Fig. 3. Neuronal progenitor cells from Epor–/– cortex. (A) At E9.5, nestin expression is comparable in cortices of the Epor+/+ and Epor+/– (n=5), and Epor–/– embryos (n=3). (B) Single-cell suspension isolated from E10.5 Epor–/– cerebral cortices (n=6) showed marked reduction in total cell numbers compared with Epor+/+ and Epor+/– cortices (n=13). (C) By E10.5, nestin mRNA level was downregulated by four times in the Epor–/– cortex (n=4) compared with Epor+/+ and Epor+/– (n=6). (D,E) A marked decrease in the numbers and proportion of nestin positive progenitor cells acutely isolated from the E10.5 Epor–/– cortex (n=4) was also observed compared with Epor+/+ and Epor+/– cortices (n=12). (F,J,K) Fewer neurons were produced from in vitro cultures of Epor–/– cortical cells. Cells were stained for MAP2 (F) or ß-tubulin III (red) (J,K) and with DAPI (blue) after 4 days of culture in NBM. The proportions of cells with MAP2 positive staining are indicated in (F) for Epor–/– (n=5) and for Epor+/+ and Epor+/– (n=6). Representative fields for Epor+/+ cultures with 41% of 200 cells (J) and for Epor–/– cultures with 23% of 199 cells (K) with ß-tubulin III positive staining are shown. (G-I,L,M) EpoR production improved neuronal cell survival under hypoxia. Six and 24 hours after cells were switched to Locke’s solution and cultured under the hypoxic condition of 2% oxygen tension, cells were stained with MAP2 (red), TUNEL (green) and DAPI (blue). After 6 hours, the number of surviving neurons was markedly decreased (G) and apoptotic neurons were significantly increased (H) in Epor–/– cultures (n=3). No surviving Epor–/– neurons were observed after 24 hours exposure (n=3) (G). About 10% of the surviving cells were neurons in the Epor+/+ cultures. Erythropoietin addition increased the survival of Epor+/+ neurons producing EpoR (I); no erythropoietin response was observed in Epor–/– cultures. In all experiments (E-M), cells were initially plated at the same density. For cell enumeration, the percentage of nestin (E) and MAP2 (F-I) positive cells isolated for each individual embryo were determined by an investigator blind to the genotypes by quantifying 20 microscopic fields documented by digital camera images. A total of 2000-4000 cells were counted. Scale bars: 0.025 mm in L,M; 0.05 mm in J,K. (B-F) P < 0.01. Open bars represent Epor+/+ or Epor+/+ and Epor+/– cultures, as indicated, and solid bars represent Epor–/– cultures.
