Yafeng Zhou

Mesenchymal Stem Cells as a Gene Delivery System to Create Biological Pacemaker Cells in vitro

Pacemaker cells differ from common cardiomyocytes due to the presence of a spontaneous depolarization process during the diastolic phase of the cardiac cycle. This is due to hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which are responsible for providing an inward current. Genetically engineered mesenchymal stem cells (MSCs) were transfected with hHCN4 genes using lentiviral transfection, and their potential use as biological pacemaker cells was investigated. In addition to expressing an anticipated high level of the hHCN4 gene, MSCs transfected with hHCN4 genes also expressed characteristic hHCN4 protein, a cardiac pacemaker-like current and were capable of increasing the spontaneous beating rate of co-cultured cardiac myocytes. Control MSCs did not exert these effects. It is hypothesized that genetically engineered MSCs transfected with hHCN4 genes by lentiviral transfection can be modified to be cardiac pacemaker cells in vitro.

Genetically-engineered mesenchymal stem cells transfected with human HCN1 gene to create cardiac pacemaker cells

Objective To test the proof-of-principle that genetically-engineered mesenchymal stem cells (MSCs) transfected with the human hyperpolarization-activated cyclic nucleotide-gated channel 1 (hHCN1) gene can be modified to become cardiac pacemaker cells. Methods MSCs were transfected with the hHCN1 gene using lentiviral-based transfection. The expressed pacemaker current (If) in hHCN1-transfected MSCs was recorded using whole-cell patch-clamp analysis. The effect of the hHCN1-transfected MSCs on cardiomyocyte excitability was determined by coculturing the MSCs with neonatal rabbit ventricular myocytes (NRVM). The spontaneous action potentials of the NRVM were recorded by whole-cell current-clamp analysis. Results A high level time- and voltage-dependent inward hyperpolarization current that was inhibited by 4 mM caesium chloride was detected in hHCN1-transfected MSCs, suggesting that the HCN1 proteins acted as If channels in MSCs. The mean ± SE beating frequency in NRVMs cocultured with control MSCs transfected with the pcDNA3 plasmid control was 82 ± 8 beats/min (n = 5) compared with 129 ± 11 beats/min (n = 5) in NRVMs cocultured with hHCN1-transfected MSCs. Conclusions Genetically-engineered MSCs transfected with the hHCN1 gene can be modified to become cardiac pacemaker cells.

GATA-4 induces changes in electrophysiological properties of rat mesenchymal stem cells.

BACKGROUND Transplanted mesenchymal stem cells (MSC) can differentiate into cardiac cells that have the potential to contribute to heart repair following ischemic injury. Overexpression of GATA-4 can significantly increase differentiation of MSC into cardiomyocytes (CM). However, the specific impact of GATA-4 overexpression on the electrophysiological properties of MSC-derived CM has not been well documented. METHODS Adult rat bone marrow MSC were retrovirally transduced with GATA-4 (MSC(GATA-4)) and GFP (MSC(Null)) and subsequently co-cultured with neonatal rat ventricular cardiomyocytes (CM). Electrophysiological properties and mRNA levels of ion channels were assessed in MSC using patch-clamp technology and real-time PCR. RESULTS MSC(GATA-4) exhibited higher levels of the TTX-sensitive Na(+) current (INa.TTX), L-type calcium current (ICa.L), transient outward K(+) current (Ito), delayed rectifier K(+) current (IKDR) and inwardly rectifying K(+) current (IK1) channel activities reflective of electrophysiological characteristics of CM. Real-time PCR analyses showed that MSC(GATA-4) exhibited upregulated mRNA levels of Kv1.2, Kv2.1, SCN2a1, CCHL2a, KV1.4 and Kir1.1 channels versus MSC(Null). Interestingly, MSC(GATA-4) treated with IGF-1 neutralizing antibodies resulted in a significant decrease in Kir1.1, Kv2.1, KV1.4, CCHL2a and SCN2a1 channel mRNA expression. Similarly, MSC(GATA-4) treated with VEGF neutralizing antibodies also resulted in an attenuated expression of Kv2.1, Kv1.2, Kv1.4, Kir1.1, CCHL2a and SCN2a1 channel mRNAs. CONCLUSIONS GATA-4 overexpression increases Ito, IKDR, IK1, INa.TTX and ICa.L currents in MSC. Cytokine (VGEF and IGF-1) release from GATA-4 overexpressing MSC can partially account for the upregulated ion channel mRNA expression. GENERAL SIGNIFICANCE Our results highlight the ability of GATA4 to boost the cardiac electrophysiological potential of MSC.

Genetically engineered mesenchymal stem cells to create cardiac pacemaker cells

Objective The study was to test proof-of-principle if genetically engineered mesenchymal stem cells (MSCs) transfected with HCN2 genes can be modified to be cardiac pacemaker cells. Methods (1) MSCs of rabbit were isolated from the posterior iliac crest of rabbit and were used from passages 2 to 4. (2) The self-inactivating HIV1-based lentiviral vector (LentiV) was used as transgene delivery, which was constructed with plasmid hHCN2/pcDNA3. (3) Total RNA was extracted from control MSCs and those transfected with hHCN2, and RT-PCR was performed. (4) Membrane proteins were extracted from control MSCs and those transfected with hHCN2. Western blot analysis was performed. (5) Whole-cell patch clamp was used to study membrane currents. Results (1) In addition to expressing characteristic hHCN2 protein, mHCN2-transfected hMSCs also express an anticipated high level of hHCN2 gene by RT-PCR and Western blot analysis. (2) If was elicited using hyperpolarising steps in 10-mV increments from −40to −140 mV, and it was significantly inhibited by 4 mM cesium chloride. (3) The coculture beating rate of cardiac myocytes was 87±11 bpm when MSCs were transfected with control plasmid (expressing only GFP) and 149±14 bpm when MSCs were expressing both GFP+hHCN2 (p<0.05). Conclusions The MSC expressing hHCN2 is a demonstration of feasibility of preparing MSC-based biological pacemaker cells. MSCs transfected with hHCN2 genes by LentiV are capable of actively pacing ventricular cardiac myocytes and can be modified to be cardiac pacemaker cells.