Hematopoietic Progenitor Cells

3.5.1. Gene Transfer Efficiency by Flow Cytometry

1. Collect cells 48 h after retroviral exposure, count them and determine how many cells you have available for flow cytometry (see Note 16). Stain 10,000-20,000 cells.

2. Resuspend 10,000-20,000 cells in 200 |L of PBS + 3% FBS in a 5-mL polystyrene tube.

3. Add 5 |L of mouse anti-human CD34-PE and 2 |L mouse anti-human CD45-APC. As a negative control, add 2 |L of mouse IgG-PE and 2 |L of mouse IgG-APC into a separate 5-mL polystyrene tube containing 5000 cells in 200 |L of 1X PBS + 3% FBS. Incubate for 30 min at 4°C.

4. Wash the cells with 3 mL 1X PBS + 3% FBS. Centrifuge the tubes for 5 min at 453g. Aspirate off the supernatant.

5. Resuspend the cells in 300 |L 1X PBS + 3% FBS. Add 2 |L of 7-AAD and stain for 10 min at room temperature to exclude dead cells.

6. Run the cells in a FACSCalibur flow cytometer. Set up quadrants based on mock retroviral exposure for GFP expression and based on respective IgG isotype controls for CD34 and CD45. The percent of GFP+ cells as compared to mock represents the overall transduction efficiency. The percent of GFP+CD45+ cells represents the transduction efficiency into CD45negPFV-derived hematopoietic cells and the percent of GFP + CD45 + CD34+ indicates the transduction efficiency into CD45negPFV-derived cells with phenotype of hematopoietic progenitor (see Fig. 1).

3.5.2. Detection of Transduced Hematopoietic Progenitors by CFU Assay

1. Collect cells 48 h after retroviral exposure, count them, and determine the number of cells available for CFU assay (see Note 16). Plate 10,000 cells.

2. Using a 1-mL syringe coupled to a 16-gage needle, add 0.6 mL of H4230 methylcellulose media supplemented with growth factors (see Subheading 2.5.2., item 13) to 1.5-mL microtube.

3. Take 14,000 cells and bring them to a final volume of 100 ||L in FBS-free IMDM. Mix cell suspension and 0.6 mL of H4230 methylcellulose media.

4. Vortex the tube vigorously and let it stand for 5 min to get rid of the bubbles.

5. Using a 1-mL syringe coupled to a 16-gage needle, plate 0.5 mL of the plating mixture in a nontreated 12-well plate.

6. Distribute methylcellulose over entire well surface avoiding bubbles.

7. Incubate cells at 37°C and 5% CO2 in a humidified atmosphere.

8. Score the colonies after 12-14 d using standard morphological criteria in an inverted microscope. Then, use a fluorescence microscope to assess transduction into hematopoietic progenitors by calculating the frequency of GFP+ CFU (see Fig. 2).

3.5.3. Confirmation of Proviral Integration by PCR

3.5.3.1. Isolation of Individual Colonies From Methylcellulose

1. Pick a well-isolated GFP+ CFU under the microscope with a P-20 pipetman, and place into 300 ||L PBS in 1.5-mL microtube, incubate for 20 min. Special care must be taken to ensure that cells from adjacent colonies do not contaminate the sample.

3. Aspirate off the PBS.

4. Snap freeze and store at -80°C until ready to extract DNA.

3.5.3.2. Genomic DNA Extraction From Individual Isolated Colonies

1. Thaw frozen colonies on ice.

2. Resuspend very well in 100 |L DNA A + 50 |g Proteinase K (5 |L of stock 10 mg/mL).

3. Add 100 |L DNA B and place in 56°C water bath for 1 h (see Note 17).

4. Add 200 |L Tris-buffered phenol and rotate the tube for 15 min (see Note 18). Do not vortex or pipet the DNA.

5. Spin tubes at maximum speed for 5 min.

6. Transfer the upper aqueous phase (containing genomic DNA) into clean 1.5-mL microtube. Ensure you do not transfer the white pellet containing the proteins.

7. Add 200 |L of 1:1 phenol:chloroform isoamyl alcohol solution and rotate the tubes for 15 min.

Fig. 1. Retroviral transduction of human embryonic stem cells (hESC)-derived hematopoietic cells. (A) Isolation of CD45negPECAM1+Flk1+, CD45negPECAM1+ or CD45negFlk1+ cells from hEBs demonstrated that VE-cadherin segregated with either of these populations and produced identical results (14), indicating that any of these sorting strategies isolate a functionally identical subpopulation in which 90% of the cells are CD45negPFVpos. Therefore, flow cytometric isolation of CD45negPECAM1+ cells represents a simpler and accurate strategy for purification of CD45negPFV precursors. (B,C) Flow cytometric analyses of transduction efficiency into CD45negPFV (B) and remaining EB cells (C). Phenotype of the transduced (GFP+) progeny arising from CD45negPFV precursors (D) and remaining EB cells (E) showing that transduced hematopoietic cells exclusively arise from hESC-derived CD45negPFV precursors.

Fig. 1. Retroviral transduction of human embryonic stem cells (hESC)-derived hematopoietic cells. (A) Isolation of CD45negPECAM1+Flk1+, CD45negPECAM1+ or CD45negFlk1+ cells from hEBs demonstrated that VE-cadherin segregated with either of these populations and produced identical results (14), indicating that any of these sorting strategies isolate a functionally identical subpopulation in which 90% of the cells are CD45negPFVpos. Therefore, flow cytometric isolation of CD45negPECAM1+ cells represents a simpler and accurate strategy for purification of CD45negPFV precursors. (B,C) Flow cytometric analyses of transduction efficiency into CD45negPFV (B) and remaining EB cells (C). Phenotype of the transduced (GFP+) progeny arising from CD45negPFV precursors (D) and remaining EB cells (E) showing that transduced hematopoietic cells exclusively arise from hESC-derived CD45negPFV precursors.

Fig. 2. Assessment of transduction efficiency into hematopoietic progenitors by using fluorescent microscopy. Bright-field (left panels) and fluorescence (right panels) microscopy pictures of representative colonies formed from CD45negPFV precursors following mock transduction (A,B) or GALV-pseudotyped retroviral transduction (C-F).

Fig. 2. Assessment of transduction efficiency into hematopoietic progenitors by using fluorescent microscopy. Bright-field (left panels) and fluorescence (right panels) microscopy pictures of representative colonies formed from CD45negPFV precursors following mock transduction (A,B) or GALV-pseudotyped retroviral transduction (C-F).

8. Spin tubes at maximum speed for 5 min.

9. Transfer the upper aqueous phase (containing genomic DNA) into a clean 1.5-mL microtube.

10. Add 200 |L of chloroform isoamyl alcohol 24:1.

11. Spin tubes at maximum speed for 10 min.

12. Transfer the upper aqueous phase into a 1.5-mL microtube containing 30 ||L 5 M NaCl and 20 |g glycogen at 20 mg/mL. Then add 500 |L 100% ethanol (two volumes).

13. Incubate overnight at -30°C.

14. Spin tubes at maximum speed for 30 min at 4°C.

15. Remove supernatant completely and dry the pellet in the fume hood.

16. Resuspend the pellet in 30 ||L TE.

17. Measure DNA concentration using a spectrophotometer.

18. Store the DNA at 4°C until ready to use for PCR.

1. Prepare the PCR reaction mix (50 |L final volume): for GFP gene: 38.5 |L PCR dH2O, 5 |L 10X PCR buffer, 1 |L 50 mM MgCl2, 2 |L 20 mM dNTPs, 0.5 |L 50 |M forward primer, 0.5 |L 50 |M reverse primer, 0.5 |L Taq DNA polymerase, and 2 | L template DNA (approx 10 ng).

For hCART-1 gene: 37.5 |L PCR dH2O, 5 |L 10X PCR buffer, 2 |L 50 mM MgCl2, 2 |L 20 mM dNTPs, 0.5 |L 50 |M forward primer, 0.5 |L 50 |M reverse primer, 0.5 |L Taq DNA polymerase, and 2 |L template DNA (approx 10 ng).

2. Run samples as per the following conditions: for GFP amplification: 1 cycle 96°C for 5 min, 35 cycles 96°C for 40 s, 65°C for 40 s, 72°C for 1 min, 1 cycle 72°C for 10 min, then hold at 4°C.

For hCART-1 amplification: 1 cycle 96°C for 2 min, 35 cycles 94°C for 30 s, 60°C for 30 s, 72°C for 15 s, 1 cycle 72°C for 5 min, then hold at 4°C.

3. Resolve the PCR products on a 1% agarose gel. Only those colonies containing amplified hCART-1 template are used for determination of gene transfer efficiency into CD45negPFV-derived hematopoietic progenitors (see Fig. 3).

Was this article helpful?

0 0

Post a comment