Figure 1.
Modification of ESC gene and surface marker expression during in vitro differentiation in fully defined medium.
(A) Oct-4 and Brachyury mRNA levels were assessed by quantitative RT-PCR at the onset of the differentiation (d0) and 4 (d4) and 8 (d8) days later. Data were normalized with Gus-B housekeeping gene expression (Representative data of 4 independent experiments). (B) CD34 expression was assessed by flow cytometry upon 8 days of differentiation before (black line, left panel) and after (black line, right panel) MACS selection. Grey profiles represent CD34 expression before hematopoietic differentiation. (C) Analysis of relevant markers was performed on MACS-enriched ES34+ after 8 days of differentiation and compared to CB34+ sorted cells. (D) SCL/Tal1 and Runx1 mRNA levels on indicated populations were assessed by quantitative RT-PCR and RT-PCR, respectively. Data are representative of 4 independent experiments.
Figure 2.
Cells differentiated from ESC are functional hematopoietic progenitors.
(A) Colony morphology upon 14 days of methylcellulose culture. Erythroid CFU (upper panels) and myeloid CFU (lower panels) are shown (initial magnification ×100). (B) CFU frequencies of CB34+, ES34+ and ES34−. Overall frequencies (left panel) of CB and ESC-derived cells, and a close-up of ES34+ and ES34− frequencies (right panel) displayed with an enlarged scale to help reading are depicted. Grey columns represent erythroids and white columns, myeloids. Bars are SD's. n = 8, 12 and 5 for CB, ES34+ and ES34− respectively. (C) FACS analysis of cells recovered after the CFU assay derived from CB34+ (upper panels) and ES34+ (lower panels). Myeloid (CD45, CD11b, CD14) and erythroid (GPA) cell surface markers are depicted. Quadrants were established using appropriate isotype controls and percentages are indicated. (D) Qualitative analysis of embryonic (ε), fetal (γ) and adult (β) globin mRNAs expression by RT-PCR of cell suspensions recovered after the CFU assays, derived from the indicated populations.
Figure 3.
Hematopoietic differentiation of human iPSC.
(A) Oct-4 and Brachyury mRNA expression were assessed by quantitative PCR at the onset of the differentiation (d0), and 4 (d4) and 8 (d8) days later. The expression of the Gus-B housekeeping gene was used for normalization (representative data of 3 experiments). (B) CD34 and KDR expression was assessed by flow cytometry. ESC are shown in the upper panels and iPSC in the lower panels. Expression prior to differentiation (d0, left panel) and after 8 days (d8, right panel) is depicted. (C) CFU morphology was assessed upon 14 days of methylcellulose culture of iPSC-derived cells. Erythroid CFUs (upper panels) and myeloid CFUs (lower panels) were observed in these cultures and CB-derived cells are shown (initial magnification ×100).
Table 1.
Indicative frequencies of hematopoietic colonies recovered after clonogenic cultures of sorted iPSC.