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only.
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
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 CD341, CD341/CD382,
and CD341/CD382/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% 6 8.4% of CD341 cells and 42% 6
14% of CD341/CD382cells still fluorescent 48 hours after injection. Single-cell
transfer and culture of injected cells has
demonstrated long-term survival and proliferation of CD341 and CD341/CD382 cells,
and retention of the ability of CD341/
CD382 cells to generate progenitor cells.
Delivery of DNA constructs (currently I
19.6 kb) and fluorescently labeled proteins into CD341 and CD341/CD382 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)
r 2000 by The American Society of Hematology
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 CD341, CD341/CD382, and
CD341/CD382/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.
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.
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.
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.
BLOOD, 15 JANUARY 2000 • VOLUME 95, NUMBER 2
Materials and methods
Isolation and further fractionation of human CD341 cells
CD341 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 3 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
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.
r 2000 by The American Society of Hematology
437
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BLOOD, 15 JANUARY 2000 • VOLUME 95, NUMBER 2
DAVIS et al
(Stem Cell Medium). Subpopulations of CD341 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 CD341/
CD382 cells and anti-CD34-PerCP, anti-CD38-PE, and anti-Thy-1-FITC
for CD341/CD382/Thy-1lo cells.
CD341 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-GlyAsp-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
and CD341/CD382/Thy-1lo cells
CD341,
CD341/CD382,
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 3 100). 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.
intravenously (iv; tail vein) with purified CD341 cells (2 3 104 cells/
mouse). CD341 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 fluorescenceactivated cell sorter (FACS) analysis using antihuman CD45FITC and
antihuman CD34 PE (Becton Dickinson). Percent CD451 cells represents
the sum of the frequency of human CD451/CD342 and CD451/CD341
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.
Results
Immobilization of human blood stem/progenitor cells
on matrix-coated dishes
Hematopoietic stem/progenitor cells in the bone marrow express
a4b1 and a5b1 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 FNcoated plates was sufficient for glass needle–mediated injection.
Attachment of primary CD341 cells to plates coated with FN
resulted in only tethering of the cells rather than solid attachment
(Figure 1B; unattached CD341 cells in suspension culture are
shown in Figure 1A); the cells dislodged during injection. However, when CD341 cells were treated with specific anti-b1 integrin
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-Prkcscid/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
Figure 1. Photomicrographs of attached and injected CD341 cells. Represented
are CD341 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 CD341 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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BLOOD, 15 JANUARY 2000 • VOLUME 95, NUMBER 2
MICROINJECTION OF MACROMOLECULES AND TRANSGENES
mAb, they attached strongly to FN-coated plates (Figure 1C). Up to
100% of CD341 cells were immobilized in this way and acquired a
more flattened morphology and extended micropodia (Figure 1C).
CD341/CD382 and CD341/CD382/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-b1 mAb. For both methods of attachment, cells
withstood both manual (Figure 1E) and semiautomatic injection
without becoming dislodged.
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% 6 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 CD341 and CD341/
CD382 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 6 1.1 hours and 23.2 6 1.7 hours
were obtained for untreated control CD341 and TS2/16 mAbtreated treated CD341 cells grown in liquid culture, respectively.
Similarly, doubling rates of 21.6 6 2.5 hours and 21.6 6 2.1 hours
were obtained for CD341 cells attached for 2 hours to either FN or
RN, respectively, released by peptide and grown in liquid culture.
Because CD341 cells are highly enriched in progenitor cells,18
the effect of temporary immobilization on colony-forming activity
was evaluated. CD341 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).
439
Table 2. NOD/SCID engrafting ability of CD341 cells after attachment and
detachment
Matrix
Presence of
TS2/16 mAb
Method of
Detachment
Engrafted per
Injected Mice
% of Mice
Engrafted*
—†
no
Pipetting
16/17
94
FN‡
no
Pipetting
16/19
84
FN‡
yes
Pipetting
12/16
75
RN‡
no
Pipetting
5/6
83
RN‡
no
Peptides
9/11
82
RN (16 h)
no
Peptides
9/9
100
*Values represent total mice engrafted/total mice injected 3 100.
†Cells were incubated in suspension.
‡Cells were incubated on matrix for 75 to 120 minutes.
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 CD341 cells that had been immobilized
on FN (with activating mAb) or RN and detached or with control
CD341 cells. Control cells for the FN plus mAb experimental
group consisted of CD341 cells incubated on FN-coated dishes in
the absence of activating mAb. Controls for the RN-attached cells
consisted of CD341 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 CD451 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% 6 0.6% human CD451 cells; mean 6
SD) as compared with control cells incubated on FN
Table 1. Immobilization on FN or RN and subsequent detachment does not affect the colony forming ability of CD341 cells
Colony Formation*
Number of Colonies Per 2000 Cells
Percent of Nonimmobilized Control
Method of
Attachment†
Method of
Release
Erythroid
Myeloid
Erythroid
Myeloid
—
—
70 6 5
52 6 6
100 6 5
100 6 6
Con A
Pipetting
75 6 16
26 6 2
108 6 20
54 6 4
FN 1 TS2/16.2.1 mAb
Pipetting
67 6 4
43 6 8
98 6 6
84 6 19
RN
Peptide
81 6 18
53 6 8
117 6 23
102 6 14
*Colonies formed were evaluated and designated as erythroid- or myeloid-containing. Colony formation is represented as number of colonies formed per 2000 cells plated
and percentage of the nonimmobilized control (number of colonies formed by treated cells per number of colonies formed by control suspension cells 3 100) 6 SD for 4
separate experiments.
†CD34 cells were incubated in Stem Cell Media 48 hours before onset of assay and then attached to matrix coated plates for 45 minutes. Cells were detached,
resuspended in MethoCult GF Kit (StemCell Technologies, Vancouver, BC, Canada) (1000 cells/mL/plate), and incubated for 14 days in a humidified incubator at 37°C,
6% CO2.
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BLOOD, 15 JANUARY 2000 • VOLUME 95, NUMBER 2
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Figure 2. Extent of CD341 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 CD451 cells was determined on
ungated samples. (A) Plot of the percentage CD451 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 CD451 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 CD451 cells in each group.
in the absence of activating mAb (1.8% 6 3.7%). Engraftment
levels with RN-attached cells (1.9% 6 2.3%) were equivalent to
those of control cells (2.4% 6 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.
Microinjection-mediated delivery of fluorescencelabeled dextrans into CD341, CD341/CD382, and
CD341/CD382/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
CD341 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 CD341 cells during and after injection with OGdextran 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 CD341 cells using semiautomatic injection.
Results for CD341/CD382and CD341/CD382/Thy-1lo cells
injected with fluorescent dextrans are also shown in Table 3. The
disparate viabilities observed between the 2 CD341/CD382/Thy1lo 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 CD341 cells and
CD341/CD382 cells (using needles of 0.22 µ OTD), cell viabilities
2 hours after OG-dextran injections were 84% 6 3.3% and
87% 6 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 CD341 and CD341/CD382cells
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, CD341 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% 5 86%; attached/detached: 96%/96% 5 100%; injected: 72%/80% 5 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
Table 3. Microinjection of fluorescent compounds into CD341, CD341/CD382, and CD341/CD382/Thy-1lo cells results in high cell viability
Cells
Matrix
Material
Injected
Mode of
Injection
CD341
RN
OG-dextran
Manual
CD341
RN
OG-dextran
Semiautomatic
CD341
RN
Cy-3 IgG
CD341/CD382
RN
CD341/CD382/Thy-1lo
FN
Percent Viability*†
2-4 h
24 h
48 h
67 6 13
55 6 8.5
52 6 8.4
(n 5 63)
(n 5 45)
(n 5 45)
n.d.
56 6 5.2
54 6 5.2
(n 5 6)
(n 5 6)
Manual
70 6 7.5
53 6 6.2
44 6 5.4
(n 5 3)
(n 5 3)
(n 5 3)
OG-dextran
Manual
58 6 23
41 6 17
42 6 14
(n 5 15)
(n 5 9)
(n 5 9)
FITC-dextran
Manual
28, 54
9, 52
3, 32
*Values represent mean 6 SD for number of separate plates (n), each containing 50 injected cells per plate.
†Values shown in last row are, respectively, for 2 separate experiments of 32 and 50 injected cells per plate.
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BLOOD, 15 JANUARY 2000 • VOLUME 95, NUMBER 2
MICROINJECTION OF MACROMOLECULES AND TRANSGENES
441
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).
Similar results were obtained in CD341/CD382 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 CD341/CD382 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 CD341 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% 5 100%; injected: 84%/87% 5 97%). As can be seen in
Figure 4. Injected CD341/CD382 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.
Figure 3. Microinjected CD341 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 CD341 cells were transferred.
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.
To determine whether injection of blood stem cells had any
impact on their hematopoietic activity, we evaluated whether
injected CD341/CD382 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 CD341/CD382 cells does not adversely
affect their ability to generate CFCs or their selection of differentiation program.
Delivery of fluorescently labeled protein to CD341 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 CD341
cells surviving injection with Cy-3 IgG is similar to that for
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BLOOD, 15 JANUARY 2000 • VOLUME 95, NUMBER 2
DAVIS et al
Table 4. Immobilization on RN followed by microinjection and detachment
does not affect the ability of CD341/CD382 cells to produce colony forming
cells
Method of
Attachment*
Method
of Release
Microinjection
% Wells Producing
Colony-Forming
Cells*
—
—
2
74
Colony Formation
(% of Nonimmobilized
Control)†
Erythroid
Myeloid
100
100
79
86
132
100
n 5 42
RN
Peptide
2
RN
Peptide
1
68
n 5 40
77
n 5 64
*Individual cells were transferred to wells of 96-well plates using the Quixell
Transfer unit. To allow for the development and expansion of colony forming cells,
transferred cells were cultured in Stem Cell Medium for 10 days before the addition of
MethoCult GF media. After 14 days incubation in methylcellulose, the individual wells
were evaluated for colony growth and phenotype. The total number of wells
containing either erythroid or myeloid growth was determined for each condition.
†Colony formation is represented as the % of nonimmobilized control (number of
wells giving rise to colony growth for treated cells 4 number of wells giving rise to
colony growth for suspension cells). Values represent combined results of 2
microinjection sessions in which the number of wells specified (n) were analyzed.
OG-dextran (Table 3), demonstrating efficient protein delivery to
primitive CD341 cord blood cells.
Transient expression of GFP by microinjected CD341
and CD341/CD382 cells
To examine transient reporter gene expression, CD341 and CD341/
CD382 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
CD341 and CD341/CD382 cells successfully injected with pGreen
Lantern was 47% 6 16% and 76% 6 9%, respectively (Table 5).
GFP expression was detected in 68% of the CD341/CD382 cells
successfully injected with the linearized pgkGFP DNA. OGdextran injection results from experiments performed during the
same period demonstrated that , 84% and 87% of injected CD341
and CD341/CD382 cells, respectively, survived quantitative delivery of injected material at 2 hours after injection (n 5 5 most recent
experiments). Using these values, we have estimated that ,56%
(47%/84%) and ,87% (76%/87%) of viable injected CD341 or
CD341/CD382 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
Table 5. CD341 and CD341/CD382 cells express green fluorescent protein
after injection with GFP constructs
Cells*
DNA Construct
Injected
% Expression
at 5 h†
CD341
pGreen Lantern
47 6 16‡
CD341/CD382
pGreen Lantern
76 6 9‡
CD341/CD382
Linearized pgkGFP
68§
*Cells attached to RN-coated dishes.
†Values represent number of expressing cells/number of injected cells 3 100.
‡Values represent the mean 6 SD of 3 experiments of 100 cells per experiment.
§Value represents 1 experiment of 110 injected cells.
preliminary experiments, CD341/CD382 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.
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.
CD341 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 CD342 cells,22,23 CD341 cells also contain all
measurable human stem cell activity.18 The CD341/CD382 (, 5%
to 10% of CD341 cells) and CD341/lineage2/Thy-1lo phenotype
characterize more primitive subpopulations.2, 24-27 CD341 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 CD341
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-b1 integrin antibodies, which induce low avidity b1-containing
integrin heterodimers (eg, a4b1, a5b1) 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 CD451 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 CD341 cells (unpublished data). This increased viability using ultrafine needles can be attributed to
reduced physical damage to the cell during needle insertion and
From www.bloodjournal.org at UNIV OF KANSAS MEDICAL CENTER on November 24, 2010. For personal use
only.
BLOOD, 15 JANUARY 2000 • VOLUME 95, NUMBER 2
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 CD341 and
CD341/CD382 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 CD341 and CD341/CD382 cells, respectively. Furthermore, there was no observed change in the frequency
of surviving CD341 or CD341/CD382 cells undergoing proliferation at 6 to 10 days after transfer. Finally, injection had no impact
on the ability of CD341/CD382 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 CD341 and CD341/CD382 cells. Interestingly, the more primitive CD341/CD382 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 CD341/CD382 versus CD341/CD381 cells (B.R.D., unpublished results). In preliminary experiments, transient GFP expression was also seen in injected CD341/CD382/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 reconsti-
MICROINJECTION OF MACROMOLECULES AND TRANSGENES
443
tuted with as little as 30 mL of transplanted cord blood32 containing
approximately 1.5 to 3 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 microinjectionmediated delivery of macromolecules (protein, DNA, and dextrans) into human CD341, CD341/CD382, and CD341/CD382/Thy1lo cord blood cells. Moreover, our immobilization and injection
methods are applicable to quiescent blood stem cells because
immunomagnetically isolated CD341 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 CD341/CD382 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.
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