Expression of Rh30 and Rh-related glycoproteins during erythroid differentiation in a two-phase liquid culture system

To gain insight into the formation of the Rh complex during erythroid differentiation, the ways in which Rh30 and Rh-related glycoproteins, especially Rh50, were produced in a modified two-phase liquid culture system were studied.

A mononuclear cell fraction from fresh peripheral blood was first cultured in a medium supplemented with conditioned medium collected from the culture of a bladder carcinoma cell line (5637) for 7 days. Nonadherent cells were then collected for culture in a secondary medium containing 2 U per mL of erythropoietin to initiate erythroid differentiation. The expression of Rh30 and Rh50 during secondary culture (16 days) was monitored by flow cytometry.

D+ cells appeared after Day 4 and increased to 70 percent by Day 8. On Day 12, 90 percent of the total cells became D+ and remained so until the end of the culture. A similar expression profile was obtained for Rh50. As determined from mean fluorescence intensities recorded in flow cytometry, the number of both D and Rh50 antigenic sites per cell increased as the differentiation progressed. Rh-related glycoprotein, CD47, had expression patterns significantly different from those of Rh30 and Rh50. In addition, the cultured cells produced partially glycosylated protein (approx. 32 kDa) in Rh50.

Expressions of Rh30 and Rh50 occur simultaneously during erythroid differentiation, and both proteins are most actively synthesized at the last stage of the differentiation. In contrast, CD47 may be involved in expression of Rh30 in a different manner from Rh50. The two-phase liquid culture system will be an excellent model for studying the interaction among the components of the Rh complex during protein synthesis and complex assembly on the cell membrane.