Introduction

Human embryonic stem (hES) cells are derived from the pluripotent cells of the inner cell mass of the blastocyst^1,2. These cells can potentially proliferate indefinitely in culture, yet still retain a normal karyotype and the potential to differentiate into any cell type. Hence hES cells are expected to have far-reaching applications in regenerative medicine and basic research. However, the exploitation of the remarkable potential of hES cells largely depends on the development of technologies that will allow efficient manipulation of the cells in vitro. The objective of this study was to develop an effective strategy for stable genetic modification of hES cells. We have chosen to use vectors that are derived from human immunodeficiency virus type 1 (HIV-1) lentiviruses, given their potential to promote efficient transduction and stable transgene expression in a variety of cell types in vivo and in vitro^3,4,5. The lentiviruses offer advantages over other members of the retrovirus family. First, whereas most retroviruses infect only dividing cells, the lentiviruses can also infect nondividing cells. Second, although the use of retroviral vectors for gene delivery into murine ES cells proved ineffective because of silencing of transgene expression^6,7, 'gene silencing' was not observed upon transduction of murine ES cells by an HIV-1 vector^8,9. Here, we report that modified self-inactivating (SIN) derived vectors HIV-1 are efficient tools for stable genetic modification of hES cells. Transduction of hES cells by these vectors facilitates transgene expression that is maintained throughout prolonged cultivation as well as differentiation in vitro and in vivo.

Results

Transduction of hES cells by a modified HIV-1-based SIN18 vector

In our initial attempts, we evaluated the potential of lentiviral vectors, pseudotyped with vesicular stomatitis virus-G protein (VSV-G), to genetically modify hES cells by using the SIN18 HIV-1 vector pRLLSIN18.hPGKp.EGFP, which harbors the reporter gene enhanced green fluorescent protein (EGFP) under the control of the human phosphoglycerate kinase (hPGK) promoter. The viral promoter/enhancer at the long terminal repeat (LTR) regions are deleted in this vector to increase its safety and to prevent potential interference between the LTR and the internal promoter^10,11 (Fig. 1A). Using transient co-transfection of 293T cells with three plasmids, we have generated vectors at titers of 2–3 10⁷ transducing units (TU)/ml. Transduction of hES cell colonies that were cultured in serum-containing medium on mouse embryonic fibroblasts² with 10-fold concentrated virus resulted in the expression of the transgene by 7% of the hES cells (Fig. 1B). It should be noted that the feeder layer displayed intense transgene expression due to absorption of the viral particles (data not shown).

Figure 1.

Transduction of hES cells by modified HIV-1-based SIN18 vectors. (A) Schematic representation of the HIV-1 SIN18 vectors. The vector pRLLSIN18.hPGK.EGFP, which harbors EGFP under the control of the hPGK promoter, was modified to make pSIN18.cPPT.hEF1.EGFP.WPRE by (1) insertion of WPRE downstream of the reporter gene, (2) replacement of the hPGK promoter by the hEF1 promoter, and (3) re-introduction of cPPT upstream of the promoter. (B) FACS analysis of hES cells 7 days after transduction with the nonmodified vector (middle panel) and the modified vector using an improved transduction protocol (right panel). The level of spontaneous differentiation was determined by analysis of the percentage of cells that were immunoreactive with GCTM2 antibody. The percentages of cells are indicated in each quadrant together with the mean fluorescence intensity (in parentheses) of EGFP expression. The upper and lower right quadrants represent the undifferentiated and differentiated transduced hES cells, respectively. The dot-plot analyses show the results of one representative experiment out of three experiments.

Full figure and legend (231K)

To optimize the transduction and to eliminate possible unwanted effect of the feeders, we have modified the transduction protocol as follows: First, we used a serum-free KnockOut medium supplemented with 20% serum replacer (SR; Gibco-BRL) during the preparation of the recombinant virions as well as during transduction. The use of serum-free medium increased transduction efficiencies by 2-fold, compared with transduction with serum-containing medium. Second, we carried out 'double transduction' for short consecutive periods (1.5 hours each) on clumps of mainly undifferentiated hES cells (150–200 cells) that were incubated in suspension with the viral particles without the support of feeders. Immediately following the short period of transduction (3 hours), we further cultured the hES cell clumps on feeders. Concurrent with optimizing the transduction protocol, we have modified the HIV-1 SIN18 vector to include the post-transcriptional regulatory element from woodchuck hepatitis virus (WPRE)¹² and the central polypurine tract (cPPT) sequence of HIV-1^13,14. These elements were shown to increase transgene expression and transduction efficiency, respectively. Insertion of WPRE increased transgene expression by 2-fold, whereas the introduction of the cPPT sequence increased transduction efficiencies by 3-fold. In addition, the hPGK promoter was replaced by the eukaryotic translation elongation factor-1 (hEF1) promoter¹⁵, which was shown to be active in both undifferentiated mouse ES cells and their differentiated derivatives¹⁶. The modified vector pSIN18.cPPT.hEF1.EGFP.WPRE is depicted in Figure 1A. Transduction of hES cells by this vector using the improved protocol significantly increased transduction efficiency by 5- to 6-fold compared with the transduction efficiency that was achieved with the original HIV-1 SIN18 vector, using the nonmodified protocol. Moreover, the intensity of transgene expression increased by 2-fold (Fig. 1B). Using the modified vector and improved protocol, we have obtained transduction efficiencies of 30–48% in six independent experiments. One week after transduction, the percentage of cells that were immunoreactive with GCTM-2 antibody, which detects an epitope on the cell surface of undifferentiated hES cells^2,17, was similar in transduced and nontransduced control cells (Fig. 1B), suggesting that the transduction protocol did not induce differentiation.

Stable transgene expression during prolonged cultivation

We enriched the hES cells transduced by pSIN18.cPPT.hEF1p.EGFP.WPRE for cells expressing the transgene by mechanical selective weekly passage of regions of hES colonies that showed EGFP expression. Four weeks after transduction (after four selective passages), we visualized an intense near-homogeneous expression of the transgene by the majority of cells within the colonies by fluorescence microscopy (Fig. 2A). Analysis by fluorescence-activated cell sorting (FACS) 7 weeks after transduction revealed that 80% of the hES cells expressed high levels of the transgene. Transgene expression was stable during prolonged nonselective cultivation (38 weeks) of transduced hES cells. FACS analysis at 36 weeks after transduction demonstrated no significant reduction in the percentage of cells expressing EGFP or in the intensity of expression (Fig. 2B). Furthermore, at that time point, the percentage of transduced cells that were immunoreactive with the GCTM2 antibody was similar to nontransduced hES cells (Fig. 2B). Hence it seems that the transduced undifferentiated cells retained the property of self-renewal during prolonged cultivation in vitro. To assess whether the stable expression of the transgene resulted from integration of the provirus into the host DNA, we conducted Southern blot analysis of genomic DNA prepared from transduced cells 29 weeks after transduction. The analysis revealed that the viral vector had integrated into the host cells. Because five distinct bands were detected, it appears that the transduced hES cell population was mainly composed of a few overrepresented clones that have integrated a maximum of five copies of the provirus per cell (Fig. 2C).

Figure 2.

Stable transgene expression following transduction by a lentiviral vector. (A) Phase-contrast (left) and fluorescence microscopy (right) images of a section of a hES cell colony 4 weeks (four selective passages) after transduction. Scale bars represent 100 m. (B) FACS analysis of hES cells 36 weeks after transduction. The analysis was done as described in Figure 1B. The dot-plot analysis shows the results of one representative experiment out of three experiments. (C) Southern blot analysis of transduced hES cells (at 29 weeks). Genomic DNA that was prepared from nontransduced (lanes 1, 3) and transduced (lanes 2, 4) hES cells was digested with EcoRV (cleaves viral DNA once, therefore detects integrated virus; lanes 1, 2), or with EcoRV and EcoRI (cleave viral DNA on both sides of the EGFP, therefore detect a 1.56-kb internal viral fragment shared by integrated or nonintegrated virus; lanes 3, 4). The digested DNA was subjected to hybridization with EGFP probe. Schematic representations of the integrated viral DNA and the position of the probe are shown above the blot.

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Transgene expression is retained throughout differentiation in vitro

To examine whether transgene expression is retained during differentiation in vitro, we cultured the transduced hES cells (11 weeks after transduction) continuously for 4 weeks on feeders without passage. We have already demonstrated that under these culture conditions, hES cells differentiate into extraembryonic and somatic lineages². FACS analyses revealed that after 4 weeks of culture without passage, the percentage of EGFP-expressing cells and the intensity of transgene expression were similar to control cultures examined a week after passage (Fig. 3A). We further studied the potential of the transduced hES cells to differentiate in vitro during differentiation within embryoid bodies (EBs). EBs were generated from transduced undifferentiated cells that were cultivated for at least 15 weeks. Fluorescence microscopy analysis of cystic EBs 20 days after transfer of transduced hES cell clumps to suspension culture conditions revealed intense expression of EGFP (Figs. 3B and 3C).

Figure 3.

Transgene expression throughout differentiation of transduced hES cells in vitro. (A) FACS analyses of transduced hES cells that were cultured for 1 and 4 weeks without passage. The percentages of EGFP-expressing cells are indicated together with the mean fluorescence intensity (in parentheses). (B, C) Phase-contrast (B) and fluorescence microscopy (C) images of cystic EBs, 20 days after generation from transduced hES cells. (D-H) Immunophenotyping of differentiated cells from disaggregated cystic EBs demonstrating co-expression of EGFP (green fluorescence) and indirect immunofluorescence staining (red) for the mesodermal markers muscle actin (D) and desmin (E), and the endodermal markers -fetoprotein (F), laminin (G), and LMW cytokeratin (H). (I-J) Phase-contrast (I) and fluorescence microscopy (J) images of neural progenitor spheres generated from transduced hES cells. (K-R) Indirect immunofluorescence analyses of neural progenitors and their differentiated progeny demonstrating co-expression of EGFP (green) and markers (red) of the neural progenitors nestin (K), PSA-NCAM (L), and A2B5 (M); early neurons (-tubulin III (N)); mature neurons (MAP 2a,b, (O) glutamate (P), NF-160 (Q)); and astrocytes (GFAP (R)). Arrows indicate cells co-expressing EGFP and markers. Scale bars represent 100 m (B, C, I, J), 20 m (D-H), and 10 m (K-R).

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To determine whether the transduced hES cells retain their pluripotent potential and whether transgene expression is sustained during differentiation in vitro into progeny of all three germ layers, we partially disaggregated the cystic EBs, then plated and subjected them to indirect immunofluorescence analysis. The analysis revealed differentiated cells co-expressing EGFP and mesodermal markers (muscle actin and desmin) (Figs. 3D and 3E) as well as endodermal markers (-fetoprotein, laminin, and low-molecular-weight (LMW) cytokeratin) (Figs. 3F-H). To examine transgene expression during differentiation into the neuroectodermal lineage, we derived neural progenitor spheres from transduced hES cells and propagated them in culture as described¹⁸. Fluorescence microscopy analysis revealed an intense expression of EGFP within the neural spheres (Figs. 3I and 3J). Indirect immunofluorescence analysis of progenitor cells from spheres that have been propagated for 4 weeks¹⁸ demonstrated co-expression of EGFP and markers of primitive neuroectoderm (nestin, PSA-NCAM, and A2B5) (Figs. 3K-3M). After induction of differentiation of the neural progenitors¹⁸, we have observed EGFP⁺ cells that displayed the morphology and markers of early neurons such as -tubulin III (Fig. 3N); mature neurons (MAP 2a,b, glutamate, and NF-160) (Figs. 3O-3Q); and astrocytes (glial fibrillary acidic protein; GFAP) (Fig. 3R). Taken together, these results indicated that the transduced hES cells retained their pluripotent potential and maintained transgene expression during differentiation into progeny of the three embryonic germ layers.

Transgene expression in teratoma tumors

To confirm further the pluripotent potential of transduced hES cells and to examine whether the expression of transgene is retained during differentiation in vivo, we engrafted clumps of undifferentiated transduced hES cells (20 and 23 weeks after transduction) into the testis of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. We have earlier demonstrated that when hES cells are engrafted into the testis of SCID mice, they form benign teratomas that contain a variety of cell types and structures that represent all three germ layers². All mice (n = 4) that were inoculated with transduced hES cells developed teratomas. Histological analysis of the teratomas 8 weeks after engraftment demonstrated tissues representing all three germ layers. Differentiated tissues seen included muscle, cartilage, and bone (mesoderm), primitive neuroectoderm (ectoderm), glandular structures and primitive bronchi formation (endoderm). We observed an extensive diffuse expression of EGFP within the teratomas both by direct analysis of fluorescence as well as by indirect immunofluorescence staining with anti-GFP antibodies. Tissues representing all three germ layers expressed the transgene (Fig. 4). These data suggested that transduced hES cells retained their pluripotency and that transgene expression was sustained during differentiation in vivo.

Figure 4.

Transgene expression in teratoma tumors. Histology and transgene expression in differentiated elements found in teratomas formed in the testis of NOD/SCID mice 8 weeks after implantation of lentiviral-transduced hES cells. Hematoxylin-eosin staining images of cartilage (A, E), primitive bronchus (C), neural rosettes (G), and glandular structures (I) are shown. The cells within these structures are expressing EGFP as demonstrated in adjacent sections by fluorescence microscopy (B, D) or by indirect immunohistochemistry with anti-EGFP (F, H, J). Scale bars represent 50 m.

Full figure and legend (593K)

Discussion

Our results demonstrate that HIV-1-based vectors are a powerful and efficient tool for stable genetic modification of hES cells. Transduction of hES cells by the modified self-inactivating HIV-1 vector containing the WPRE and cPPT sequences led to stable transgene expression that was maintained throughout prolonged cultivation as well as differentiation in vitro and in vivo.

When oncoretroviral vectors were used to transduce mouse ES cells, a 'gene silencing' phenomenon was observed upon differentiation¹⁹. Our results indicate that when HIV-derived vectors are used to deliver genes into human ES cells, transgene expression is sustained during stem cell proliferation and differentiation. A recent report on the transduction of mouse ES cells by a similar HIV-1 SIN18 vector supports our results. The transduced mouse ES cells, like the human ES cells, maintained transgene expression during differentiation in vitro and in vivo⁹.

Initial data highlighting the potential of lentiviral vectors to serve as a tool for gene delivery into hES cells was recently reported by Pfeifer and his colleagues⁹. Human ES cells were transduced by HIV-1-derived vector and expressed the transgene over several passages. However, the pluripotent nature of the transduced cells as well as the stability of transgene expression along prolonged cultivation or differentiation were not demonstrated. Genetic modification of human ES cells may be accomplished by transfection²⁰. Yet, in contrast to the efficient introduction of stable genetic modification using lentiviral vectors, the efficiency of deriving stable clones from transfected hES cells is relatively low (a clone per 100,000 cells). In addition, the potential of transfected hES cells to retain transgene expression during differentiation into progeny of all three germ layers in vitro and in vivo in teratomas was not studied.

Genetic modification of hES cells with lentivirus vectors may have far-reaching applications, including the study of gene function in early human development, genetic selection of either undifferentiated stem cells or differentiated cells of specific lineages²¹, and forced expression of transcriptional factors that could direct the differentiation of hES cells into a specific cell type²². The ability to direct the differentiation of hES cells or to select a specific lineage may be highly valuable for the study of human embryogenesis and could potentially provide an unlimited supply of a pure population of various cell types for transplantation therapy. Furthermore, transplantation of genetically modified hES cells may potentially allow the delivery and expression of foreign DNA within target organs in the course of gene therapy.

We conclude that HIV-1-derived vectors are efficient tools for the delivery and stable expression of transgenes by hES cells. Genetic modifications by these vectors may offer new avenues for the study and use of hES cells in basic and applied scientific research.

Materials and methods

Vector plasmid construction

pSIN18.cPPT.hEF-1p.EGFP.WPRE was constructed as follows: First, the WPRE component was amplified by PCR from plasmid pHR'-CMV-GFP using primers 5'-ACGCGTCGACAATCAACCTCTGGATTACAA-3' (SalI) and 5'-ACGCGTCGACAAGGCGGGGAGGCGG-3' (SalI), and inserted into pRLLSIN18.hPGK.EGFP^10,11 that had been digested with SalI. Second, the hEF1 promoter was amplified from plasmid pEF-BOS (a gift of S Nagata, Osaka University Medical School, Japan) by PCR using primers 5'-GCGATATCGGCTCCGGTGCCCGTCAG-3' (EcoRV) and 5'-CGGGATCCTGTGTTCTGGCGGCAAAC-3' (BamHI). After amplification, the PCR product and pSIN18.hPGKp.EGFP.WPRE were digested with EcoRV and BamHI and then ligated to generate plasmid pSIN18.hEF-1p.EGFP.WPRE. Third, the cPPT element was amplified from plasmid pCMV89.1 using primers 5'-CCGCTCGAGACAAATGGCAGTATTCATCCA-3' (XhoI) and 5'-CCGCTCGAGCCAAAGTGGATTTCTGCTGTC-3' (XhoI), and inserted into plasmid pRLLSIN18.hEF-1p.EGFP.WPRE digested with XhoI.

Virus production

Viruses were produced by transient co-transfection of three plasmids into 293T cells as described earlier^3,10, with several modifications. Briefly, 1.5 10⁶ 293T cells were transfected using the FuGENE6 Transfection Reagent (Roche Molecular Biochemicals, Mannheim, Germany) with a total of 20 g of plasmid DNA: 3.5 g of the envelope plasmid pMD.G harboring the gene encoding VSV-G, 6.5 g of the packaging plasmid pCMVR8.91, and 10 g of the transfer vector. The medium was replaced 20–24 hours after transfection with KnockOut medium supplemented with 20% KnockOut SR (Gibco-BRL, Gaithersburg, MD). The medium containing the viral particles was collected 48 and 72 hours after transfection and filtered through 0.45-m filters (Sartorius, Goettingen, Germany). Virus was concentrated by ultracentrifugation at 50,000g at 4°C for 1.5 hours. Viral titers (TU/ml) were determined by transduction of 293T cells with serial dilutions of the viral supernatant and FACS analysis of the percentage of EGFP-expressing cells.

Transduction of human ES cells

Human ES cells (HES-1 cell line²) were cultured on mouse embryonic fibroblasts, as earlier described². At the time of routine passage, clumps of 200 undifferentiated cells were isolated from hES colonies by mechanical slicing followed by dispase digestion (10 mg/ml; Gibco-BRL). The clumps were transferred into the medium containing the viral particles and incubated with the viral particles in the presence of 5 g/ml Polybrene (Sigma, St Louis, MO), at 37°C for 1.5 hour. Fresh virus was then added, and the incubation continued for another 1.5 hour. The transduced hES cell clumps were then washed briefly with PBS and replated on a fresh mouse feeder layer. Measurement of transduction efficiency was carried out by FACS analysis 7 days after transduction.

FACS analysis

The percentage of cells and intensity of transgene expression were analyzed on a FACSCalibur system (Becton-Dickinson, San Jose, CA). The proportion of undifferentiated cells among the transduced and control hES cells was determined by immunoreactivity with the mouse monoclonal antibody GCTM2¹⁷. For FACS analysis the transduced hES colonies were treated with 10 mg/ml dispase (Gibco-BRL), washed with PBS, trypsinized, and then incubated with the GCTM-2 antibody. Primary antibody was detected using rabbit anti-mouse immunoglobulins conjugated with RPE-Cy5 (1:10; Dako, Carpinteria, CA). Following immunostaining, the cells were resuspended in FACS buffer.

Detection and quantification of integrated viral DNA

Southern hybridization analysis was done with genomic DNA prepared from nontransduced and transduced hES cells. The DNA was digested with EcoRV, or with EcoRV and EcoRI. The digested DNA was separated on 1% agarose gel, transferred to a nylon membrane, and hybridized with [³²P]-dCTP-labeled EGFP fragment.

Differentiation in vitro

To induce differentiation of human ES cells as EBs, clumps of 200 undifferentiated hES cells were transferred to petri dishes (Becton Dickinson) and cultured in suspension in KnockOut medium supplemented with 20% KnockOut SR (Gibco-BRL) for 3 weeks. Neural spheres were derived from hES cell colonies as described¹⁸.

Immunohistochemistry studies

In general, for immunophenotyping, disaggregated EBs, neural progenitor cells, differentiated neurons, and glia cells were fixed with 4% paraformaldehyde for 20 minutes at room temperature. It was followed by blocking and permeabilization with 0.2% Triton X (Sigma) and 5% normal goat serum (Dako) in PBS for 30 minutes. Samples were incubated with the primary antibodies at room temperature for 30 minutes, washed, incubated with the secondary antibodies for the same amount of time, counterstained, and mounted with Vectashield mounting solution with DAPI (Vector Laboratories, Burlingame, CA). Primary antibody localization was done using mouse anti-rabbit IgG and goat anti-mouse IgG conjugated to Cy3 (1:100; Jackson Lab, West Grove, PA). Proper controls for primary and secondary antibodies revealed neither nonspecific staining nor antibody cross-reactivity. For the analysis of EBs, 3 weeks after their generation they were mechanically partially disaggregated, plated on poly-D-lysine (30–70 kDa, 10 g/ml; Sigma) and laminin (4 g/ml; Sigma) or fibronectin (5 g/ml; Sigma) coated coverslips, further cultured for 3–5 days in KnockOut medium supplemented with 20% KnockOut SR (Gibco-BRL), fixed and examined by indirect immunofluorescence analysis for the expression of -fetoprotein (1:500; Sigma), laminin (1:1000; Sigma), LMW cytokeratin (Becton Dickinson), desmin (1:50; Dako), and muscle actin (1:50; Dako). For the analysis of neural progenitors, neural spheres were disaggregated into single cells that were plated on coverslips coated with poly-D-lysine and laminin, fixed after 4–12 hours, and examined by indirect immunofluorescence analysis for the expression of PSA-NCAM (DSHB, Iowa City, IA), nestin (1:200; Chemicon, Temecula, CA), and A2B5 (1:20; American Type Culture Collection, ATCC, Manassas, VA). Differentiation of the neural progenitors was induced as described¹⁷. Differentiated neural cells were analyzed by indirect immunofluorescence for the expression of -tubulin III (1:1000; Sigma), MAP 2a,b (1:50; NeoMarkers, Fremont, CA), Glutamate (1:1000; Sigma), NF-160 (1:50; Chemicon), and GFAP (1:200; Dako).

Teratoma formation in NOD/SCID mice

Clumps of 200 transduced hES cells with an undifferentiated morphology were injected into the testis of 6-week-old NOD scid mice (10–15 clumps per testis; Harlan, Jerusalem, IL). The resulting tumors were removed 8–12 weeks later. To detect EGFP expression directly, samples from the tumors were fixed in 4% paraformaldehyde, frozen, and sectioned by a cryostat. Serial frozen sections (8 m) were examined by fluorescence microscopy or counterstained with hematoxylin and eosin. For immunohistochemical analysis of EGFP expression, sections from the tumors were fixed in 10% neutral buffered formalin, embedded in paraffin, and examined by indirect immunofluorescence for GFP expression. Briefly, the samples were incubated at 56°C for 1 hour, and then deparaffinized by washing with xylene. The samples were treated with 0.1% protease type XXIV (Sigma), washed with PBS, and then blocked and permeabilized with 0.5% Triton X (Sigma) and 1% BSA in PBS at room temperature (RT) for 1 hour. Samples were incubated with the primary antibodies (1:100; Santa Cruz Biotechnology, Santa Cruz, CA) at 4°C for 48 hours, washed with PBS, and incubated with goat anti-rabbit IgG conjugated to fluorescein isothiocyanate (FITC, 1:100; Jackson) for 1 hour at RT. The samples were then counterstained and mounted with Vectashield mounting solution with DAPI (Vector Laboratories).

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Acknowledgements

We gratefully acknowledge Neri Laufer for his generous support, and Vitaly Ablamunits for his help with the animal study. The monoclonal antibody for PSA-NCAM was obtained from the Developmental Studies Hybridoma Bank, developed under the auspices of the NICHD, and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, Iowa 52242. The study was supported by a grant from Embryonic Stem Cells International (ESI) Pte Ltd.