Glass Needle–Mediated Microinjection of Macromolecules and Transgenes Into Primary Human Blood Stem/Progenitor Cells

From www.bloodjournal.org at UNIV OF KANSAS MEDICAL CENTER on November 24, 2010. For personal use 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 microin- cell function (colony forming ability), or 19.6 kb) and fluorescently labeled pro- jection method for delivery of macromol- stem cell function (NOD/SCID reconstitut- teins into CD34؉ and CD34؉/CD38؊ cells ecules, including proteins and larger ing ability). Delivery of fluorescent dex- was achieved with transient expression transgene DNAs, into the nuclei of blood trans to stem/progenitor cells was achieved of green fluorescent protein observed in stem/progenitor cells was developed. Tem- with 52% ؎ 8.4% of CD34؉ cells and 42% ؎ up to 75% of injected cells. These data -porary immobilization of cells to extracellu- 14% of CD34؉/CD38Ϫcells still fluores- indicate that glass needle–mediated deliv lar matrix–coated dishes has enabled rapid cent 48 hours after injection. Single-cell ery of macromolecules into primitive hema- and consistent injection of macromol- transfer and culture of injected cells has topoietic cells is a valuable method for -ecules into nuclei of CD34؉, CD34؉/CD38؊, demonstrated long-term survival and pro- studies of stem cell biology and a promis and CD34؉/CD38؊/Thy-1lo human cord blood liferation of CD34؉ and CD34؉/CD38؊ cells, ing method for human blood stem cell (cells. Immobilization and detachment proto- and retention of the ability of CD34؉/ gene therapy. (Blood 2000;95:437-444 .cols were identified, which had no ad- CD38؊ cells to generate progenitor cells verse effect on cell survival, progenitor Delivery of DNA constructs (currently I ௠ 2000 by The American Society of Hematology Introduction

Hematopoietic stem cells are a major focus of investigation in both First, we have developed a method for attaching human blood developmental biology and clinical medicine. The stem cell stem/progenitor cells to extracellular matrix–coated dishes, which compartment consists of rare, long-lived, self-renewing, predomi- does not affect cell function. Second, we have developed injection nantly quiescent cells capable of long-term reconstitution of the needles with very small outer tip diameters (OTD; ϳ 0.2 µ), which complete hematopoietic system in transplanted hosts.1 It is of have excellent flow properties and result in excellent postinjection significant biologic and clinical interest to elucidate the mecha- viabilities. nisms controlling stem cell self-renewal, commitment to matura- We demonstrate that glass needle–mediated microinjection can tion, and selection of differentiation program. The potential to be used effectively for the introduction of macromolecules (pro- genetically correct the complete hematopoietic system by success- tein, DNA, and dextrans) into human CD34ϩ, CD34ϩ/CD38Ϫ, and fully modifying only the stem cell compartment has made these CD34ϩ/CD38Ϫ/Thy-1lo cord blood cells. Importantly, macromol- cells a primary target for gene therapy. ecule delivery is accomplished with high postinjection cell viabil- We have sought to develop a method that allows for the ity, no discernible impact on stem/progenitor cell proliferation or introduction of macromolecules into stem/progenitor cells. Current biologic activity, and a high frequency of cells expressing injected technologies (eg, standard retroviruses, adeno-associated viruses, transgenes. 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 Materials and methods independently by Diacumakos6 and Graessman,7 has proven to be a powerful approach for analyzing the biologic activity of specific Isolation and further fractionation of human CD34؉ cells molecules (eg, peptide inhibitors, purified active proteins, neutral- CD34ϩ cells were immunomagnetically purified from human umbilical izing antibodies, RNA, and DNA) in adherent cells.8,9 However, cord blood (University of Texas Medical Branch) using either the Progeni- this approach has rarely been used for primary hematopoietic tor or Multisort Kits (Miltenyi Biotec, Auburn, CA), then cultured in 10 cells because immobilization of hematopoietic cells has generally Iscoves’ Modified Dulbecco’s Medium without phenol red (IMDM) with been ineffective and standard injection needles cause significant 100 µg/mL glutamine/penicillin/streptomycin, 1 ϫ BIT 9500 (StemCell damage to these relatively small cells (ϳ 6 µ diameter for stem Technologies, Vancouver, BC), 20 ng/mL each of human Interleukin (II)-3, cells11). Two developments have allowed us to apply glass needle– flt-3 ligand, and stem cell factor (PeproTech, Inc, Rocky Hill, NJ), mediated microinjection technology to blood stem/progenitor cells. 40 µg/mL low-density lipoprotein (Sigma), and 50 µM 2-mercaptoethanol

From Sealy Center for Oncology and Hematology, Department of Microbiology Reprints: Brian R. Davis, Sealy Center for Oncology and Hematology, MRB and Immunology, Department of Human Biological Chemistry and Genetics, 9.104, University of Texas Medical Branch, Galveston, TX 77555-1048; or University of Texas Medical Branch, Galveston; Gene-Cell, Inc, Houston, TX; David B. Brown, Department of Human Biological Chemistry and Genetics, and Frederick Cancer Research and Development Center, National Cancer BSB 506H, University of Texas Medical Branch, Galveston, TX 77555-0645. Institute, Frederick, MD. The publication costs of this article were defrayed in part by page charge Submitted March 1, 1999; accepted September 1, 1999. payment. Therefore, and solely to indicate this fact, this article is hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734. Partly supported by NIH R21DK53923 to B.R.D. and Sealy and Smith Endowment grants to J. Y.-B. and B.R.D. ௠ 2000 by The American Society of Hematology

BLOOD, 15 JANUARY 2000 • VOLUME 95, NUMBER 2 437 From www.bloodjournal.org at UNIV OF KANSAS MEDICAL CENTER on November 24, 2010. For personal use only. 438 DAVIS et al BLOOD, 15 JANUARY 2000 • VOLUME 95, NUMBER 2

(Stem Cell Medium). Subpopulations of CD34ϩ cells were isolated by flow intravenously (iv; tail vein) with purified CD34ϩ cells (2 ϫ 104 cells/ cytometry on a FACS Vantage (Becton Dickinson, San Jose, CA) after mouse). CD34ϩ cells had either been maintained in suspension or first immunostaining with anti-CD34-FITC and anti-CD38-PE for CD34ϩ/ immobilized on RN or FN, then detached by pipetting or treatment with CD38Ϫ cells and anti-CD34-PerCP, anti-CD38-PE, and anti-Thy-1-FITC peptides. At 6 weeks inoculation, marrow cells from both femurs of the for CD34ϩ/CD38Ϫ/Thy-1lo cells. mice were analyzed for the presence of human cells by fluorescence- activated cell sorter (FACS) analysis using antihuman CD45FITC and CD34؉ cell matrix attachment and detachment antihuman CD34 PE (Becton Dickinson). Percent CD45ϩ cells represents the sum of the frequency of human CD45ϩ/CD34Ϫ and CD45ϩ/CD34ϩ Dishes were coated overnight at 4°C with either Fibronectin (FN) or FN cells. When the level of human cell engraftment was low (Ͻ 1% human fragment CH-296 (Retronectin (RN); TaKaRa Biomedicals, Panvera, cells), semiquantitative DNA polymerase chain reaction amplification for Madison, WI; 15 µg/mL) in phosphate-buffered saline (PBS) and washed human Cart-1 sequences was performed to confirm engraftment.15 Statisti- with IMDM containing glutamine/penicillin/streptomycin. Cells were added cal analysis was performed with the Student’s t test (unless otherwise noted) to cloning rings and attached to the plates for 45 minutes at 37°C. When using Statview 4.1 (Abacus Concepts, Inc, Berkeley, CA) and Microsoft using FN, the integrin-activating monoclonal antibody (mAb) TS2/16.2.1 Excel 8.0 (Microsoft, Redmond, WA); P values were considered to be 12 IgG purified from ascites (1 µg/mL; ATCC, HB-243 ) was added to the statistically significant if less than .05. 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 Results stream of medium. Immobilization of human blood stem/progenitor cells Injection of macromolecules into CD34؉, CD34؉/CD38؊, on matrix-coated dishes and CD34؉/CD38؊/Thy-1lo cells Hematopoietic stem/progenitor cells in the bone marrow express ␣ ␤ ␣ ␤ Injection needles were pulled from 10 cm borosilicate capillaries with a 1.2 4 1 and 5 1 integrins, allowing them to interact with FN through 16 mm outer/0.94 mm inner diameter using a Flaming/Brown Micropipette the CS-1 region and RGDS sequence, respectively. We evaluated Puller Model P-97 (Sutter Instrument Co, Novato, CA) and had a range of whether the attachment of blood stem/progenitor cells to FN- 0.17 to 0.25 µ OTD, as determined by scanning electron microscopy. Cells coated plates was sufficient for glass needle–mediated injection. were visualized using a Nikon Eclipse TE300 microscope equipped with a Attachment of primary CD34ϩ cells to plates coated with FN Fryer A-50 temperature-controlled stage (set at 37°C) and injected using the resulted in only tethering of the cells rather than solid attachment electronically interfaced Eppendorf Micromanipulator (Model 5171) and (Figure 1B; unattached CD34ϩ cells in suspension culture are Transjector (Model 5246). Manual injections were performed, except where shown in Figure 1A); the cells dislodged during injection. How- noted as semiautomatic injections. In some cases, manual injections were ever, when CD34ϩ cells were treated with specific anti-␤1 integrin 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 ϫ 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 cytomegalovi- rus (CMV) enhancer/promoter sequences were replaced with the pgk promoter sequences.13 Linear pgkGFP sequences were obtained by digest- ing pgkGFP with Pac I and Asc I and isolating the resulting 1.7 kb fragment after agarose gel electrophoresis. Figure 1. Photomicrographs of attached and injected CD34؉ cells. Represented ϩ NOD/SCID engraftment assay 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 ϩ Nonobese diabetic and severe combined immmunodeficient NOD/LtSz-Prkc- of attached CD34 cells is shown in panel E. Note the injection needle (arrow) and scid/scid (NOD/SCID) mice were obtained from Jackson Laboratory (Bar cells injected with OG-dextran (arrowheads). A fluorescence micrograph (F) of the identical field shown in panel E demonstrates the successful delivery of fluorescent 14 Harbor, ME ) and were bred and maintained in microisolator cages on material (arrowheads). Images in panels E-F were captured from videotape during a laminar flow racks. NOD/SCID mice of 6 to 8 weeks of age were live injection session and, therefore, display a decreased resolution, compared with sublethally irradiated with 350 or 400 rad (137Cs source) and injected images in panels A-D. 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 MICROINJECTION OF MACROMOLECULES AND TRANSGENES 439

mAb, they attached strongly to FN-coated plates (Figure 1C). Up to Table 2. NOD/SCID engrafting ability of CD34؉ cells after attachment and 100% of CD34ϩ cells were immobilized in this way and acquired a detachment more ﬂattened morphology and extended micropodia (Figure 1C). Presence of Method of Engrafted per % of Mice CD34ϩ/CD38Ϫ and CD34ϩ/CD38Ϫ/Thy-1lo cells, believed to rep- Matrix TS2/16 mAb Detachment Injected Mice Engrafted* resent increasingly enriched populations of blood stem cells, were —† no Pipetting 16/17 94 also immobilized using this method (data not shown). Up to 100% FN‡ no Pipetting 16/19 84 of primary blood stem/progenitor cells also attached ﬁrmly to RN FN‡ yes Pipetting 12/16 75 (Figure 1D), a recombinant derivative of FN. RN contains only the RN‡ no Pipetting 5/6 83 RN‡ no Peptides 9/11 82 heparin-binding, CS-1, and RGDS-containing cell association RN (16 h) no Peptides 9/9 100 regions of FN.17 Attachment to RN occurred even in the absence of activating anti-␤1 mAb. For both methods of attachment, cells *Values represent total mice engrafted/total mice injected ϫ 100. withstood both manual (Figure 1E) and semiautomatic injection †Cells were incubated in suspension. ‡Cells were incubated on matrix for 75 to 120 minutes. without becoming dislodged.

Biologic activity of human blood stem/progenitor cells after We also examined whether there was any adverse effect of immobilization on FN and RN immobilization on stem cell function using the NOD/SCID engraft- Despite their strong attachment to FN (in the presence of activating ment model. NOD/SCID mice demonstrate active human blood TS2/16.2.1 mAb) and RN, cells could be recovered with vigorous cell development in the marrow of transplanted mice after intrave- pipetting. However, this resulted in decreased viability nous delivery of total marrow, total cord blood, or primitive (64% Ϯ 12%) and incomplete recovery (Յ 85%) of cells. Virtually hematopoietic subpopulations.15,19 Sublethally irradiated mice were 100% of cells were recovered from FN and RN by competing for injected intravenously with CD34ϩ cells that had been immobilized attachment with peptides corresponding to the CS-1 and RGDS on FN (with activating mAb) or RN and detached or with control regions of FN. In all cases, detached cells were Ͼ 95% viable and CD34ϩ cells. Control cells for the FN plus mAb experimental rapidly reacquired their normal rounded morphology and nonadher- group consisted of CD34ϩ cells incubated on FN-coated dishes in ent behavior. the absence of activating mAb. Controls for the RN-attached cells We evaluated the effects of in vitro attachment on stem/ consisted of CD34ϩ cells incubated on noncoated plastic dishes or progenitor cell function. Immobilization of CD34ϩ and CD34ϩ/ FN-coated dishes without activating mAb. No statistical difference CD38Ϫ cells to FN or RN for 2 hours and release with peptide had was detected when comparing the percentage of mice engrafting no effect on viability or proliferative capacity of cells in liquid (P Ͼ .42) or the extent of engraftment (P Ͼ .35) with either control culture. Doubling rates of 21.2 Ϯ 1.1 hours and 23.2 Ϯ 1.7 hours group. As shown in Table 2, there was no evidence for any adverse were obtained for untreated control CD34ϩ and TS2/16 mAb- effect on the engraftment ability of the human NOD/SCID reconsti- treated treated CD34ϩ cells grown in liquid culture, respectively. tuting cells after attachment to RN. Although a slight decrease in Similarly, doubling rates of 21.6 Ϯ 2.5 hours and 21.6 Ϯ 2.1 hours engraftment ability was seen in the cells attached to FN with were obtained for CD34ϩ cells attached for 2 hours to either FN or activating mAb (75% compared with the control value of 94% for RN, respectively, released by peptide and grown in liquid culture. suspension cells or 84% for cells incubated on FN in the absence of Because CD34ϩ cells are highly enriched in progenitor cells,18 activating mAb), this difference was not statistically significant the effect of temporary immobilization on colony-forming activity (P Ͼ .19 and P Ͼ .22, respectively; Student’s paired t test). The was evaluated. CD34ϩ cells were allowed to attach for 2 hours to extent of engraftment was determined for each mouse by quantitat- either FN, RN, or Con A lectin, detached, then plated in methylcel- ing via FACS analysis the percentage of human CD45ϩ cells lulose with appropriate cytokines to compare their colony-forming present in the bone. As can be seen in Figure 2, there was activity (erythroid and myeloid) to nonattached suspension cells considerable variability in the range of engraftment levels obtained (Table 1). FN- or RN-immobilized cells demonstrated no signifi- for each of the experimental conditions. Although not statistically cant alteration in either frequency (Table 1) or size (data not shown) significant (P Ͼ .13), there was a distinct trend toward lower levels of erythroid or myeloid colonies. In contrast, Con A immobilization of engraftment when cells were attached to FN in the presence of lead to a significant reduction in myeloid colony formation activating antibody (0.4% Ϯ 0.6% human CD45ϩ cells; mean Ϯ (Table 1). 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 CD34؉ cells Colony Formation* Number of Colonies Per 2000 Cells Percent of Nonimmobilized Control Method of Method of Attachment† Release Erythroid Myeloid Erythroid Myeloid

——70Ϯ 552Ϯ 6 100 Ϯ 5 100 Ϯ 6 Con A Pipetting 75 Ϯ 16 26 Ϯ 2 108 Ϯ 20 54 Ϯ 4 FN ϩ TS2/16.2.1 mAb Pipetting 67 Ϯ 443Ϯ 898Ϯ 684Ϯ 19 RN Peptide 81 Ϯ 18 53 Ϯ 8117Ϯ 23 102 Ϯ 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 ϫ 100) Ϯ 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. From www.bloodjournal.org at UNIV OF KANSAS MEDICAL CENTER on November 24, 2010. For personal use only. 440 DAVIS et al BLOOD, 15 JANUARY 2000 • VOLUME 95, NUMBER 2

successful delivery of OG-dextran to CD34ϩ cells using semiautomatic injection. Results for CD34ϩ/CD38Ϫand CD34ϩ/CD38Ϫ/Thy-1lo cells injected with fluorescent dextrans are also shown in Table 3. The disparate viabilities observed between the 2 CD34ϩ/CD38Ϫ/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 Figure 2. Extent of CD34؉ cell engraftment in NOD/SCID mice after attachment and detachment. Mouse bone marrow mononuclear cells were immunostained with resultant fluorescence intensity was low, indicating the delivery of antihuman CD45FITC and CD34PE mAbs and analyzed by FACS by using Cell Quest a relatively small volume of material. In subsequent experiments, ϩ Software (Becton Dickinson). The percentage CD45 cells was determined on care was taken to ensure injections were consistent and delivery of ungated samples. (A) Plot of the percentage CD45ϩ cells in mice injected with cells plated on FN in the presence (diamonds) or absence (control; triangles) of TS2/16.2.1 material was minimal (ie, low levels of fluorescence). For example, ϩ activating mAb. (B) Scatter plot of the percentage of CD45ϩ cells in mice injected with in the last 5 experiments performed with CD34 cells and RN-attached cells (circles) or control cells (ie, cells maintained in suspension or CD34ϩ/CD38Ϫ cells (using needles of 0.22 µ OTD), cell viabilities plated on FN in the absence of activating mAb; inverted triangles). Horizontal lines Ϯ represent the mean percentage CD45ϩ cells in each group. 2 hours after OG-dextran injections were 84% 3.3% and 87% Ϯ 2.8%, respectively. These results demonstrate that macromolecules can be successfully delivered to various populations of in the absence of activating mAb (1.8% Ϯ 3.7%). Engraftment immobilized primitive, human cord blood cells via glass needle– levels with RN-attached cells (1.9% Ϯ 2.3%) were equivalent to mediated injection. those of control cells (2.4% Ϯ 4.3%). Thus, primitive cord blood cells temporarily attached to RN are neither affected in their Survival, proliferation, and hematopoietic activity of individual ؉ ؉ ؊ proliferation, colony-forming activity, nor NOD/SCID engrafting microinjected CD34 and CD34 /CD38 cells ability. In contrast, primitive cord blood cells attached to FN in the It was critical that we follow the fate of individual injected cells for presence of activating mAb may have impaired NOD/SCID subsequent survival and biologic activity. Therefore, 2 hours after engrafting ability, while still retaining control levels of proliferative injection with OG-dextran, CD34ϩ cells were detached with and colony-forming activity. In light of these observations, subse- peptides and individual fluorescent cells were transferred, as single quent experiments used RN attachment. cells, into individual wells of 96-well plates. Individual wells were monitored for the number of surviving and proliferating cells. The Microinjection-mediated delivery of fluorescence- frequency of surviving cells in the microinjected group (80%) was labeled dextrans into CD34؉, CD34؉/CD38؊, and marginally lower than the attached/detached cells (96%) or suspen- CD34؉/CD38؊/Thy-1lo cells sion controls (92%) at day 6 (Figure 3, closed bars). Because the To optimize injection conditions for primitive blood cells, it was cells were transferred promptly after microinjection, the slight critical to directly monitor the flow of material from the injection decrease (20%) in overall cell survival is likely due to the increased needle and the fate of individual cells after injection. Therefore, we fragility of the cells immediately after microinjection. The fre- used OG-dextran or FITC-dextran when optimizing blood stem/ quency of proliferating microinjected and attached/detached cells progenitor cell injections.20 was not obviously different from controls by day 6 (Figure 3, open CD34ϩ cells were immobilized on RN and manually injected bars). This was especially evident when the frequency of proliferat- with OG-dextran. Immediately after injection, only 5% to 10% of ing cells was calculated, based on the actual number of surviving the injected cells exhibited damage visible by light microscopy. cells (ie, frequency of proliferation/frequency of survival; control: Examples of CD34ϩ cells during and after injection with OG- 79%/92% ϭ 86%; attached/detached: 96%/96% ϭ 100%; in- dextran are shown in Figure 1E and F, respectively. Approximately jected: 72%/80% ϭ 90%). However, it appeared that microinjec- 67%, 55%, and 52% of cells were fluorescent 2, 24, and 48 hours, tion induced a delay in the average time before proliferation respectively, after injection (Table 3). Table 3 also shows the (Figure 3, open bars). The observed delay in proliferation was

Table 3. Microinjection of ﬂuorescent compounds into CD34؉, CD34؉/CD38؊, and CD34؉/CD38؊/Thy-1lo cells results in high cell viability Percent Viability*† Material Mode of Cells Matrix Injected Injection 2-4 h 24 h 48 h

CD34ϩ RN OG-dextran Manual 67 Ϯ 13 55 Ϯ 8.5 52 Ϯ 8.4 (n ϭ 63) (n ϭ 45) (n ϭ 45) CD34ϩ RN OG-dextran Semiautomatic n.d. 56 Ϯ 5.2 54 Ϯ 5.2 (n ϭ 6) (n ϭ 6) CD34ϩ RN Cy-3 IgG Manual 70 Ϯ 7.5 53 Ϯ 6.2 44 Ϯ 5.4 (n ϭ 3) (n ϭ 3) (n ϭ 3) CD34ϩ/CD38Ϫ RN OG-dextran Manual 58 Ϯ 23 41 Ϯ 17 42 Ϯ 14 (n ϭ 15) (n ϭ 9) (n ϭ 9) CD34ϩ/CD38Ϫ/Thy-1lo FN FITC-dextran Manual 28, 54 9, 52 3, 32

*Values represent mean Ϯ 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. 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 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 Ͼ .3). Similar results were obtained in CD34ϩ/CD38Ϫ 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 CD34ϩ/CD38Ϫ 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 CD34ϩ 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% ϭ 100%; injected: 84%/87% ϭ 97%). As can be seen in

Figure 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.

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 Ͼ .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 CD34ϩ/CD38Ϫ cells retained the ability to generate colony-forming cells (CFCs).21 Gross microscopic analysis re- vealed 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 line- ages when comparing injected to control suspension cells (see Table 4). Microinjection of CD34ϩ/CD38Ϫ cells does not adversely affect their ability to generate CFCs or their selection of differentia- .Figure 3. Microinjected CD34؉ cells are viable and retain their ability to tion program proliferate after single cell transfer. Cells were attached to RN and then ؉ microinjected with OG-dextran. Two hours after injection, cells were detached with Delivery of fluorescently labeled protein to CD34 cells 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 Glass needle–mediated microinjection of other cell types (eg, with peptide (B) were also transferred as single cells. Survival and proliferation of the fibroblasts) with proteins (eg, recombinant proteins or antibodies to cells was monitored at 0, 2, and 6 days after transfer. Values shown represent the specific intracellular proteins) has been a powerful method for percentage of total wells transferred that displayed cells surviving (closed bar) and ϩ proliferating (open bar) for 1 experiment in which 30 suspension, 30 RN attached/ assaying the function of specific proteins. The frequency of CD34 peptide detached, and 45 OG-dextran microinjected CD34ϩ cells were transferred. cells surviving injection with Cy-3 IgG is similar to that for From www.bloodjournal.org at UNIV OF KANSAS MEDICAL CENTER on November 24, 2010. For personal use only. 442 DAVIS et al BLOOD, 15 JANUARY 2000 • VOLUME 95, NUMBER 2

Table 4. Immobilization on RN followed by microinjection and detachment preliminary experiments, CD34ϩ/CD38Ϫ cells were injected with a ؉ ؊ does not affect the ability of CD34 /CD38 cells to produce colony forming 19.6 kb GFP construct (5 copies/fL) 13.3% and 54.5% of injected cells cells expressed GFP at 5 hours after injection. Thus, injection Colony Formation (% of Nonimmobilized yields transient transgene (GFP) expression in primitive blood cell % Wells Producing Control)† populations with DNAs as large as 19.6 kb in size. Method of Method Micro- Colony-Forming Attachment* of Release injection Cells* Erythroid Myeloid

——Ϫ 74 100 100 n ϭ 42 Discussion RN Peptide Ϫ 68 79 86 n ϭ 40 To our knowledge, this is the ﬁrst report of glass needle–mediated RN Peptide ϩ 77 132 100 microinjection of DNA, protein, and/or dextran into primary n ϭ 64 human stem/progenitor cells. The difficulties in injecting hemato-

*Individual cells were transferred to wells of 96-well plates using the Quixell poietic cells, which are normally nonadherent and very small, were Transfer unit. To allow for the development and expansion of colony forming cells, overcome by development of improved attachment protocols and transferred cells were cultured in Stem Cell Medium for 10 days before the addition of ultrafine microinjection needles. We have identified conditions MethoCult GF media. After 14 days incubation in methylcellulose, the individual wells were evaluated for colony growth and phenotype. The total number of wells whereby primary human cord blood stem/progenitor cells may be containing either erythroid or myeloid growth was determined for each condition. temporarily immobilized in a manner sufficiently strong to with- †Colony formation is represented as the % of nonimmobilized control (number of stand microinjection without altering cell function/biology. wells giving rise to colony growth for treated cells Ϭ number of wells giving rise to ϩ ϳ colony growth for suspension cells). Values represent combined results of 2 CD34 cells represent 0.5% to 1.0% of nucleated umbilical microinjection sessions in which the number of wells specified (n) were analyzed. cord blood and bone marrow cells and comprise all measurable human progenitor activity.18 With the possible exception of a Ϫ ϩ OG-dextran (Table 3), demonstrating efficient protein delivery to limited subset of CD34 cells,22,23 CD34 cells also contain all ϩ Ϫ primitive CD34ϩ cord blood cells. measurable human stem cell activity.18 The CD34 /CD38 (ϳ 5% to 10% of CD34ϩ cells) and CD34ϩ/lineageϪ/Thy-1lo phenotype ؉ Transient expression of GFP by microinjected CD34 characterize more primitive subpopulations.2, 24-27 CD34ϩ cells, and ؉ ؊ and CD34 /CD38 cells subsets thereof, attach strongly to plates coated with a recombinant To examine transient reporter gene expression, CD34ϩ and CD34ϩ/ derivative of FN, CH-296 known as RN. The RN attachment CD38Ϫ cells were injected with various expression constructs method had no effect on survival, proliferative potential, or ϩ containing the humanized rsGFP protein under control of either NOD/SCID engrafting ability. In contrast, attachment of CD34 CMV (pGreen Lantern plasmid DNA) or pgk regulatory sequences cells to Con A adversely affected myeloid colony formation (see (linearized pgk-GFP). GFP expression at 5 hours after injection in Table 1), which is consistent with previous reports suggesting that CD34ϩ and CD34ϩ/CD38Ϫ cells successfully injected with pGreen attachment of transformed hematopoietic cells via lectins (eg, Con Lantern was 47% Ϯ 16% and 76% Ϯ 9%, respectively (Table 5). A28-30) can have mitogenic or inhibitory effects on hematopoietic GFP expression was detected in 68% of the CD34ϩ/CD38Ϫ cells cells. Strong attachment to FN requires treatment with specific successfully injected with the linearized pgkGFP DNA. OG- anti-␤1 integrin antibodies, which induce low avidity ␤1-containing 16,31 dextran injection results from experiments performed during the integrin heterodimers (eg, ␣4␤1, ␣5␤1) to a high avidity state. same period demonstrated that ϳ 84% and 87% of injected CD34ϩ Activation of the integrins with the TS2/16.2.1 activating antibody, and CD34ϩ/CD38Ϫ cells, respectively, survived quantitative deliv- followed by attachment to FN, may have an effect on NOD/SCID ery of injected material at 2 hours after injection (n ϭ 5 most recent reconstituting activity in the cells. A decrease in the number of mice experiments). Using these values, we have estimated that ϳ56% demonstrating human cell engraftment was noted with the cells ϩ (47%/84%) and ϳ87% (76%/87%) of viable injected CD34 or attached to FN in the presence of activating mAb compared with ϩ Ϫ CD34 /CD38 cells transiently expressed GFP 5 hours after control cells (FN with activating mAb). Although this difference injection (as estimated for pGreen Lantern). Expression frequen- was not statistically significant, it was consistent with a reduction cies gradually decreased with increasing time of culture (data not in the number of human CD45ϩ cells in the mice injected with cells shown); this was not unexpected, because almost all expression attached to FN with activating mAb. Because RN attachment of should be transient, due to unintegrated DNA copies. However, we cells had no effect on cell function, we chose to use RN for cell have observed GFP-expressing cells (and a smaller number of immobilization in all subsequent studies. dividing, expressing cells) as late as 4 to 5 days after injection. In 2 Previous reports of the successful injection of transformed hematopoietic cells are limited.28,29 Attempted injection of small, ؉ ؉ ؊ Table 5. CD34 and CD34 /CD38 cells express green fluorescent protein normally nonadherent cells (eg, primary hematopoietic cells) has after injection with GFP constructs resulted in extremely low cell survival.30 Injection needles with a DNA Construct % Expression Cells* Injected at5h† minimal OTD had to be developed to achieve high viability of blood stem/progenitor cells. We have demonstrated increased ϩ Ϯ CD34 pGreen Lantern 47 16‡ viability of injected fibroblasts when using needles with decreased CD34ϩ/CD38Ϫ pGreen Lantern 76 Ϯ 9‡ CD34ϩ/CD38Ϫ Linearized pgkGFP 68§ OTD (Brown et al, unpublished data). Reducing the injection needle OTD to 0.2 µ did indeed yield a considerable increase in the *Cells attached to RN-coated dishes. viability of injected CD34ϩ cells (unpublished data). This in- †Values represent number of expressing cells/number of injected cells ϫ 100. ‡Values represent the mean Ϯ SD of 3 experiments of 100 cells per experiment. creased viability using ultrafine needles can be attributed to §Value represents 1 experiment of 110 injected cells. 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 MICROINJECTION OF MACROMOLECULES AND TRANSGENES 443

retraction and minimized injection volumes due to finer control of tuted with as little as 30 mL of transplanted cord blood32 containing sample flow from the injection needle. approximately 1.5 to 3 ϫ 108 nucleated cells. If the frequency of Our technology has allowed for efficient macromolecule deliv- stem cells is 1 in 105 to 106, then successful engraftment could ery and excellent postinjection viabilities of both CD34ϩ and occur with as few as 150 to 3000 stem cells. Although enriched ϩ Ϫ CD34 /CD38 cells. Optimization of injection conditions (re- stem cell–containing populations of primitive human hematopoi- flected in the last 5 experiments) and observation of individual etic cells have already been described,11,23,26,27 further definition of injected cells resulted in calculated actual long-term survival the human stem cell phenotype(s) is necessary. Indeed, determina- frequencies at 10 days after injection of 73% and 67% for tion of the mouse stem cell phenotype has reached the point where ϩ ϩ Ϫ successfully injected CD34 and CD34 /CD38 cells, respec- delivery of as few as 10 cells,33 or even 1 marrow cell34 is sufficient tively. Furthermore, there was no observed change in the frequency ϩ ϩ Ϫ for total hematopoietic reconstitution. of surviving CD34 or CD34 /CD38 cells undergoing prolifera- Both manual (100-200 injected cells/h) and semiautomated tion at 6 to 10 days after transfer. Finally, injection had no impact (200-400 cells/h) modes of injection were used in this study; rates on the ability of CD34ϩ/CD38Ϫ cells to generate or expand progeny likely insufficient for feasible therapeutic time constraints. Fully CFCs, an in vitro measure of primitive hematopoietic activity. automated systems, capable of 1500 cell injections per hour,20 Excellent transgene delivery and transient expression was would likely be used to inject a sufficient number of stem cells for observed in injected CD34ϩ and CD34ϩ/CD38Ϫ cells. Interest- transplantation. Any significant in vitro or in vivo expansion of ingly, the more primitive CD34ϩ/CD38Ϫ population displayed an stem cells,35 perhaps together with selection for marked cells, increased rate of CMV-driven GFP expression. Although not statistically significant, this trend is consistent with an observed would further decrease the number of injected stem cells required increase in frequency of pGreen Lantern expression in electropor- for engraftment. ated CD34ϩ/CD38Ϫ versus CD34ϩ/CD38ϩ cells (B.R.D., unpub- In summary, we have demonstrated glass needle microinjection- lished results). In preliminary experiments, transient GFP expres- mediated delivery of macromolecules (protein, DNA, and dex- ϩ ϩ Ϫ ϩ Ϫ sion was also seen in injected CD34ϩ/CD38Ϫ/Thy-1lo cells (data trans) into human CD34 , CD34 /CD38 , and CD34 /CD38 /Thy- not shown). Future experiments will focus on assaying and 1lo cord blood cells. Moreover, our immobilization and injection optimizing long-term transgene maintenance in primitive hemato- methods are applicable to quiescent blood stem cells because ϩ poietic stem/progenitor cells. immunomagnetically isolated CD34 cells are capable of quantita- We have achieved delivery of DNAs 19.6 kb in length and tive attachment to RN immediately after isolation without cytokine subsequent expression of GFP in the injected cells demonstrating prestimulation (data not shown). It has been demonstrated that 95% that glass needle–microinjection is a powerful tool for delivering of cord blood CD34ϩ/CD38Ϫ cells are quiescent at time of large DNAs to cells. These DNAs can accommodate the regulatory purification.24,36 The demonstration that these cells can be injected elements and intron/exon structure necessary for long-term, cell with high viability without any observable adverse effect on type-specific, integration site-independent expression of trans- proliferation or biologic function strongly supports our intended genes. Thus, injection may be preferable for developmental or gene application of this technology to experimental studies and gene therapeutic applications requiring tightly regulated gene expression therapeutic applications of hematopoietic stem/progenitor cells. in progeny hematopoietic cells. An evaluation of glass needle–mediated microinjection as a potential method for stem cell gene therapy must take into account Acknowledgments the number of stem cells required for transplantation, the frequency of actual stem cells in the injected population, and the total time We thank Gina Barron, Mark Griffin, Aqing Yao, Jianming Lu, and required to perform the injections. Children have been reconsti- Fuming Pan for expert technical assistance.

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