Project description:Normal donors BM CD34+ cells were differentiated in thrombopoietin-treated liquid suspension cultures. Briefly, CD34+ cells (80,000 /mL) were resuspended in a serum-free medium in the presence of 100 ng/mL of human recombinant thrombopoietin (TPO; Genzyme, Boston, MA). Every 3 days, viable cells were scored by Trypan blue dye exclusion and cultures were amplified with fresh serum-free medium. Each well was then supplemented with 100 ng/mL TPO. After 14 to 16 days of liquid culture, CD34-derived megakaryocytes (MKs) were purified by means of an anti-CD41a monoclonal antibody (MoAb) (Dako, Milan, Italy) directed against the glycoproteic *IIb-*3 complex and immunobeads (MPC 450 Dynabeads; Dynal, Oslo, Norway), as previously described (27). The purity of MKs was determined for each isolation by indirect immunofluorescence, using an anti-CD41b MoAb which reacts with a different epitope of *IIb-*3 subunit (Immunotech Inc., Westbrook, ME), followed by a goat anti-mouse IgG, covalently linked to fluorescein (Becton Dickinson, San Jose, CA). Essential Throbocytemia BM CD34+ cells were differentiated in thrombopoietin-treated liquid suspension cultures. Briefly, CD34+ cells (80,000 /mL) were resuspended in a serum-free medium in the presence of 100 ng/mL of human recombinant thrombopoietin (TPO; Genzyme, Boston, MA). Every 3 days, viable cells were scored by Trypan blue dye exclusion and cultures were amplified with fresh serum-free medium. Each well was then supplemented with 100 ng/mL TPO. After 14 to 16 days of liquid culture, CD34-derived megakaryocytes (MKs) were purified by means of an anti-CD41a monoclonal antibody (MoAb) (Dako, Milan, Italy) directed against the glycoproteic *IIb-*3 complex and immunobeads (MPC 450 Dynabeads; Dynal, Oslo, Norway), as previously described (27). The purity of MKs was determined for each isolation by indirect immunofluorescence, using an anti-CD41b MoAb which reacts with a different epitope of *IIb-*3 subunit (Immunotech Inc., Westbrook, ME), followed by a goat anti-mouse IgG, covalently linked to fluorescein (Becton Dickinson, San Jose, CA). Keywords: other

Project description:Normal donors BM CD34+ cells were differentiated in thrombopoietin-treated liquid suspension cultures. Briefly, CD34+ cells (80,000 /mL) were resuspended in a serum-free medium in the presence of 100 ng/mL of human recombinant thrombopoietin (TPO; Genzyme, Boston, MA). Every 3 days, viable cells were scored by Trypan blue dye exclusion and cultures were amplified with fresh serum-free medium. Each well was then supplemented with 100 ng/mL TPO. After 14 to 16 days of liquid culture, CD34-derived megakaryocytes (MKs) were purified by means of an anti-CD41a monoclonal antibody (MoAb) (Dako, Milan, Italy) directed against the glycoproteic *IIb-*3 complex and immunobeads (MPC 450 Dynabeads; Dynal, Oslo, Norway), as previously described (27). The purity of MKs was determined for each isolation by indirect immunofluorescence, using an anti-CD41b MoAb which reacts with a different epitope of *IIb-*3 subunit (Immunotech Inc., Westbrook, ME), followed by a goat anti-mouse IgG, covalently linked to fluorescein (Becton Dickinson, San Jose, CA). Essential Throbocytemia BM CD34+ cells were differentiated in thrombopoietin-treated liquid suspension cultures. Briefly, CD34+ cells (80,000 /mL) were resuspended in a serum-free medium in the presence of 100 ng/mL of human recombinant thrombopoietin (TPO; Genzyme, Boston, MA). Every 3 days, viable cells were scored by Trypan blue dye exclusion and cultures were amplified with fresh serum-free medium. Each well was then supplemented with 100 ng/mL TPO. After 14 to 16 days of liquid culture, CD34-derived megakaryocytes (MKs) were purified by means of an anti-CD41a monoclonal antibody (MoAb) (Dako, Milan, Italy) directed against the glycoproteic *IIb-*3 complex and immunobeads (MPC 450 Dynabeads; Dynal, Oslo, Norway), as previously described (27). The purity of MKs was determined for each isolation by indirect immunofluorescence, using an anti-CD41b MoAb which reacts with a different epitope of *IIb-*3 subunit (Immunotech Inc., Westbrook, ME), followed by a goat anti-mouse IgG, covalently linked to fluorescein (Becton Dickinson, San Jose, CA).

Project description:Background: Children with Down syndrome (DS) are predisposed to acute megakaryoblastic leukemia (AMKL, or AML M7) and a related myeloid disorder, referred to as transient myeloproliferative disorder (TMD), or transient leukemia (TL). Recently, acquired mutations in the erythroid/megakaryocytic lineage-specific transcription factor GATA-1 have been identified in megakaryoblasts from virtually all DS patients with AMKL (DS-AMKL), as well as in nearly all DS neonates with TMD. These mutations prevent synthesis of full-length GATA-1, but lead to synthesis of a truncated GATA-1 protein (GATA-1s) that lacks the N-terminal transactivation domain. To determine the in vivo requirement of GATA-1 N-terminal domain in hematopoiesis and to model DS megakaryocytic leukemia initiated by GATA-1 mutation, we generated a GATA-1 knockin allele with a truncation at this domain (GATA-1deltaN). ES cells bearing this mutant allele generated a unique type of large Thrombopoietin (Tpo)-dependent megakaryocytes-containing colonies, when differentiated in vitro. Fetal livers from mice carrying this mutant allele contained elevated numbers of CD41+ cells throughout embryonic development, especially during mid-gestation. In in vitro hematopoietic colony assays, mutant cells from yolk sacs and mid-gestation fetal livers gave rise to a large number of macroscopic hyperproliferative megakaryocytic colonies (CFU-MKs). Consistent with that, when mutant fetal liver progenitors were cultured in liquid medium in the presence of Tpo, the derived megakaryocytes displayed marked hyperproliferation and grew for longer period of time than WTs. Specific aims: To understand the molecular basis for the megakaryocytic hyperproliferation phenotype of the GATA-1deltaN mutants, we plan to profile gene expression patterns of fetal liver-derived primary megakaryocytes and megakaryocytic progenitors from the GATA-1deltaN mutants and compare them with WTs, as well as GATA-1deltaNeodeltaHS mutants (GATA-1– megakaryocytes). Experimental design: E12.5 fetal livers were harvested from either GATA-1 WT and mutant embryos. Single cell suspensions were made from these livers and the cells were cultured in liquid medium in the presence of Tpo for 3 days. On day 3, primary megakaryocytes were enriched by a BSA gradient, and further enriched by anti-CD41 antibody and magnetic microbeads. Megakaryocytic progenitors were collected by FACS sorting for small cells in the same culture that express low level of CD41. Total RNAs were then prepared from these two populations and submitted for microarray profiling. Conclusions: When examining genes that are normally upregulated upon megakaryocyte differentiation (many of this class are megakaryocyte-specific genes), we found in GATA-1deltaN megakaryocytes, the majority of these genes are upregulated, although not to the full extent as seen in WTs. In contrast, this class of genes is largely not upregulated in GATA-1deltaNeodeltaHS megakaryocytes, which are blocked at an immature stage of differentiation. Genes that are downregulated during megakaryocyte differentiation include those that are normally repressed by GATA-1. Among genes that are not properly downregulated in GATA-1deltaN megakaryocytes, five transcription factors (Myc, Myb, GATA-2, PU.1, Ikaros) are of particular interest. Inadequate GATA-1s-mediated repression of transcription factors required for proliferation of hematopoietic progenitors (Myc, Myb and GATA-2) and for specifying lineage choices other than megakaryocyte/erythrocyte (PU.1 and Ikaros) may contribute to hyperproliferation of GATA-1deltaN megakaryocytes. Keywords = GATA-1 Keywords = megakaryocyte Keywords = mouse model Keywords: other

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Comparison of Mature Megakaryocytes Differentiated by TPO or Small Molecule TPO Mimetics

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