title: Cytokine-free directed differentiation of human pluripotent stem cells efficiently produces hemogenic endothelium with lymphoid potential.
abstract: The robust generation of human hematopoietic progenitor cells from induced or embryonic pluripotent stem cells would be beneficial for multiple areas of research, including mechanistic studies of hematopoiesis, the development of cellular therapies for autoimmune diseases, induced transplant tolerance, anticancer immunotherapies, disease modeling, and drug/toxicity screening. Over the past years, significant progress has been made in identifying effective protocols for hematopoietic differentiation from pluripotent stem cells and understanding stages of mesodermal, endothelial, and hematopoietic specification. Thus, it has been shown that variations in cytokine and inhibitory molecule treatments in the first few days of hematopoietic differentiation define primitive versus definitive potential of produced hematopoietic progenitor cells. The majority of current feeder-free, defined systems for hematopoietic induction from pluripotent stem cells include prolonged incubations with various cytokines that make the differentiation process complex and time consuming. We established that the application of Wnt agonist CHIR99021 efficiently promotes differentiation of human pluripotent stem cells in the absence of any hematopoietic cytokines to the stage of hemogenic endothelium capable of definitive hematopoiesis.
  
  
  
  The hemogenic endothelium differentiation was accomplished in an adherent, serum-free culture system by applying CHIR99021. Hemogenic endothelium progenitor cells were isolated on day 5 of differentiation and evaluated for their endothelial, myeloid, and lymphoid potential.
  
  
  
  Monolayer induction based on GSK3 inhibition, described here, yielded a large number of CD31+CD34+hemogenic endothelium cells. When isolated and propagated in adherent conditions, these progenitors gave rise to mature endothelium. When further cocultured with OP9 mouse stromal cells, these progenitors gave rise to various cells of myeloid lineages as well as natural killer lymphoid, T-lymphoid, and B-lymphoid cells.
  
  
  
  The results of this study substantiate a method that significantly reduces the complexity of current protocols for hematopoietic induction, offers a defined system to study the factors that affect the early stages of hematopoiesis, and provides a new route of lymphoid and myeloid cell derivation from human pluripotent stem cells, thus enhancing their use in translational medicine.
authors.affiliation: Developmental Biology Program, Stanley Manne Children's Research Institute, Ann & Robert H. Lurie Children's Hospital of Chicago, Chicago, IL, USA.
authors.forename: Yekaterina
authors.initials: Y
authors.lastname: Galat
authors.affiliation: Department of Microbiology and Immunology, Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA.
authors.forename: Svetlana
authors.initials: S
authors.lastname: Dambaeva
authors.affiliation: Developmental Biology Program, Stanley Manne Children's Research Institute, Ann & Robert H. Lurie Children's Hospital of Chicago, Chicago, IL, USA.|Present Address: Department of Pediatrics, Division of Hematology & Oncology, Penn State Hershey College of Medicine, Hershey, PA, USA.
authors.forename: Irina
authors.initials: I
authors.lastname: Elcheva
authors.affiliation: Department of Pathology, Stanley Manne Children's Research Institute, Ann & Robert H. Lurie Children's Hospital of Chicago, Northwestern University Feinberg School of Medicine, Chicago, IL, USA.
authors.forename: Aaruni
authors.initials: A
authors.lastname: Khanolkar
authors.affiliation: Department of Microbiology and Immunology, Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA.
authors.forename: Kenneth
authors.initials: K
authors.lastname: Beaman
authors.affiliation: Department of Pediatrics, Developmental Biology Program, Stanley Manne Children's Research Institute, Ann & Robert H. Lurie Children's Hospital of Chicago, Northwestern University Feinberg School of Medicine, Chicago, IL, USA.
authors.forename: Philip M
authors.initials: PM
authors.lastname: Iannaccone
authors.affiliation: Department of Pathology, Developmental Biology Program, Stanley Manne Children's Research Institute, Ann & Robert H. Lurie Children's Hospital of Chicago, Northwestern University Feinberg School of Medicine, Chicago, IL, USA. v-galat@northwestern.edu.
authors.forename: Vasiliy
authors.initials: V
authors.lastname: Galat
mesh_terms: D000818:Animals; D001402:B-Lymphocytes / Q000166:cytology / Q000187:drug effects / Q000276:immunology; D002448:Cell Adhesion / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D018920:Coculture Techniques; D016895:Culture Media, Serum-Free / Q000737:chemistry / Q000494:pharmacology; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects* / Q000276:immunology; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D006410:Hematopoiesis / Q000235:genetics; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000276:immunology; D006801:Humans; D051379:Mice; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000276:immunology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011725:Pyridines / Q000494:pharmacology*; D011743:Pyrimidines / Q000494:pharmacology*; D013601:T-Lymphocytes / Q000166:cytology / Q000187:drug effects / Q000276:immunology
chemical_list: C473711:Chir 99021; D016895:Culture Media, Serum-Free; D047428:Protein Kinase Inhibitors; D011725:Pyridines; D011743:Pyrimidines; D038362:Glycogen Synthase Kinase 3
keywords: Glycogen synthase kinase 3 inhibitor; Hematopoietic cells; Hemogenic endothelium; Lymphocytes; Pluripotent stem cells