A feeder-free and efficient production of functional neutrophils from human embryonic stem cells.
A novel, feeder-free hematopoietic differentiation protocol was established for highly efficient production of neutrophils from human embryonic stem cells (hESCs). For the induction of differentiation, spheres were generated in the presence of serum and cytokine cocktail and subjected to attachment culture on gelatin-coated plates. After approximately 2 weeks, a sac-like structure filled with abundant round cells emerged at the center of flattened spheres. After cutting off this sac-like structure, round cells actively proliferated, either floating in the supernatant or associated weakly with the adherent cells. Almost all of these round cells were CD45-positive hematopoietic cells with myeloid phagocytic markers (CD33 and CD11b), and approximately 30%-50% of the round cells were mature neutrophils, as judged from morphology, cytochemical characteristics (myeloperoxidase and neutrophil alkaline phosphatase), and neutrophil-specific cell surface markers (CD66b, CD16b, and GPI-80). In addition, hESC-derived neutrophils had chemotactic capacity in response to the bacterial chemotactic peptide formyl-methionyl-leucyl-phenylalanine and neutrophil-specific chemokine interleukin (IL)-8. Using "semipurified" neutrophils migrated to IL-8, both phagocytic and respiratory burst activities were demonstrated. Finally, it was shown that hESC-derived neutrophils had chemotactic activity in vivo in a murine air-pouch inflammatory model. The present results indicate successful induction of functional mature neutrophils from hESCs via highly efficient feeder-free differentiation culture system of human hematopoietic cells.
Saeki
Koichi
K
Department of Hematology, Research Institute, International Medical Center of Japan, Tokyo, Japan.
Saeki
Kumiko
K
Nakahara
Masako
M
Matsuyama
Satoko
S
Nakamura
Naoko
N
Yogiashi
Yoshiko
Y
Yoneda
Asako
A
Koyanagi
Makoto
M
Kondo
Yasushi
Y
Yuo
Akira
A
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D048430:Cell Shape / Q000187:drug effects; D002633:Chemotaxis / Q000187:drug effects; D003114:Colony-Forming Units Assay; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D005434:Flow Cytometry; D006410:Hematopoiesis / Q000187:drug effects; D006801:Humans; D007249:Inflammation / Q000473:pathology; D016209:Interleukin-8 / Q000494:pharmacology; D007621:Karyotyping; D051379:Mice; D016513:Mice, SCID; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D010587:Phagocytosis / Q000187:drug effects; D016897:Respiratory Burst / Q000187:drug effects; D014158:Transcription, Genetic / Q000187:drug effects
D016209:Interleukin-8; D009240:N-Formylmethionine Leucyl-Phenylalanine