Fig. 4. The pre-fusion allantois and chorion have hematopoietic potential. (A) Explant of ConA 488 (red) labeled chorion following 48 hours of culture on OP9 stromal cells. ConA 488 positive cells that crawled off the explant are boxed. (B) Explant of ConA 594 labeled visceral endoderm (ve) after 48 hours on OP9 cells. (C) Allantois cultured for 7 days on OP9 cells in the presence of SCF, IL3, IL6, Flt3-L, G-CSF, GM-CSF and 2-ME. Note the presence of abundant, nonadherent round cells. (D) Allantois OP9 culture from a Runx1 deficient (Runx1^lz/rd) conceptus. No nonadherent round cells are present. (E) Chorion OP9 explant culture, same conditions as in panel C. (F) Chorion OP9 culture from a Runx1 deficient (Runx1^lz/rd) conceptus. (G) Allantois OP9 culture from a ß-actin/GFP transgenic conceptus, showing that nonadherent cells originate from the conceptus. (H) Allantois OP9 culture from ß-actin/GFP transgenic conceptus, stained with DiI-Ac-LDL (red) and anti-CD45 (blue). GFP⁺ DiI-Ac-LDL⁺ cells are yellow and GFP⁺ CD45⁺ cells are cyan. (I) Chorion OP9 culture from a ß-actin/GFP transgenic conceptus. (J) Chorion OP9 culture from a ß-actin/GFP transgenic conceptus, labeled with DiI-Ac-LDL and anti-CD45 as in panel H. (K) Representative FACS analysis of three pooled allantois and chorion explants from +/+ and Runx1 deficient (Runx1^rd/rd) conceptuses cultured on OP9 cells for 14 days in the presence of SCF, IL3, IL6, Flt3-L, G-CSF, GM-CSF and 2-ME and stained for CD45 and c-kit. The explants were cultured separately, and three allantoises or chorions were pooled for FACS analysis in these plots. Cells in each gate were analyzed in the plots below. The experiment was performed three times, with a total of ten +/+ and seven Runx1^rd/rd allantoises and chorions.