© The American Society of Gene & Cell Therapy
Molecular Therapy vol. 17 no. 9, 1585–1593 sep. 2009
Transient genetic manipulation of human neurons
without chromosomal integration of the transgene would
be valuable but has been challenging due to the quies-
cent nature of these postmitotic cells. In this study, we
developed a set of baculoviral vectors for transient trans-
duction in nondividing neurons derived from human
embryonic stem cells (hESCs). Using a baculoviral vec-
tor equipped with the woodchuck hepatitis virus post-
transcriptional regulatory element (WPRE), we observed
a quick onset of transgene expression as early as day 1
after baculoviral transduction and a high efficiency of
up to 80%. Strong transgene expression in the cultured
human neurons was observed for more than 1 month
and the signal was easily detectable even after 3 months.
Using two baculoviral vectors carrying different trans-
genes, we found that co-transduction at a single neuron
level was possible. After transplantation into the brain of
nude mice, the baculovirus-transduced human neurons
were integrated into the mouse brain and maintained
transgene expression for at least 4 weeks, portending
the usefulness of this technique in assisting neural trans-
plantation. Therefore, by mediating efficient transient
gene expression, baculoviral vectors can provide useful
tools for both basic gene function studies in human neu-
rons and therapeutic applications of these cells.
Received 15 December 2008; accepted 9 May 2009; published online
16 June 2009. doi:10.1038/mt.2009.124
Self-renewable and pluripotent human embryonic stem cells
(hESCs) are a reliable and accessible source to provide unlimited
amounts of human neurons that would otherwise be very difficult to
obtain for basic studies and clinical applications.1 Transplantation
of these human neurons in the diseased or injured central nervous
system provides the potential for treatment of a broad spectrum
of neurological diseases. At present, hurdles that limit the use of
these hESC-derived cells for clinical transplantation include the
poor survival and inappropriate functional recovery of grafted
hESC-derived neurons.2,3 Genetic manipulation of these human
neurons through gene transfer in culture prior to transplantation
would be a possible way to overcome the problems, holding out
the prospect of enhancing the survival, maturation, integration,
and many other cellular properties of transplanted neurons in
One of the possible genetic manipulation strategies for hESC-
derived neurons is integrating a transgene into the hESC genome
and then deriving human neurons from the stably modified
hESCs, in which the transgene expression is retained.4 This is a
highly valuable approach for the purpose of using hESC-derived
cells to deliver therapeutic genes for the applications that require
persistent expression. However, as an approach for improving
transplantation efficiency, stable expression of certain genes, for
example, anti-apoptosis genes, poses the risk of tumor formation
after cell transplantation.5 In addition, stable expression is not a
physiological feature for other genes, like those encoding tran-
scriptional factors, and the stable expression of these genes could
be harmful to cells.6,7 In the case of using antibiotic selection to
generate stably modified hESCs, the potential immune responses
to the products of integrated antibiotic-resistance gene may
impede the clinical use of these hESC-derived cells.8 Therefore,
transient gene transfer to hESC-derived neurons is a more prefer-
able alternative to improve transplantation efficiency. The success
of this strategy depends on the availability of suitable vectors for
human neurons that provide a transgene expression profile includ-
ing quick onset expression, expression at a functional level, and
transient expression but long enough to support neuronal trans-
plantation. It would also be beneficial to have concomitant expres-
sion of multiple genes in view of complex processes involved in
functional recovery of grafted neurons.
To develop a vector suitable for transient gene transfer into hESC-
derived neurons, we tested in the current study the insect baculo-
virus Autographa californica multiple nucleopolyhedrovirus–based
vectors. These vectors have been introduced as an effective delivery
vehicle for transgene expression in a wide variety of mammalian
cells.9,10 Upon transduction, the viruses exist mainly as episomes
in the host cells and are considered as a nonintegrating vector.
Recently, baculoviral vectors have also been applied in stem cell
research, such as in transduction of human mesenchymal stem
cells11 and the progenitor cells derived from them.12 In our lab, we
have used baculoviral vectors for transient transduction of hESCs.4
The high transduction efficiency of the baculoviral vectors in
Correspondence: Shu Wang, Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos #04-01, Singapore 138669.
High-efficiency Transient Transduction
of Human Embryonic Stem Cell–derived
Neurons With Baculoviral Vectors
Jieming Zeng1, Juan Du1,2, Jiakai Lin1, Xiao Ying Bak1, Chunxiao Wu1 and Shu Wang1,2
1Institute of Bioengineering and Nanotechnology, Singapore; 2Department of Biological Sciences, National University of Singapore, Singapore
www.moleculartherapy.org vol. 17 no. 9 sep. 2009
© The American Society of Gene & Cell Therapy
Baculoviral Transduction of Human Neurons
hESCs prompts us to investigate the feasibility of using baculoviral
vectors for transgene expression in hESC-derived neurons. As the
first proof-of-concept study, we focused on the gene encoding
enhanced green fluorescent protein (eGFP), using it as a reporter
gene to evaluate four different expression cassettes in the context
of baculoviral vectors.
Generation of human neurons from Hes-1 and Hes-3
To test the baculoviral transduction of hESC-derived human neu-
rons, two National Institutes of Health–recognized hESC lines,
HES-1 (Figure 1a) and HES-3, were used to generate neurons
using a well-established protocol.1 After neural differentiation
was initiated with prolonged culturing of hESCs at high density,
the areas containing neural progenitors were identified by their
uniformly white-gray and opaque appearance under dark-field
stereomicroscope. To obtain neural progenitors of high purity,
these identified areas were further selected under phase-contrast
microscope for their containment of rosettes-like structures
(Figure 1b), before being cut into small cell clumps for expansion.
The neural precursors were expanded for 6 weeks in the presence
of basic fibroblast growth factor and epidermal growth factor in
order to increase cell homogeneity. After that, the resulting neu-
ral spheres (Figure 1c) were dissected into small cell clumps and
plated on culture dishes in medium without the growth factors
for differentiation. These cell clumps attached to the coated plates
rapidly and neurite outgrowth from the cell clumps was observed
as early as day 1 after plating. After another 6-week culturing,
differentiated cells showed typical neuronal morphology with
very long extending neurites (Figure 1d). By using an anti-
body against NCAM, a surface marker for late stage of neuronal
differentiation,13 flow cytometric analysis showed up to 80% of the
cells derived from neural progenitor differentiation were NCAM
positive (Figure 1e). These differentiated cells were stained posi-
tively by antibodies against neuronal markers such as βIII-tubulin
(Figure 1f), MAP2ab (Figure 1g), and NF200 (Figure 1h), con-
firming further that large majority of the cells derived from our
process were neurons. Similarly, homogenous human neurons
derived from HES-3 (Figure 2f) were also generated using the
Baculoviral transduction of hesc-derived human
We first tested baculoviral vector BV-CMV.eGFP carrying an
eGFP gene driven by cytomegalovirus (CMV) promoter, a strong
viral promoter that has been widely used for transgene expression
in many cell types. In Figure 2a, live cell fluorescence image shows
Figure 1 Production of homogenous human neurons from human
embryonic stem cells. (a) HES-1 grown on feeder cells. (b) HES-1
overgrown on feeder cells for 4 weeks. The areas containing rosette-
like structures are selected to produce neural spheres. (c) Six-week-old
neural spheres. (d) Neurons derived by plating dissected neural spheres
and allowing them to grow without growth factors for 6 weeks. (e) Flow
cytometric analysis showing the NCAM expression in human neurons
derived from HES-1. (f–h) Immunofluorescence staining showing the
expression of neuronal marker (f) βIII tubulin in the periphery of a cluster
of neurons and the expression of (g) MAP2ab and (h) NF200 in human
neurons with complex multipolar morphology. Bar = 500 µm (a); 200 µm
(b,c); 100 µm (d,f–h).
Figure 2 transgene expression in human neurons mediated by
baculoviral vectors. (a) Phase contrast and fluorescence images of a
group of live human neuron clusters transduced by baculoviral vectors
carrying the expression cassette CMV.eGFP at a multiplicity of infec-
tion of 100 plaque-forming units per cell. The neurons were derived
from HES-1. The pictures were taken 2 days after transduction.
(b–e) Immunofluorescence staining showing the colocalization of neu-
ronal markers (b) βIII tubulin, (c) MAP2ab, (d) NCAM, (e) NF200 with
eGFP in HES-1–derived human neurons transduced by baculoviral vec-
tors. (f) Immunofluorescence staining showing the colocalization of
neuronal marker βIII tubulin and eGFP in human neurons derived from
HES-3. The neurons were transduced by baculoviral vectors as described
in (a). Bar = 500 µm (a); 200 µm (b,f); 100 µm (c–e). eGFP, enhanced
green fluorescent protein; GFP, green fluorescent protein.
Molecular Therapy vol. 17 no. 9 sep. 2009
© The American Society of Gene & Cell Therapy
Baculoviral Transduction of Human Neurons
Alexa Fluor 488–labeled rabbit anti-GFP antibody at 4 °C overnight.
Sections were rinsed three times in PBS before being mounted on slides
and were observed under a fluorescence microscope or a confocal
microscope. Hematoxylin and eosin staining was used for brain sections
of immunocompetent mice.
All the handling and care of animals were carried out by following the
Guidelines on the Care and Use of Animals for Scientific Purposes issued
by the National Advisory Committee for Laboratory Animal Research,
Singapore. The current study experimental protocols were approved by the
Institutional Animal Care and Use Committee, Biological Resource Center,
and the Agency for Science, Technology and Research of Singapore.
We thank other lab members for helpful discussion and support. The
work was supported by Institute of Bioengineering and Nanotechnology,
Biomedical Research Council, and Agency for Science, Technology and
1. Reubinoff, BE, Itsykson, P, Turetsky, T, Pera, MF, Reinhartz, E, Itzik, A et al. (2001).
Neural progenitors from human embryonic stem cells. Nat Biotechnol 19:
2. Li, JY, Christophersen, NS, Hall, V, Soulet, D and Brundin, P (2008). Critical issues
of clinical human embryonic stem cell therapy for brain repair. Trends Neurosci 31:
Brederlau, A, Correia, AS, Anisimov, SV, Elmi, M, Paul, G, Roybon, L et al. (2006).
Transplantation of human embryonic stem cell-derived cells to a rat model of
Parkinson’s disease: effect of in vitro differentiation on graft survival and teratoma
formation. Stem Cells 24: 1433–1440.
4. Zeng, J, Du, J, Zhao, Y, Palanisamy, N and Wang, S (2007). Baculoviral vector-
mediated transient and stable transgene expression in human embryonic stem cells.
Stem Cells 25: 1055–1061.
5. Shim, JW, Koh, HC, Chang, MY, Roh, E, Choi, CY, Oh, YJ et al. (2004). Enhanced
in vitro midbrain dopamine neuron differentiation, dopaminergic function, neurite
outgrowth, and 1-methyl-4-phenylpyridium resistance in mouse embryonic stem cells
overexpressing Bcl-XL. J Neurosci 24: 843–852.
Cai, L, Morrow, EM and Cepko, CL (2000). Misexpression of basic helix-loop-helix
genes in the murine cerebral cortex affects cell fate choices and neuronal survival.
Development 127: 3021–3030.
7. Geoffroy, CG and Raineteau, O (2007). A Cre-lox approach for transient transgene
expression in neural precursor cells and long-term tracking of their progeny in vitro
and in vivo. BMC Dev Biol 7: 45.
8. Yates, F and Daley, GQ (2006). Progress and prospects: gene transfer into embryonic
stem cells. Gene Ther 13: 1431–1439.
9. Kost, TA, Condreay, JP and Jarvis, DL (2005). Baculovirus as versatile vectors for
protein expression in insect and mammalian cells. Nat Biotechnol 23: 567–575.
10. Hu, YC (2008). Baculoviral vectors for gene delivery: a review. Curr Gene Ther 8:
11. Ho, YC, Chung, YC, Hwang, SM, Wang, KC and Hu, YC (2005). Transgene expression
and differentiation of baculovirus-transduced human mesenchymal stem cells. J Gene
Med 7: 860–868.
12. Ho, YC, Lee, HP, Hwang, SM, Lo, WH, Chen, HC, Chung, CK et al. (2006). Baculovirus
transduction of human mesenchymal stem cell-derived progenitor cells: variation of
transgene expression with cellular differentiation states. Gene Ther 13: 1471–1479.
13. Pruszak, J, Sonntag, KC, Aung, MH, Sanchez-Pernaute, R and Isacson, O (2007).
Markers and methods for cell sorting of human embryonic stem cell-derived neural
cell populations. Stem Cells 25: 2257–2268.
14. Zufferey, R, Donello, JE, Trono, D and Hope, TJ (1999). Woodchuck hepatitis virus
posttranscriptional regulatory element enhances expression of transgenes delivered by
retroviral vectors. J Virol 73: 2886–2892.
15. Loeb, JE, Cordier, WS, Harris, ME, Weitzman, MD and Hope, TJ (1999). Enhanced
expression of transgenes from adeno-associated virus vectors with the woodchuck
hepatitis virus posttranscriptional regulatory element: implications for gene therapy.
Hum Gene Ther 10: 2295–2305.
16. Detrait, ER, Bowers, WJ, Halterman, MW, Giuliano, RE, Bennice, L, Federoff, HJ et al.
(2002). Reporter gene transfer induces apoptosis in primary cortical neurons. Mol Ther
17. Slack, RS and Miller, FD (1996). Viral vectors for modulating gene expression in
neurons. Curr Opin Neurobiol 6: 576–583.
18. Washbourne, P and McAllister, AK (2002). Techniques for gene transfer into neurons.
Curr Opin Neurobiol 12: 566–573.
19. Zeitelhofer, M, Vessey, JP, Xie, Y, Tübing, F, Thomas, S, Kiebler, M et al. (2007).
High-efficiency transfection of mammalian neurons via nucleofection. Nat Protoc
20. Tenenbaum, L, Chtarto, A, Lehtonen, E, Velu, T, Brotchi, J and Levivier, M (2004).
Recombinant AAV-mediated gene delivery to the central nervous system. J Gene Med
6 (suppl. 1): S212–S222.
21. Mandel, RJ, Manfredsson, FP, Foust, KD, Rising, A, Reimsnider, S, Nash, K et al. (2006).
Recombinant adeno-associated viral vectors as therapeutic agents to treat neurological
disorders. Mol Ther 13: 463–483.
22. Davidson, BL and Breakefield, XO (2003). Viral vectors for gene delivery to the
nervous system. Nat Rev Neurosci 4: 353–364.
23. McCarty, DM (2008). Self-complementary AAV vectors; advances and applications.
Mol Ther 16: 1648–1656.
24. Wang, S, Rosengren, L, Hamberger, A and Haglid, K (2001). Antisense inhibition
of BCL-2 expression induces retinoic acid-mediated cell death during differentiation
of human NT2N neurons. J Neurochem 76: 1089–1098.
25. Derrington, EA, López-Lastra, M, Chapel-Fernandez, S, Cosset, FL, Belin, MF,
Rudkin, BB et al. (1999). Retroviral vectors for the expression of two genes in human
multipotent neural precursors and their differentiated neuronal and glial progeny.
Hum Gene Ther 10: 1129–1138.
26. Roy, NS, Wang, S, Jiang, L, Kang, J, Benraiss, A, Harrison-Restelli, C et al. (2000).
In vitro neurogenesis by progenitor cells isolated from the adult human hippocampus.
Nat Med 6: 271–277.
27. Keyoung, HM, Roy, NS, Benraiss, A, Louissaint, A Jr., Suzuki, A, Hashimoto, M et al.
(2001). High-yield selection and extraction of two promoter-defined phenotypes of
neural stem cells from the fetal human brain. Nat Biotechnol 19: 843–850.
28. Sarkis, C, Serguera, C, Petres, S, Buchet, D, Ridet, JL, Edelman, L et al. (2000). Efficient
transduction of neural cells in vitro and in vivo by a baculovirus-derived vector. Proc
Natl Acad Sci USA 97: 14638–14643.
29. Li, Y, Wang, X, Guo, H and Wang, S (2004). Axonal transport of recombinant
baculovirus vectors. Mol Ther 10: 1121–1129.
30. Li, Y, Yang, Y and Wang, S (2005). Neuronal gene transfer by baculovirus-derived
vectors accommodating a neurone-specific promoter. Exp Physiol 90: 39–44.
31. Wang, X, Wang, C, Zeng, J, Xu, X, Hwang, PY, Yee, WC et al. (2005). Gene transfer
to dorsal root ganglia by intrathecal injection: effects on regeneration of peripheral
nerves. Mol Ther 12: 314–320.
32. Wang, J, Li, B, Cai, C, Zhang, Y, Wang, S, Hu, S et al. (2007). Efficient transduction of
spiral ganglion neurons in vitro by baculovirus vectors. Neuroreport 18: 1329–1333.
33. Merrihew, RV, Clay, WC, Condreay, JP, Witherspoon, SM, Dallas, WS and Kost, TA
(2001). Chromosomal integration of transduced recombinant baculovirus DNA in
mammalian cells. J Virol 75: 903–909.
34. Donello, JE, Loeb, JE and Hope, TJ (1998). Woodchuck hepatitis virus contains a
tripartite posttranscriptional regulatory element. J Virol 72: 5085–5092.
35. Higashimoto, T, Urbinati, F, Perumbeti, A, Jiang, G, Zarzuela, A, Chang, LJ et al.
(2007). The woodchuck hepatitis virus post-transcriptional regulatory element reduces
readthrough transcription from retroviral vectors. Gene Ther 14: 1298–1304.
36. Paterna, JC, Moccetti, T, Mura, A, Feldon, J and Büeler, H (2000). Influence of
promoter and WHV post-transcriptional regulatory element on AAV-mediated
transgene expression in the rat brain. Gene Ther 7: 1304–1311.
37. Glover, CP, Bienemann, AS, Heywood, DJ, Cosgrave, AS and Uney, JB (2002).
Adenoviral-mediated, high-level, cell-specific transgene expression: a SYN1-WPRE
cassette mediates increased transgene expression with no loss of neuron specificity.
Mol Ther 5(5 Pt 1): 509–516.
38. Xu, ZL, Mizuguchi, H, Mayumi, T and Hayakawa, T (2003). Woodchuck hepatitis
virus post-transcriptional regulation element enhances transgene expression from
adenovirus vectors. Biochim Biophys Acta 1621: 266–271.
39. Sims, K, Ahmed, Z, Gonzalez, AM, Read, ML, Cooper-Charles, L, Berry, M et al.
(2008). Targeting adenoviral transgene expression to neurons. Mol Cell Neurosci
40. Mähönen, AJ, Airenne, KJ, Purola, S, Peltomaa, E, Kaikkonen, MU, Riekkinen, MS et al.
(2007). Post-transcriptional regulatory element boosts baculovirus-mediated gene
expression in vertebrate cells. J Biotechnol 131: 1–8.
41. Carson, CT, Aigner, S and Gage, FH (2006). Stem cells: the good, bad and barely in
control. Nat Med 12: 1237–1238.
42. Roy, NS, Cleren, C, Singh, SK, Yang, L, Beal, MF and Goldman, SA (2006). Functional
engraftment of human ES cell-derived dopaminergic neurons enriched by coculture
with telomerase-immortalized midbrain astrocytes. Nat Med 12: 1259–1268.
43. Fukuda, H, Takahashi, J, Watanabe, K, Hayashi, H, Morizane, A, Koyanagi, M et al.
(2006). Fluorescence-activated cell sorting-based purification of embryonic stem cell-
derived neural precursors averts tumor formation after transplantation. Stem Cells 24:
44. Hedlund, E, Pruszak, J, Ferree, A, Viñuela, A, Hong, S, Isacson, O et al. (2007).
Selection of embryonic stem cell-derived enhanced green fluorescent protein-positive
dopamine neurons using the tyrosine hydroxylase promoter is confounded by
reporter gene expression in immature cell populations. Stem Cells 25: 1126–1135.
45. Hedlund, E, Pruszak, J, Lardaro, T, Ludwig, W, Viñuela, A, Kim, KS et al. (2008).
Embryonic stem cell-derived Pitx3-enhanced green fluorescent protein midbrain
dopamine neurons survive enrichment by fluorescence-activated cell sorting and
function in an animal model of Parkinson’s disease. Stem Cells 26: 1526–1536.
46. Callaway, EM (2005). A molecular and genetic arsenal for systems neuroscience.
Trends Neurosci 28: 196–201.
47. Janson, CG, McPhee, SW, Leone, P, Freese, A and During, MJ (2001). Viral-based gene
transfer to the mammalian CNS for functional genomic studies. Trends Neurosci 24:
48. Cezar, GG (2007). Can human embryonic stem cells contribute to the discovery of
safer and more effective drugs? Curr Opin Chem Biol 11: 405–409.
49. Pouton, CW and Haynes, JM (2007). Embryonic stem cells as a source of models for
drug discovery. Nat Rev Drug Discov 6: 605–616.
50. Hoffman, RM and Yang, M (2006). Whole-body imaging with fluorescent proteins.
Nat Protoc 1: 1429–1438.