STEM CELLS AND DEVELOPMENT 17:441–450 (2008)
© Mary Ann Liebert, Inc.
Original Research Report
Optimized Lentiviral Transduction of Mouse Bone
Marrow-Derived Mesenchymal Stem Cells
DAVID M. RICKS,1–3ROBERT KUTNER,1,3XIAN-YANG ZHANG,1
DAVID A. WELSH,2and JAKOB REISER1
Mesenchymal stem cells (MSCs) have attracted much attention as potential platforms for transgene
delivery and cell-based therapy for human disease. MSCs have the capability to self-renew and re-
tain multipotency after extensive expansion in vitro, making them attractive targets for ex vivo mod-
ification and autologous transplantation. Viral vectors, including lentiviral vectors, provide an effi-
cient means for transgene delivery into human MSCs. In contrast, mouse MSCs have proven more
difficult to transduce with lentiviral vectors than their human counterparts, and because many stud-
ies use mouse models of human disease, an improved method of transduction would facilitate stud-
ies using ex vivo-modified mouse MSCs. We have worked toward improving the production of hu-
man immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors and optimizing transduction
conditions for mouse MSCs using lentivirus vectors pseudotyped with the vesicular stomatitis virus
G glycoprotein (VSV-G), the ecotropic murine leukemia virus envelope glycoprotein (MLV-E), and
the glycoproteins derived from the Armstrong and WE strains of lymphocytic choriomeningitis virus
(LCMV-Arm, LCMV-WE). Mouse MSCs were readily transduced following overnight incubation
using a multiplicity of infection of at least 40. Alternatively, mouse MSCs in suspension were read-
ily transduced after a 1-h exposure to lentiviral pseudotypes immediately following trypsin treat-
ment or retrieval from storage in liquid nitrogen. LCMV-WE pseudotypes resulted in efficient trans-
duction of mouse MSCs with less toxicity than VSV-G pseudotypes. In conclusion, our improved
production and transduction conditions for lentiviral vectors resulted in efficient transduction of
mouse MSCs, and these improvements should facilitate the application of such cells in the context
of mouse models of human disease.
vectors, vectors based on adeno-associated virus (AAV),
and retroviral vectors are commonly used . The abil-
OR TRANSGENE DELIVERYinto mesenchymal stem cells
(MSCs), viral vector systems including adenoviral
ity of MSCs to self-renew at a high proliferation rate led
to the prediction that they would be ideal targets for trans-
gene delivery strategies involving retroviral vectors.
However, a major limitation of transduction approaches
involving oncogenic retroviral vectors. such as Moloney
murine leukemia virus, is a general lack of long-term
1Gene Therapy Program, Department of Medicine, LSU Health Sciences Center, New Orleans, LA 70112.
2Pulmonary Critical Care, Department of Medicine, LSU Health Sciences Center, New Orleans, LA 70112.
3The first two authors contributed equally to this work.
transgene expression [2,3]. Vectors based on murine stem
cell virus appear to be less prone to transcriptional si-
lencing of gene expression, and thus appear to be more
Recent results from several labs have indicated that hu-
man immunodeficiency virus type 1 (HIV-1)-based vec-
tors are very efficient at delivering and expressing trans-
genes in human MSCs [5–8]. For example, a single round
of transduction using unconcentrated HIV-1-based
lentiviral vectors led to the efficient transduction of hu-
man MSCs and sustained transgene expression up to at
least 5 months . An advantage of lentiviral vectors over
vectors based on oncogenic retroviruses is their ability to
transduce nondividing cells . This property is impor-
tant given that a relatively large subset (20%) of mes-
enchymal progenitor cells (MPCs) has been described to
be quiescent .
Transgene delivery strategies using lentiviral vectors
involving MSCs from other species have also been re-
ported. For example, Lee et al.  have used self-inac-
tivating HIV-1-based lentiviral vectors to transduce
MSCs derived from fetal rhesus monkey bone marrow.
Flow cytometric analyses indicated an 8- to 10-fold
greater quantity of green fluorescent protein (GFP)-ex-
pressing rhesus MSCs when cells were transduced with
vectors bearing the cytomegalovirus immediate early
(CMV) or translation elongation factor-1? (EF-1?) pro-
moters as compared to vectors bearing the phosphoglyc-
erate kinase (PGK) promoter. Transduced rhesus MSCs
differentiated toward an osteogenic lineage comparable
to untransduced MSCs. In agreement with the reports
published by Zhang et al. , these findings suggested
that HIV-1-derived lentiviral vectors can efficiently
transduce rhesus MSCs in vitro without inhibiting their
In a recent report, McMahon et al.  performed a
direct comparison of different vectors on rat MSCs, in-
cluding lentiviral vectors, vectors based on adenovirus or
AAV, as well as nonviral vectors. The results indicated
that VSV-G pseudotyped HIV-1-based vectors were the
vector of choice for rat MSCs. Furthermore, it was shown
that the transduction process and the high levels of re-
porter gene expression achieved did not have any dele-
terious effect on the ability of the cells to differentiate
down the adipogenic pathway. Efficient lentivirus-medi-
ated gene transfer into mouse MSCs has been more
challenging, in part because of host range barriers for
HIV-1 in such cells, including tissue-specific restriction
factors  and blocks in the nuclear uptake of the prein-
tegration complex .
In this study, we present improved protocols for the
transduction of mouse MSCs with pseudotyped lentiviral
vectors that result in significant transduction efficiency
and reduced cell toxicity. We investigated alternative
transduction conditions, such as varying transduction
length and transducing cells immediately following
trypsin treatment or retrieval from storage in liquid nitro-
gen. We also considered aspects of lentivirus production
and concentration to maximize viral titers and minimize
potential contaminants of vector stocks. Investigation of
alternative pseudotypes led us to develop and test a mod-
ified titration method based on quantifying lentiviral RNA
content of vector preparations. Using this titration method
to normalize vector quantity, we compared the ability of
vectors pseudotyped with vesicular stomatitis virus G gly-
coprotein (VSV-G), murine leukemia virus envelope gly-
coprotein (MLV-E), and glycoproteins derived from the
Armstrong and WE strains of lymphocytic choriomenin-
gitis virus (LCMV-Arm and LCMV-WE).
MATERIALS AND METHODS
Production and propagation of murine MSCs
Murine MSCs were harvested from the tibias and femurs of
C57/BL6 mice, as previously described  and provided by
the Tulane Center for Gene Therapy. The plastic-adherent pop-
ulation was grown in complete culture medium, consisting of
Iscove’s modified Dulbecco’s medium (IMDM; Invitrogen,
Carlsbad, CA), 10% fetal bovine serum (FBS; Atlanta Biolog-
icals, Lawrenceville, GA), 10% horse serum (HS; Hyclone, Lo-
gan, UT), 2 mM L-glutamine (Invitrogen), and 100 U/ml peni-
cillin/streptomycin (Pen/Strep; Invitrogen) and passaged at low
density (50 cells/cm2). For long-term storage, cells were resus-
pended in IMDM with 20% FBS, 20% HS, 5% dimethylsul-
foxide (DMSO; Sigma-Aldrich, St. Louis, MO), frozen at
?1°C/min until reaching ?80°C, and transferred to liquid ni-
trogen. Passage-five cells were used for transduction experi-
Design of lentiviral vectors
To construct the pNL-EGFP/CMV/WPRE?U3 lentiviral
vector plasmid, a 591-bp woodchuck hepatitis virus post-tran-
scriptional element (WPRE) was added between the unique Xho
I and Kpn I sites, downstream of the enhanced green fluores-
cent protein (EGFP) coding region present in pNL-EGFP/CMV/
?U3  (see http://www.medschool.lsuhsc.edu/reiser). The
WPRE fragment was generated by PCR amplification using
pWHV8 (ATCC, Manassas, VA) as a template. Primers used
were WPRE-S (5?-AAC TCG AGA ATC AAC CTC TGG ATT
ACA A-3?) and WPRE-A (5?-AAG GTA CCC AGG CGG
GGA GGC GGC CCA A-3?). The pNL-EGFP/EF-1?/
WPRE?U3 plasmid was constructed by PCR amplifying the
EF-1?/human T cell leukemia virus (HTLV) type 1 long ter-
minal repeat (LTR) hybrid promoter from pGT70LacZ (In-
vivogen, San Diego, CA) and inserting it between the Hinc II
and Nhe I sites of pNL-EGFP/CMV/WPRE?U3. The pNL-
hCXCR2/EF-1?/WPRE?U3 plasmid was constructed by ex-
cising the human CXCR2 open reading frame (ORF) from
pCRII-hCXCR2 (kindly provided by Dr. Tim Sparer)  and
subcloning it into pNL-EGFP/EF-1?/WPRE?U3.
RICKS ET AL.
Production of lentiviral vectors
Lentiviral vectors pseudotyped with the VSV-G  MLV-E
, LCMV-Arm , and LCMV-WE glycoproteins  were
generated by calcium phosphate-mediated transfection of 293T
cells with modifications as described [20,21]. 293T cells were
plated in 150-cm2plates at a density of 8 ? 106cells in 25 ml
of Dulbecco’s modified Eagle’s medium (DMEM, high-glu-
cose) supplemented with 10% FBS, 1% Glutamax (Invitrogen),
1% Pen/Strep, with or without 0.3% HyQ LipiMate (Hyclone),
Chemically Defined Lipid Concentrate (Invitrogen), or choles-
terol (Sigma). Twenty four hours later, chloroquine (Sigma) was
added to the medium at a final concentration of 25 ?M.
Lentivirus vector, packaging, and envelope glycoprotein plas-
mids were mixed together in 3 ml of 0.25 M CaCl2per plate,
added to 3 ml of 2? HEPES-buffered saline (HBS) under gen-
tle vortexing, and pipetted into the medium. The amount of
DNA used per plate was 21 ?g of lentiviral vector plasmids
pNL-EGFP/EF-1?/WPRE ?U3, pNL-EGFP/CMV/WPRE?U3,
or pNL-hCXCR2/EF-1?/WPRE ?, 14 ?g of packaging plas-
mid pCD/NL-BH*??? , and either 7 ?g pLTR-G  or 21
?g pCAGGS-LCMV-WE, pCAGGS-LCMV-Arm (provided by
Dr. Juan Carlos de la Torre, The Scripps Research Institute), or
pLTR-MLV-E. The pLTR-MLV-E plasmid was derived from
the pLTR 4070A Env plasmid  by replacing the 4070A Env
sequence with a fragment encoding the MLV ecotropic Env.
The medium was removed 16 h after transfection and replaced
with 17 ml of fresh DMEM, 10% FBS, and 1% Glutamax. Forty
eight hours after transfection, the vector-containing medium
was collected and spun at 500 ? g for 5 min, filtered through
a 0.45-? pore size filter (Corning, Corning, NY) and stored at
?80°C. For vector concentration by ultracentrifugation, the
vector-containing medium from two plates was underlaid with
4 ml of 20% sucrose and centrifuged for 2 h at 25,000 rpm,
4°C using a Beckman SW28 ultracentrifuge rotor (Beckman
Coulter, Fullerton, CA). The resulting pellet was dissolved in
100 ?l of PBS without calcium or magnesium (Invitrogen) for
2 h at 4°C. Vector aliquots were stored at ?80°C.
Analysis of vector supernatants using
Four milliliters of vector-containing medium or 100 ?l of
concentrated vectors diluted in 3.9 ml of PBS were loaded into
Beckman SW28 UltraClear tubes (Beckman Coulter) and un-
derlaid with a continuous gradient of 10–30% OptiPrep (Axis-
Shield, Oslo, Norway) in 20 mM Tris-HCl, 1 mM EDTA, and
0.85 (wt/vol) NaCl, pH 7.5. The gradient was spun at 25,000
rpm, for 4 h at 4°C, using an SW28 rotor. Eighteen 2-ml frac-
tions were collected, and each fraction was titrated by end-point
dilution on human osteosarcoma (HOS) cells , or measured
for p24 antigen levels using a HIV-1 p24 Antigen ELISA kit
(Zeptometrix, Buffalo, NY) .
Concentration of lentivirus vectors using Mustang Q
For lentiviral vector concentration using Mustang Q
Acrodisks (PALL, East Hills, NY), vector-containing super-
natants were adjusted to 25 mM Tris-HCl, pH 8.0, 0.3 M NaCl
and loaded onto a Mustang Q Acrodisk (bed volume 0.18 ml).
The membrane was washed with loading buffer, and the
flowthrough was discarded. Vectors were eluted with 10 ml of
25 mM Tris-HCl, pH 8.0, and 1.5 M NaCl directly into 25 ml
of PBS. To concentrate the vectors further and to remove any
residual salt, the diluted eluate was loaded into a Beckman SW28
UltraClear tube and centrifuged at 25,000 rpm for 2 h at 4°C
and the pellets were resuspended in PBS as described above.
Vector copy number determination using quantitative
Quantitative real-time PCR analysis of proviral copy num-
bers was similar to that reported before . The sequences of
the WPRE-specific primers were: 5?-CCT TTC CGG GAC TTT
CGC TTT-3? (forward primer); 5?-GCA GAA TCC AGG TGG
CAA CA-3? (reverse primer), and 5?-FAM-ACT CAT CGC
CGC CTG CCT TGC C-TAMRA-3? (probe). The cycling con-
ditions were 10 min at 95°C, then 40 cycles of 95°C for 15 sec
and 60°C for 1 min. Genomic vector copies in each sample
were normalized to human RNaseP gene copies using specific
primers and probes (TaqMan DNA Template Reagent Kit, Ap-
plied Biosystems, Foster City, CA)
Vector RNA isolation and quantitative
A 2?l lentiviral vector sample was diluted into 398 ?l of 25
mM Tris-HCl, pH 8.0, and treated with 50 pg of RNase A (USB,
Cleveland, OH) at 37°C for 10 min. Then 20 ?l of SUPERase-
In (Ambion, Austin, TX) was added prior to performing RNA
extraction using the PureLink Viral RNA/DNA kit (Invitrogen).
The final elution volume was 30 ?l. Reverse transcription PCR
was performed using an iScript cDNA synthesis kit (BioRad,
Hercules, CA) on half of the RNA. The other half was used in
control reactions lacking reverse transcriptase. Five microliters
of each reaction was used for quantitative real-time PCR as de-
Transduction of MSCs
For transduction comparisons using preplated, trypsinized,
and thawed mouse MSCs, preplated cells were plated 24 h be-
fore transduction. Trypsinized cells were detached using 0.25%
trypsin and 1 mM EDTA (Invitrogen), inactivated with com-
plete culture medium, washed, and counted immediately before
transduction. Thawed cells were removed from liquid nitrogen,
thawed rapidly, washed with complete culture medium,
counted, and used directly for transduction. Equal numbers of
cells were used for each experiment. Transductions were per-
formed in 0.5 ml of transduction medium (IMDM ?10% heat-
inactivated FBS) in the presence of 8 ?g/ml Polybrene (Sigma),
and medium was changed to complete culture medium 1 or 16
h after addition of lentivirus.
Analysis of transgene expression by flow cytometry
Transduced cells expressing EGFP were trypsinized, inacti-
vated with complete culture medium, washed twice with PBS
containing 2% FBS, and analyzed using a FACSCalibur sys-
tem (BD Biosciences, San Jose, CA). For antibody staining of
LENTIVIRAL TRANSDUCTION OF MOUSE MSCs
hCXCR2-transduced cells, cells were resuspended in PBS and
2% FBS containing either phycoerythrin (PE)-labeled anti-hu-
man CXCR2 monoclonal antibody (Clone 48311, R&D Sys-
tems, Inc., Minneapolis, MN) or mouse immunoglobulin G2A
(IgG2A) PE-labeled isotype control (Clone 20102, R&D) and
incubated for 30 min at 4°C. Cells were washed twice with PBS
and 2% FBS and analyzed by flow cytometry.
Colony-forming unit assay
To assay the proliferation rate of MSCs that were transduced
overnight, 100 viable cells were added to six-well plates (Corn-
ing) 48 h before transduction. Following overnight transduc-
tion, the medium was removed, the cells were washed once with
PBS, and the wells were filled with 2 ml of freshly prepared
complete culture medium. The plates were then placed in a
37°C, 5% CO2humidified incubator for 14 days, after which
the medium was removed and the wells washed with PBS.
Colonies were stained for 10 min with 3.0% crystal violet in
100% methanol, and after washing the wells three times with
deionized water, the colonies that were 1 mm or larger in di-
ameter were counted. Assay of MSCs transduced for 1 h was
performed as described, with the exception that cells were trans-
duced in suspension after trypsin treatment and subsequently
plated into 10-cm dishes (Corning) filled with 10 ml of medium.
Statistical analyses were performed using one-way analysis
of variance (ANOVA) followed by multiple paired comparisons
(Student’s t-test). * represents p ? 0.05; ** represents p ? 0.01;
*** represents p ? 0.001.
Rapid lentiviral vector transduction protocol for
To increase the participation of MSCs in site-directed
tissue repair, we are using lentiviral vector-mediated gene
delivery strategies to modify MSC trafficking and
achieve higher rates of engraftment in damaged tissues.
RICKS ET AL.
efficiency using mouse MSCs transduced at an MOI of 40 and 80. Mouse MSCs were preplated 24 h before transduction and
transduced for 16 h (overnight). (B) Transduction efficiency using mouse MSCs transduced for 1 h at an MOI of 40 and 80. Four
days after transduction, transduction efficiencies were measured by analyzing EGFP expression of transduced cells. The gray-
shaded histograms represent mock-transduced controls and open histograms represent cells transduced with EGFP vector. The
percentages of EGFP?cells are indicated.
Representative flow cytometric analysis of lentiviral vector-mediated transduction of mouse MSCs. (A) Transduction
of retroviral vectors have previously been reported. For
example, pharmacological disruption of lipid rafts and
cholesterol depletion of producer cells has been shown
to interfere with virus particle formation . We sur-
mised that decreased cholesterol and lipid levels of pro-
ducer cells is a limiting step in lentiviral vector produc-
tion and thus evaluated the effects of various lipid
additives on the relative titers of such vector stocks. The
results presented in Fig. 3A show significant increases in
vector titers as a result of lipid additives during vector
production. Vector titers on HOS cells increased 3.7-fold
in the presence of cholesterol, 4.8-fold in the presence of
chemically defined lipid concentrate, and 11.2-fold in the
presence of LipiMate, relative to vector stocks produced
in the absence of lipid additives. Similar increases in vec-
LENTIVIRAL TRANSDUCTION OF MOUSE MSCs
Previously, we have described conditions allowing ef-
ficient lentiviral vector-mediated transgene delivery into
human MSCs, and we revealed that efficient lentiviral
vector-mediated gene transfer was possible using a mul-
tiplicity of infection (MOI) of 1 [5,7]. At similar low
MOIs, transduction of mouse MSCs was found to be in-
efficient, consistent with the view that there are impedi-
ments to efficient lentiviral vector-mediated transduction
of murine cells. However, at MOIs of 40 and 80, up to
50.3% and 60.5% of the cells were GFP?following ex-
posure to the lentiviral vectors overnight (Fig. 1A). We
also observed modest transduction of mouse MSCs fol-
lowing exposure to the vectors for just 1 h (Fig. 1B). Fur-
thermore, transduction strategies using suspended mouse
MSCs immediately after retrieval of such cells from stor-
age in liquid nitrogen or after trypsin treatment were sig-
nificantly more efficient as those using preplated MSCs
(Fig. 2). Thus, there does not appear to be a need for
MSCs to be plated prior to transduction, and using MSCs
in suspension may increase transduction efficiency.
Improved lentiviral vector production
using lipid additives
Because efficient transduction of mouse MSCs re-
quires high MOIs, it is important to improve lentivirus
yields. Several factors that affect the stability and/or yield
ing various transduction conditions. The graphs shown repre-
sent relative transduction efficiencies using mouse MSCs trans-
duced 24 h after plating (Pre-Plated), immediately after
passaging with trypsin (Trypsinized), and immediately after
thawing from storage in liquid nitrogen (Thawed). MSCs were
transduced for 1 h or 16 h (overnight) at an MOI of 40 and 80.
Four days after transduction, transduction efficiency was mea-
sured by analyzing EGFP expression of transduced cells. The
bars represent % EGFP?cells. Error bars represent the SEM
resulting from three to four independent transductions per-
formed for each group.
Quantitative analysis of mouse MSC transduction us-
duction on vector titers. Relative differences in vector titers cul-
tured with various additives prior to and during vector produc-
tion in 293T cells. Control represents no additives. The
additives included chemically defined lipid concentrate (CDL),
cholesterol, and LipiMate. Titers were determined by FACS
analysis of transduced HOS cells (A) or mouse MSCs (B). Val-
ues represented in A are the average ? SEM of three individ-
ual productions. Results shown in B are the average ? SEM of
three individual transductions.
Effects of lipid addition during lentiviral vector pro-
tor titers as a result of lipid additives were seen with
mouse MSCs (Fig. 3B).
Alternative lentiviral vctor pseudotypes
for mouse MSC transduction
We next tested the ability of lentiviral vectors bear-
ing glycoproteins other than VSV-G to transduce
mouse MSCs. Park et al.  have demonstrated that
lentiviral vectors pseudotyped with the LCMV-WE
glycoprotein displayed reduced liver toxicity in mice
compared to VSV-G pseudotypes. These findings
prompted us to investigate alternative envelopes, in-
cluding LCMV-WE and LCMV-Arm pseudotypes
[18,19]. Preliminary titration of alternative pseudo-
types based on p24 levels generated ambiguous results,
possibly caused by the existence of unincorporated p24
in the vector stocks. To determine the relationships be-
tween p24 levels and transducing units in more detail,
we subjected a crude lentivirus vector preparation to
rate-zonal ultracentrifugation using a 10– 30% gradi-
ent of OptiPrep (Fig. 4A). A total of 18 fractions were
collected and individual fractions were tested for vec-
tor transducing units and p24 content using a p24 en-
zyme-linked immunoassay (ELISA) test. The results
presented in Fig. 4A show that a relatively large frac-
tion of the total p24 did not co-sediment with infec-
tious vector particles. However, concentrating the
lentivirus preparation prior to rate-zonal ultracentrifu-
gation improved the correlation between p24 levels and
transducing units (Fig. 4B). These results indicate that
titer determinations based on p24 content are unreli-
able for crude vector stocks but may be applicable for
vectors concentrated by ultracentrifugation over a su-
crose cushion. As an alternative to p24-based titration,
we used a modified version of the protocol based on
virion RNA originally described by Sastry et al. 
to adjust titers of lentiviral vectors bearing alternative
The results presented in Fig. 5 show the transduction
efficiencies of lentiviral vectors pseudotyped with vari-
ous envelope glycoproteins including VSV-G, MLV-E,
LCMV-Arm, and LCMV-WE. In all cases, titers were
adjusted by quantitative RT-PCR based on virion RNA.
A total of 7.9 ? 109virus particles were used to trans-
duce 5 ? 104mouse MSCs. It is evident that VSV-G
pseudotypes were most efficient both during short-term
transduction (1 h) and after overnight transduction (16
h). However, vectors pseudotyped with the LCMV-WE
glycoprotein were also efficient, particularly after
overnight transduction (Fig. 5).
Table 1 presents our findings regarding the toxicity
of VSV-G-pseudotyped vectors before and after Mus-
tang Q anion-exchange chromatography and compared
to MLV-E, LCMV-Arm, and LCMV-WE pseudotypes.
Both Mustang Q chromatography and pseudotyping
with alternative envelopes resulted in higher colony
formation than VSV-G, indicating less toxicity to pro-
genitor cells. We also found that transduction for 1 h
did not result in any differences in colony formation
between groups, indicating a low toxicity associated
with short exposure.
Lentiviral vector-mediated expression of human
CXCR2 receptor in mouse MSCs
Having optimized lentiviral vector production and
transduction for mouse MSCs, we validated our findings
with a transgene encoding a cell-surface protein. Ex-
pression of human chemokine receptor CXCR2 was as-
sessed in mouse MSCs following lentiviral vector-medi-
ated gene transfer. The results shown in Fig. 6
demonstrate that, using our improved transduction con-
ditions, up to 88.4% of mouse MSCs expressed hCXCR2
after being transduced overnight at an MOI of 90.
RICKS ET AL.
centrifugation. (A) Unconcentrated virus (4 ml) was subjected
to ultracentrifugation using a linear gradient ranging from 10%
to 30% OptiPrep. Each fraction was titrated by end-point dilu-
tion on HOS cells (diamonds), or measured for p24 antigen lev-
els using a HIV-1 p24 Antigen ELISA kit (circles). (B) Con-
centrated virus (100 ?l of virus diluted into 3.9 ml of PBS) was
subjected to the same analysis.
p24 antigen distribution following rate zonal ultra-
Optimized conditions for mouse
In an attempt to optimize HIV-1-based lentiviral vec-
tors for efficient transgene delivery into mouse MSCs,
we have investigated various transduction protocols and
have tested several vector pseudotypes. In general, mouse
cells do not support HIV-1 replication because of host
range barriers at various steps including virus entry, nu-
clear import , RNA splicing , polyprotein pro-
cessing, assembly, and release. For example, Noser et al.
 reported that HIV-1 infection of murine cells is in-
hibited by dominant factors related to immunophilins and
that competitive inhibitors of cyclophilins, including cy-
closporin and the related compound Debio-025, stimu-
lated HIV-1 vector transduction of primary murine bone
marrow-derived cells and macrophages up to 20-fold. On
the basis of these findings we tested the impact of cy-
closporin A on mouse MSCs and did not observe im-
provements in transduction efficiencies (Ricks, unpub-
Our results indicate that lentivirus-mediated transduc-
tion of mouse MSCs was robust, provided that high
enough MOIs were used. We were able to boost vector
titers substantially by adding lipids such as LipiMate dur-
ing virus production. Increased vector titers were ob-
served both using HOS cells (Fig. 3A) and mouse MSCs
(Fig. 3B). This is consistent with the view that lipid ad-
ditives primarily affected virus production. It was is also
evident from the results shown in Fig. 2 that transduc-
tion efficiencies involving cells in suspension were sig-
nificantly higher than those involving adherent cells.
However, it was apparent from the CFU assay displayed
in Table 1 that overnight transductions at high MOIs re-
sulted in VSV-G-mediated cell toxicity . This tox-
icity issue could be partially overcome by reducing the
time of vector exposure to 1 h, or by using alternative
pseudotypes including vectors bearing the LCMV-WE
glycoprotein. These findings are in line with those re-
ported earlier by Park et al. , which showed that
LCMV-pseudotyped lentiviral vectors resulted in re-
duced systemic or hepatic injury compared to VSV-G
pseudotypes after in vivo administration.
Methods to titrate lentiviral vectors
Our attempts to compare the transduction efficiencies
of the various pseudotypes tested revealed two technical
issues in quantitating viral titers: (1) assay selection, and
(2) cell line transduction efficiency. Different methods
have been used to determine lentiviral vector titers, in-
cluding measures based on the number of vector parti-
cles present in a virus stock and measures derived from
the number of proviral copies in transduced target cells.
Virus particle numbers can be determined using real-time
PCR based on strong-stop cDNA present in virions .
Alternatively, the amount of a virus core protein present
in the vector preparation, such as p24 Gag, is determined
by ELISA to arrive at relative particle titers . Func-
tional titration assays are based on vector-encoded re-
porter gene expression. For example, vectors encoding
LENTIVIRAL TRANSDUCTION OF MOUSE MSCs
pseudotyped with alternative glycoproteins. MOIs were ad-
justed using vector particle titers, which were determined based
on virion RNA copies. A total of 7.9 ? 109virus particles were
added for each pseudotype. MSCs (5 ? 104cells per well) were
transduced for either 1 h or overnight, and transduction effi-
ciencies were analyzed by flow cytometry 5 days following
transduction. VSV-G (MustQ) refers to concentrated virus that
was purified using Mustang Q Acrodisks.
Transduction of mouse MSCs using lentiviral vectors
TABLE 1.EFFECT OF VECTOR PSEUDOTYPE
AND TRANSDUCTION TIME ON THE
PROLIFERATION ABILITY OF MOUSE MSCS
Colony-forming units ? SDb
VSV-G (Mustang Q)c
31.33 ? 2.89
9.67 ? 1.53d
15.67 ? 0.58
19.00 ? 1.00
17.67 ? 0.58
16.67 ? 2.08
32.67 ? 2.89
34.00 ? 1.00
38.00 ? 1.73
36.67 ? 2.52
36.67 ? 3.06
34.67 ? 5.77
aMouse MSCs (5 ? 104) were transduced for either 1 h or
16 h (overnight) using 7.9 ? 109virus particles, and 100 cells
were plated for colony-forming unit assay.
bAfter 2 weeks, the number of colonies greater than 1 mm
was counted for each group.
cVSV-G (Mustang Q) refers to concentrated virus which was
purified using Mustang Q Acrodisks.
dDifferences between VSV-G and all other glycoproteins in-
cluding VSV-G (Mustang Q) are significant.
GFP have been titrated using fluorescence-activated cell
sorting (FACS) analysis [5,30]. For vectors that do not
contain a reporter gene, proviral DNA copy numbers de-
termined by real-time PCR using DNA extracted from
transduced cells have been used to measure titers .
The cell line used for titration is significant, as receptors
for a given pseudotype may vary among cell lines, pos-
sibly producing a falsely depressed titer .
To adjust titers of pseudotyped vectors, we decided to
pursue particle-based titration methods. The results pre-
sented in Fig. 4A show that the correlation between p24
levels and transduction units was poor for unconcentrated
vector stocks. Thus, this avenue was abandoned in favor
of a modified particle assay that is based on virion RNA
. The performance of titer-adjusted vectors including
VSV-G, MLV-E, LCMV-Arm, and LCMV-WE pseudo-
types is displayed in Fig. 5. It is evident from this anal-
ysis that VSV-G pseudotypes were the most efficient fol-
lowed by LCMV-WE pseudotypes. An attractive feature
of LCMV-WE pseudotypes is that they appear to be less
toxic as judged by the CFU assay.
Concentration of lentiviral vectors
We have also investigated a scaleable protocol for
lentiviral vector concentration based on strong anion ex-
change membranes, such as Mustang Q Acrodisks, that
allows viral concentration with little volume limitation
and improved purification from serum proteins and cel-
lular contaminants (Kutner, unpublished). Mustang Q-
treated VSV-G pseudotypes were less toxic compared to
VSV-G pseudotypes that had been concentrated by ul-
tracentrifugation, as judged by the CFU assay.
In conclusion, our results show that HIV-1-based vec-
tors appear to be efficient for delivering and expressing
transgenes in mouse MSCs, provided that high MOIs and
transduction protocols involving suspended cells are
used. Our results also indicate that lipid additives such
RICKS ET AL.
transduced 24 h after plating (A) or immediately after passaging (B) at an MOI of 30 and 90 for 16 h. The gray-shaded his-
tograms represent mock-transduced controls and open histograms represent cells transduced with hCXCR2 vector.
Overexpression of hCXCR2 in mouse MSCs following lentiviral vector-mediated gene transfer. Mouse MSCs were
as LipiMate helped boost vector titers, thus simplifying
the production of high-titer vector stocks. Finally, cell
toxicity was lowest with LCMV-WE pseudotypes. Thus,
for optimal transduction of mouse MSCs we recommend
the use of LCMV-WE pseudotypes produced in the pres-
ence of LipiMate.
The preclinical utility of genetically modified mouse
MSCs and their progenitors in the context of mouse mod-
els of human disease requires stable and long-term ex-
pression of the desired gene product as well as regula-
tion of gene expression according to disease status. These
goals may be achieved using lentiviral vectors.
We are grateful to Dr. Juan Carlos de la Torre for pro-
viding us with plasmids encoding LCMV-WE and
LCMV-Arm glycoproteins and to Dr. Tim Sparer for pro-
viding the plasmid encoding hCXCR2. We thank Con-
nie Porretta for assistance with FACS. Some of the ma-
terials employed in this work were provided by the
Tulane Center for Gene Therapy through a grant from
the National Center for Research Resources (NCRR) of
the National Institutes of Health (NIH), grant
P40RR017447. This work was supported by NIH grants
NS044832, HL075161, and HL073770.
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Address reprint requests to:
Dr. Jakob Reiser
Gene Therapy Program
LSU Health Sciences Center
533 Bolivar Street, CSRB 606
New Orleans, LA 70112
Received for publication September 8, 2007; accepted af-
ter revision November 6, 2007.
RICKS ET AL.