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. Author manuscript; available in PMC: 2012 Nov 27.
Published in final edited form as: Reprod Biomed Online. 2010 Apr 24;21(2):196–205. doi: 10.1016/j.rbmo.2010.04.017

Figure 5.

Figure 5

Expression of embryonic, fetal and adult haemoglobins in differentiated primitive and definitive pluripotent preimplantation genetic diagnosis-derived human embryonic stem cell line SC233 erythroid cells. (A) Comparative haemoglobin gene expression analysis of normal (H1) and mutant (SC233) erythroid cells by quantitative real-time PCR (qRT-PCR). Day-10 embryoid body primitive colonies (EryP), day-17 embryoid body/human mesenchymal cell co-culture erythroid colonies (BFU-e) and BFU-e colonies derived from control CD34+ neonatal cord blood cells were picked from methylcellulose cultures and pooled for RNA isolation and qRT-PCR. Expression of embryonic haemoglobins (epsilon, zeta), fetal haemoglobins (alpha, gamma) and adult haemoglobins (alpha, beta) was quantified using the 2 ΔT method (Zambidis et al., 2005, 2008). Results are shown as fold differences relative to haemoglobin expressions in control cord blood BFU-e (normalized to a value of 1). Left inset = EryP; right inset = BFU-e. (B) Expression of sickle cell haemoglobin in SC233 erythroid cells. RNA derived from SC233 BFU-e, H1 BFU-e, normal donor peripheral blood and SCD patient peripheral blood was isolated and analysed by RT-PCR. A 687 bp PCR product was amplified using beta-globin locus primers. PCR products digested with DdeI and fractionated on agarose gels with or without enzyme digestion (–/+). (C) Haematopoietic differentiation of control H1 or SC233 ACE/CD143+ day-10 embryoid body cells on OP9 stromal layer cells produced morphologically comparably definitive-type erythro-myeloid differentiation several weeks later. The amounts of CD45+ myeloid and megakaryocytic and CD235a+ definitive-type erythroid cells are comparably robust from both H9 and SC233 embryoid body cells. Both embryoid body cells generated definitive-type erythrocytes on OP9 stromal cells (some with evidence of enucleation; green arrow) that had increased liver-like fetal haemoglobin expression, but minor amounts of adult haemoglobin expression. BFU-e = burst-forming unit erythroid; EryP = primitive erythroid CFU; H1 = WA01 control; HbA = adult haemoglobin; HbF = fetal haemoglobin; HbS = sickle cell haemoglobin; hEB = human embryoid body; hMSC – human mesenchymal cell; SCD = sickle cell disease; WT = wild type.