RESUMEN
Allogeneic hematopoietic stem cell (HSC) transplantations from umbilical cord blood or autologous HSCs for gene therapy purposes are hampered by limited number of stem cells. To test the ability to expand HSCs in vitro prior to transplantation, two growth factor cocktails containing stem cell factor, thrombopoietin, fms-related tyrosine kinase-3 ligand (STF) or stem cell factor, thrombopoietin, insulin-like growth factor-2, fibroblast growth factor-1 (STIF) either with or without the addition of angiopoietin-like protein-3 (Angptl3) were used. Culturing HSCs in STF and STIF media for 7 days expanded long-term repopulating stem cells content in vivo by â¼6-fold and â¼10-fold compared to freshly isolated stem cells. Addition of Angptl3 resulted in increased expansion of these populations by â¼17-fold and â¼32-fold, respectively, and was further supported by enforced expression of Angptl3 in HSCs through lentiviral transduction that also promoted HSC expansion. As expansion of highly purified lineage-negative, Sca-1+, c-Kit+ HSCs was less efficient than less pure lineage-negative HSCs, Angptl3 may have a direct effect on HCS but also an indirect effect on accessory cells that support HSC expansion. No evidence for leukemia or toxicity was found during long-term follow up of mice transplanted with ex vivo expanded HSCs or manipulated HSC populations that expressed Angptl3. We conclude that the cytokine combinations used in this study to expand HSCs ex vivo enhances the engraftment in vivo. This has important implications for allogeneic umbilical cord-blood derived HSC transplantations and autologous HSC applications including gene therapy.
Asunto(s)
Angiopoyetinas/genética , Proliferación Celular/genética , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Proteína 3 Similar a la Angiopoyetina , Proteínas Similares a la Angiopoyetina , Angiopoyetinas/metabolismo , Angiopoyetinas/farmacología , Animales , Antígenos Ly/metabolismo , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Femenino , Factor 1 de Crecimiento de Fibroblastos/farmacología , Citometría de Flujo , Vectores Genéticos/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Células HeLa , Trasplante de Células Madre Hematopoyéticas/métodos , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Factor II del Crecimiento Similar a la Insulina/farmacología , Lentivirus/genética , Masculino , Proteínas de la Membrana/metabolismo , Ratones Endogámicos BALB C , Proteínas Proto-Oncogénicas c-kit/metabolismo , Factor de Células Madre/farmacología , Trombopoyetina/farmacología , TransfecciónRESUMEN
Here, we show that transcription factors bound to regulatory sequences can be identified by purifying these unique sequences directly from mammalian cells in vivo. Using targeted chromatin purification (TChP), a double-pull-down strategy with a tetracycline-sensitive "hook" bound to a specific promoter, we identify transcription factors bound to the repressed γ-globin gene-associated regulatory regions. After validation of the binding, we show that, in human primary erythroid cells, knockdown of a number of these transcription factors induces γ-globin gene expression. Reactivation of γ-globin gene expression ameliorates the symptoms of ß-thalassemia and sickle cell disease, and these factors provide potential targets for the development of therapeutics for treating these patients.
