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Blood, Vol. 106, Issue 3, 860-870, August 1, 2005
Hematopoietic differentiation of human embryonic stem cells progresses through sequential hematoendothelial, primitive, and definitive stages resembling human yolk sac development
Blood Zambidis et al.
106: 860
Supplemental materials for: Zambidis et al, Vol. 106, Issue 3, 922-924
Files in this Data Supplement:
- Figure S1. hESCs differentiate into cystic human embryoid bodies (hEBs) capable of efficient hematopoietic differentiation (JPEG, 67 KB)
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(A) Differentiation schematic: hESCs were grown to ~80%-90% confluency in standard serum-free conditions, cultured an additional 24 hours with “adaptation medium” as described in “Materials and methods” (1), and then transferred to nonadherent plates in semisolid methylcellulose differentiation medium at high density (2). Four days later, formed hEBs were collected, replated in liquid differentiation medium, and (3) cultured an additional 4-30 days with medium changes every 3-4 days. Pooled hEBs were enzymatically-dissociated into single-cell suspensions (4), analyzed by FACS or qRT-PCR, or assayed in serum-free semisolid medium (5) with hematopoietic growth factors for CFC potential. (B) hEBs mature over 2-25 days into large complex cellular aggregates, which develop cystic internal structures by 8-12 days. (C) Kinetic qRT-PCR analysis of OCT-3/4 expression during progressive hEB differentiation. Expression levels of OCT-3/4 in hEB cells were compared to RNA from purified, feeder-free hESCs. Expression levels were normalized/quantitated using the 2-ΔΔCT method as described in “Materials and methods.” Day 0 represents values for undifferentiated hESCs.
- Figure S2. hEB cells with a CD45-CD31+CD34+ phenotype possess definitive hematopoietic potential (JPEG, 113 KB)
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CD34 expression peaks in hEB cells between days ~12 and 15 of differentiation, approximately one week prior to detectable CD45 hEB expression (days 16-30; Figure 1). CD34 expression in native (fixed with 4% paraformaldehyde and sectioned) hEBs is found primarily on elongated cells (A, top), and often line hEB cysts (A, bottom). CD31 expression in native hEBs revealed a similar pattern (not shown). The majority of CD34+ hEB cells costained for CD31 (B, top: horizontal axis, FITC; vertical axis, PE) at all time points of differentiation. FACS-sorted CD45-CD34+CD31+ from day 9-10 hEB (CD45-negativity verified by RT-PCR; B, bottom), were assayed for hematopoietic potential by coculture on OP9 feeder cells29 in the presence of human growth factors. The use of an H1 hESC line constitutively expressing EF1α-EGFP (lentivirally transduced)45,73 allowed discrimination between hEB-derived and murine stromal cells (C, top). Day 9-10 CD45-CD34+ hEB cells were cocultured in liquid differentiation medium (see “Materials and methods”) on irradiated (3000 rad) OP9 layers with a growth factor cocktail (GF) consisting of 100 ng/mL Flt3L, 100 ng/mL SCF, 100 ng/mL TPO, 20 ng/mL IL-3, 20 ng/mL IL-6. 50 ng/mL GM-CSF, 50 ng/mL G-CSF, 5 ng/mL IL-2, 20 ng/mL IL-7, 10 ng/mL IL-15, and 2 U/mL EPO for one week and differentiating hematopoietic clusters were pooled and assayed for CFC potential (C, bottom). Alternatively, hEB/OP9 cocultures were further passaged onto fresh OP9 layers and allowed to further differentiate another week in the presence of GF cocktail (D, right). Differentiating cultures were then evaluated for expression of CD45 (panhematopoietic), CD13/CD33 (myeloid), CD71/glycophorin A (erythroid), or CD56 (NK cell). Cells from differentiating liquid hEB/OP9 cultures were sorted on CD45 (D, left) and evaluated for morphology by Wright-Giemsa on cytospun slides, which revealed definitive-type myeloid cells.
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