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. Author manuscript; available in PMC: 2011 May 1.
Published in final edited form as: Exp Hematol. 2010 Mar 3;38(5):403–416. doi: 10.1016/j.exphem.2010.02.011

Figure 2.

Figure 2

Modeling human hematopoiesis in vitro with human embryonic stem cell culture. (A) Hematopoietic development in embryoid bodies. Bright field images showing development of hEBs from undifferentiated hESCs (day 0) over time at 4-day intervals. Red squares outline merged inset images. (B) Real-time PCR analysis of hEB differentiation. The pluripotency markers OCT4, NANOG and SOX2 decrease in expression over time as pluripotent hESCs differentiate. The mesoderm markers T and MIXL1 show a dramatic spike in expression between days 4–8 before downregulating, implying that mesoderm is generated between these timepoints in this culture system. The human HSC markers CD34, CD43 and TAL1 peak in expression at day 12 (enclosed by dashed lines) of culture indicating definitive HSCs are being generated during this time window. Analysis of the expression of RUNX1 isoforms during hematopoietic development show that total RUNX1 expression and the RUNX1b isoform are consistent throughout the differentiation process but expression of RUNX1c is dynamic, peaking at day 12 in a similar pattern to that of HSC markers. (C) Wholemount in situ hybridization analysis of RUNX1 isoforms in hEBs. Sense controls show no background staining, but on a gross level no discernable difference can be discriminated between RUNX1b and RUNX1c. (D) Section analysis of stained embryoid bodies showed that at day 12 when there is the most differential expression, RUNX1b is broadly expressed at low levels while RUNX1c is specifically expressed in cells lining the developing cavities in the hEBs. By day 16, the expression profiles of RUNX1b and RUNX1c become highly overlapping. (E) Co-immunofluorescence analysis of stained day 12 hEBs for vascular (VE-CADHERIN) and HSC (CD34) markers. While RUNX1b was broadly expressed throughout the day 12 hEBs, and its expression did not generally correlate with VE-CADHERIN or CD34 expression. RUNX1c however was expressed in a subpopulation of CD34+ cells. (F,G) hEB differentiation in vitro mimics mouse hematopoietic development in vivo. (F) Merged RUNX1b in situ hybridization (purple staining) and immunofluorescence (CD34 – red; VE-CADHERIN – green; DAPI – blue) images of day 20 hEB showing clusters of hematopoietics cells budding off into the lumen. (G) Section of RUNX1b-stained in situ hybridization of AGM region of E11.5 mouse embryo showing presumptive HSCs budding off into the dorsal aorta.