ERYTHROPOIETIN (EPO) AND FUNCTIONAL EPO RECEPTORS ARE EXPRESSED IN THE DEVELOPING HUMAN FETAL CNS † 266

We have previously demonstrated Epo in the spinal fluid of normal preterm and term infants, and EpoR in the spinal cords of human fetuses. Our present objective was to determine which cell types within the fetal CNS express Epo and EpoR, and to assess the function of these receptors. Expression of mRNA for Epo and EpoR was sought by RTPCR in mixed primary cultures of fetal spinal cords as well as in human cell lines of neuronal precursers (NT2) and mature neurons (HNT). Epo was measured by ELISA in spent media from primary cell culture, and immunohistochemistry was used to identify EpoR in cultured neurons and brain sections. Changes in Epo and EpoR expression were sought in spinal cords and brains from fetuses of 7 to 24 weeks postconception by semiquantitative PCR. Differential display PCR was used to determine changes in gene expression resulting from Epo stimulation of the EpoR. Epo and EpoR mRNA were expressed in mixed primary cultures of neural tissues, NT2 and HNT cells, and low levels of Epo (2 mU/mL) were detected by ELISA in media removed from mixed cell cultures. The presence of EpoR was confirmed by immunohistochemical staining of neurons and glia in culture, as well as in fetal brain. Semiquantitative PCR revealed no change in EpoR expression in spinal cords and brain between 7 and 24 weeks gestation. A trend towards decreased Epo expression occurred in spinal cords from 7 to 17 weeks gestation, with an increase in brain Epo noted between 17 and 24 weeks gestation. Epo mRNA transcripts doubled from baseline levels in neurons cultured in conditions of hypoxia. Stimulation of cultured neurons and glial cells by rEpo resulted in changes in gene expression as determined by differential banding patterns. We have isolated and cloned 8 differentially expressed bands resulting from rEpo stimulation, which will require further characterization. We conclude that neurons and glial cells in the developing human fetus express Epo and EpoR, and these receptors appear to be functional.