After sorting

1041

R2

R3

aïïLi

'-v.-

PECAM-li,

PECAM-li,

R1+R2 Gated

R1+R3 Gated

1 04l

^9-100%

PECAM-1

100 io4

PECAM-1

Fig. 2. Isolation of CD45-PECAM1+ precursors. Hematopoietic precursor CD45-PECAM+ cells are isolated from d 10 human embryoid bodies by a fluorescence activated cell sorting Vantage SE. Dead cells are excluded by positive staining for 7AAD. The sorting purity are 99-100% for CD45-PECAM1- (R1+R2), and 92-99% for CD45-PFV+ (R1+R3) subpopulations determined using the same sorting gate settings.

4. Notes

1. MEF-CF quality is crucial for maintenance of undifferentiated hESC and subsequent formation and development of hEB. Because MEF-CM quality varies among different batches of MEF cells and MEF-CM preparations, assessing each batch of MEF-CM before use is required (see Subheading 2.1.1.).

Fig. 3. Hematopoietic colonies from human embryoid bodies (hEB)-derived CD45+ cells. Representative colonies from hEB-derived CD45+ cells are depicted, including erythroid colony (A), multipotent colony (B), macrophage colony (C), and granulocyte colony (D). Cells comprising the erythroid colonies express erythroid marker Glycophorin A, but lack pan-leukocyte marker CD45 and myelomonocytic markers CD33 and CD 13 (E). Cells from the macrophage colonies or granulocyte colonies express CD45, CD33, and CD13, but lack glycophorin A (F). (Please see the companion CD for the color version of this figure.)

Fig. 3. Hematopoietic colonies from human embryoid bodies (hEB)-derived CD45+ cells. Representative colonies from hEB-derived CD45+ cells are depicted, including erythroid colony (A), multipotent colony (B), macrophage colony (C), and granulocyte colony (D). Cells comprising the erythroid colonies express erythroid marker Glycophorin A, but lack pan-leukocyte marker CD45 and myelomonocytic markers CD33 and CD 13 (E). Cells from the macrophage colonies or granulocyte colonies express CD45, CD33, and CD13, but lack glycophorin A (F). (Please see the companion CD for the color version of this figure.)

2. All tissue culture protocols must be performed under sterile conditions. Prewarm all media required at 37°C before use. Do not warm unnecessary media, because certain components in the media might be sensitive to temperature. Keep all fluoro-chrome-conjugated MAbs in the dark. All staining steps using fluorochromes should be performed under protection from light. For all PCR experiments, wear gloves, clean bench and all equipment with RNase away solution before use, and employ new stocks of disposable RNase-free materials (tips, tubes) and reagents (see Subheading 2.7.).

3. Compact hESC colonies are crucial for hEB formation. When dissociation of hESC to form hEBs, determine the appropriate incubation time by examining the colonies under microscope, as incubation time will vary between different batches of collagenase IV and hESC lines. In addition, avoid vigorous pipetting (see Subheading 3.1.).

4. Do not fix the cells. Fixation step will increase background and cause artefacts (see Subheading 3.5.1.1.).

5. To avoid extrusion of the nucleus and distortion of cells' morphology, do not use higher speed or longer centrifugation time (see Subheading 3.5.2.2.).

6. We use GeneQuant II to measure total amount of RNA (see Subheading 3.5.3.2.1.).

7. Careful removal of supernatants and air-dry steps are critical to eliminate previously used reagents that could inhibit the reverse transcription reaction (see Subheading

8. For each PCR reaction, P-actin PCR product is amplified in parallel (see Subheading

9. Before injection, shake Avertin (2.5% working solution) vigorously because the solution is water insoluble (see Subheading 3.6.1.).

10. The IBMT technique requires practice. Most failures come from: inserting the needle when your index finger fails to identify the femur orientation; moving the 27-gage needle under the skin before "drilling" the femur (poor alignment of the entrance of skin and bone) causing the 28-gage needle to miss the bone entrance of the femur channel; or changing the femur position during the procedure (see Subheading 3.6.1.).

11. The criteria for mouse engraftment are the presence of human DNA in both the transplanted femur and nontransplanted bone marrows (see Subheading 3.6.4.).

References

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2 Schuldiner, M., Yanuka, O., Itskovitz-Eldor, J., Melton, D. A, and Benvenisty, N. (2000) Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells. Proc. Natl. Acad. Sci. USA 97,11307-11312.

3 Itskovitz-Eldor, J., Schuldiner, M., Karsenti, D., et al. (2000) Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers. Mol. Med. 6, 88-95.

4 Bhatia, M. (2003) The ultimate source of human hematopoietic stem cells: thinking outside the marrow. Cloning Stem Cells 5, 89-97.

5 Chadwick, K., Wang, L., Li, L., et al. (2003) Cytokines and BMP-4 promote hematopoietic differentiation of human embryonic stem cells. Blood 102, 906-915.

6 Cerdan, C., Rouleau, A., and Bhatia, M. (2004) VEGF-A165 augments erythropoietic development from human embryonic stem cells. Blood 103, 2504-2512.

7 Wang, L., Li, L., Shojaei, F., et al. (2004) Endothelial and hematopoietic cell fate of human embryonic stem cells originates from primitive endothelium with hemangioblastic properties. Immunity 20, 31-41.

8 Dick, J. E., Bhatia, M., Gan, O., Kapp, U., and Wang, J. C. (1997) Assay of human stem cells by repopulation of NOD/SCID mice. Stem Cells 15,199-203; discussion 204-207.

9 Wang, L., Menendez, P., Shojaei, F., et al. (2005) Generation of hematopoietic repopulating cells from human embryonic stem cells independent of ectopic HOXB4 expression. J. Exp. Med. 201, 1603-1614.

10 Bhatia, M., Bonnet, D., Kapp, U., Wang, J. C., Murdoch, B., and Dick, J. E. (1997) Quantitative analysis reveals expansion of human hematopoietic repopulating cells after short-term ex vivo culture. J. Exp. Med. 186, 619-624.

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