Erythropoietic activity in mouse spleen cell-conditioned medium detected by erythroid colony formation of fetal mouse liver cells in serum-free cultures

A modified serum-free soft agar culture system for the colony formation by late erythroid progenitor cells (CFU-E) of fetal mouse liver is described. Fetal calf serum (FCS) was replaced by bovine serum albumin, human transferrin, cholesterol, and L-α-phosphatidylcholine. The plating efficiency was about 70% of that for the control serum-supplemented cultures. This serum-free culture system makes it possible to detect very low concentrations of erythropoietin because no erythroid colonies were observed without the addition of exogenous erythropoietin. Using this culture system, we investigated whether or not medium conditioned by pokeweed mitogen-stimulated mouse spleen cells (SPCM) prepared in the absence of FCS contained erythropoietic activity. SPCM stimulated the colony formation by fetal liver CFU-E without the addition of exogenous erythropoietin. A study using a Sephadex G-100 column demonstrated that the apparent molecular weight of the active substance(s) was around 40,000 daltons. The erythropoietin-like activity was different from erythropoietic activities in human urine, human serum, and mouse serum. Although both erythropoietin-like and burst-promoting activities were detected in SPCM, they were not separated from each other on the Sephadex G-100 column.