Isolation of CD45negPFV Precursors Cells

1. 1X PBS Ca2+ and Mg2+ free (PBS) (500-mL; Gibco; cat. no. 11965-092).

2. Nonheat-inactivated fetal bovine serum (FBS) characterized and screened for hESC growth (500 mL; Hyclone, Logan, UT; cat. no. 30071-03).

3. Allophycocyanin (APC)-conjugated anti-human CD45 monoclonal antibody (clone HI30; Becton Dickinson; cat. no. 555485). It should be used at 5 |g/mL. Protect from light.

4. PE-conjugated mouse anti-human PECAM-1-PE monoclonal antibody (clone WM59; Becton Dickinson; cat. no. 555446). It should be used at 5 |g/mL. Protect from light.

5. 7-aminoactinomycin D (7-AAD) (Immunotech, Marseille, France; cat. no. PN IM3422). Protect from light.

6. PE-conjugated mouse immunoglobulin (Ig)G isotype (Becton Dickinson; cat. no. 340043). Protect from light.

7. APC-conjugated mouse IgG isotype (Becton Dickinson; cat. no. 340442). Protect from light.

8. Trypan blue viability reagent (100 mL; Gibco; cat. no. 1691049).

9. FACS Vantage SE sorter (Becton Dickinson).

10. 5-mL polystyrene tubes (Becton Dickinson; cat. no. 352054).

11. Hematocytometer (Hausser Scientific, Horsman, CA; cat. no. 177569).

2.4. Retroviral Exposure of CD45negPFV Precursors Cells

1. Retroviral vector containing the gene of interest (GFP reporter) under the control of viral long terminal repeat promoter.

2. PG13 retroviral packaging cell line (ATCC, Manassas, VA; cat. no. CRL-10686) (see Note 1) stably expressing the provirus containing the gene of interest with an appropriate functional titer (see Note 2) (34-36) (see Note 3).

3. T-25 tissue culture flasks (Becton Dickinson; cat. no. 353109).

4. 96-well fibronectin-coated plates (Becton Dickinson; cat. no. 354409).

5. 96-well nontreated plates (Becton Dickinson; cat. no. 15705-066).

6. Fibronectin solution 1 |g/|L (5 mL; Sigma, Oakville, Canada; cat. no. F0895).

7. 5X serum-free BIT (bovine serum albumin-insulin-transferrin) media (Stem Cell Technologies, Vancouver, Canada; cat. no. 09500).

10. 14.3 M 2-betamercaptoethanol (100 mL; Sigma; cat. no. M7522).

11. 55 mM 2-betamercaptoethanol: prepare 1.43 M solution by diluting 1:10 in PBS the 14.3 M solution and then dilute 10 |L of 1.43 M solution in 250 |L of IMDM. 1.43 M and 55 mM solutions of 2-betamercaptoethanol can be stored at 4°C for 1 mo and 2 wk, respectively.

12. rhuSCF 2500 |g powder (Amgen Inc., Thousands Oaks, CA; donated). Prepare a working concentration of 1.5 x 103 ng/|L: dissolve the powder in 1.67 mL PBS + 2% FBS. Make 50-|L aliquots and store at -30°C. Once thawed, keep the vials at 4°C for no longer than 1 mo.

13. rhuFlt-3 Ligand 250|g powder (R&D Systems Inc, Minneapolis, MN; cat. no. 308-FK/CF). Prepare a working concentration of 250 ng/|L: dissolve the powder in 1 mL of PBS + 2%FBS. Make 100-|L aliquots and store at -30°C. Once thawed, keep the vials at 4°C for no longer than 1 mo.

14. rhuIL-3 50 |g powder (R&D Systems Inc; cat. no. 203-IL-050). Prepare a working concentration of 25 ng/|L: dissolve the powder in 2 mL of PBS + 2% FBS. Make 100-|L aliquots and store at -30°C. Once thawed, keep the vials at 4°C for no longer than 1 mo.

15. rhuIL-6 50 |g powder (R&D Systems Inc; cat. no. 206-IL-050). Prepare a working concentration of 50 ng/|L: dissolve the powder in 1 mL of PBS + 2% FBS. Make

50-|L aliquots and store at -30°C. Once thawed, keep the vials at 4°C for no longer than 1 mo.

16. rhuG-CSF 300 |g/mL (Amgen Inc; cat. no. 3105100). Make 100 |L aliquots and use as it is at 300 ng/|L. Aliquots must be stored at -30°C. Once thawed, keep them at 4°C for up to 1 mo.

17. 1X complete serum-free media for liquid culture of CD45negPFV precursors cells. The 1X complete serum-free media is composed of 1X BIT media made up in IMDM supplemented with 2 mM l-Glu, 0.1 mM 2-betamercaptoethanol, 300 ng/mL SCF, 300 ng/mL Flt-3L, 10 ng/mL interleukin (IL)-3, 10 ng/mL IL-6, and 50 ng/mL G-CSF. To prepare 10 mL of 1X complete serum-free media: combine 2 mL 5X BIT media, 7.86 mL IMDM, 2 |L rhuSCF at 1.5 x 103 ng/|L, 12 |L rhuFlt-3L at 250 ng/|L, 4 |L rhuIL-3 at 25 ng/|L, 2 |L rhuIL-6 at 50 ng/|L, 1.64 |L rhuG-CSF at 300 ng/|L, 20 |L 2-betamercaptoethanol at 55 mM and 100 |L l-Glu at 200 mM. The media must be prepared fresh.

18. Millex-GP 0.22-|im low binding protein filter (Millipore, Nepean, Canada; cat. no. SLGP033RS).

19. Millex-GP 0.45-|im low binding protein filter (Millipore; cat. no. SLHVR25LS).

20. 0.25% Trypsin-1 mM EDTA (100-mL; Gibco; cat. no. 25300-054).

21. 1-mL syringes (Becton Dickinson; cat. no. 309602).

22. 5-mL syringes (Becton Dickinson; cat. no. 309585).

23. 10-mL syringes (Becton Dickinson; cat. no. 309604).

24. Protamine sulfate (1 g; Sigma, cat. no. P40201). For a 1 |g/|L working solution: reconstitute 1 g in 1 L distilled water. The working solution is stored at 4°C for up to 1 yr.

25. Glucose 450 g/L solution (100 mL; Sigma; cat. no. G8769).

27. a-minimum essential medium (a-MEM; Gibco; cat. no. 12200-028). a-MEM needs to be supplemented with 7% of FBS and 4.5 g/L glucose. To prepare 500 mL: mix 460 mL of a-MEM with 35 mL of FBS and 5 mL of 450 g/L glucose. a-MEM supplemented with FBS and glucose can be store at 4°C for up to 3 mo.

28. IMDM supplemented with 2.5% FBS. To prepare 500 mL: mix 487.5 mL of a-MEM with 12.5 mL of FBS.

29. IMDM supplemented with 10% FBS. To prepare 500 mL: mix 450 mL of a-MEM with 50 mL of FBS.

30. Flow buffer: PBS + 3% FBS. For 100 mL buffer preparation: mix 97 mL of 1XPBS and 3 mL of FBS.

2.5. Assessment of Transduction Efficiency Into hESC-Derived

Hematopoietic Progenitor Cells

2.5.1. Gene Transfer Efficiency by Flow Cytometry

1. FACSCalibur (Becton Dickinson).

2. 5-mL polystyrene tubes (Becton Dickinson; cat. no. 352054).

3. PE-conjugated mouse IgG isotype (Becton Dickinson; cat. no. 340043). Protect from light.

4. APC-conjugated mouse IgG isotype (Becton Dickinson; cat. no. 340442). Protect from light.

5. 7-AAD (Immunotech; cat. no. PN IM3422). Protect from light.

6. PE-conjugated anti-human CD34 monoclonal antibody (clone HPCA-2; Becton Dickinson; cat. no. 555822). Use at 5 |ig/mL and protect from light.

7. APC-conjugated anti-human CD45 monoclonal antibody (clone HI30; Becton Dickinson, cat. no. 340943). It should be used at 5 |g/mL. Protect it from light.

8. Flow buffer: PBS + 3% FBS (see Subheading 2.4., item 30).

2.5.2. Detection of Transduced Hematopoietic Progenitors by Colony-Forming Unit Assay

1. Hematocytometer (Hausser Scientific; cat. no. 177569).

2. 12-well nontreated plates (Becton Dickinson; cat. no. 351143).

3. 1-mL syringes (Becton Dickinson; cat. no. 309602).

4. 16-gage, 1.5-in. bevel needles (Becton Dickinson; cat. no. 305198).

5. Sterile water.

6. Trypan blue viability reagent (100 mL; Gibco; cat. no. 1691049).

8. Inverted microscope.

9. Fluorescence microscope.

14. H4230 methylcellulose culture (80 mL; Stem Cell Technologies; cat. no. 04230). H4230 has to be supplemented with growth factors. Thaw H4230 overnight at 4°C and add directly into the H4230 bottle: 20 mL of sterile IMDM, 50 ng/mL SCF (3.7 |L at 1.5 x 103 ng/|L), 10 ng/mL IL-3 (44 |L at 25 ng/|L), 3 U/mL EPO (33 |L at 10,000 U/mL) and 10 ng/mL GM-CSF (3.7 |L at 1:10 dilution of 3 x 103 ng/|L). Shake vigorously and leave overnight to settle. Make 5- or 10-mL aliquots and store at -30°C. Once thawed, keep at 4°C for no longer than 1 wk.

15. 1.5-mL microtubes (Sarstedt, Montreal, Canada; cat. no. 72690).

2.5.3. Polymerase Chain Reaction Detection of Proviral Integration

1. P-20 pipetman and tips.

7. 20% SDS (1 L; Bio-Rad, Hercules, CA; cat. no. 161-0418).

8. DNA-A solution (10 mM Tris-HCl, 10 mM EDTA, 10 mM NaCl). To prepare: mix 1 mL 1 M Tris-HCl pH 7.6, 2 mL 0.5 M EDTA, 0.2 mL 5 M NaCl, and 96.8 mL H2O.

9. Proteinase K 10 mg/mL (Sigma; cat. no. P2308). Make 100 |L aliquots and store at -30°C.

10. DNA-B solution: mix 90 mL DNA-A solution and 10 mL 20% SDS. Store at room temperature.

12. Fume hood.

13. Tris-buffered phenol (100 mL; Invitrogen, Mississauga, Canada; cat. no. 15513-039). Store at 4°C.

14. Benchtop microcentrifuge.

15. Shaker for 1.5-mL microtubes.

16. Chloroform isoamyl alcohol 24:1 (1 L; Merck, Montreal, Canada; cat. no. CX1054-6). Store at room temperature.

17. 1:1 (v:v) phenol:chloroform isoamyl alcohol solution.

18. 100% ethanol.

19. 5 M sodium acetate.

20. Glycogen 20 mg/mL aqueous solution (1 mL; Fermentas, Burlington, Canada; cat. no. R0561).

21. TE buffer pH 8.0. To prepare: mix 100 mM Tris-HCl and 10 mM EDTA.

22. GeneAmp polymerase chain reaction (PCR) system 9000 (Applied Biosystems; Foster City, CA).

23. 0.2-mL PCR microtubes (Applied Biosystems; cat. no. N801-0840).

24. Distilled water DNAse, RNAse-free (500 mL; Gibco; cat. no. 10977-015).

25. 10X PCR buffer (Invitrogen; cat. no. Y02028).

27. Taq DNA polymerase (Invitrogen; cat. no. 18038-042).

28. 100 mM dNTP set (Invitrogen; cat. no. 55082-85).

29. 20 mM dNTPs mixture (5 mM each): combine 20 |L of each dNTP at 100 mM with 320 |L of distilled water DNAse, RNAse-free water. Store at -30°C.

30. PCR primers diluted to 50 | M with sterile distilled water DNAse, RNAse-free water. Primers used: eGFP (forward: 5'-GAC TTC AAG GAG GAC GGC AAC-3'; reverse: 5'-TAG AAA CTG CTG AGG GCG GG-3'); human CART-1 (forward: 5'-AGC TGC TGT GTG TGG AAT TG-3'; reverse: 5'-ACA GGG TTT GTG GAG ACT GG-3').

31. Spectrophotometer.

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