Erythroblasts derived in vitro from embryonic stem cells in the presence of erythropoietin do not express the TER-119 antigen.
OBJECTIVE
In this study, we analyzed murine primitive erythropoiesis by coculturing Flk-1+ ES-derived cells with OP9 to find efficient culture conditions for erythroid cell induction. We utilized a nonserum culture system and EPO (erythropoietin) and found that this cytokine had unique properties.


METHODS
ES cells (E14.1) were first differentiated to Flk-1+ cells and then cocultured with OP9 stromal cells. BIT9500 was used as a serum replacement. The erythroid morphology, hemoglobin types, and TER-119 expression levels were analyzed.


RESULTS
Primitive erythroid cells with embryonic hemoglobin were generated very efficiently when the serum-containing culture was converted to the nonserum system. In this serum-free culture, TER-119+ erythroblasts appeared first on day 2 and maturation proceeded until day 7. When EPO was added to this coculture, the number of induced floating cells increased twofold to threefold. Unexpectedly, the erythroid-specific antigen TER-119 expression of these cells was drastically reduced. Since reduced TER-119 expression is usually interpreted as maturation arrest, we examined the phenotypic features of the EPO-treated cells. We found, however, no evidence of maturation arrest in the aspects of morphology and hemoglobin content. EPO did not suppress TER-119 expression of erythroblasts derived from fetal liver or adult bone marrow.


CONCLUSIONS
Our results showed that EPO had the unusual property of inducing TER-119- erythroblasts in ES-derived primitive erythropoiesis. It is likely that this effect is unique to primitive erythropoiesis.
Otani
Takeshi
T
Fujisaki Cell Center, Hayashibara Biochemical Labs Inc, Okayama, Japan. fcch@hayashibara.co.jp
Nakamura
Shuji
S
Inoue
Toshiya
T
Ijiri
Yoshihiro
Y
Tsuji-Takayama
Kazue
K
Motoda
Ryuichi
R
Orita
Kunzo
K
D000818:Animals; D001483:Base Sequence; D001789:Blood Group Antigens / Q000032:analysis* / Q000235:genetics; D002454:Cell Differentiation; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D018920:Coculture Techniques; D017931:DNA Primers; D004900:Erythroblasts / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D004920:Erythropoiesis / Q000187:drug effects / Q000502:physiology; D004921:Erythropoietin / Q000494:pharmacology*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D051379:Mice; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000502:physiology*; D017154:Stromal Cells / Q000166:cytology
D001789:Blood Group Antigens; D017931:DNA Primers; D008164:Luminescent Proteins; C489122:TER-119 antigen, mouse; D004921:Erythropoietin; D049452:Green Fluorescent Proteins