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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Circulation. Author manuscript; available in PMC 2013 September 11.
Published in final edited form as:
PMCID: PMC3481839

Safe Genetic Modification of Cardiac Stem Cells Using a Site-Specific Integration Technique

Feng Lan, PhD,1,2,* Junwei Liu, MD,1,2,* Kazim H. Narsinh, MD,1,2,* Shijun Hu, PhD,1,2 Leng Han, PhD,1,2 Andrew S. Lee, BS,1,2 Marisa Karow, PhD,3 Patricia K. Nguyen, MD,1,2 Divya Nag,1,2 Michele P. Calos, PhD,3 Robert C. Robbins, MD,4,5 and Joseph C. Wu, MD, PhD1,2,5,6



Human cardiac progenitor cells (hCPCs) are a promising cell source for regenerative repair after myocardial infarction. Exploitation of their full therapeutic potential may require stable genetic modification of the cells ex vivo. Safe genetic engineering of stem cells, using facile methods for site-specific integration of transgenes into known genomic contexts, would significantly enhance the overall safety and efficacy of cellular therapy in a variety of clinical contexts.

Methods and Results

We employed the phiC31 site-specific recombinase to achieve targeted integration of a triple fusion reporter gene into a known chromosomal context in hCPCs and human endothelial cells (hECs). Stable expression of the reporter gene from its unique chromosomal integration site resulted in no discernible genomic instability or adverse changes in cell phenotype. Namely, phiC31-modified hCPCs were unchanged in their differentiation propensity, cellular proliferative rate, and global gene expression profile when compared to unaltered control hCPCs. Expression of the triple fusion reporter gene enabled multimodal assessment of cell fate in vitro and in vivo using fluorescence microscopy, bioluminescence imaging (BLI), and positron emission tomography (PET). Intramyocardial transplantation of genetically modified hCPCs resulted in significant improvement in myocardial function two weeks after cell delivery, as assessed by echocardiography (P = 0.002) and magnetic resonance imaging (P = 0.001). We also demonstrated the feasibility and therapeutic efficacy of genetically modifying differentiated hECs, which enhanced hindlimb perfusion (P<0.05 at day 7 and 14 after transplantation) on laser Doppler imaging.


The phiC31 integrase genomic modification system is a safe, efficient tool to enable site-specific integration of reporter transgenes in progenitor and differentiated cell types.

Keywords: cell therapy, stem cells, imaging, cardiovascular disease


Cardiovascular disease remains one of the leading causes of morbidity and mortality in the United States and globally. After a myocardial infarction, the limited proliferative ability of the surviving myocardial cells renders the heart susceptible to morbid sequelae such as unfavorable remodeling and ischemic cardiomyopathy. Recently, stem cell therapy has emerged as a promising adjunct to existing pharmacologic and device treatment options 1, 2. In particular, human cardiac progenitor cells (hCPCs) are attractive candidates for clinical translation of cell therapy 3. hCPCs can be autologously derived, obviating the need for immune suppression after transplantation, and are pre-programmed for differentiation into the three principal cardiovascular lineages: cardiomyocytes, vascular endothelial cells, and smooth muscle cells. Pre-clinical studies in small and large animals have thus far affirmed the beneficial effect of CPC transplantation on recipient myocardial function 47. More recently, phase I clinical trials demonstrated that infusion of hCPCs resulted in a mean improvement of ~8% in left ventricular ejection fraction (LVEF) in treated patients compared to controls after 4 months 8.

Despite these promising initial results, little is known regarding important questions such as the optimal cell dose or delivery method, the rate of in vivo cellular survival, proliferation, engraftment and differentiation, or the mechanism(s) of potential therapeutic benefit in clinical hCPC trials. Translation of stem cell therapy has also been limited by poor engraftment and survival 9, 10 as well as unpredictable differentiation in vivo 11, 12. In recent years, stem cell imaging has emerged as a valuable tool to quantitatively address some of these variables, and its implementation in clinical trials could add novel insight into the determinants of successful stem cell therapy 13. Genetic engineering of stem cells is currently being explored as a means to image transplanted cells as well as overcome the aforementioned challenges. Stable ex vivo gene transfer into stem cells prior to their transplantation can be used to enhance stem cell homing 14, survival 15, imaging 16, and targeted ablation 16, 17. However, genetic engineering is limited by many of the challenges facing the gene therapy field at large. In particular, several current gene therapy approaches rely on retrovirus, lentivirus, or transposon elements, which catalyze random transgene integration into many potential sites. Randomly integrating vectors are not safe for clinical use because insertional mutagenesis can cause oncogenic activation and malignant transformation of cells 18. Safe techniques for site-specific integration of transgenes into the human genome are therefore needed to overcome these shortcomings.

The phiC31 integrase is a site-specific recombinase that can be used to integrate transgenes into specific loci of the human genome. Specifically, the phiC31 integrase catalyzes recombination between attB and attP attachment sites, which are DNA recognition sequences of substantial size (~17bp). This enzyme can therefore be used to integrate a transgene-containing plasmid carrying an attB site into pseudo-attP sites in the human genome. These specific loci of the human genome are known to produce robust, long-term transgene expression without risking proto-oncogene activation 19. Here we employed this site-specific integration system to safely derive hCPC clones capable of long-term in vivo monitoring by using positron emission tomography (PET) or bioluminescence imaging (BLI). The resulting hCPCs are safe for use in humans because of their precisely known genetic makeup. Importantly, in addition to reporter gene imaging, this technique can be used to introduce a variety of genetic constructs safely and efficiently for enhanced cardiac stem cell therapy.


Isolation, culture, and characterization of human cardiac progenitor cells

Human cardiac progenitor cells (hCPCs) were isolated based on a previously described protocol using magnetic separation in conjunction with a Sca-1 antibody 2022. The study protocol was approved by the Stanford Institutional Review Board (IRB). In brief, human fetal hearts (StemExpress, Diamond Springs, CA) were perfused using a Langendorff apparatus using Tyrode’s solution containing collagenase and protease. hCPCs were isolated by magnetic cell sorting (MACS, Miltenyl Biotec, Sunnyvale, CA) using Sca-1-coupled magnetic beads, according to the manufacturer’s protocol. Sca-1+ cells were eluted from the column by washing with PBS supplemented with 2% FBS and cultured on 0.1% gelatin-coated dishes in M199 (Gibco)/EGM-2 (3:1) supplemented with 10% FCS (Gibco), 10 ng/ml basic fibroblast growth factor (bFGF), 5 ng/ml epithelial growth factor (EGF), 5 ng/ml insulin-like growth factor (IGF-1), and 5 ng/ml hepatocyte growth factor (HGF). For isolation of human endothelial cells (hECs), please see Supplemental Methods for details. All hCPCs (unmodified and genetically-modified) used for in vitro experiments and intramyocardial injection were at passage 10–16.

Flow cytometry analysis

Please see Supplemental Methods for details.

Plasmid constructs and generation of site-specific integrated cell lines

The donor construct pDB-FRT was generated by replacing the cassette coding GFP in a donor vector pDB2 23 containing an attB site with a triple fusion gene 24. The plasmid pCSI expressing phiC31 integrase has been described 25. To construct the donor plasmid pDB-FRT, a triple fusion gene coding firefly luciferase (Fluc), monomeric red fluorescent protein (mRfp), and herpes simplex virus truncated thymidine kinase (HSVttk) was cloned downstream of the CMV promoter. The hCPCs and hECs were grown and transfected with Fugene6 according to the manufacturer’s instructions using 3 μl of reagent per 1 μg of DNA. Transfections yielding integrase-derived clones used 10 ng of pDB-FRT and 5 μg of pCSI. After 24 h, cells were split 1:30 to three 10 cm plates. At 48 h after transfection, cells were selected in medium containing 350 μg/ml of G418 (Invitrogen). Selection was continued for 10–14 days, at which time colonies became visible. Individual colonies were picked with a pipet tip and transferred to individual wells of a 24-well plate. Surviving colonies were expanded for PCR analysis. To screen for site-specific integrations, a nest PCR method using primers targeting 11 most frequent integration sites was used as described previously 26. Briefly, a set of 11 primers were used for the first round PCR to amplify the integration genes from the genomic DNA extracted from individual clones. The clones with site-specific integration demonstrated the expected size second round PCR product and were selected for further characterization and functional studies.

Effect of reporter gene expression on hCPC viability, proliferation, and differentiation

Please see Supplemental Methods for details.

In vitro [18F]-FHBG accumulation assay

Please see Supplemental Methods for details.

Microarray hybridization and data analysis

Please see Supplemental Methods for details.

Surgical model of mice myocardial infarction and hCPC delivery

In adult female SCID Beige mice (Charles River Laboratories, Wilmington, MA), myocardial infarction (MI) was induced by ligation of the left coronary artery under 1.5–2% inhaled isoflurane anesthesia, and confirmed by myocardial blanching and EKG changes. Animals were randomized into experimental groups receiving phiC31-hCPCs (n=30) or unmodified hCPCs (n=15) and a control group receiving PBS (n=15). All experimental animals were injected with 1×106 cells using a 31-gauge Hamilton syringe immediately after MI. In all groups, the volume of injection was 20 μl at 3 sites near the peri-infarct border zone. All surgical procedures and injections were performed by a blinded experienced microsurgeon (Y.G.).

In vivo optical bioluminescence imaging (BLI) of cell survival in transplanted animals

Please see Supplemental Methods for details.

[18F]-FHBG positron emission tomographic (PET) imaging of cell survival in transplanted animals

Please see Supplemental Methods for details.

Analysis of left ventricular function with echocardiogram and magnetic resonance imaging (MRI)

Echocardiography was performed before (day -7) and after (day 2, week 1, week 2, week 3 and week 4) the LAD ligation. The Siemens-Acuson Sequoia C512 system equipped with a multi-frequency (8–14 MHZ) 15L8 transducer was used by an experienced operator (P.K.N.) blinded to the group designation. Analysis of M-mode images was performed using Siemens built-in software. Left ventricular volume at end of diastolic diameter (EDD) and end-systolic diameter (ESD), and volume blood at end of diastolic (BED) were measured and used to calculate left ventricular ejection fraction by the formula: LVFS = [EDD-ESD]/BED. The MRI imaging was performed on a Sigma 3.0T Excite HD scanner (GE Healthcare Systems, Milwaukee, Wisconsin) with a Mayo Clinic T/R MRI coil (Mayo Clinic Medical Devices, Rochester, Minnesota) in a subset of animals (n=5/group). Mice were anesthetized with 2% isoflurane with oxygen (1 l/min) and placed in the prone position for imaging. A small animal electrocardiogram and respiratory gating system (Small Animal Instruments, Stony Brook, New York) was used to acquire images as previously described 27.

Delivery of human endothelial cells (hECs) in a hindlimb ischemia model

Please see Supplemental Methods for details.

Laser Doppler perfusion imaging

Blood flow to the ischemic or normal (nonischemic) hindlimb was assessed using a PeriScan PIM3 laser Doppler system (Perimed AB, Sweden) as described previously 28. Each animal was prewarmed to a core temperature of 37°C, and hindlimb blood flow was measured pre- and post-operatively on day 0 and again on days 3, 7, and 14. Regions of interest of the ischemic or nonischemic hindlimb were drawn in a standard fashion. The level of perfusion in the ischemic and normal hindlimbs was quantified using the mean pixel value within the region of interest, and the relative changes in hindlimb blood flow were expressed as the ratio of the left (ischemic) over right (normal) mean pixel value.

Histological examination

Please see Supplemental Methods for details.

Statistical analysis

Statistics were calculated using SPSS 12.0 (SPSS Inc, Chicago, Ill). Descriptive statistics included mean and SD. One-way analysis of variance and 2-way repeated-measures analysis of variance with post hoc testing were used. Differences were considered significant at P-values of <0.05.


phiC31 integrase mediates site-specific integration of the triple fusion reporter gene

A donor vector containing the triple fusion reporter gene was designed as shown in Figure 1A. The triple fusion reporter gene encodes firefly luciferase (Fluc), monomeric red fluorescent protein (mRFP), and herpes simplex virus type truncated thymidine kinase (HSVttk) to enable multimodal imaging using bioluminescence, fluorescence, and positron emission tomography (PET), respectively. This vector was co-transfected with the integration vector containing the phiC31 integrase into 293 cells to validate the site-specific integration system. The transfected cells were cultured in neomycin for three weeks to select stably-integrated cell clones. Genomic DNA was isolated from these clones, and primers for three typical integration sites were used to screen for site-specific integration of the triple fusion reporter transgene (Supplemental Figure 1). As expected, only the cells transfected with phiC31 integrase showed an appropriately-sized PCR product, indicating unique transgene insertion at the intended genomic site.

Figure 1
Site-specific integration of triple fusion reporter gene into human cardiac progenitor cells

Isolation and derivation of genetically modified human cardiac progenitor cells

We first isolated Sca-1+ hCPCs from human fetal hearts and clonally expanded them using a previously described protocol 21. The purity of the isolated progenitor cells was confirmed by cell surface marker expression (Supplemental Figure 2). Then, one 60-mm cell culture dish containing approximately 1×106 hCPCs was co-transfected with the donor and integration plasmids as shown in Figure 1A. After 3 weeks of growth in selection media containing neomycin, an average of 57 colonies was obtained (See Supplemental Methods). Control transfections of the donor plasmid only (without the integration plasmid) yielded only 1–2 neomycin-resistant colonies, suggesting that transient expression of the phiC31 integrase substantially improved the rate of site-specific recombination. We then screened the genomic DNA of hCPCs for integration using a set of 44 pairs of primers 19. The genomic DNA of pooled neomycin-resistant hCPCs reliably demonstrated integration in 3 well-characterized integration sites on chromosomes 19, 21, and X (19q13, 21q21 and Xq22; Figure 1B; See Supplemental Methods). We screened 20 arbitrarily selected clones with 8p22, 19q13, 21q21, and Xq22 primers. From these 20 clones, we obtained 2 clones with a single integration: clone 16 contained a single integration at the 19q13 site and clone 7 contained a single integration at the 21q21 site (Figure 1C). Of note, only single integration sites were found in the clones obtained using phiC31 integration, whereas clones derived in parallel using lentiviral vectors displayed multiple integration sites, as evaluated by Southern blotting (Supplemental Figure 3). We then chose the hCPC clone with known integration at the 19q13 site (Figure 1D) for subsequent functional experiments (designated phiC31-hCPCs).

Site-specific integration of the triple fusion reporter gene does not alter hCPC phenotype and enables robust transgene expression

Expression of the reporter gene from the “safe harbor” locus on chromosome 19 did not affect the cellular phenotype of hCPCs. Namely, integration of the reporter gene using phiC31 caused no discernible adverse effects on cellular proliferation (Supplemental Figure 4), karyotype (Supplemental Figure 5), or gene expression profile (Supplemental Figure 6). Of note, hCPC clones derived using lentivirus did display a relative deficit in cellular proliferation capacity at 72 and 96 hours compared to untransduced hCPCs (Supplemental Figure 4). Most importantly, derivation of hCPC clones using phiC31 did not appreciably affect the differentiation propensity of hCPCs. phiC31-hCPCs retained expression of the Mef2C and Nkx2.5 cardiac transcription factors, as well as their ability to differentiate into cardiomyocyte, smooth muscle, and endothelial cell lineages, as evidenced by expression of the α-actinin, smooth muscle actin (SMA), or CD31 markers, respectively (Figure 2A, Supplemental Figure 7). The gene expression profile of phiC31-hCPCs, as assessed by transcriptome microarray, was no different than that of untransduced hCPCs (Figure 2B). Finally, expression of the triple fusion reporter gene (Fluc-mRFP-HSVttk) from the 19q13 site was stable and robust. Fluc activity correlated linearly with cell number (R2 = 0.99) as shown in Figure 2C and Supplemental Figure 8. Bioluminescent signal intensity did not decrease appreciably over multiple passages (Supplemental Figure 9), indicating that “gene silencing” played a negligible role in interpretation of the results. Uptake of 9-[4-[18F]fluoro-3-(hydroxymethyl)butyl]guanine ([18F]-FHBG) radiotracer increased linearly over time in both phiC31-modified hCPCs and lentivirally-modified hCPCs, signifying robust HSVttk activity (Figure 2D, Supplemental Figure 8). As expected, HSVttk activity was diminished in phiC31-modified hCPCs containing a single integration copy of the reporter gene, compared to lentivirally-modified hCPCs that contained multiple randomly integrated copies of the reporter gene (Supplemental Figure 3).

Figure 2
In vitro comparison of triple fusion reporter gene-expressing hCPCs derived using site-specific phiC31 integrase recombination versus random lentiviral infection

Intramyocardial transplantation of human cardiac progenitor cells improves left ventricular contractility

We next tested the efficacy of hCPC transplantation in ameliorating the effects of myocardial infarction in mice. Adult NOD SCID mice were subjected to LAD artery ligation followed by injection with 1×106 phiC31-hCPCs (n=30), 1×106 unmodified-hCPCs (n=15), or PBS control (n=15). Myocardial left ventricular (LV) function after hCPC transplantation was assessed by echocardiography (Figure 3A) and cardiac MRI (Supplemental Figure 10A) over the 8-week study period. At week 2 post-transplant, phiC31-hCPC-treated hearts demonstrated a statistically significant improvement in fractional shortening (24.9±1.34% vs. 19.8±0.99%; P=0.0017; Figure 3B) and ejection fraction (54.3±1.82% vs. 45.6±1.51%; P=0.0008; Supplemental Figure 10B) compared to PBS controls. However, no statistically significant differences in contractility were observed between the hCPC-treated and untreated PBS controls at week 8 post-transplant. As expected, the phiC31-hCPC treated group did not display statistically significant differences from the unmodified hCPC-treated group at any of the tested time points.

Figure 3
Improvement in myocardial contractility after hCPC transplant as assessed by echocardiography and MRI

Molecular imaging of human cardiac progenitor cell survival in ischemic myocardium

We then evaluated the temporal kinetics of hCPC engraftment using BLI and PET. We carried out serial acquisitions concurrently over the 8-week study period, and observed loss of both BLI and PET signal intensities over time, reflecting death of the transplanted cells (Figure 4A). When normalized to BLI signal intensity at day 1, the percentage of cells that survived was 22.7 ±11.5% at day 7, 15.7 ± 7.7% at day 14, 10.6 ± 5.06% at day 28, and 0.8 ± 0.4% at day 56 (Figure 4B). These results were confirmed using PET imaging (Figure 4C and 4D), and are in agreement with the results of other groups showing death of the majority of transplanted stem cells within 4–6 weeks after intramyocardial delivery 9, 2931. To confirm the results of our PET and BLI data, we also measured cell engraftment using real-time quantitative polymerase chain reaction (RT-qPCR). To this end, we utilized human-specific primers against Alu sequences to quantify the number of human genomes present within murine hearts at 14 and 28 days after transplantation. Our results using RT-qPCR were concordant with data obtained using PET and BLI. Namely, at days 14 and 28 after transplantation, 12.12 ± 3.33% and 6.36 ± 2.35% of unmodified hCPCs had successfully engrafted into murine myocardium. Similarly, at days 14 and 28 after transplantation, 11.37 ± 3.55% and 6.65 ± 2.46% of phiC31-hCPCs had successfully engrafted (Supplemental Figure 11).

Figure 4
Temporal kinetics of hCPC survival in infarcted hearts

Isolation and site-specific genetic modification of differentiated human endothelial cells

hCPCs represent a multipotent cell type with significant proliferative potential, which may account for the relative ease with which site-specific hCPC integrants can be obtained. We therefore wished to demonstrate site-specific integration in another cell type; in particular, a more differentiated cell source with therapeutic potential such as human endothelial cells (hECs). We generated human ECs containing site-specific integration of the triple-fusion reporter gene by co-transfection of donor and integration vectors (Figure 1A). The transfected cells were clonally expanded and screened for integration at known pseudo-attP sites 19. One out of 20 clones was shown to have a single integration at the 8p22 site and was expanded for further characterization and functional experiments phiC31-hECs were identical to untransduced hECs in their phenotype, as assessed by their expression of the endothelial cell marker CD31 (>90%), LDL uptake, and ability to form tubules in vitro (Supplemental Figure 12).

Transplantation of human endothelial cells improves ischemic limb perfusion

To assess cellular engraftment over time, we used BLI to evaluate the temporal kinetics of hEC survival (Figure 5A). Normalized to day 1, the BLI signals were 37.6±5.5% at day 4, 17.7±3.5% at day 7, and 11.3±2.1% at day 14 (Figure 5B). These results suggest acute death of hECs during the first week, but relatively stable engraftment thereafter. We subsequently carried out laser Doppler blood perfusion (LDBP) monitoring to examine whether hECs delivery improved ischemic hindlimb perfusion (Figure 5C). The hindlimb perfusion ratio (ischemic/control hindlimb) was significantly improved in the hindlimbs transplanted with hECs compared to PBS at day 7 (0.41±0.051 vs. 0.32±0.035, P<0.005) and day 14 (0.54±0.071 vs. 0.41±0.036; P<0.005) after transplantation (Figure 5D). The hindlimb perfusion ratios of phiC31-hECs and the unmodified hECs were similar. These data show that phiC31-hECs, despite their genetic modification, can be safely transplanted in vivo and retain their therapeutic capability to restore perfusion.

Figure 5
Improvement in hindlimb perfusion after transplantation of human endothelial cells


In recent years, stem cell therapy has emerged as a promising approach to promote tissue repair after myocardial infarction. Despite encouraging initial results, clinical translation of cellular therapy has been impeded by several challenges, including poor engraftment 32, malignant transformation 33, or acute death of the transplanted cells 34. Innovative approaches to overcome these challenges have relied on genetic modification of the delivered stem cells to enable expression of transgenes. Stable expression of such transgenes can enable a variety of valuable enhancements to cellular therapy, including enhanced vasculogenesis 35, improved cell survival 15, 36, cellular tracking using clinical imaging modalities 37, and the availability of a “safety switch” to induce apoptosis in the event of cellular misbehavior 17. However, commonly employed gene therapy vectors such as lentivirus and retrovirus can produce random integrations that are subject to the unpredictable effects of insertional mutagenesis. Hence site-specific integration techniques are needed to safely translate gene therapy into the clinical setting.

In recent years, stem and progenitor cells have emerged as excited new tools for ameliorating left ventricular dysfunction following myocardial infarction. hCPCs hold considerable potential in this regard, as they are a clonogenic, self-renewing population that is pre-programmed for differentiation into cells of all three cardiac lineages in vitro: myocytes, smooth muscle cells, and endothelial cells. Several parameters for hCPC identification are currently found in the literature, including isolation of c-kit+ cells 3, cardiosphere-derived cells 38, Sca-1+ cells 39, and Isl1+ cells 40. These various hCPC formulations have all demonstrated a degree of efficacy in animal studies, although evaluation of the degree of overlap between these various populations and their comparative efficacy is still needed. We employed Sca-1+ hCPCs in this study, as we and others have found that hCPCs isolated using the Sca-1 antibody consistently provide high yields of homogeneous cells with identical morphology, differentiation, and surface marker expression 39, 41, 42. However, the human equivalent for murine Sca-1 has not yet been identified. Presumably, the Sca-1 antibody cross-reacts with a human protein to yield a homogeneous population, but further work is needed to elucidate the identity of this epitope.

Here, we employed the phiC31 integrase to achieve site-specific integration of a triple fusion reporter gene into a known chromosomal context in hCPCs and hECs. This proof-of-principle study demonstrates the utility of the phiC31 integration technique in producing both progenitor cells (hCPCs) and differentiated cells (hECs) that have been genetically modified to stably express a transgene of interest. Moreover, the techniques described herein are easily applied in any context with just basic molecular biology expertise and plasmid manipulation tools 43. Genomic modification of hCPCs did not appreciably alter the progenitor cell phenotype, as assessed by global transcriptome microarray analysis and immunohistochemical analysis of differentiating progenitor cells. Indeed, phiC31-modified hCPCs retained their ability to improve myocardial contractility by a statistically significant margin compared to controls when assessed two weeks after intramyocardial injection. Likewise, phiC31-modified hECs maintained their ability to improve ischemic hindlimb perfusion after genomic modification.

We have the found the phiC31 integrase technique a facile and efficient approach for generating site-specific integrants in progenitor and differentiated cells. Alternate site-specific integration techniques are significantly less efficient than phiC31-integrase mediated recombination because they often rely on successful activation of mammalian cells’ homologous recombination machinery. One such technique involves the introduction of a double-strand break at the desired genomic location by a tandem pair of zinc-finger nucleases (ZFNs) 44. Although ZFNs allow targeting of a wider variety of genomic loci, their design and validation require extensive time and expertise. In addition to verifying proper targeting, cells’ exposure to ZFNs must be minimized in order to prevent any off-target cleavage events. The phiC31 integrase system is easily implemented using common plasmid manipulation and molecular biology techniques.

In summary, our report establishes for the first time the feasibility of site-specific genetic integration in human cardiac progenitor cells and human endothelial cells using non-viral vectors. This type of genetic engineering of human stem cells enables a multitude of improvements to overcome many of cellular therapy’s current limitations. The ease, efficiency, and safety of the phiC31 integrase system make it an appealing approach to such attempts at stable genetic modification of therapeutic cell populations.

Supplementary Material


Funding sources. This work was supported in part by grants from Burroughs Wellcome Function, NIH HL093172, and NIH EB009689 (JCW) and NIH HL099776 (RCR).

We would like to acknowledge Fangjun Jia, Yongquan Gong, and Li Wang for helpful discussions as well as assistance with experimental protocols.


Statement of responsibility. The authors had full access to and take full responsibility for the integrity of the data. All authors have read and accept the manuscript as written.

Conflict of interest. M.P.C. is an inventor on Stanford-owned patents covering phiC31 integrase. All other authors indicate no potential conflicts of interest.


1. Rasmussen TL, Raveendran G, Zhang J, Garry DJ. Getting to the heart of myocardial stem cells and cell therapy. Circulation. 2011;123:1771–1779. [PMC free article] [PubMed]
2. Jameel MN, Zhang J. Stem cell therapy for ischemic heart disease. Antioxid Redox Signal. 2010;13:1879–1897. [PMC free article] [PubMed]
3. Bearzi C, Rota M, Hosoda T, Tillmanns J, Nascimbene A, De Angelis A, Yasuzawa-Amano S, Trofimova I, Siggins RW, LeCapitaine N, Cascapera S, Beltrami AP, D’Alessandro DA, Zias E, Quaini F, Urbanek K, Michler RE, Bolli R, Kajstura J, Leri A, Anversa P. Human cardiac stem cells. Proceedings of the National Academy of Sciences. 2007;104:14068–14073. [PubMed]
4. Tang X-L, Rokosh G, Sanganalmath SK, Yuan F, Sato H, Mu J, Dai S, Li C, Chen N, Peng Y, Dawn B, Hunt G, Leri A, Kajstura J, Tiwari S, Shirk G, Anversa P, Bolli R. Intracoronary administration of cardiac progenitor cells alleviates left ventricular dysfunction in rats with a 30-day-old infarction. Circulation. 2010;121:293–305. [PMC free article] [PubMed]
5. Cheng K, Li T-S, Malliaras K, Davis DR, Zhang Y, Marban E. Magnetic targeting enhances engraftment and functional benefit of iron-labeled cardiosphere-derived cells in myocardial infarction. Circulation Research. 2010;106:1570–1581. [PMC free article] [PubMed]
6. Lee S-T, White AJ, Matsushita S, Malliaras K, Steenbergen C, Zhang Y, Li T-S, Terrovitis J, Yee K, Simsir S, Makkar R, Marban E. Intramyocardial injection of autologous cardiospheres or cardiosphere-derived cells preserves function and minimizes adverse ventricular remodeling in pigs with heart failure post-myocardial infarction. Journal of the American College of Cardiology. 2011;57:455–465. [PubMed]
7. Smith RR, Barile L, Cho HC, Leppo MK, Hare JM, Messina E, Giacomello A, Abraham MR, Marban E. Regenerative potential of cardiosphere-derived cells expanded from percutaneous endomyocardial biopsy specimens. Circulation. 2007;115:896–908. [PubMed]
8. Bolli R, Chugh AR, D’Amario D, Loughran JH, Stoddard MF, Ikram S, Beache GM, Wagner SG, Leri A, Hosoda T, Sanada F, Elmore JB, Goichberg P, Cappetta D, Solankhi NK, Fahsah I, Rokosh DG, Slaughter MS, Kajstura J, Anversa P. Cardiac stem cells in patients with ischaemic cardiomyopathy (scipio): Initial results of a randomised phase 1 trial. The Lancet. 2011;378:1847–1857. [PMC free article] [PubMed]
9. Li Z, Lee A, Huang M, Chun H, Chung J, Chu P, Hoyt G, Yang P, Rosenberg J, Robbins RC, Wu JC. Imaging survival and function of transplanted cardiac resident stem cells. Journal of the American College of Cardiology. 2009;53:1229–1240. [PMC free article] [PubMed]
10. Cao F, Wagner RA, Wilson KD, Xie X, Fu J-D, Drukker M, Lee A, Li RA, Gambhir SS, Weissman IL, Robbins RC, Wu JC. Transcriptional and functional profiling of human embryonic stem cell-derived cardiomyocytes. PLoS One. 2008;3:e3474. [PMC free article] [PubMed]
11. Balsam LB, Wagers AJ, Christensen JL, Kofidis T, Weissman IL, Robbins RC. Haematopoietic stem cells adopt mature haematopoietic fates in ischaemic myocardium. Nature. 2004;428:668–673. [PubMed]
12. Noiseux N, Gnecchi M, Lopez-Ilasaca M, Zhang L, Solomon SD, Deb A, Dzau VJ, Pratt RE. Mesenchymal stem cells overexpressing akt dramatically repair infarcted myocardium and improve cardiac function despite infrequent cellular fusion or differentiation. Mol Ther. 2006;14:840–850. [PubMed]
13. Nguyen PK, Lan F, Wang Y, Wu JC. Imaging: Guiding the clinical translation of cardiac stem cell therapy. Circulation Research. 2011;109:962–979. [PMC free article] [PubMed]
14. Huang J, Zhang Z, Guo J, Ni A, Deb A, Zhang L, Mirotsou M, Pratt RE, Dzau VJ. Genetic modification of mesenchymal stem cells overexpressing ccr1 increases cell viability, migration, engraftment, and capillary density in the injured myocardium. Circulation Research. 2010;106:1753–1762. [PMC free article] [PubMed]
15. Fischer KM, Cottage CT, Wu W, Din S, Gude NA, Avitabile D, Quijada P, Collins BL, Fransioli J, Sussman MA. Enhancement of myocardial regeneration through genetic engineering of cardiac progenitor cells expressing pim-1 kinase. Circulation. 2009;120:2077–2087. [PMC free article] [PubMed]
16. Cao F, Lin S, Xie X, Ray P, Patel M, Zhang X, Drukker M, Dylla SJ, Connolly AJ, Chen X, Weissman IL, Gambhir SS, Wu JC. In vivo visualization of embryonic stem cell survival, proliferation, and migration after cardiac delivery. Circulation. 2006;113:1005–1014. [PubMed]
17. Di Stasi A, Tey S-K, Dotti G, Fujita Y, Kennedy-Nasser A, Martinez C, Straathof K, Liu E, Durett AG, Grilley B, Liu H, Cruz CR, Savoldo B, Gee AP, Schindler J, Krance RA, Heslop HE, Spencer DM, Rooney CM, Brenner MK. Inducible apoptosis as a safety switch for adoptive cell therapy. New England Journal of Medicine. 2011;365:1673–1683. [PMC free article] [PubMed]
18. Hacein-Bey-Abina S, Von Kalle C, Schmidt M, McCormack MP, Wulffraat N, Leboulch P, Lim A, Osborne CS, Pawliuk R, Morillon E, Sorensen R, Forster A, Fraser P, Cohen JI, de Saint Basile G, Alexander I, Wintergerst U, Frebourg T, Aurias A, Stoppa-Lyonnet D, Romana S, Radford-Weiss I, Gross F, Valensi F, Delabesse E, Macintyre E, Sigaux F, Soulier J, Leiva LE, Wissler M, Prinz C, Rabbitts TH, Le Deist F, Fischer A, Cavazzana-Calvo M. Lmo2-associated clonal t cell proliferation in two patients after gene therapy for scid-x1. Science. 2003;302:415–419. [PubMed]
19. Chalberg TW, Portlock JL, Olivares EC, Thyagarajan B, Kirby PJ, Hillman RT, Hoelters J, Calos MP. Integration specificity of phage œïc31 integrase in the human genome. Journal of Molecular Biology. 2006;357:28–48. [PubMed]
20. Goumans MJ, de Boer TP, Smits AM, van Laake LW, van Vliet P, Metz CH, Korfage TH, Kats KP, Hochstenbach R, Pasterkamp G, Verhaar MC, van der Heyden MA, de Kleijn D, Mummery CL, van Veen TA, Sluijter JP, Doevendans PA. Tgf-beta1 induces efficient differentiation of human cardiomyocyte progenitor cells into functional cardiomyocytes in vitro. Stem Cell Res. 2007;1:138–149. [PubMed]
21. Smits AM, van Vliet P, Metz CH, Korfage T, Sluijter JP, Doevendans PA, Goumans MJ. Human cardiomyocyte progenitor cells differentiate into functional mature cardiomyocytes: An in vitro model for studying human cardiac physiology and pathophysiology. Nat Protoc. 2009;4:232–243. [PubMed]
22. van Vliet P, Roccio M, Smits AM, van Oorschot AA, Metz CH, van Veen TA, Sluijter JP, Doevendans PA, Goumans MJ. Progenitor cells isolated from the human heart: A potential cell source for regenerative therapy. Neth Heart J. 2008;16:163–169. [PMC free article] [PubMed]
23. Keravala A, Portlock JL, Nash JA, Vitrant DG, Robbins PD, Calos MP. Phic31 integrase mediates integration in cultured synovial cells and enhances gene expression in rabbit joints. J Gene Med. 2006;8:1008–1017. [PubMed]
24. Ray P, Tsien R, Gambhir SS. Construction and validation of improved triple fusion reporter gene vectors for molecular imaging of living subjects. Cancer Res. 2007;67:3085–3093. [PubMed]
25. Olivares EC, Hollis RP, Chalberg TW, Meuse L, Kay MA, Calos MP. Site-specific genomic integration produces therapeutic factor ix levels in mice. Nat Biotechnol. 2002;20:1124–1128. [PubMed]
26. Chalberg TW, Portlock JL, Olivares EC, Thyagarajan B, Kirby PJ, Hillman RT, Hoelters J, Calos MP. Integration specificity of phage phic31 integrase in the human genome. J Mol Biol. 2006;357:28–48. [PubMed]
27. Rosenzweig A. Cardiac cell therapy--mixed results from mixed cells. N Engl J Med. 2006;355:1274–1277. [PubMed]
28. Niiyama H, Kai H, Yamamoto T, Shimada T, Sasaki K, Murohara T, Egashira K, Imaizumi T. Roles of endogenous monocyte chemoattractant protein-1 in ischemia-induced neovascularization. J Am Coll Cardiol. 2004;44:661–666. [PubMed]
29. Terrovitis J, Lautamaki R, Bonios M, Fox J, Engles JM, Yu J, Leppo MK, Pomper MG, Wahl RL, Seidel J, Tsui BM, Bengel FM, Abraham MR, Marban E. Noninvasive quantification and optimization of acute cell retention by in vivo positron emission tomography after intramyocardial cardiac-derived stem cell delivery. Journal of the American College of Cardiology. 2009;54:1619–1626. [PMC free article] [PubMed]
30. van der Bogt KE, Sheikh AY, Schrepfer S, Hoyt G, Cao F, Ransohoff KJ, Swijnenburg RJ, Pearl J, Lee A, Fischbein M, Contag CH, Robbins RC, Wu JC. Comparison of different adult stem cell types for treatment of myocardial ischemia. Circulation. 2008;118:S121–129. [PMC free article] [PubMed]
31. Johnston PV, Sasano T, Mills K, Evers R, Lee S-T, Smith RR, Lardo AC, Lai S, Steenbergen C, Gerstenblith G, Lange R, Marbán E. Engraftment, differentiation, and functional benefits of autologous cardiosphere-derived cells in porcine ischemic cardiomyopathy. Circulation. 2009;120:1075–1083. [PMC free article] [PubMed]
32. Schachinger V, Aicher A, Dobert N, Rover R, Diener J, Fichtlscherer S, Assmus B, Seeger FH, Menzel C, Brenner W, Dimmeler S, Zeiher AM. Pilot trial on determinants of progenitor cell recruitment to the infarcted human myocardium. Circulation. 2008;118:1425–1432. [PubMed]
33. Amariglio N, Hirshberg A, Scheithauer BW, Cohen Y, Loewenthal R, Trakhtenbrot L, Paz N, Koren-Michowitz M, Waldman D, Leider-Trejo L, Toren A, Constantini S, Rechavi G. Donor-derived brain tumor following neural stem cell transplantation in an ataxia telangiectasia patient. PLoS Med. 2009;6:e1000029. [PMC free article] [PubMed]
34. Freyman T, Polin G, Osman H, Crary J, Lu M, Cheng L, Palasis M, Wilensky RL. A quantitative, randomized study evaluating three methods of mesenchymal stem cell delivery following myocardial infarction. European Heart Journal. 2006;27:1114–1122. [PubMed]
35. Yang F, Cho S-W, Son SM, Bogatyrev SR, Singh D, Green JJ, Mei Y, Park S, Bhang SH, Kim B-S, Langer R, Anderson DG. Genetic engineering of human stem cells for enhanced angiogenesis using biodegradable polymeric nanoparticles. Proceedings of the National Academy of Sciences. 2010;107:3317–3322. [PubMed]
36. Tang YL, Tang Y, Zhang YC, Qian K, Shen L, Phillips MI. Improved graft mesenchymal stem cell survival in ischemic heart with a hypoxia-regulated heme oxygenase-1 vector. Journal of the American College of Cardiology. 2005;46:1339–1350. [PubMed]
37. Chen IY, Wu JC. Cardiovascular molecular imaging. Circulation. 2011;123:425–443. [PMC free article] [PubMed]
38. Davis DR, Zhang Y, Smith RR, Cheng K, Terrovitis J, Malliaras K, Li T-S, White A, Makkar R, Marban E. Validation of the cardiosphere method to culture cardiac progenitor cells from myocardial tissue. PLoS ONE. 2009;4:e7195. [PMC free article] [PubMed]
39. Smits AM, van Vliet P, Metz CH, Korfage T, Sluijter JPG, Doevendans PA, Goumans M-J. Human cardiomyocyte progenitor cells differentiate into functional mature cardiomyocytes: An in vitro model for studying human cardiac physiology and pathophysiology. Nat Protocols. 2009;4:232–243. [PubMed]
40. Bu L, Jiang X, Martin-Puig S, Caron L, Zhu S, Shao Y, Roberts DJ, Huang PL, Domian IJ, Chien KR. Human isl1 heart progenitors generate diverse multipotent cardiovascular cell lineages. Nature. 2009;460:113–117. [PubMed]
41. van Oorschot AAM, Smits AM, Pardali E, Doevendans PA, Goumans M-J. Low oxygen tension positively influences cardiomyocyte progenitor cell function. Journal of Cellular and Molecular Medicine. 2011;15:2723–2734. [PubMed]
42. Ryzhov S, Goldstein AE, Novitskiy SV, Blackburn MR, Biaggioni I, Feoktistov I. Role of a2b adenosine receptors in regulation of paracrine functions of stem cell antigen 1-positive cardiac stromal cells. Journal of Pharmacology and Experimental Therapeutics. 2012;341:764–774. [PubMed]
43. Keravala A, Calos MP. Site-specific chromosomal integration mediated by phic31 integrase. Methods Mol Biol. 2008;435:165–173. [PubMed]
44. Urnov FD, Rebar EJ, Holmes MC, Zhang HS, Gregory PD. Genome editing with engineered zinc finger nucleases. Nat Rev Genet. 2010;11:636–646. [PubMed]