Characterization of hESCDerived Hematopoietic Cells In Vivo

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Despite in vitro analysis, bona fide hematopoietic stem cells can only be functionally defined by sustained multi-lineage in vivo reconstitution upon transplantation. Experimentally, the NOD/SCID xenotransplant assay has provided a powerful tool to functionally define candidate human hematopoietic stem cells, defined as SCID-repopulating cells (8). However, conventional intravenous transplantation of hESC-derived hematopoietic cells causes mouse mortality owing to emboli formed from rapid cellular aggregation in response to mouse serum (9). IBMT is required for functional in vivo assessment of hESC-derived hematopoietic cells (9).

1. Collect CD45negPFV-derived hematopoietic cells as described previously. Resuspend the cells in IMDM medium.

2. Transfer 5 x 105-1 x 106 cells to 1.5-mL Eppendorf tubes. Prepare one tube for each mouse IBMT. Centrifuge the tubes for 2 min at 453g. Carefully discard supernatant without disturbing the cell pellet.

3. Resuspend the cells in 25 |L of IMDM medium, avoiding bubbles.

4. Sublethally irradiate (3.25 Gy) 8- to 12-wk old NOD/SCID-P2m-/- mice.

5. Weigh the animal (see Note 9). Inject Avertin intraperitoneally at a dose of 0.014-0.018 mL/g of body weight. Use lower dosage for small females and higher dosage for large males. Leave the animal undisturbed until anaesthetic plane has been attained.

6. Fill a 3-mL syringe coupled to a 27-gage needle with 2 mL D-PBS. Rinse a 28-gage insulin syringe (0.5 in., 0.3 mL) with IMDM medium to fill the dead space, then carefully fill the barrel with the cell suspension.

7. Put the mouse on sterilized gauze. Gently place a small amount of sterile eye ointment on the mouse's eyes using sterile gauze.

8. Sterilize the knee with Proviodine detergent, alcohol wipe, and Proviodine disinfectant. Start sterilization at the incision site and move toward the periphery. Allow final solution to dry in order to create a bacteriostatic barrier.

9. Flex the mouse's leg to move the femur and tibia to a vertical position. Brace the animal's femur with your index finger, the tibia with your thumb, and use your middle finger for additional stabilization where needed. Make sure that your index finger can feel the femur and guide the direction.

10. "Drill" (do not push) a 27-gage needle vertically into the femoral cavity through the patellar tendon to form a channel.

11. Gently withdraw the 27-gage needle and keep the leg position unmoved. Insert a 28-gage insulin syringe into the channel made by the 27-gage needle. Inject 25 |L of cell suspension.

12. Gently remove the needle while straightening the leg and holding it in that position for several seconds to stop leakage of the injected cells.

13. Subcutaneously inject 0.3 mL buprenorphine (0.05-0.1 mg/kg of body weight) and 1 mL 0.9% D-PBS into the scruff.

14. Wrap the mouse in a sterile gauze. Return the mouse to the cage and keep it in a warm (under a heating lamp) and quiet environment until full recovery.

15. Administer the second dose of buprenorphine (0.3 mL) 24 h after IBMT. Monitor the mice daily for the first 3 wk (see Note 10).

3.6.2. Collect Bone Marrow of NOD/SCID-$2m-/- Mice Post-IBMT

for Analysis of SCID-Repopulating Cells

1. Euthanize the mouse 8 wk after IBMT. Dip the mouse in 70% ethanol and dissect the mouse bones using sterile scissors and forceps.

2. Place injected femur, contralateral femur, or two tibias and two iliac crests separately into 35-mm dishes containing 2 mL of DMEM.

3. Flush bone marrow into a FACS tube with an insulin syringe containing 1 mL of DMEM.

4. For analysis of human white cells, lyse red cells with cold 0.8% ammonium chloride solution for 5 min at 4°C. Wash the remaining cells twice in D-PBS containing 3% FBS.

3.6.3. FACS Analysis of Engrafted Human Hematopoietic Cells in the Bone Marrow of NOD/SCID-$2m-/- Mice

1. Resuspend the cells in FACS buffer (D-PBS containing 3% FBS) at a concentration of 2.5 x 106 cells/mL. Distribute 200 |L of cell suspension (5 x 105 cells) to each FACS tube. To analyze human myeloid and lymphoid lineages, add 5 | L of FITC-conjugated MAb against human CD45 and 5 | L of PE-conjugated MAbs against either human CD19 (lymphoid) or CD33 (myeloid) to each tube. To analyze human erythroid lineage, add 5 | L of PE-conjugated MAb against human glycophorin-A and 5 |L of FITC-conjugated MAb against human CD36. Simultaneously set up corresponding IgG isotype MAb controls.

2. Incubate the tube for 30 min at 4°C.

3. Add 3 mL of FACS buffer to each tube. Centrifuge at 453g for 3 min. Aspirate the supernatant.

4. Repeat the wash step once. Leave 0.2 mL of supernatant after aspiration. Add 4 |L of 7-AAD. Incubate at room temperature for 10 min in the dark.

5. Analyze cell surface markers with a FACSCalibur and Cell Quest software (B&D).

3.6.4. Southern Blot and PCR Analysis to Detect Human DNA in Mouse Bone Marrow

1. In parallel, perform Southern blot and PCR analyses to detect human DNA in mouse bone marrow.

2. Isolate high-molecular-weight DNA using phenol/chloroform extraction or DNAzol reagent, according to the manufacturer's instructions.

3. Digest 1 |g of DNA with £coRI restriction enzyme at 37°C overnight and separate on a 1.0% agarose gel.

4. Transfer the DNA to Hybond-N+ nylon membrane and hybridize with a 32P-labeled human chromosome 17-specific a-satellite probe.

5. The level of human cell engraftment is quantified using a phosphorimager and ImageQuant software (Molecular Dynamics, Sunnyvale, CA) by comparing the characteristic 2.7-kb band to human:mouse DNA mixture controls (limit of detection, approx 0.1% of human DNA).

6. If the level of human DNA is less than 0.1%, perform PCR to detect the human-specific chromosome 17-specific a-satellite. Forward primer 5'-ACACTCTTTTTGCAGGATCTA-3' and reverse primer 5'-AGCAATGT-GAAACTCTGGGA-3' are used to amplify an 1171 bp sequence (40 cycles, 94°C 30 s, 60°C 30 s, 72°C 15 s + 72°C 10 min). The PCR products are separated on 1.0% agarose gels (see Note 11).

Fig. 1. Development of human embryoid bodies (hEBs) in the presence of hematopoietic cytokines and bone morphogenetic protein-4. Debris and single cells can be seen after hEB formation (d 0). Cystic hEBs can be observed after d 7 hEB development. Numerous bright single cells found in 22 hEBs are CD45+ cells that have been confirmed by FACS analysis. Bar = 50 |im. (Please see the companion CD for the color version of this figure.)

Fig. 1. Development of human embryoid bodies (hEBs) in the presence of hematopoietic cytokines and bone morphogenetic protein-4. Debris and single cells can be seen after hEB formation (d 0). Cystic hEBs can be observed after d 7 hEB development. Numerous bright single cells found in 22 hEBs are CD45+ cells that have been confirmed by FACS analysis. Bar = 50 |im. (Please see the companion CD for the color version of this figure.)

Isotype FITC

Isotype FITC

100 io4

7AAD

1041

100 io4

7AAD

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