A definitive role of Shp-2 tyrosine phosphatase in mediating embryonic stem cell differentiation and hematopoiesis.
Homozygous mutant (Shp-2Delta46-110) 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-2Delta46-110 ES cells by examining early points of differentiation, conducting leukemia inhibitory 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-2Delta46-110, and reconstituted ES cells to mesoderm, by measuring brachyury expression, to hemangioblasts, 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 hemangioblasts was lower in Shp-2Delta46-110 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-2Delta46-110 ES cells than in WT and reconstituted cells. The increased phospho-Stat3 levels correlated with increased Shp-2Delta46-110 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.
Chan
Rebecca J
RJ
Department of Pediatrics, Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, USA.
Johnson
Scott A
SA
Li
Yanjun
Y
Yoder
Mervin C
MC
Feng
Gen-Sheng
GS
D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D002454:Cell Differentiation / Q000502:physiology; D002460:Cell Line; D002470:Cell Survival / Q000502:physiology; D050822:Cytokine Receptor gp130; D004268:DNA-Binding Proteins / Q000378:metabolism; D005333:Fetus; D006131:Growth Inhibitors / Q000378:metabolism*; D006410:Hematopoiesis / Q000502:physiology*; D015850:Interleukin-6; D047908:Intracellular Signaling Peptides and Proteins; D053605:Leukemia Inhibitory Factor; D008222:Lymphokines / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism; D008648:Mesoderm / Q000166:cytology / Q000201:enzymology; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D016296:Mutagenesis / Q000502:physiology; D010641:Phenotype; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D017027:Protein Tyrosine Phosphatases / Q000235:genetics / Q000378:metabolism*; D050796:STAT3 Transcription Factor; D013234:Stem Cells / Q000166:cytology* / Q000201:enzymology*; D015534:Trans-Activators / Q000378:metabolism
D015703:Antigens, CD; D004268:DNA-Binding Proteins; D006131:Growth Inhibitors; C494200:Il6st protein, mouse; D015850:Interleukin-6; D047908:Intracellular Signaling Peptides and Proteins; D053605:Leukemia Inhibitory Factor; C507838:Lif protein, mouse; D008222:Lymphokines; D008562:Membrane Glycoproteins; D050796:STAT3 Transcription Factor; C494087:Stat3 protein, mouse; D015534:Trans-Activators; D050822:Cytokine Receptor gp130; D020928:Mitogen-Activated Protein Kinases; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D017027:Protein Tyrosine Phosphatases; C516725:Ptpn11 protein, mouse