Adapted from Gene Transfer: Delivery and Expression of DNA and RNA (eds. Friedmann and Rossi). CSHL Press, Cold Spring Harbor, NY, USA, 2007.
INTRODUCTION
In the protocol presented here, hematopoietic stem cells (HSCs) are specifically transduced with a vector displaying the HSC-activating polypeptides, stem cell factor (SCF) and thrombopoietin (TPO). Targeted HSC transduction is evaluated in the non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mouse model.
RELATED INFORMATION
An overview of approaches to modify lentivirus vectors for use in gene transfer can be found in Engineering the Surface Glycoproteins of Lentiviral Vectors for Targeted Gene Transfer (Verhoeyen and Cosset 2009).
MATERIALS
Reagents
293T cells
Antibodies (BD Pharmingen or equivalent)
anti-hCD13-phycoerythrin (PE)
anti-hCD14-PE
anti-hCD19-PE
anti-hCD34-PE
anti-hCD45-CyChrome
Use corresponding PE-conjugated mouse IgG controls to evaluate specific labeling (see Step 19).
CalPhos Mammalian Transfection Kit (Clontech)
CD34+ cell separation kit (CD34 MicroBead Kit containing anti-human CD34 MicroBeads and Blocking Reagent; Miltenyi Biotec)
Cellgro medium (serum-free medium; Cellgenix)
DMEM for HSC
Fetal calf serum (FCS; sterile)
Ficoll-Paque PLUS (sterile; GE Healthcare Life Sciences)
HeLa cells
Hematopoietic stem cells (HSCs; purified from fresh neonatal cord blood as described below)
Many studies suggest that HSCs reside in a cell population expressing CD34+ antigen. Approximately 1% of mononuclear cells from cord blood are CD34+.
Neonatal human cord blood (CB)
NOD/SCID mice (sublethally irradiated [3.5 Gy])
Nucleic acids
Lentiviral vector DNA encoding an HIV-1-derived self-inactivating vector with the internal EF1-
promoter driving the green fluorescent protein (GFP) reporter gene
Envelope glycoprotein-expressing plasmids
Fusion glycoprotein: VSV-G
Activating and targeting glycoproteins for HSCs: (1) TPOHA (TPO fused to HA envelopeglycoprotein) and (2) SCFSUx (SCF fused to MLV envelope glycoprotein)
Virus structural protein (Gag-Pol)-expressing plasmid (pCMV8.91) (HIV packaging construct)
Neuraminidase-expressing plasmid pCMV-NA
Phosphate-buffered saline (PBS) (without calcium, magnesium, or sodium bicarbonate; sterile)
Reagents for harvesting bone marrow from mouse femurs
Trypsin/EDTA (0.5%)
Equipment
Centrifuge
Centrifuge tubes (50-mL)
Equipment for harvesting bone marrow from mouse femurs
Filter membranes (0.45-µm)
Flow cytometry system capable of cell sorting and three-color analysis
Flow cytometry tubes
Incubator preset to 37ºC
MACS cell separation columns (Miltenyi Biotec)
MACS Separator (Miltenyi Biotec)
Pipettes
Rocking platform
Standard tissue-culture equipment (including 100-mm, six-well, and 48-well tissue-culture plates)
Syringe with needle for injecting mice tail veins (see Step 17)
METHOD
Production of Lentiviral Vectors Displaying Activating Polypeptides
1. The day before transfection, seed 293T cells in DMEM for HSC at a density of 2.5 x 106 cells per 100-mm tissue-culture dish in a final volume of 10 mL/dish.
2. On the day of transfection, use the CalPhos Mammalian Transfection Kit to cotransfect 8.6 µg of the HIV packaging construct with 8.6 µg of the lentiviral gene-transfer vector and the two glycoproteins: VSV-G (1.5 µg) and TPOHA (1.5 µg) or VSV-G (1.5 µg) and SCFSUx (1.5 µg).
Cotransfect TPOHA with the neuraminidase-expressing plasmid (pCMV-NA) to allow efficientrelease of virus from the producer cell. Otherwise, the vector particles bind to the producer cellsvia an interaction between the HA envelope and the receptor sialic acid.
3. Fifteen hours after transfection, replace the medium with 6 mL of fresh Cellgro medium.
This medium is adapted for culture of CD34+ cells and allows the cytokines displayed on the vector’s surface to maintain their functionality.
4. Thirty-six hours after transfection, harvest the vectors, filter through a 0.45-µm membrane, and store at –80°C.
Vectors can be stored for 2-3 mo.
Immunoselection of Human CD34+ Cells
5. Dilute CB 1:1 with PBS. Gently layer 35 mL of the diluted blood on 15 mL of Ficoll-Paque PLUS in a 50-mL tube.
6. Centrifuge the cells at 850g for 30 min at 20°C with no brake. Collect the layer that contains the mononuclear cells.
The mononuclear cells are found in a white band at the top of the Ficoll layer.
7. Prepare PBS/2% FCS. Wash the collected mononuclear cells in PBS/2% FCS and centrifuge at 850g for 10 min at 20°C.
8. Resuspend the cells to a concentration of 1 x 108 to 2 x 108 cells/mL in PBS/2% FCS. To magnetically label the cells using the CD34 MicroBead Kit, add Blocking Reagent and then add anti-hCD34+ MicroBeads according to the manufacturer’s instructions. Incubate while rocking for 30 min at 4°C.
9. Wash the cells to remove unbound antibody and resuspend in PBS/2% FCS.
10. Separate the CD34+ cells as follows:
i. Wash a MACS cell separation column with 200 µL of PBS/2% FCS.
ii. Add the labeled cells to the column and place it in the MACS separator.
iii. Allow the unlabeled cells to pass through the column.
iv. Wash once with PBS/2% FCS.
v. Remove the column from the separator to elute the CD34+ cells.
vi. Repeat this procedure once.
The purity of the CD34+ cells is routinely 90%-95%.
Titer Determination
11. Day –1: Seed HeLa cells in DMEM for HSC at a density of 2 x 105 cells/well in six-well plates in a final volume of 2 mL/well. Incubate overnight.
12. Day 0: Prepare serial dilutions of vector preparations, add to the HeLa cells and incubate overnight.
13. Day 1: Replace the medium on the cells with 2 mL of fresh DMEM for HSC and incubate for 72 h.
14. Day 3: Trypsinize the cells and transfer to flow cytometry tubes. Determine the percentage of GFP-positive cells by flow cytometry.
Infectious titer is expressed as transducing units (TU)/mL and is calculated by the following formula:
Titer = % GFP-positive cells (number of cells at the time of infection/100) x dilution
Multiplicity of infection (moi) is the ratio of infectious particles to target cells.
Transduction of Human CD34+ Cells
15. Seed CD34+ CB cells in CellGro medium at a density of 5 x 104 cells/well in 48-well plates. Transduce with fresh lentiviral vector supernatant at an moi of 20 or 4. Incubate for 24 h.
16. Wash the transduced cells and resuspend in CellGro medium. Incubate for 48 h. Determine transduction efficiency by analyzing enhanced GFP expression by flow cytometry after immunolabeling the cells with an anti-hCD34-PE antibody.
NOD/SCID Repopulating Assay
17. After a 24-h (moi of 4) transduction with TPOHA-, SCFSUx-displaying lentiviral vectors, inject CD34+ CB cells into the tail veins of sublethally irradiated (3.5 Gy) NOD/SCID mice without in vivo administration of cytokines.
18. Harvest the bone marrow from femurs 6-8 wk after transplantation.
19. Use three-color flow cytometry to detect GFP+ human cells of various lineages in the NOD/SCID bone marrow using anti-hCD45-CyChrome and anti-hCD19-PE, anti-hCD14-PE, anti-hCD13-PE, and anti-hCD34-PE antibodies. In all cases, use corresponding PE-conjugated mouseIgG controls to evaluate specific labeling.
REFERENCES
Verhoeyen E, Cosset F-L. 2009. Engineering the surface glycoproteins of lentiviral vectors for targeted gene transfer. Cold Spring Harb Protoc (this issue). doi: 10.1101/pdb.top59.
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