of objective gene increased over time after transduced, which reached the top after 7 d, and then decreased. The cell viability and cell cycle of ADSCs didn't change after transduced by Ad.HCN2, which showed Ad.HCN2 was no harm to ADSCs. Recombinant adenovirus carrier Ad.HCN2 was constructed by cloning HCN2 into adenovirus vector. CPE was not observed in infected Hela cells, which demonstrates absence of wild type virus which indicates that the prep is safe to infect cells in vitro. In order to get the best maximal protein expression and minimal cytotoxicity a MOI=50 is used. The GFP fluorescence intensity increased over time, reached peak at day 7, and then decreased. Cell viability and cell cycle of ADSCs didn't change after Ad.HCN2 transduction, which demonstrated absence of negative effects from Ad.HCN2 on these parameters.3. Construction of pacemaker cells from Ad.HCN2-transduced ADSCsRT-PCR demonstrated the expression of HCN2 mRNA in Ad.HCN2 transduced ADSCs. The incubation test further showed that the RNA samples were of high purity. In addition, we demonstrated HCN2 immunofluorescence on the membrane of Ad.HCN2-transduced ADSCs. Expression of HCN2 channel protein was most quantity in the Ad.HCN2 transduced-ADSCs with FCM and Western blot. The patch clamp showed that Ad.HCN2 transduced-ADSCs could produce If. Ad.HCN2 transduced-ADSCs could pace the NRCs effectually after cocultured them, and they showed positive to isoprenaline. Cx43 could be detected between ADSCs and NRCs after cocultured.4. Transplantation of autologous pace cells in treatment of complete atrioventricular blockThe pacemaker cells could pace the ventricle effectively after its autotransplantation. The ECG showed the ventricular heart rhythm of experimental group was from the injection region of cells. The result of 24 h Holter showed that the heart rate increased first and then decreased. The heart rate of experimental group showed positive to isoprenaline and negative to. HE staining showed ADSCs survived after implantation and there was no significant inflammatory reaction in injection region. Immunochemistry showed Ad.GFP transduced-ADSCs could express HCN2 channel protein in myocardium. |
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