title: Generation of megakaryocytic progenitors from human embryonic stem cells in a feeder- and serum-free medium.
abstract: BACKGROUND
  The production of human platelets from embryonic stem cells in a defined culture system is a prerequisite for the generation of platelets for therapeutic use. As an important step towards this goal, we report the differentiation of human embryonic stem cells (hESCs) towards the megakaryocyte (Mk) lineage using a 'spin embryoid body' method in serum-free differentiation medium.
  
  
  RESULTS
  Immunophenotypic analyses of differentiating hESC identified a subpopulation of cells expressing high levels of CD41a that expressed other markers associated with the Mk lineage, including CD110, CD42b and CD61. Differentiated cells were sorted on the basis of their expression of CD41a, CD34 and CD45 and assessed for Mk colony formation, expression of myeloid and Mk genes and ability to endoreplicate DNA. In a collagen-based colony assay, the CD41a⁺ cells sorted from these differentiation cultures produced 100-800 Mk progenitors at day 13 and 25-160 Mk progenitors at day 20 of differentiation per 100,000 cells assayed. Differentiated Mk cells produced platelet-like particles which expressed CD42b and were activated by ADP, similar to platelets generated from precursors in cord blood. These studies were complemented by real time PCR analyses showing that subsets of cells enriched for CD41a⁺ Mk precursors expressed high levels of Mk associated genes such as PF4 and MPL. Conversely, high levels of myeloid and erythroid related transcripts, such as GATA1, TAL1/SCL and PU.1, were detected in sorted fractions containing CD34⁺ and CD45⁺ cells.
  
  
  CONCLUSIONS
  We describe a serum- and feeder-free culture system that enabled the generation of Mk progenitors from human embryonic stem cells. These cells formed colonies that included differentiated Mks that fragmented to form platelet-like particles. This protocol represents an important step towards the generation of human platelets for therapeutic use.
authors: Monash Immunology and Stem Cell Laboratories, Monash University, Clayton, Victoria, Australia. Marjorie@cc.huji.ac.il
authors: Marjorie
authors: M
authors: Pick
authors: Lisa
authors: L
authors: Azzola
authors: Elissa
authors: E
authors: Osborne
authors: Edouard G
authors: EG
authors: Stanley
authors: Andrew G
authors: AG
authors: Elefanty
mesh_terms: D000244:Adenosine Diphosphate / Q000494:pharmacology; D015703:Antigens, CD / Q000378:metabolism; D001792:Blood Platelets / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000187:drug effects; D016895:Culture Media, Serum-Free; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D061252:Feeder Cells; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D008533:Megakaryocytes / Q000166:cytology* / Q000187:drug effects; D010978:Platelet Factor 4 / Q000235:genetics; D011123:Polyploidy; D053628:Receptors, Thrombopoietin / Q000235:genetics; D013997:Time Factors
chemical_list: D015703:Antigens, CD; D016895:Culture Media, Serum-Free; D053628:Receptors, Thrombopoietin; C082218:MPL protein, human; D010978:Platelet Factor 4; D000244:Adenosine Diphosphate