- Email: [email protected]
lin− hemopoietic progenitors by lentivirus vectors
Cytotherapy (1999) Vol. 1, No. 6, 433–438
Transduction of CD34Q and CD342/lin2 hemopoietic progenitors by lentivirus vectors K Moriwaki1,2, RE Sutton1,3, M Perez1,2, MK Brenner1,2,4 and HE Heslop1,2,4 1
Center for Cell and Gene Therapy, 2Department of Pediatrics, 3Division of Molecular Virology and 4
Department of Medicine, Baylor College of Medicine, Houston, Texas, USA
Background
populations. Mean transduction rates on fibronectin plates,
While hemopoietic stem cells have been thought to reside
analyzed by flow cytometry, were 14% and 5% for CD34þ cells
predominantly in the CD34þ population, recent data suggests
and lin/CD34 cells respectively, without prestimulation, and
31% and 20% with prestimulation. By contrast, a murine
population. Transduction of both these populations by murine
retroviral vector transduces CD34þ cells with lower efficiency
retroviral vectors is limited by quiescence of hemopoietic stem cells.
(mean 16.1% with prestimulation) and does not induce any
that repopulating cells may, in fact, also reside in the lin /CD34
significant transduction of the CD34/lin population (mean Methods
< 2%). When lentiviral transduction was assayed in short- and
We therefore sought to transduce these populations using a VSV-G
long-term clonogenic assays there was minimal transduction of
pseudotyped, HIV-based, human lentiviral vector, encoding
CD34 cells without prestimulation, increasing to 20% with
eGFP. CD34þ cells and lin/CD34 cells were selected from
prestimulation.
the same BM samples by immunomagnetic beads (StemSep) to deplete lineage-positive cells and by CD34 selection columns
Discussion
(Miltenyi) to separate CD34þ and CD34 populations. We
Lentiviral eGFP vectors can transduce hematopoietic progenitors
transduced target cells, with or without prestimulation with
effectively and efficiency is improved by cytokine prestimulation and
cytokines, using conventional suspension culture, or fibronectin
the use of fibronectin. Moreover, the human viral vectors can
plates, or flow-through transduction. Transduction efficiency was
transduce a candidate stem-cell population that is resistant to
analyzed by flow cytometry and clonogenic assay.
murine retroviral transduction.
Results
Keywords
We found that transduction on fibronectin plates was more efficient
CD34þ, hemopoietic stem cell, lentiviral vector, gene therapy,
than flow-through transduction, or suspension cultures, for both cell
lin/CD34
Introduction
[3] and in vivo gene transfer in clinical studies has been
Hemopoietic stem cells are an attractive target for gene
disappointingly low, perhaps because the cytokines used
therapy as gene transfer to stem cells could, theoretically,
have failed to induce true repopulating cells into the cycle, or
provide lifelong correction for many gene defects of
because these cells may lack receptors for murine retro-
marrow-derived cells. Advantages of using hemopoietic
viruses [4]. In addition, many hemopoietic progenitors
progenitors as targets for gene transfer include ease of
remain quiescent and are not susceptible to transduction.
procurement and manipulation ex vivo and the possibility of
Finally, there is also the possibility of silencing with murine
gene transfer to multiple cell lineages [1,2]. This strategy
retrovirus vectors [5,6].
could, therefore, replace allogeneic BMT as therapy for
There has been recent interest in the possibility that a
some genetic diseases. To provide long-term correction, it
significant proportion of marrow repopulating cells may be
will be necessary to transduce hemopoietic stem cells with an
CD34-negative, lineage-negative [7–10]. These cells have
integrating vector, to obtain long-term persistent expres-
been well characterized in the murine system and, more
sion. However, such vectors can only transduce dividing cells
recently, a primitive CD34– population has been shown to
Correspondence to: Helen Heslop, Center for Cell and Gene Therapy, Baylor College of Medicine, 1102 Bates St, Suite 1140, Houston, TX 77030, USA ß 1999 ISHAGE
433
434
K Moriwaki et al.
exist in humans [8,10]. Zanjani et al. [11] reported that transplantation of human lineage (lin)/CD34 cells into fetal sheep could give rise to CD34þ cells. Moreover, this very primitive population may give rise to nonhemopoietic mesenchymal precursor cells [12,13]. If the CD34 population does indeed contain a population of cells with broader potential than the hemopoietic precursors in the CD34þ population, its transduction will be essential for many gene-therapy applications. Lentiviruses can integrate the gene of interest into the target cell genome for persistent expression and may have advantages over murine retroviral vectors [14, 15]. They
Figure 1. Outline of methodology used for separation of CD34þ
have a preintegration complex that can traverse the nuclear
and CD34, lin populations.
pore of non-dividing cells so that, unlike murine retroviruses, they can also transduce neural cells, muscle cells and hepatocytes [16]. Vesicular stomatitis virus type-G (VSV-G)
Miltenyi Biotec), according to the manufacturer’s instruc-
pseudotyping of lentiviruses allows concentration by ultra-
tions. During this procedure, the flow-through of the
centrifugation, resulting in the production of high titer
separation column was collected as the CD34 fraction.
vectors. Hence, a potential advantage for this delivery system
From this fraction, we separated lin/CD34 cells using the
is that they may be able to transduce quiescent hemopoietic
StemSep kit (StemCell Technologies, Vancouver, Canada)
progenitor cells. Recently, several investigators have
and MACS depletion column (type BS, Miltenyi Biotec),
reported that lentiviral vectors can transduce hemopoietic
according to manufacturer’s instruction. The StemSep kit
progenitors derived from BM, cord blood or mobilized PB
contains the Abs for glycophorin A, CD2, CD3, CD14,
[17-21]. We have sought to determine the optimum
CD16, CD19, CD24, CD56, and CD66b. After collection,
conditions for transduction of hemopoietic progenitors,
the cells were resuspended in RPMI-1640 (BioWhittaker,
using strategies that have improved retroviral transduction,
Walkersville, Maryland) supplemented with 10% FBS
such as fibronectin [22], filter transduction [23] and cytokine
(Summit, Ft Collins, Colorado) and 1% i-glutamine
prestimulation [24], and to evaluate whether lentiviral
(GibcoBRL, Grand Island, New York) (RPMI complete
vectors could also transduce the more primitive CD34/
medium) (Figure 1).
lin populations.
Materials and methods
HIV-eGFP (VSV-G) vector and retrovirus vector
Cells
The construction of the lentiviral vector has been reported
Transduction experiments were performed on hemopoietic
previously [18]. In brief, the vif, vpr, vpu, and env genes of
progenitors selected from human BM samples. Samples
HIV are deleted and the eGFP gene inserted (HIV-eGFP).
were obtained on Institutional Review Board approved
The plasmids which contain HIV-eGFP gene and VSV-G
protocols, either from normal donors, or from patients in
env gene are co-transfected into 293T cell line and super-
remission of malignant disease, who were undergoing
natant was collected. Functional titration was performed on
autologous BMT.
the HT1080 cell line (American Type Culture Collection, Rockville, Maryland) and the titer was 2 107 IU/mL. To
Progenitor selection
compare the efficiency of pseudotyped lentivirus transduc-
The mononuclear cell fraction was obtained from BM by
tion with the most active murine retrovirus, we used a
density gradient centrifugation on Lymphoprep (Nycomed
gibbon ape leukemia virus pseudotyped Moloney vector,
Pharma, Oslo, Norway). After obtaining mononuclear cells,
containing the eGFP gene (a kind gift from Dr Robert
we separated CD34þ cells using a CD34 progenitor cell
Hawley). We have found this transduced hemopoietic cells
isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany)
at a higher efficiency than VSV-G pseudotyped Moloney
þ
and MACS separation columns (type MS
þ
or type LS ,
vectors.
Hemopoietic progenitors by lentivirus vectors
435
Transduction
For clonogenic assays, 1000 cells were plated with
Samples were transduced, with or without prestimulation
MethoCultþH4435 (StemCell Technologies, Vancouver,
with cytokines. For the prestimulation sample, cells were
Canada) in a 35 mm plate with grid (Nalge Nunc).
placed in RPMI complete medium, at a concentration of 5
Colonies were counted 2 weeks later under fluorescent
5
10 cell/mL in conventional culture plates. Cytokines were
microscopy.
added as follows; recombinant human thrombopoietin (rhTPO, R&D Systems, Minneapolis, Minnesota) 50 ng/
Statistics
mL, recombinant human stem cell factor (rhSCF, R&D
Data are shown as meanSEM for all experiments.
Systems) 50 ng/mL, recombinant human Flt3-ligand (rhFL,
Intergroup comparisons were made by t tests or rank sum
R&D Systems) 10 ng/mL. Conventional transduction was
test, as appropriate.
performed in a 35 mm tissue culture plate (FALCON3001, Becton Dickinson, Franklin Lakes, New Jersey), using target
Results
cells at a concentration of 2–4 105. 2 mL of vector were
Selection of hemopoietic progenitors
placed in the plate (MOI¼100200). For fibronectin
We first tested the purity of the lin/CD34 cell population
transduction, Retronectiu dishes (Takara Shuzo, Ohtsu,
by staining the selected cells for expression of CD34, CD3,
Japan) were used instead of normal tissue culture plates.
CD16, CD19 and CD56 Ags. The cells were consistently
Polybrene (hexadimethrine bromide, Sigma Chemical, St
<2% positive for any of these markers. The CD34-selected
Louis, Missouri) was added (6 mg/mL) to conventional, but
samples were consistently > 90% CD34þ
not to fibronectin-transduced cultures. For filter transduc-
Nalge Nunc, Rochester, New York) in the upper chamber
Effect of prestimulation, fibronectin and filter transduction on transduction of CD34þ cells with lentiviral vectors
of a container. Target cells were placed on the filter and
When CD34þ cell transduction with the lentivirus vector
vector added. After the vector had passed through the filter
was performed on conventional tissue culture plates,
under gravity, complete medium supplemented with cyto-
prestimulation had a significant effect on transduction
kines and polybrene was added.
efficiency, measured by flow cytometry. Transduction rates
tion, we used a modification of a previously-described method [23], placing a cellulose acetate filter (0.2 mm,
For all transduction methods, after overnight transduction, the cell suspension was centrifuged and resuspended in
were 15.7 2.0% (n ¼ 6) and 1.8 1.0%, with and without prestimulation respectively ( p ¼ 0.002) (Figure 2a).
RPMI complete medium, supplemented with cytokines in
We next determined whether transduction of prestimu-
the same dose and combination as during prestimulation. In
lated cells by the lentiviral vector could be further increased
the experiments without prestimulation, X-VIVO 15
by the addition of fibronectin. Transduction rates, by flow
(BioWhittaker) was used instead of RPMI complete
cytometry, were 33.54.5% (n ¼ 6) and 15.7 2.0% (n ¼ 6)
medium for the culture after transduction. For the murine
for fibronectin and conventional transduction respectively.
retrovirus transduction, target cells were prestimulated by
Transduction rates by clonogenic assay were 12.92.2%,
the same combination of cytokines for 24h and transduction
(n ¼ 5) and 2.5 0.9% (n ¼ 5) for fibronectin and
was performed for 2 consecutive days. The vector was
conventional transduction respectively. Hence, a signifi-
changed after the first overnight transduction. Comparisons
cantly higher transduction efficiency is seen with fibronectin
þ
between CD34 cells and lin /CD34 cells and among the
transduction than conventional transduction, both by
transduction methods were performed with the paired
flow cytometry and in clonogenic assay ( p ¼ 0.002 and
samples from the same source.
0.008 respectively) (Figure 2b). As we have previously observed that transduction on a
Assessment of trransduction
filter enhances murine retroviral transduction of CD34þ
After 48–120 h of culture with cytokines, cells were collected
cells [23], we compared fibronectin-assisted, with filter
and resuspended in appropriate volumes of RPMI complete
transduction, using our lentivirus vector. There was a
medium. For flow cytometry, cells are washed twice with
significant difference between fibronectin transduction
0.2% PBSA (PBS with 0.2% BSA and 0.2% sodium azide)
(15.51.4%, n ¼ 4) and filter transduction (1.6 0.6%,
and analyzed by FACScan (Becton Dickinson) for GFP.
n¼4) as assayed by flow cytometry ( p < 0.001) (Figure 2c).
436
K Moriwaki et al.
Figure 2. (a) Comparison of transduction of CD34þ progenitors with the lentivirus vector, with and without prestimulation with cytokines, analyzed by flow cytometry. (b) Comparison of transduction of prestimulated CD34þ progenitors with the lentivirus vector on conventional or fibronectincoated plates, analyzed by flow cytometry or clonogenic assay. (c) Comparison of transduction of CD34þ progenitors with the lentivirus vector on fibronectin-coated plates, or by filter transduction, analyzed by flow cytometry.
Transduction of CD34Q cells and lin2/CD342 cells We next used the lentivirus to compare transduction of CD34þ cells and CD34/lin cells from the same sample. When target cells were transduced on fibronectin with prestimulation, transduction rates analyzed by flow cytometry were 31.34.4% and 20.13.0% for CD34þ cells and lin/CD34 cells respectively (n ¼ 7). When transduced without prestimulation, transduction rates analyzed by flow cytometry were 13.63.7% and 5.22.7% for CD34þ cells and lin/CD34 cells respectively. Hence, with and without prestimulation, transduction of CD34þ cells is higher than CD34/lin cells, but lentivirus vectors nonetheless retain the ability to transduce CD34/lin cells under both sets of conditions. By contrast, when we use murine retroviral
Figure 3. Comparison of transduction of CD34þ and CD34/
vectors, we fail to transduce the CD34/lin population.
lin progenitors on fibronectin plates with the lentivirus vector,
Transduction rates on fibronectin with murine retroviral
with and without prestimulation and with the retrovirus vector.
Hemopoietic progenitors by lentivirus vectors
437
vectors after prestimulation on fibronectin were 16.1% and
the use of a different vector backbone, or it may relate to
1.2% for CD34þ cells and CD34/lin cells respectively on
differences between the cord blood CD34þ cells used by
flow cytometry ( p ¼0.016) (Figure 3).
Miyoshi et al. and our marrow-derived CD34þ progenitors.
When lentiviral transduction was assayed in short- and
The difference may also have come about because Miyoshi
long-term clonogenic assays, there was minimal transduc-
et al. transplanted these cells into NOD-SCID mice, where
tion of CD34 cells without prestimulation, increasing to
they would be subject to proliferative pressure. Of note,
20% with prestimulation. As previously reported, CD34 /
however, is the fact that, in the presence of fibronectin,
lin cells are too primitive to be clonogenic ex vivo, so the
modest levels of lentiviral transduction were obtained even
numbers of colonies from these cells were too low to be able
in the absence of prestimulation. This observation is of
to assess transduction rates by such assays.
potential importance since there is concern that the growth factors used to induce progenitor cells into cycling may
Discussion
also cause them to differentiate and lose their self-renewal
We have investigated whether or not human lentiviral
capacity [27]. Hence, it is possible that there will be a trade-
þ
vectors can transduce CD34 or CD34 /lin hemopoietic
off between transduction efficiency and self-renewal capa-
stem cells and if the degree of transduction can be increased
city, and that a lower level of transduction of unstimulated
by approaches designed to activate the target cells, or to
cells may have a more favorable in vivo outcome than a
bring them into apposition with the lentiviral particles. We
higher rate of transduction of activated progenitors.
found that lentiviral, but not murine retroviral vectors could
Two groups have recently shown that lentiviral vectors
transduce both subsets of progenitor cells and that levels of
can transduce a primitive CD34þCD38 population that is
gene transfer could be increased, either by fibronectin or by
resistant to transduction by retroviral vectors [19,21]. In our
prestimulation with cytokines. The two in combination
study, we extend these results by showing transduction of the
produced optimal results.
even-more primitive CD34/lin subpopulation. This
For murine retroviral vectors, it is well established that
subpopulation apparently contains a high proportion of
the efficiency of transduction can be improved if it is per-
repopulating cells for both hemopoietic and non-hemopoie-
formed on fibronectin support [22,24]. It is suggested that
tic mesenchymal lineages [7,8,10,12,13] and is resistant to
the benefit is produced because fibronectin can bind residues
transduction by retroviral vectors. Although the level of
on the retroviral coat proteins and the VLA-5 molecule on
transduction for this primitive subset was lower than for the
the target cell. In this way, the two components are brought
CD34 positive subset, it was nonetheless at a level that would
into physical apposition and transduction is favored. It had
be of value if it were also attainable for therapeutic genes.
not been clear whether the same effects would be obtained
Finally, in all our studies, the level of gene transfer to
for lentiviral vectors. Most studies have either not used
clonogenic cells was lower than the level of gene transfer
fibronectin at all, or have not compared transduction with
measured by gene expression in total cell populations.
and without this material [21]. In our study, fibronectin
Unfortunately, there is no good surrogate assay for human
þ
clearly enhanced transduction of CD34 cells and, to a lesser
repopulating cells, but an analysis of gene transfer into whole
extent, CD34/lin cells. The lesser increment seen with
populations of cells that have been phenotypically defined as
fibronectin for lin /CD34
cells may reflect the lower
density of VLA-5 molecules on the surface of such cells.
primitive, is probably as accurate a measure of transfer into repopulating cells as any other available surrogate. In
Several previous reports have examined the effects of
contrast, there has proved to be little relationship between
cytokine prestimulation on the efficiency of lentiviral vector
measurements of gene transfer to clonogenic cells and
transduction. Sutton et al. reported that cytokine prestimu-
subsequently-detected levels of gene transfer in vivo [28,29].
lation improved transduction [18], which may be because
We have nonetheless included this measure, to permit
lentiviral vectors more readily transduce cells in either the
comparisons with published data using other gene transfer
G1 or S/G2/M fraction than G0 cells [25]. However,
vectors.
Miyoshi et al. reported good transduction of SCID mice,
In summary, a VSV-G pseudotyped human lentivirus
repopulating cells without prestimulation [26]. Our results
vector will transduce both CD34þ and the more primitive
support the use of prestimulation to increase transduction
CD34/lin population, a subset not amenable to transduc-
efficiency. The difference may have occurred because of
tion by murine retroviral vectors. Levels of gene transfer
438
K Moriwaki et al.
can be enhanced in the presence of fibronectin and the addition of growth factors increases transduction still further. These vectors may, therefore, prove useful for transduction of both heinopoietic and non-hernopoietic mesenchymal precursor cells.
Acknowledgement This work was supported by grant HL 55703 from the National Institutes of Health.
References 1 2 3
4
5
6
7
8
9
10
11
12
13
14
Brenner MK. Gene transfer to hematopoietic cells. N Engl J Med 1996;335(5):337–9. Anderson WF. Human gene therapy. Nature 1998;392(6679 Suppl):25–30. Miller DG, Adam MA, Miller AD. Gene transfer by retrovirus vectors occurs only in cells that are actively replicating at the time of infection. Mol Cell Biol 1990;10(8):4239–42. Orlic D, Girard LJ, Jordan CT et al. The level of mRNA encoding the amphotropic retroviral receptor in mouse and human hematopoietic stem cells is low and correlates with the efficiency of retrovirus transduction. Proc Natl Acad Sci USA 1996;93:11097–102. Challita P-M, Kohn DB. Lack of expression from a retroviral vector after transduction of murine hematopoietic stem cells is associated with methylation in vivo. Proc Natl Acad Sci USA 1994;91:2567–71. Lange C, Blankenstein T. Loss of retroviral gene expression in bone marrow reconstituted mice correlates with down-regulation of gene expression in long-term culture initiating cells. Gene Tber 1997;4(4):303–8. Osawa M, Hanada K, Hamada H, Nakauchi H. Long-term lymphohematopoietic reconstitution by a single CD34 low/ negative hematopoietic stem cell. Science 1996;273(5272):242–5. Bhatia M, Bonnet D, Murdoch B, Gan OI, Dick JE. A newly discovered class of human hematopoietic cells with SCIDrepopulating activity. Nature Med 1998;(4):1038–45. Goodell MA, Brose K, Paradis G et al. Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J Exp Med 1996;184(4):1797–806. Goodell MA, Rosenzweig M, Kim H et al. Dye efflux studies suggest that hematopoietic stem cells expressing low or undetectable levels of CD34 antigen exist in multiple species. Nature Med 1997; 3(12):1337–45. Zanjani ED, Almedia-Porada G, Livingston AG et al. Human bone marrow CD34 cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34þ cells [sec comments]. Exp Hematol 1998;26(4):353–60. Gussoni E, Soneoka Y, Strickland CD et al. Dystrophin expression in the mdx mouse restored by stem cell transplantation. Nature 1999;401(6751):390–4. Jackson KJ, Mi T, Goodell MA. Hematopoietic potential of stem cells isolated from murine skeletal muscle. Proc Natl Acad Sci USA 1999;96(25):14482–6. Amado RG, Chen IS. Lentiviral vectors – the promise of gene therapy within reach? Science 1999;285(5428):674–6.
15 Naldini L. Lentiviruses as gene transfer agents for delivery to non-dividing cells. Curr Opin Biotechnol 1998;9(5):457–63. 16 Naldini L, Blomer U, Gallay P et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 1996;272:263–7. 17 Akkina RK, Walton RM, Chen ML et al. High-efficiency gene transfer into CD34þ cells with a human immunodeficiency virus type 1-based retroviral vector pseudotyped with vesicular stomatitis virus envelope glycoprotein G. J Virol 1996;70(4): 2581–5. 18 Sutton RE, Wu HT, Rigg R et al. Human immunodeficiency virus type 1 vectors efficiently transduce human hematopoietic stem cells. J Virol 1998;72(7):5781–8. 19 Uchida N, Sutton RE, Friera AM et al. HIV, but not murine leukemia virus, vectors mediate high efficiency gene transfer into freshly isolated G0/G1 human hematopoietic stem cells. Proc Natl Acad Sci USA 1998;95(20):11939–44. 20 Douglas J, Kelly P, Evans JT, Garcia JV. Efficient transduction of human lymphocytes and CD34þ cells via human immunodeficiency virus-based gene transfer vectors. Hum Gene Tber1999;10(6): 935–45. 21 Case SS, Price MA, Jordan CT et al. Stable transduction of quiescent CD34(þ)CD38() human hematopoietic cells by HIV-1-based lentiviral vectors. Proc Natl Acad Sci USA 1999;96(6): 2988–93. 22 Hanenberg H, Xiao XL, Dilloo D et al. Co-localization of retrovirus and target cells on specific fibronectin adhesion domains for increased genetic transduction of mammalian cells. Nature Med 1996;2(8):876–82. 23 Hutchings M, Moriwaki K, Dilloo D et al. Increased transduction efficiency of primary hematopoietic cells by physical co-localization of retrovirus and target cells. J Hematother 1998;7:217–24. 24 Kiem HP, Andrews RG, Morris J et al. Improved gene transfer into baboon marrow, repopulating cells using recombinant human fibronectin fragment CH-296 in combination with interleukin-6, stem cell factor, FLT-3 ligand, and megakaryocyte growth and development factor. Blood 1998;92(6): 1878–86. 25 Sutton RE, Reitsma MJ, Uchida N, Brown PO. Transduction of human progenitor hematopoietic stem cells by human immunodeficiency virus type 1-based vectors is cell cycle dependent. J Virol 1999;73(5):3649–60. 26 Miyoshi H, Smith KA, Mosier DE et al. Transduction of human CD34þ cells that mediate long-term engraftment of NOD/SLID mice by HIV vectors. Science 1999;283(5402): 682–6. 27 Tisdale JF, Hanazono Y, Sellers SE et al. Ex vivo expansion of genetically marked rhesus peripheral blood progenitor cells results in diminished long-term repopulating ability. Blood 1998;92(4): 1131–41. 28 Brenner MK, Rill DR, Holladay MS et al. Gene marking to determine whether autologous marrow infusion restores longterm haemopoiesis in cancer patients. Lancet 1993;342:1134–7. 29 Hanania EG, Giles RE, Kavanagh J et al. Results of MDR-1 vector modification trial indicate that granulocyte/macrophage colony-forming unit cells do not contribute to post transplant hematopoietic recovery following intensive systemic therapy. Proc Natl Acad Sci USA 1996;93:15346–51.
Transduction of CD34Q and CD342/lin2 hemopoietic progenitors by lentivirus vectors K Moriwaki1,2, RE Sutton1,3, M Perez1,2, MK Brenner1,2,4 and HE Heslop1,2,4 1
Center for Cell and Gene Therapy, 2Department of Pediatrics, 3Division of Molecular Virology and 4
Department of Medicine, Baylor College of Medicine, Houston, Texas, USA
Background
populations. Mean transduction rates on fibronectin plates,
While hemopoietic stem cells have been thought to reside
analyzed by flow cytometry, were 14% and 5% for CD34þ cells
predominantly in the CD34þ population, recent data suggests
and lin/CD34 cells respectively, without prestimulation, and
31% and 20% with prestimulation. By contrast, a murine
population. Transduction of both these populations by murine
retroviral vector transduces CD34þ cells with lower efficiency
retroviral vectors is limited by quiescence of hemopoietic stem cells.
(mean 16.1% with prestimulation) and does not induce any
that repopulating cells may, in fact, also reside in the lin /CD34
significant transduction of the CD34/lin population (mean Methods
< 2%). When lentiviral transduction was assayed in short- and
We therefore sought to transduce these populations using a VSV-G
long-term clonogenic assays there was minimal transduction of
pseudotyped, HIV-based, human lentiviral vector, encoding
CD34 cells without prestimulation, increasing to 20% with
eGFP. CD34þ cells and lin/CD34 cells were selected from
prestimulation.
the same BM samples by immunomagnetic beads (StemSep) to deplete lineage-positive cells and by CD34 selection columns
Discussion
(Miltenyi) to separate CD34þ and CD34 populations. We
Lentiviral eGFP vectors can transduce hematopoietic progenitors
transduced target cells, with or without prestimulation with
effectively and efficiency is improved by cytokine prestimulation and
cytokines, using conventional suspension culture, or fibronectin
the use of fibronectin. Moreover, the human viral vectors can
plates, or flow-through transduction. Transduction efficiency was
transduce a candidate stem-cell population that is resistant to
analyzed by flow cytometry and clonogenic assay.
murine retroviral transduction.
Results
Keywords
We found that transduction on fibronectin plates was more efficient
CD34þ, hemopoietic stem cell, lentiviral vector, gene therapy,
than flow-through transduction, or suspension cultures, for both cell
lin/CD34
Introduction
[3] and in vivo gene transfer in clinical studies has been
Hemopoietic stem cells are an attractive target for gene
disappointingly low, perhaps because the cytokines used
therapy as gene transfer to stem cells could, theoretically,
have failed to induce true repopulating cells into the cycle, or
provide lifelong correction for many gene defects of
because these cells may lack receptors for murine retro-
marrow-derived cells. Advantages of using hemopoietic
viruses [4]. In addition, many hemopoietic progenitors
progenitors as targets for gene transfer include ease of
remain quiescent and are not susceptible to transduction.
procurement and manipulation ex vivo and the possibility of
Finally, there is also the possibility of silencing with murine
gene transfer to multiple cell lineages [1,2]. This strategy
retrovirus vectors [5,6].
could, therefore, replace allogeneic BMT as therapy for
There has been recent interest in the possibility that a
some genetic diseases. To provide long-term correction, it
significant proportion of marrow repopulating cells may be
will be necessary to transduce hemopoietic stem cells with an
CD34-negative, lineage-negative [7–10]. These cells have
integrating vector, to obtain long-term persistent expres-
been well characterized in the murine system and, more
sion. However, such vectors can only transduce dividing cells
recently, a primitive CD34– population has been shown to
Correspondence to: Helen Heslop, Center for Cell and Gene Therapy, Baylor College of Medicine, 1102 Bates St, Suite 1140, Houston, TX 77030, USA ß 1999 ISHAGE
433
434
K Moriwaki et al.
exist in humans [8,10]. Zanjani et al. [11] reported that transplantation of human lineage (lin)/CD34 cells into fetal sheep could give rise to CD34þ cells. Moreover, this very primitive population may give rise to nonhemopoietic mesenchymal precursor cells [12,13]. If the CD34 population does indeed contain a population of cells with broader potential than the hemopoietic precursors in the CD34þ population, its transduction will be essential for many gene-therapy applications. Lentiviruses can integrate the gene of interest into the target cell genome for persistent expression and may have advantages over murine retroviral vectors [14, 15]. They
Figure 1. Outline of methodology used for separation of CD34þ
have a preintegration complex that can traverse the nuclear
and CD34, lin populations.
pore of non-dividing cells so that, unlike murine retroviruses, they can also transduce neural cells, muscle cells and hepatocytes [16]. Vesicular stomatitis virus type-G (VSV-G)
Miltenyi Biotec), according to the manufacturer’s instruc-
pseudotyping of lentiviruses allows concentration by ultra-
tions. During this procedure, the flow-through of the
centrifugation, resulting in the production of high titer
separation column was collected as the CD34 fraction.
vectors. Hence, a potential advantage for this delivery system
From this fraction, we separated lin/CD34 cells using the
is that they may be able to transduce quiescent hemopoietic
StemSep kit (StemCell Technologies, Vancouver, Canada)
progenitor cells. Recently, several investigators have
and MACS depletion column (type BS, Miltenyi Biotec),
reported that lentiviral vectors can transduce hemopoietic
according to manufacturer’s instruction. The StemSep kit
progenitors derived from BM, cord blood or mobilized PB
contains the Abs for glycophorin A, CD2, CD3, CD14,
[17-21]. We have sought to determine the optimum
CD16, CD19, CD24, CD56, and CD66b. After collection,
conditions for transduction of hemopoietic progenitors,
the cells were resuspended in RPMI-1640 (BioWhittaker,
using strategies that have improved retroviral transduction,
Walkersville, Maryland) supplemented with 10% FBS
such as fibronectin [22], filter transduction [23] and cytokine
(Summit, Ft Collins, Colorado) and 1% i-glutamine
prestimulation [24], and to evaluate whether lentiviral
(GibcoBRL, Grand Island, New York) (RPMI complete
vectors could also transduce the more primitive CD34/
medium) (Figure 1).
lin populations.
Materials and methods
HIV-eGFP (VSV-G) vector and retrovirus vector
Cells
The construction of the lentiviral vector has been reported
Transduction experiments were performed on hemopoietic
previously [18]. In brief, the vif, vpr, vpu, and env genes of
progenitors selected from human BM samples. Samples
HIV are deleted and the eGFP gene inserted (HIV-eGFP).
were obtained on Institutional Review Board approved
The plasmids which contain HIV-eGFP gene and VSV-G
protocols, either from normal donors, or from patients in
env gene are co-transfected into 293T cell line and super-
remission of malignant disease, who were undergoing
natant was collected. Functional titration was performed on
autologous BMT.
the HT1080 cell line (American Type Culture Collection, Rockville, Maryland) and the titer was 2 107 IU/mL. To
Progenitor selection
compare the efficiency of pseudotyped lentivirus transduc-
The mononuclear cell fraction was obtained from BM by
tion with the most active murine retrovirus, we used a
density gradient centrifugation on Lymphoprep (Nycomed
gibbon ape leukemia virus pseudotyped Moloney vector,
Pharma, Oslo, Norway). After obtaining mononuclear cells,
containing the eGFP gene (a kind gift from Dr Robert
we separated CD34þ cells using a CD34 progenitor cell
Hawley). We have found this transduced hemopoietic cells
isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany)
at a higher efficiency than VSV-G pseudotyped Moloney
þ
and MACS separation columns (type MS
þ
or type LS ,
vectors.
Hemopoietic progenitors by lentivirus vectors
435
Transduction
For clonogenic assays, 1000 cells were plated with
Samples were transduced, with or without prestimulation
MethoCultþH4435 (StemCell Technologies, Vancouver,
with cytokines. For the prestimulation sample, cells were
Canada) in a 35 mm plate with grid (Nalge Nunc).
placed in RPMI complete medium, at a concentration of 5
Colonies were counted 2 weeks later under fluorescent
5
10 cell/mL in conventional culture plates. Cytokines were
microscopy.
added as follows; recombinant human thrombopoietin (rhTPO, R&D Systems, Minneapolis, Minnesota) 50 ng/
Statistics
mL, recombinant human stem cell factor (rhSCF, R&D
Data are shown as meanSEM for all experiments.
Systems) 50 ng/mL, recombinant human Flt3-ligand (rhFL,
Intergroup comparisons were made by t tests or rank sum
R&D Systems) 10 ng/mL. Conventional transduction was
test, as appropriate.
performed in a 35 mm tissue culture plate (FALCON3001, Becton Dickinson, Franklin Lakes, New Jersey), using target
Results
cells at a concentration of 2–4 105. 2 mL of vector were
Selection of hemopoietic progenitors
placed in the plate (MOI¼100200). For fibronectin
We first tested the purity of the lin/CD34 cell population
transduction, Retronectiu dishes (Takara Shuzo, Ohtsu,
by staining the selected cells for expression of CD34, CD3,
Japan) were used instead of normal tissue culture plates.
CD16, CD19 and CD56 Ags. The cells were consistently
Polybrene (hexadimethrine bromide, Sigma Chemical, St
<2% positive for any of these markers. The CD34-selected
Louis, Missouri) was added (6 mg/mL) to conventional, but
samples were consistently > 90% CD34þ
not to fibronectin-transduced cultures. For filter transduc-
Nalge Nunc, Rochester, New York) in the upper chamber
Effect of prestimulation, fibronectin and filter transduction on transduction of CD34þ cells with lentiviral vectors
of a container. Target cells were placed on the filter and
When CD34þ cell transduction with the lentivirus vector
vector added. After the vector had passed through the filter
was performed on conventional tissue culture plates,
under gravity, complete medium supplemented with cyto-
prestimulation had a significant effect on transduction
kines and polybrene was added.
efficiency, measured by flow cytometry. Transduction rates
tion, we used a modification of a previously-described method [23], placing a cellulose acetate filter (0.2 mm,
For all transduction methods, after overnight transduction, the cell suspension was centrifuged and resuspended in
were 15.7 2.0% (n ¼ 6) and 1.8 1.0%, with and without prestimulation respectively ( p ¼ 0.002) (Figure 2a).
RPMI complete medium, supplemented with cytokines in
We next determined whether transduction of prestimu-
the same dose and combination as during prestimulation. In
lated cells by the lentiviral vector could be further increased
the experiments without prestimulation, X-VIVO 15
by the addition of fibronectin. Transduction rates, by flow
(BioWhittaker) was used instead of RPMI complete
cytometry, were 33.54.5% (n ¼ 6) and 15.7 2.0% (n ¼ 6)
medium for the culture after transduction. For the murine
for fibronectin and conventional transduction respectively.
retrovirus transduction, target cells were prestimulated by
Transduction rates by clonogenic assay were 12.92.2%,
the same combination of cytokines for 24h and transduction
(n ¼ 5) and 2.5 0.9% (n ¼ 5) for fibronectin and
was performed for 2 consecutive days. The vector was
conventional transduction respectively. Hence, a signifi-
changed after the first overnight transduction. Comparisons
cantly higher transduction efficiency is seen with fibronectin
þ
between CD34 cells and lin /CD34 cells and among the
transduction than conventional transduction, both by
transduction methods were performed with the paired
flow cytometry and in clonogenic assay ( p ¼ 0.002 and
samples from the same source.
0.008 respectively) (Figure 2b). As we have previously observed that transduction on a
Assessment of trransduction
filter enhances murine retroviral transduction of CD34þ
After 48–120 h of culture with cytokines, cells were collected
cells [23], we compared fibronectin-assisted, with filter
and resuspended in appropriate volumes of RPMI complete
transduction, using our lentivirus vector. There was a
medium. For flow cytometry, cells are washed twice with
significant difference between fibronectin transduction
0.2% PBSA (PBS with 0.2% BSA and 0.2% sodium azide)
(15.51.4%, n ¼ 4) and filter transduction (1.6 0.6%,
and analyzed by FACScan (Becton Dickinson) for GFP.
n¼4) as assayed by flow cytometry ( p < 0.001) (Figure 2c).
436
K Moriwaki et al.
Figure 2. (a) Comparison of transduction of CD34þ progenitors with the lentivirus vector, with and without prestimulation with cytokines, analyzed by flow cytometry. (b) Comparison of transduction of prestimulated CD34þ progenitors with the lentivirus vector on conventional or fibronectincoated plates, analyzed by flow cytometry or clonogenic assay. (c) Comparison of transduction of CD34þ progenitors with the lentivirus vector on fibronectin-coated plates, or by filter transduction, analyzed by flow cytometry.
Transduction of CD34Q cells and lin2/CD342 cells We next used the lentivirus to compare transduction of CD34þ cells and CD34/lin cells from the same sample. When target cells were transduced on fibronectin with prestimulation, transduction rates analyzed by flow cytometry were 31.34.4% and 20.13.0% for CD34þ cells and lin/CD34 cells respectively (n ¼ 7). When transduced without prestimulation, transduction rates analyzed by flow cytometry were 13.63.7% and 5.22.7% for CD34þ cells and lin/CD34 cells respectively. Hence, with and without prestimulation, transduction of CD34þ cells is higher than CD34/lin cells, but lentivirus vectors nonetheless retain the ability to transduce CD34/lin cells under both sets of conditions. By contrast, when we use murine retroviral
Figure 3. Comparison of transduction of CD34þ and CD34/
vectors, we fail to transduce the CD34/lin population.
lin progenitors on fibronectin plates with the lentivirus vector,
Transduction rates on fibronectin with murine retroviral
with and without prestimulation and with the retrovirus vector.
Hemopoietic progenitors by lentivirus vectors
437
vectors after prestimulation on fibronectin were 16.1% and
the use of a different vector backbone, or it may relate to
1.2% for CD34þ cells and CD34/lin cells respectively on
differences between the cord blood CD34þ cells used by
flow cytometry ( p ¼0.016) (Figure 3).
Miyoshi et al. and our marrow-derived CD34þ progenitors.
When lentiviral transduction was assayed in short- and
The difference may also have come about because Miyoshi
long-term clonogenic assays, there was minimal transduc-
et al. transplanted these cells into NOD-SCID mice, where
tion of CD34 cells without prestimulation, increasing to
they would be subject to proliferative pressure. Of note,
20% with prestimulation. As previously reported, CD34 /
however, is the fact that, in the presence of fibronectin,
lin cells are too primitive to be clonogenic ex vivo, so the
modest levels of lentiviral transduction were obtained even
numbers of colonies from these cells were too low to be able
in the absence of prestimulation. This observation is of
to assess transduction rates by such assays.
potential importance since there is concern that the growth factors used to induce progenitor cells into cycling may
Discussion
also cause them to differentiate and lose their self-renewal
We have investigated whether or not human lentiviral
capacity [27]. Hence, it is possible that there will be a trade-
þ
vectors can transduce CD34 or CD34 /lin hemopoietic
off between transduction efficiency and self-renewal capa-
stem cells and if the degree of transduction can be increased
city, and that a lower level of transduction of unstimulated
by approaches designed to activate the target cells, or to
cells may have a more favorable in vivo outcome than a
bring them into apposition with the lentiviral particles. We
higher rate of transduction of activated progenitors.
found that lentiviral, but not murine retroviral vectors could
Two groups have recently shown that lentiviral vectors
transduce both subsets of progenitor cells and that levels of
can transduce a primitive CD34þCD38 population that is
gene transfer could be increased, either by fibronectin or by
resistant to transduction by retroviral vectors [19,21]. In our
prestimulation with cytokines. The two in combination
study, we extend these results by showing transduction of the
produced optimal results.
even-more primitive CD34/lin subpopulation. This
For murine retroviral vectors, it is well established that
subpopulation apparently contains a high proportion of
the efficiency of transduction can be improved if it is per-
repopulating cells for both hemopoietic and non-hemopoie-
formed on fibronectin support [22,24]. It is suggested that
tic mesenchymal lineages [7,8,10,12,13] and is resistant to
the benefit is produced because fibronectin can bind residues
transduction by retroviral vectors. Although the level of
on the retroviral coat proteins and the VLA-5 molecule on
transduction for this primitive subset was lower than for the
the target cell. In this way, the two components are brought
CD34 positive subset, it was nonetheless at a level that would
into physical apposition and transduction is favored. It had
be of value if it were also attainable for therapeutic genes.
not been clear whether the same effects would be obtained
Finally, in all our studies, the level of gene transfer to
for lentiviral vectors. Most studies have either not used
clonogenic cells was lower than the level of gene transfer
fibronectin at all, or have not compared transduction with
measured by gene expression in total cell populations.
and without this material [21]. In our study, fibronectin
Unfortunately, there is no good surrogate assay for human
þ
clearly enhanced transduction of CD34 cells and, to a lesser
repopulating cells, but an analysis of gene transfer into whole
extent, CD34/lin cells. The lesser increment seen with
populations of cells that have been phenotypically defined as
fibronectin for lin /CD34
cells may reflect the lower
density of VLA-5 molecules on the surface of such cells.
primitive, is probably as accurate a measure of transfer into repopulating cells as any other available surrogate. In
Several previous reports have examined the effects of
contrast, there has proved to be little relationship between
cytokine prestimulation on the efficiency of lentiviral vector
measurements of gene transfer to clonogenic cells and
transduction. Sutton et al. reported that cytokine prestimu-
subsequently-detected levels of gene transfer in vivo [28,29].
lation improved transduction [18], which may be because
We have nonetheless included this measure, to permit
lentiviral vectors more readily transduce cells in either the
comparisons with published data using other gene transfer
G1 or S/G2/M fraction than G0 cells [25]. However,
vectors.
Miyoshi et al. reported good transduction of SCID mice,
In summary, a VSV-G pseudotyped human lentivirus
repopulating cells without prestimulation [26]. Our results
vector will transduce both CD34þ and the more primitive
support the use of prestimulation to increase transduction
CD34/lin population, a subset not amenable to transduc-
efficiency. The difference may have occurred because of
tion by murine retroviral vectors. Levels of gene transfer
438
K Moriwaki et al.
can be enhanced in the presence of fibronectin and the addition of growth factors increases transduction still further. These vectors may, therefore, prove useful for transduction of both heinopoietic and non-hernopoietic mesenchymal precursor cells.
Acknowledgement This work was supported by grant HL 55703 from the National Institutes of Health.
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