title: Inducible Gata1 suppression expands megakaryocyte-erythroid progenitors from embryonic stem cells.
abstract: Transfusion of donor-derived platelets is commonly used for thrombocytopenia, which results from a variety of clinical conditions and relies on a constant donor supply due to the limited shelf life of these cells. Embryonic stem (ES) and induced pluripotent stem (iPS) cells represent a potential source of megakaryocytes and platelets for transfusion therapies; however, the majority of current ES/iPS cell differentiation protocols are limited by low yields of hematopoietic progeny. In both mice and humans, mutations in the gene-encoding transcription factor GATA1 cause an accumulation of proliferating, developmentally arrested megakaryocytes, suggesting that GATA1 suppression in ES and iPS cell-derived hematopoietic progenitors may enhance megakaryocyte production. Here, we engineered ES cells from WT mice to express a doxycycline-regulated (dox-regulated) shRNA that targets Gata1 transcripts for degradation. Differentiation of these cells in the presence of dox and thrombopoietin (TPO) resulted in an exponential (at least 10¹³-fold) expansion of immature hematopoietic progenitors. Dox withdrawal in combination with multilineage cytokines restored GATA1 expression, resulting in differentiation into erythroblasts and megakaryocytes. Following transfusion into recipient animals, these dox-deprived mature megakaryocytes generated functional platelets. Our findings provide a readily reproducible strategy to exponentially expand ES cell-derived megakaryocyte-erythroid progenitors that have the capacity to differentiate into functional platelet-producing megakaryocytes.
authors.forename: Ji-Yoon
authors.initials: JY
authors.lastname: Noh
authors.forename: Shilpa
authors.initials: S
authors.lastname: Gandre-Babbe
authors.forename: Yuhuan
authors.initials: Y
authors.lastname: Wang
authors.forename: Vincent
authors.initials: V
authors.lastname: Hayes
authors.forename: Yu
authors.initials: Y
authors.lastname: Yao
authors.forename: Paul
authors.initials: P
authors.lastname: Gadue
authors.forename: Spencer K
authors.initials: SK
authors.lastname: Sullivan
authors.forename: Stella T
authors.initials: ST
authors.lastname: Chou
authors.forename: Kellie R
authors.initials: KR
authors.lastname: Machlus
authors.forename: Joseph E
authors.initials: JE
authors.lastname: Italiano
authors.forename: Michael
authors.initials: M
authors.lastname: Kyba
authors.forename: David
authors.initials: D
authors.lastname: Finkelstein
authors.forename: Jacob C
authors.initials: JC
authors.lastname: Ulirsch
authors.forename: Vijay G
authors.initials: VG
authors.lastname: Sankaran
authors.forename: Deborah L
authors.initials: DL
authors.lastname: French
authors.forename: Mortimer
authors.initials: M
authors.lastname: Poncz
authors.forename: Mitchell J
authors.initials: MJ
authors.lastname: Weiss
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D002454:Cell Differentiation / Q000187:drug effects* / Q000235:genetics; D004318:Doxycycline / Q000494:pharmacology*; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D050982:GATA1 Transcription Factor / Q000096:biosynthesis* / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics; D006801:Humans; D055015:Megakaryocyte-Erythroid Progenitor Cells / Q000166:cytology / Q000378:metabolism*; D008533:Megakaryocytes / Q000166:cytology / Q000378:metabolism; D051379:Mice; D013926:Thrombopoietin / Q000494:pharmacology*
chemical_list: D000900:Anti-Bacterial Agents; D050982:GATA1 Transcription Factor; C494689:Gata1 protein, mouse; D013926:Thrombopoietin; D004318:Doxycycline