title: A feeder-free hematopoietic differentiation system with generation of functional neutrophils from feeder- and cytokine-free primate embryonic stem cells.
abstract: We have established a novel feeder- and recombinant cytokine-free culture system for the maintenance of primate embryonic stem (ES) cells along with a feeder-free hematopoietic differentiation protocol for high efficiency CD45-positive cell production. In our system, cynomolgus monkey ES cells were properly maintained in an undifferentiated state with high immature marker expressions and teratoma-producing activities. Embryoid bodies (EBs) were generated in the presence of serum and cytokine cocktail and subjected to attachment culture on gelatin-coated plates. After about 2 weeks, a sac-like structure filled with abundant round cells emerged at the center of flattened EB. Then total cells were collected and transferred onto new gelatin-coated plates, where cells were firmly attached and actively proliferated to confluence. After another few days culture, abundant floating cells were detected in the culture supernatant. These cells expressed high levels of CD45 (>90%), while adherent cells expressed low levels of CD45 (<10%). The former consisted of various differentiated stages of myeloid cells from immature myeloblasts to mature polymorphonuclear neutrophils and macrophages. Although the percentages of neutrophils varied between 10 to 20 depending on experiments, their mature phenotype was reproducibly confirmed by specific staining and functional assays. Our protocol provides the minimum essence for primate ES cell maintenance and hematopoietic differentiation that is beneficial from economical and clinical points of view.
authors.affiliation: Department of Hematology, Research Institute, International Medical Center of Japan, Tokyo, Japan.
authors.forename: Masako
authors.initials: M
authors.lastname: Nakahara
authors.forename: Satoko
authors.initials: S
authors.lastname: Matsuyama
authors.forename: Kumiko
authors.initials: K
authors.lastname: Saeki
authors.forename: Naoko
authors.initials: N
authors.lastname: Nakamura
authors.forename: Koichi
authors.initials: K
authors.lastname: Saeki
authors.forename: Yoshiko
authors.initials: Y
authors.lastname: Yogiashi
authors.forename: Asako
authors.initials: A
authors.lastname: Yoneda
authors.forename: Makoto
authors.initials: M
authors.lastname: Koyanagi
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Kondo
authors.forename: Akira
authors.initials: A
authors.lastname: Yuo
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000502:physiology*; D002478:Cells, Cultured; D002633:Chemotaxis; D018920:Coculture Techniques; D016207:Cytokines / Q000235:genetics / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology / Q000502:physiology*; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology; D018507:Gene Expression Regulation, Developmental; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000502:physiology*; D008252:Macaca fascicularis; D051379:Mice; D009504:Neutrophils / Q000166:cytology / Q000502:physiology*; D011323:Primates
chemical_list: D016207:Cytokines