β-catenin activation enhances hemangioblast formation.

<p>(A) (i) Scoring of the number of BL-CFC compared to the number of EB’s for each condition by day 8 of differentiation shows activation of the canonical Wnt pathway (DP-βC or BIO) results in higher percentage of BL-CFC compared to control cultures (Mean %, +/- SEM, n=3). (ii) Hemangioblasts/Early hematopoietic progenitors (CONDITION 2) were analysis for expression of mesodermal markers Flk1 and Brachyury by flow cytometry. (iii) RT-PCR analysis of key self-renewal and differentiation genes. (B) Flow cytometry analysis was performed on the hemangioblast/early hematopoietic progenitors to determine the percentage of cells expressing CD41, CD45, c-Kit, Sca1 and Flt3. Gating on the CD41⁺, CD45⁺ population revealed that these early hematopoietic progenitors also expressed the HSC markers Sca1 and c-Kit (Representative images shown, n=3). (C) (i) Total cell population from CONDITION 2 were analyzed for key HSC markers Sca1 and c-Kit by flow cytometry. (ii) The early hematopoietic progenitor populations (Sca1⁺c-Kit⁺ cells) were sorted and Immunofluorescence performed to confirm higher levels of total and active β-catenin in DP-βC –tet and E14 cells + BIO. (iii) RT-PCR analysis of key hematopoietic genes within the Sca-1⁺c-kit⁺ hematopoietic progenitor cells and (iv) erythroid/globin genes. The control cells (DP-βC + tet or E14 ES cells -BIO) were used as calibrators and the fold change was calculated using the 2 ^-ΔΔCT method (Mean of fold change +/- SEM, n=3).