期刊
BLOOD
卷 102, 期 6, 页码 2074-2080出版社
AMER SOC HEMATOLOGY
DOI: 10.1182/blood-2003-04-1171
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- NCI NIH HHS [F32 CA 84677, R01 CA 78608] Funding Source: Medline
- NHLBI NIH HHS [R01 HL 63169] Funding Source: Medline
- NIGMS NIH HHS [R01 GM 53660] Funding Source: Medline
Homozygous mutant (Shp-2(Delta45-110)) embryonic stem (ES) cells exhibit decreased hematopoiesis; however, the point at which Shp-2 is critical for ES cell differentiation to hematopoietic cells is unknown. We characterized the differentiation defect of Shp-2(Delta46-110) ES cells by examining early points of differentiation, conducting leukemia inhibilory factor (LIF)-stimulated biochemical analysis, and performing in vitro reconstitution studies with wild-type (WT) Shp-2. ES cell in vitro differentiation assays were used to compare the differentiation of WT, Shp-2(Delta46-110), and reconstituted ES cells to mesoderm, by measuring brachyury expression, to hemangloblasts, by measuring blast colony-forming cell (BL-CFC) formation and flk-1 expression, and to hematopoietic progenitor colony-forming cells, by performing secondary plating assays. LIF-stimulated phospho-Stat3 (known, to be critical for ES cell self-renewal and maintenance of an undifferentiated state) and phospho-Erk levels were examined by immunoblotting. ES cell survival, using annexin V staining, and secondary embryoid body (EB) formation were also evaluated. Differentiation to both mesoderm and heman-gioblasts was lower in Shp-2(Delta46-110) cells compared to WT cells. On reconstitution with WT Shp-2, expression of brachyury and flk-1 and differentiation to hemangioblasts and primitive and definitive hematopoietic progenitors were restored. LIF-stimulated phospho-Stat3 levels were higher, whereas phospho-Erk levels were lower in Shp-2(Delta46-110) ES cells than in WT and reconstituted cells. The increased phospho-Stat3 levels correlated with increased Shpr2(Delta46-110) ES cell secondary EB formation and survival. We conclude that normal Shp-2 function is critical for the initial step of ES cell differentiation to mesoderm and to hemangioblasts and acts within the LIF-gp130-Stat3 pathway to maintain a proper balance of ES cell differentiation, pluripotency, and apoptosis.
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