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

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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 CD341 and CD341/CD382 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% 6 8.4% of CD341 cells and 42% 6 up to 75% of injected cells. These data porary immobilization of cells to extracellu- 14% of CD341/CD382cells 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 CD341, CD341/CD382, demonstrated long-term survival and pro- studies of stem cell biology and a promis- and CD341/CD382/Thy-1lo human cord blood liferation of CD341 and CD341/CD382 cells, ing method for human blood stem cell cells. Immobilization and detachment proto- and retention of the ability of CD341/ gene therapy. (Blood 2000;95:437-444) cols were identified, which had no ad- CD382 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; , 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 CD341, CD341/CD382, and fully modifying only the stem cell compartment has made these CD341/CD382/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 microin- jection 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 CD341 cells molecules (eg, peptide inhibitors, purified active proteins, neutral- CD341 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 3 BIT 9500 (StemCell damage to these relatively small cells (, 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 CD341 cells were isolated by flow intravenously (iv; tail vein) with purified CD341 cells (2 3 104 cells/ cytometry on a FACS Vantage (Becton Dickinson, San Jose, CA) after mouse). CD341 cells had either been maintained in suspension or first immunostaining with anti-CD34-FITC and anti-CD38-PE for CD341/ immobilized on RN or FN, then detached by pipetting or treatment with CD382 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 CD341/CD382/Thy-1lo cells. mice were analyzed for the presence of human cells by fluorescence- activated cell sorter (FACS) analysis using antihuman CD45FITC and CD341 cell matrix attachment and detachment antihuman CD34 PE (Becton Dickinson). Percent CD451 cells represents the sum of the frequency of human CD451/CD342 and CD451/CD341 Dishes were coated overnight at 4°C with either Fibronectin (FN) or FN cells. When the level of human cell engraftment was low (, 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 CD341, CD341/CD382, on matrix-coated dishes and CD341/CD382/Thy-1lo cells Hematopoietic stem/progenitor cells in the bone marrow express a b a b 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 CD341 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 CD341 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 CD341 cells were treated with specific anti-b1 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 3 100).
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