374. Knockdown of Shp-2 Expression by siRNA in Mouse Embryonic Stem Cell Derived EB Cells Affects Their Hematopoietic Development

Shp-2 is a member of a small subfamily of cytoplasmic Src homology 2 (SH2) domain-containing protein tyrosine phosphatases. To address the question of whether Shp-2 is required for normal hemangioblast, primitive, and definitive hematopoietic development, we used Shp-2 siRNA to knockdown Shp-2 gene expression in embryonic stem (ES) derived embryoid body (EB) cells. We found that a pool of (but not single siRNA) Shp-2 siRNA at a concentration of 50 nM/ml was efficient to knockdown Shp-2 expression in EB cells. The density of Shp-2 protein in western blot assay was decreased about 10 fold in Shp-2 siRNA treated cells compared with cells treated with a scrambled control Shp-2 siRNA. The frequency of growth factor-dependent hemangioblast formation were decreased 5 fold following cell treatment with Shp-2 siRNA (112.6 |[plusmn]| 11.7 vs 31 |[plusmn]| 1), but not by scrambled Shp-2 siRNA (112.6 |[plusmn]| 11.7 vs 114 |[plusmn]| 12). Basic fibroblast growth factor (bFGF) promotes hemangioblast development. As Shp-2 is involved in the upstream signaling of bFGF receptor activation, we then asked whether Shp-2 was involved in this bFGF mediated hemangioblast effect. Addition of bFGF at doses of 10 and 20 ng/ml significantly increased (121.6 |[plusmn]| 4.7 vs 232.3 |[plusmn]| 10) and (121.6 |[plusmn]| 4.7 vs 281.3 |[plusmn]| 4.1) (compared with control siRNA treated cells) the frequency of hemangioblast formation. However, knockdown of Shp-2 gene expression by siRNA blocked the bFGF effect on hemangioblast development. In primitive hematopoietic assays, knockdown of Shp-2 expression in day-6 EB cells resulted in a (350 |[plusmn]| 12.5 vs 173 |[plusmn]| 16.3) 2 fold decrease in EPO-dependent Ery-P colonies, a (25 |[plusmn]| 2.3 vs 7.2 |[plusmn]| 1.8) 3 fold decrease in Ery-D colonies and a (23 |[plusmn]| 1.8 vs 4.1 |[plusmn]| 0.6) 5 fold decrease in GM-CSF-dependent CFU-GM colonies. Altogether, these data demonstrate that Shp-2 is required in hemangioblast, primitive, and definitive progenitor hematopoietic development. Shp-2 is also involved in bFGF regulation of hemangioblast development. We conclude that Shp-2 plays a critical role in hemangioblast, primitive, and definitive hematopoiesis.