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Blood, Vol. 95 No. 2 (January 15), 2000:
pp. 437-444
GENE THERAPY
Glass needle-mediated microinjection of macromolecules and
transgenes into primary human blood stem/progenitor cells
Brian R. Davis,
Judith Yannariello-Brown,
Nicole
L. Prokopishyn,
Zhongjun Luo,
Mark R. Smith,
Jue Wang,
N. D. Victor Carsrud, and
David B. Brown
From Sealy Center for Oncology and Hematology, Department of
Microbiology and Immunology, Department of Human Biological Chemistry
and Genetics, University of Texas Medical Branch, Galveston; Gene-Cell,
Inc, Houston, TX; and Frederick Cancer Research and Development Center,
National Cancer Institute, Frederick, MD.
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Abstract |
A novel glass needle-mediated microinjection method for delivery of
macromolecules, including proteins and larger transgene DNAs, into the
nuclei of blood stem/progenitor cells was developed. Temporary
immobilization of cells to extracellular matrix-coated dishes
has enabled rapid and consistent injection of
macromolecules into nuclei of CD34+,
CD34+/CD38 , and
CD34+/CD38/Thy-1lo human cord
blood cells. Immobilization and detachment protocols were
identified, which had no adverse effect on cell survival, progenitor
cell function (colony forming ability), or stem cell function (NOD/SCID
reconstituting ability). Delivery of fluorescent dextrans to
stem/progenitor cells was achieved with 52% ± 8.4% of
CD34+ cells and 42% ± 14% of
CD34+/CD38cells still fluorescent
48 hours after injection. Single-cell transfer and culture of injected
cells has demonstrated long-term survival and proliferation of
CD34+ and CD34+/CD38
cells, and retention of the ability of
CD34+/CD38 cells to generate progenitor
cells. Delivery of DNA constructs (currently  19.6 kb) and
fluorescently labeled proteins into CD34+ and
CD34+/CD38 cells was achieved with
transient expression of green fluorescent protein observed in up to
75% of injected cells. These data indicate that glass needle-mediated
delivery of macromolecules into primitive hematopoietic
cells is a valuable method for studies of stem cell biology and a
promising method for human blood stem cell gene therapy.
(Blood. 2000;95:437-444)
© 2000 by The American Society of Hematology.
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Introduction |
Hematopoietic stem cells are a major focus of
investigation in both developmental biology and clinical medicine. The
stem cell compartment consists of rare, long-lived, self-renewing, predominantly quiescent cells capable of long-term reconstitution of
the complete hematopoietic system in transplanted hosts.1 It is of significant biologic and clinical interest to elucidate the
mechanisms controlling stem cell self-renewal, commitment to
maturation, and selection of differentiation program. The potential to
genetically correct the complete hematopoietic system by successfully modifying only the stem cell compartment has made these cells a primary
target for gene therapy.
We have sought to develop a method that allows for the introduction of
macromolecules into stem/progenitor cells. Current technologies (eg,
standard retroviruses, adeno-associated viruses, liposomes) have
demonstrated only limited success in efficiently transducing human stem
cells.2-5 Glass needle-mediated microinjection of
macromolecules into living mammalian cells, developed independently by
Diacumakos6 and Graessman,7 has proven to be a
powerful approach for analyzing the biologic activity of specific
molecules (eg, peptide inhibitors, purified active proteins,
neutralizing antibodies, RNA, and DNA) in adherent
cells.8,9 However, this approach has rarely been used for
primary hematopoietic cells10 because immobilization of
hematopoietic cells has generally been ineffective and standard
injection needles cause significant damage to these relatively small
cells (~ 6 µ diameter for stem cells11). Two
developments have allowed us to apply glass needle-mediated microinjection technology to blood stem/progenitor cells. First, we
have developed a method for attaching human blood stem/progenitor cells
to extracellular matrix-coated dishes, which does not affect cell
function. Second, we have developed injection needles with very small
outer tip diameters (OTD; ~ 0.2 µ), which have excellent flow
properties and result in excellent postinjection viabilities.
We demonstrate that glass needle-mediated microinjection can be used
effectively for the introduction of macromolecules (protein, DNA, and
dextrans) into human CD34+,
CD34+/CD38, and
CD34+/CD38/Thy-1lo cord
blood cells. Importantly, macromolecule delivery is accomplished with
high postinjection cell viability, no discernible impact on
stem/progenitor cell proliferation or biologic activity, and a high
frequency of cells expressing injected transgenes.
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Materials and methods |
Isolation and further fractionation of human CD34+
cells
CD34+ cells were immunomagnetically purified from human
umbilical cord blood (University of Texas Medical Branch) using either the Progenitor or Multisort Kits (Miltenyi Biotec, Auburn, CA), then
cultured in Iscoves' Modified Dulbecco's Medium without phenol red
(IMDM) with 100 µg/mL glutamine/penicillin/streptomycin,
1 × BIT 9500 (StemCell Technologies, Vancouver, BC), 20 ng/mL
each of human Interleukin (II)-3, flt-3 ligand, and stem cell factor (PeproTech, Inc, Rocky Hill, NJ), 40 µg/mL low-density
lipoprotein (Sigma), and 50 µM 2-mercaptoethanol (Stem Cell Medium).
Subpopulations of CD34+ cells were isolated by flow
cytometry on a FACS Vantage (Becton Dickinson, San Jose, CA) after
immunostaining with anti-CD34-FITC and anti-CD38-PE for
CD34+/CD38 cells and anti-CD34-PerCP,
anti-CD38-PE, and anti-Thy-1-FITC for
CD34+/CD38/Thy-1lo cells.
CD34+ cell matrix attachment and detachment
Dishes were coated overnight at 4°C with either Fibronectin (FN)
or FN fragment CH-296 (Retronectin (RN); TaKaRa Biomedicals, Panvera,
Madison, WI; 15 µg/mL) in phosphate-buffered saline (PBS) and washed
with IMDM containing glutamine/penicillin/streptomycin. Cells were
added to cloning rings and attached to the plates for 45 minutes at
37°C. When using FN, the integrin-activating monoclonal antibody
(mAb) TS2/16.2.1 IgG purified from ascites (1 µg/mL; ATCC,
HB-24312) was added to the cells. Cells were detached from
the matrix-coated dishes by either: (1) incubating in a mixture of FN
CS-1 fragment (0.42 mg/mL), H-Arg-Gly-Asp-Ser-OH (1.0 mg/mL) and
Phenylac-Leu-Asp-Phe-D-Pro-NH2 (1.0 mg/mL; Bachem BioScience, Torrance,
CA); or (2) pipetting under a stream of medium.
Injection of macromolecules into CD34+,
CD34+/CD38, and
CD34+/CD38/Thy-1lo cells
Injection needles were pulled from 10 cm borosilicate capillaries
with a 1.2 mm outer/0.94 mm inner diameter using a Flaming/Brown Micropipette Puller Model P-97 (Sutter Instrument Co, Novato, CA) and
had a range of 0.17 to 0.25 µ OTD, as determined by scanning electron
microscopy. Cells were visualized using a Nikon Eclipse TE300
microscope equipped with a Fryer A-50 temperature-controlled stage (set
at 37°C) and injected using the electronically interfaced Eppendorf
Micromanipulator (Model 5171) and Transjector (Model 5246). Manual
injections were performed, except where noted as semiautomatic
injections. In some cases, manual injections were performed with a
Narishige joystick-controlled micromanipulator. After injection, cells
were maintained on matrix-coated plates and observed using fluorescent
microscopy to determine the percentage viability (number of fluorescent
cells/number of successfully injected cells × 100). A
successfully injected cell was defined as a cell that was intact, was
alive, and remained attached to the plate after injection. For the
experiments presented in this report, the percentage of successful
injections ranged from 50% to 95%. Alternatively, injected cells were
transferred individually using a Quixell Automated Cell Selection and
Transfer Unit (Stoelting, Wood Dale, IL) to wells of 96-well plates
containing Stem Cell Medium with 50 mmol/L HEPES, pH 7.4.
Microinjection samples
Samples were dialyzed against 50 mmol/L HEPES, pH 7.4, 140 mmol/L
KCl. Oregon Green 488-dextran (OG-dextran; 70 000 MW), FITC-dextran (150 000 MW), and Cy-3 conjugated mouse immunoglobulin (Cy-3 IgG) were
adjusted to 0.15, 2.5, and 0.5 µg/mL, respectively. pGreen Lantern (5 kb; Gibco-BRL, Bethesda, MD) and linearized phosphoglycerate kinase
Green Fluorescent Protein (pgkGFP) DNA (1.7 kb) were adjusted to 5 copies per fL. Both vectors contain the humanized, red
shifted GFP from Aequorea victoria jellyfish. The linearized
pgkGFP construct is based on pCMV-Beta (CLONTECH, Palo Alto, CA). The
lacZ gene in pCMV-Beta was replaced with the GFP gene of pGreen
Lantern, and the cytomegalovirus (CMV) enhancer/promoter sequences were replaced with the pgk promoter sequences.13 Linear pgkGFP
sequences were obtained by digesting pgkGFP with Pac I and Asc I and
isolating the resulting 1.7 kb fragment after agarose gel electrophoresis.
NOD/SCID engraftment assay
Nonobese diabetic and severe combined immmunodeficient
NOD/LtSz-Prkc-scid/scid (NOD/SCID) mice were obtained from
Jackson Laboratory (Bar Harbor, ME14) and were bred and
maintained in microisolator cages on laminar flow racks. NOD/SCID mice
of 6 to 8 weeks of age were sublethally irradiated with 350 or 400 rad
(137Cs source) and injected intravenously (iv; tail vein)
with purified CD34+ cells (2 × 104
cells/mouse). CD34+ cells had either been maintained in
suspension or first immobilized on RN or FN, then detached by pipetting
or treatment with peptides. At 6 weeks inoculation, marrow cells from
both femurs of the mice were analyzed for the presence of human cells
by fluorescence-activated cell sorter (FACS) analysis using antihuman
CD45FITC and antihuman CD34 PE (Becton
Dickinson). Percent CD45+ cells represents the sum of the
frequency of human CD45+/CD34 and
CD45+/CD34+ cells. When the level of human cell
engraftment was low (< 1% human cells), semiquantitative DNA
polymerase chain reaction amplification for human Cart-1 sequences was
performed to confirm engraftment.15 Statistical analysis
was performed with the Student's t test (unless otherwise
noted) using Statview 4.1 (Abacus Concepts, Inc, Berkeley, CA) and
Microsoft Excel 8.0 (Microsoft, Redmond, WA); P values were
considered to be statistically significant if less than .05.
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Results |
Immobilization of human blood stem/progenitor cells on
matrix-coated dishes
Hematopoietic stem/progenitor cells in the bone marrow express
 4 1 and 51
integrins, allowing them to interact with FN through the CS-1 region
and RGDS sequence, respectively.16 We evaluated whether the
attachment of blood stem/progenitor cells to FN-coated plates was
sufficient for glass needle-mediated injection. Attachment of primary
CD34+ cells to plates coated with FN resulted in only
tethering of the cells rather than solid attachment (Figure
1B; unattached CD34+ cells in
suspension culture are shown in Figure 1A); the cells dislodged during
injection. However, when CD34+ cells were treated with
specific anti-1 integrin mAb, they attached strongly to
FN-coated plates (Figure 1C). Up to 100% of CD34+ cells
were immobilized in this way and acquired a more flattened morphology
and extended micropodia (Figure 1C).
CD34+/CD38 and
CD34+/CD38/Thy-1lo cells,
believed to represent increasingly enriched populations of blood stem
cells, were also immobilized using this method (data not shown). Up to
100% of primary blood stem/progenitor cells also attached firmly to RN
(Figure 1D), a recombinant derivative of FN. RN contains only the
heparin-binding, CS-1, and RGDS-containing cell association regions of
FN.17 Attachment to RN occurred even in the absence of
activating anti-1 mAb. For both methods of attachment, cells
withstood both manual (Figure 1E) and semiautomatic injection without
becoming dislodged.
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| Fig 1.
Photomicrographs of attached and injected
CD34+ cells.
Represented are CD34+ cells maintained in
suspension (A) or attached to FN-coated dishes in the absence (B) or
presence of TS2/16.2.1 mAb (C) or RN-coated dishes (D-F). Injection of
attached CD34+ cells is shown in panel E. Note the
injection needle (arrow) and cells injected with OG-dextran
(arrowheads). A fluorescence micrograph (F) of the identical field
shown in panel E demonstrates the successful delivery of fluorescent
material (arrowheads). Images in panels E-F were captured from
videotape during a live injection session and, therefore, display a
decreased resolution, compared with images in panels A-D.
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Biologic activity of human blood stem/progenitor cells after
immobilization on FN and RN
Despite their strong attachment to FN (in the presence of activating
TS2/16.2.1 mAb) and RN, cells could be recovered with vigorous
pipetting. However, this resulted in decreased viability (64% ± 12%) and incomplete recovery ( 85%) of cells.
Virtually 100% of cells were recovered from FN and RN by competing for
attachment with peptides corresponding to the CS-1 and RGDS regions of
FN. In all cases, detached cells were > 95% viable and rapidly
reacquired their normal rounded morphology and nonadherent behavior.
We evaluated the effects of in vitro attachment on stem/progenitor cell
function. Immobilization of CD34+ and
CD34+/CD38 cells to FN or RN for 2 hours
and release with peptide had no effect on viability or proliferative
capacity of cells in liquid culture. Doubling rates of 21.2 ± 1.1
hours and 23.2 ± 1.7 hours were obtained for untreated control
CD34+ and TS2/16 mAb-treated treated CD34+
cells grown in liquid culture, respectively. Similarly, doubling rates
of 21.6 ± 2.5 hours and 21.6 ± 2.1 hours were obtained for CD34+ cells attached for 2 hours to either FN or RN,
respectively, released by peptide and grown in liquid culture.
Because CD34+ cells are highly enriched in progenitor
cells,18 the effect of temporary immobilization on
colony-forming activity was evaluated. CD34+ cells were
allowed to attach for 2 hours to either FN, RN, or Con A lectin,
detached, then plated in methylcellulose with appropriate cytokines to
compare their colony-forming activity (erythroid and myeloid) to
nonattached suspension cells (Table 1). FN-
or RN-immobilized cells demonstrated no significant alteration in either frequency (Table 1) or size (data not shown) of erythroid or
myeloid colonies. In contrast, Con A immobilization lead to a
significant reduction in myeloid colony formation (Table 1).
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Table 1.
Immobilization on FN or RN and subsequent detachment
does not affect the colony forming ability of CD34+
cells
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We also examined whether there was any adverse effect of immobilization
on stem cell function using the NOD/SCID engraftment model. NOD/SCID
mice demonstrate active human blood cell development in the marrow of
transplanted mice after intravenous delivery of total marrow, total
cord blood, or primitive hematopoietic subpopulations.15,19
Sublethally irradiated mice were injected intravenously with
CD34+ cells that had been immobilized on FN (with
activating mAb) or RN and detached or with control CD34+
cells. Control cells for the FN plus mAb experimental group consisted of CD34+ cells incubated on FN-coated dishes in the absence
of activating mAb. Controls for the RN-attached cells consisted of
CD34+ cells incubated on noncoated plastic dishes or
FN-coated dishes without activating mAb. No statistical difference was
detected when comparing the percentage of mice engrafting
(P > .42) or the extent of engraftment
(P > .35) with either control group. As shown in Table
2, there was no evidence for any adverse
effect on the engraftment ability of the human NOD/SCID reconstituting cells after attachment to RN. Although a slight decrease in engraftment ability was seen in the cells attached to FN with activating mAb (75%
compared with the control value of 94% for suspension cells or 84%
for cells incubated on FN in the absence of activating mAb), this
difference was not statistically significant (P > .19 and
P > .22, respectively; Student's paired t test).
The extent of engraftment was determined for each mouse by quantitating
via FACS analysis the percentage of human CD45+ cells
present in the bone. As can be seen in Figure
2, there was considerable variability in
the range of engraftment levels obtained for each of the experimental
conditions. Although not statistically significant
(P > .13), there was a distinct trend toward lower levels
of engraftment when cells were attached to FN in the presence of
activating antibody (0.4% ± 0.6% human CD45+ cells;
mean ± SD) as compared with control cells incubated on FN in the
absence of activating mAb (1.8% ± 3.7%). Engraftment levels with
RN-attached cells (1.9% ± 2.3%) were equivalent to those of
control cells (2.4% ± 4.3%). Thus, primitive cord blood cells
temporarily attached to RN are neither affected in their proliferation,
colony-forming activity, nor NOD/SCID engrafting ability. In contrast,
primitive cord blood cells attached to FN in the presence of activating
mAb may have impaired NOD/SCID engrafting ability, while still
retaining control levels of proliferative and colony-forming activity.
In light of these observations, subsequent experiments used RN
attachment.
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| Fig 2.
Extent of CD34+ cell engraftment in
NOD/SCID mice after attachment and detachment.
Mouse bone marrow mononuclear cells were immunostained with antihuman
CD45FITC and CD34PE mAbs and analyzed by FACS
by using Cell Quest Software (Becton Dickinson). The percentage
CD45+ cells was determined on ungated samples. (A) Plot of
the percentage CD45+ cells in mice injected with cells
plated on FN in the presence (diamonds) or absence (control; triangles)
of TS2/16.2.1 activating mAb. (B) Scatter plot of the percentage of
CD45+ cells in mice injected with RN-attached cells
(circles) or control cells (ie, cells maintained in suspension or
plated on FN in the absence of activating mAb; inverted triangles).
Horizontal lines represent the mean percentage CD45+ cells
in each group.
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Microinjection-mediated delivery of fluorescencelabeled
dextrans into CD34+,
CD34+/CD38, and
CD34+/CD38/Thy-1lo cells
To optimize injection conditions for primitive blood cells, it was
critical to directly monitor the flow of material from the injection
needle and the fate of individual cells after injection. Therefore, we
used OG-dextran or FITC-dextran when optimizing blood stem/progenitor
cell injections.20
CD34+ cells were immobilized on RN and manually injected
with OG-dextran. Immediately after injection, only 5% to 10% of the injected cells exhibited damage visible by light microscopy. Examples of CD34+ cells during and after injection with OG-dextran
are shown in Figure 1E and F, respectively. Approximately 67%, 55%,
and 52% of cells were fluorescent 2, 24, and 48 hours, respectively,
after injection (Table 3). Table 3 also
shows the successful delivery of OG-dextran to CD34+ cells
using semiautomatic injection.
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Table 3.
Microinjection of fluorescent compounds into
CD34+, CD34+/CD38, and
CD34+/CD38/Thy-1lo cells
results in high cell viability
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Results for CD34+/CD38and
CD34+/CD38/Thy-1lo cells
injected with fluorescent dextrans are also shown in Table 3. The
disparate viabilities observed between the 2 CD34+/CD38/Thy-1lo
experiments likely occurred because these experiments were performed before optimization of the cell attachment protocol and injection technology.
The particular field shown in Figure 1E and F shows the range of
fluorescent intensities obtainable using the manual setting on the
injector. The most optimal injections were those in which the resultant
fluorescence intensity was low, indicating the delivery of a relatively
small volume of material. In subsequent experiments, care was taken to
ensure injections were consistent and delivery of material was minimal
(ie, low levels of fluorescence). For example, in the last 5 experiments performed with CD34+ cells and
CD34+/CD38 cells (using needles of 0.22 µ OTD), cell viabilities 2 hours after OG-dextran injections were
84% ± 3.3% and 87% ± 2.8%, respectively. These results
demonstrate that macromolecules can be successfully delivered to
various populations of immobilized primitive, human cord blood cells
via glass needle-mediated injection.
Survival, proliferation, and hematopoietic activity of individual
microinjected CD34+ and
CD34+/CD38cells
It was critical that we follow the fate of individual injected cells
for subsequent survival and biologic activity. Therefore, 2 hours after
injection with OG-dextran, CD34+ cells were detached with
peptides and individual fluorescent cells were transferred, as single
cells, into individual wells of 96-well plates. Individual wells were
monitored for the number of surviving and proliferating cells. The
frequency of surviving cells in the microinjected group (80%) was
marginally lower than the attached/detached cells (96%) or suspension
controls (92%) at day 6 (Figure 3, closed
bars). Because the cells were transferred promptly after
microinjection, the slight decrease (20%) in overall cell survival is
likely due to the increased fragility of the cells immediately after
microinjection. The frequency of proliferating microinjected and
attached/detached cells was not obviously different from controls by
day 6 (Figure 3, open bars). This was especially evident when the
frequency of proliferating cells was calculated, based on the actual
number of surviving cells (ie, frequency of proliferation/frequency of
survival; control: 79%/92% = 86%; attached/detached: 96%/96% = 100%; injected: 72%/80% = 90%). However, it
appeared that microinjection induced a delay in the average time before proliferation (Figure 3, open bars). The observed delay in
proliferation was likely due to either the effect(s) of OG-dextran or
the mechanical stress induced by the microinjection process. Finally,
microinjected cells showed no significant difference in either range or
distribution of values for the total number of progeny derived from
each well compared with control cells (F test, P > .3).
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| Fig 3.
Microinjected CD34+ cells are viable and
retain their ability to proliferate after single cell transfer.
Cells were attached to RN and then microinjected with OG-dextran. Two
hours after injection, cells were detached with peptide, and the
fluorescent cells were transferred as single cells into individual
wells of 96-well plates (C). Cells in suspension (A) and cells attached
to RN and detached with peptide (B) were also transferred as single
cells. Survival and proliferation of the cells was monitored at 0, 2, and 6 days after transfer. Values shown represent the percentage of
total wells transferred that displayed cells surviving (closed bar) and
proliferating (open bar) for 1 experiment in which 30 suspension, 30 RN
attached/peptide detached, and 45 OG-dextran microinjected
CD34+ cells were transferred.
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Similar results were obtained in
CD34+/CD38 cell experiments (Figure
4). The left panels (A and C) summarize the
frequency of wells containing surviving or proliferating cells. The
frequency of surviving cells in the injected group (84%) was
marginally lower than the suspension controls (93%) at day 10. A
slight delay in proliferation of injected
CD34+/CD38 cells was also detected.
However, by day 3 after transfer, the frequency of proliferating cells
was similar to that of the suspension control. As described previously
for CD34+ cells, this was especially evident when the
values were corrected for the actual number of surviving cells (ie,
frequency of proliferation/frequency of survival; control:
97%/97% = 100%; injected: 84%/87% = 97%). As can be seen in
the right panels (Figure 4, B and D), there was no significant
difference in either range or distribution of values for the total
number of progeny derived from each well (F test, P > .3)
(for ease of visualization, only 12 individual wells representing the
full range of proliferation rates are presented). Therefore,
attachment, injection, and detachment have no significant impact on the
survival or proliferation of cells in culture.
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| Fig 4.
Injected CD34+/CD38 cells
are viable and retain their ability to proliferate after single cell
transfer.
Cells were attached to RN and injected with OG-dextran. Two hours after
injection, cells were detached with peptide and fluorescent cells
transferred as single cells to individual wells of 96-well plates. In
total, 46 suspension and 75 injected cells were analyzed after 2 injection sessions. (A, C) Percentage of total wells transferred that
displayed cells surviving (closed bar) and proliferating (open bar)
after transfer. (B, D) Representative sampling of the survival and
proliferation of 12 individually transferred suspension (B) or injected
(D) cells. Each symbol represents the progeny generated by an
individually transferred cell.
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To determine whether injection of blood stem cells had any
impact on their hematopoietic activity, we evaluated whether injected CD34+/CD38 cells retained the ability to
generate colony-forming cells (CFCs).21 Gross microscopic
analysis revealed no apparent difference in either the number of wells
giving rise to colonies, the total amount of hematopoietic progeny
per well, or the distribution of erythroid versus myeloid
lineages when comparing injected to control suspension cells (see
Table 4). Microinjection of
CD34+/CD38 cells does not adversely
affect their ability to generate CFCs or their selection of
differentiation program.
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Table 4.
Immobilization on RN followed by microinjection and
detachment does not affect the ability of
CD34+/CD38 cells to produce colony forming
cells
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Delivery of fluorescently labeled protein to CD34+
cells
Glass needle-mediated microinjection of other cell types (eg,
fibroblasts) with proteins (eg, recombinant proteins or antibodies to
specific intracellular proteins) has been a powerful method for
assaying the function of specific proteins. The frequency of
CD34+ cells surviving injection with Cy-3 IgG is similar to
that for OG-dextran (Table 3), demonstrating efficient protein delivery to primitive CD34+ cord blood cells.
Transient expression of GFP by microinjected CD34+
and CD34+/CD38 cells
To examine transient reporter gene expression, CD34+ and
CD34+/CD38 cells were injected with
various expression constructs containing the humanized rsGFP protein
under control of either CMV (pGreen Lantern plasmid DNA) or pgk
regulatory sequences (linearized pgk-GFP). GFP expression at 5 hours
after injection in CD34+ and
CD34+/CD38 cells successfully injected
with pGreen Lantern was 47% ± 16% and 76% ± 9%,
respectively (Table 5). GFP expression was
detected in 68% of the CD34+/CD38 cells
successfully injected with the linearized pgkGFP DNA. OG-dextran injection results from experiments performed during the same period demonstrated that ~ 84% and 87% of injected CD34+ and
CD34+/CD38 cells, respectively, survived
quantitative delivery of injected material at 2 hours after injection
(n = 5 most recent experiments). Using these values, we have
estimated that ~56% (47%/84%) and ~87% (76%/87%) of viable
injected CD34+ or CD34+/CD38
cells transiently expressed GFP 5 hours after injection (as estimated for pGreen Lantern). Expression frequencies gradually decreased with
increasing time of culture (data not shown); this was not unexpected,
because almost all expression should be transient, due to unintegrated
DNA copies. However, we have observed GFP-expressing cells (and a
smaller number of dividing, expressing cells) as late as 4 to 5 days
after injection. In 2 preliminary experiments, CD34+/CD38 cells were injected with a
19.6 kb GFP construct (5 copies/fL) 13.3% and 54.5% of injected cells
expressed GFP at 5 hours after injection. Thus, injection yields
transient transgene (GFP) expression in primitive blood cell
populations with DNAs as large as 19.6 kb in size.
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Discussion |
To our knowledge, this is the first report of glass
needle-mediated microinjection of DNA, protein, and/or dextran into
primary human stem/progenitor cells. The difficulties in injecting
hematopoietic cells, which are normally nonadherent and very small,
were overcome by development of improved attachment protocols and
ultrafine microinjection needles. We have identified conditions whereby primary human cord blood stem/progenitor cells may be temporarily immobilized in a manner sufficiently strong to withstand microinjection without altering cell function/biology.
CD34+ cells represent ~ 0.5% to 1.0% of
nucleated umbilical cord blood and bone marrow cells and comprise all
measurable human progenitor activity.18 With the possible
exception of a limited subset of CD34
cells,22,23 CD34+ cells also contain all
measurable human stem cell activity.18 The
CD34+/CD38 (~ 5% to 10% of
CD34+ cells) and
CD34+/lineage/Thy-1lo
phenotype characterize more primitive subpopulations.2,
24-27 CD34+ cells, and subsets thereof, attach
strongly to plates coated with a recombinant derivative of FN, CH-296
known as RN. The RN attachment method had no effect on survival,
proliferative potential, or NOD/SCID engrafting ability. In contrast,
attachment of CD34+ cells to Con A adversely affected
myeloid colony formation (see Table 1), which is consistent with
previous reports suggesting that attachment of transformed
hematopoietic cells via lectins (eg, Con A28-30) can have
mitogenic or inhibitory effects on hematopoietic cells. Strong
attachment to FN requires treatment with specific anti-1
integrin antibodies, which induce low avidity
1-containing integrin heterodimers (eg,
41, 51) to
a high avidity state.16,31 Activation of the integrins with
the TS2/16.2.1 activating antibody, followed by attachment to FN, may
have an effect on NOD/SCID reconstituting activity in the cells. A
decrease in the number of mice demonstrating human cell engraftment was
noted with the cells attached to FN in the presence of activating mAb
compared with control cells (FN with activating mAb). Although this
difference was not statistically significant, it was consistent with a
reduction in the number of human CD45+ cells in the mice
injected with cells attached to FN with activating mAb. Because RN
attachment of cells had no effect on cell function, we chose to use RN
for cell immobilization in all subsequent studies.
Previous reports of the successful injection of transformed
hematopoietic cells are limited.28,29 Attempted injection
of small, normally nonadherent cells (eg, primary hematopoietic cells) has resulted in extremely low cell survival.30 Injection
needles with a minimal OTD had to be developed to achieve high
viability of blood stem/progenitor cells. We have demonstrated
increased viability of injected fibroblasts when using needles with
decreased OTD (Brown et al, unpublished data). Reducing the injection
needle OTD to 0.2 µ did indeed yield a considerable increase in the
viability of injected CD34+ cells (unpublished data). This
increased viability using ultrafine needles can be attributed to
reduced physical damage to the cell during needle insertion and
retraction and minimized injection volumes due to finer control of
sample flow from the injection needle.
Our technology has allowed for efficient macromolecule delivery and
excellent postinjection viabilities of both CD34+ and
CD34+/CD38 cells. Optimization of
injection conditions (reflected in the last 5 experiments) and
observation of individual injected cells resulted in calculated actual
long-term survival frequencies at 10 days after injection of 73% and
67% for successfully injected CD34+ and
CD34+/CD38 cells, respectively.
Furthermore, there was no observed change in the frequency of surviving
CD34+ or CD34+/CD38 cells
undergoing proliferation at 6 to 10 days after transfer. Finally,
injection had no impact on the ability of
CD34+/CD38 cells to generate or
expand progeny CFCs, an in vitro measure of primitive
hematopoietic activity.
Excellent transgene delivery and transient expression was observed in
injected CD34+
and CD34+/CD38 cells. Interestingly, the
more primitive CD34+/CD38 population
displayed an increased rate of CMV-driven GFP expression. Although not
statistically significant, this trend is consistent with an observed
increase in frequency of pGreen Lantern expression in electroporated
CD34+/CD38 versus
CD34+/CD38+ cells (B.R.D., unpublished
results). In preliminary experiments, transient GFP expression was also
seen in injected
CD34+/CD38/Thy-1lo cells
(data not shown). Future experiments will focus on assaying and
optimizing long-term transgene maintenance in primitive hematopoietic stem/progenitor cells.
We have achieved delivery of DNAs 19.6 kb in length and subsequent
expression of GFP in the injected cells demonstrating that glass
needle-microinjection is a powerful tool for delivering large DNAs to
cells. These DNAs can accommodate the regulatory elements and
intron/exon structure necessary for long-term, cell type-specific,
integration site-independent expression of transgenes. Thus, injection
may be preferable for developmental or gene therapeutic applications
requiring tightly regulated gene expression in progeny hematopoietic cells.
An evaluation of glass needle-mediated microinjection as a potential
method for stem cell gene therapy must take into account the number of
stem cells required for transplantation, the frequency of actual stem
cells in the injected population, and the total time required to
perform the injections. Children have been reconstituted with as little
as 30 mL of transplanted cord blood32 containing approximately 1.5 to 3 × 108 nucleated cells. If
the frequency of stem cells is 1 in 105 to 106,
then successful engraftment could occur with as few as 150 to 3000 stem
cells. Although enriched stem cell-containing populations of primitive
human hematopoietic cells have already been
described,11,23,26,27 further definition of the human stem
cell phenotype(s) is necessary. Indeed, determination of the mouse stem
cell phenotype has reached the point where delivery of as few as 10 cells,33 or even 1 marrow cell34 is sufficient
for total hematopoietic reconstitution.
Both manual (100-200 injected cells/h) and semiautomated (200-400 cells/h) modes of injection were used in this study; rates likely
insufficient for feasible therapeutic time constraints. Fully automated
systems, capable of 1500 cell injections per hour,20 would
likely be used to inject a sufficient number of stem cells for
transplantation. Any significant in vitro or in vivo expansion of stem
cells,35 perhaps together with selection for marked cells,
would further decrease the number of injected stem cells required for engraftment.
In summary, we have demonstrated glass needle microinjection-mediated
delivery of macromolecules (protein, DNA, and dextrans) into human
CD34+, CD34+/CD38, and
CD34+/CD38/Thy-1lo cord
blood cells. Moreover, our immobilization and injection methods are
applicable to quiescent blood stem cells because immunomagnetically isolated CD34+ cells are capable of quantitative attachment
to RN immediately after isolation without cytokine prestimulation (data
not shown). It has been demonstrated that 95% of cord blood
CD34+/CD38 cells are quiescent at time
of purification.24,36 The demonstration that these cells
can be injected with high viability without any observable adverse
effect on proliferation or biologic function strongly supports our
intended application of this technology to experimental studies and
gene therapeutic applications of hematopoietic stem/progenitor cells.
| |
Acknowledgments |
We thank Gina Barron, Mark Griffin, Aqing Yao, Jianming Lu, and Fuming
Pan for expert technical assistance.
| |
Footnotes |
Submitted March 1, 1999; accepted September 1, 1999.
Partly supported by NIH R21DK53923 to B.R.D. and Sealy and Smith
Endowment grants to J. Y.-B. and B.R.D.
Reprints: Brian R. Davis, Sealy Center for Oncology and
Hematology, MRB 9.104, University of Texas Medical Branch, Galveston,
TX 77555-1048; or David B. Brown, Department of Human Biological
Chemistry and Genetics, BSB 506H, University of Texas Medical Branch,
Galveston, TX 77555-0645.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction