Phenotypic and functional analyses of CD41-sorted cell fractions of embryonic HSC reservoirs. E12 placenta (A-C,H) and E11/E14 liver (D-G,I). (A,E) Flow cytometric analysis of E12 placenta and E14 fetal liver (FL) showing representative sorting gates (red represents CD41^high; green, CD41^int; and blue, CD41⁻) and percentages of cells in each fraction. (B,F) In vitro colony-forming unit in culture (CFU-C) analyses showing the total number of hematopoietic progenitors per 1000 cells in each CD41-sorted fraction of E12 placenta and E14 FL cells. Each sample was analyzed in triplicate for each dilution. n = 2 for E12 placenta, and n = 3 for E14 FL. (C,G) In vivo hematopoietic repopulation analysis of CD41-sorted fractions of E12 placenta and E14 FL 4 months after transplantation. Percentage of repopulated mice showing greater than 10% donor chimerism in peripheral blood is shown. Numbers above columns indicate the number of mice repopulated/number of mice transplanted. Dose of injected cells is indicated as embryo equivalents (ee). n = 5 for E12 placenta, and n = 3 for E14 FL. (D) CD41 immunostaining of E11 Ly6A GFP embryo section showing the liver. (Top panel) Red fluorescent CD41 expression in hematopoietic cells. (Middle panel) Green fluorescent Ly6A GFP expression in hematopoietic cells. (Bottom panel) Merged fluorescence. The lack of yellow fluorescence indicates no coexpression of CD41 and Ly6A in liver hematopoietic cells. Image acquisition was from LSM510NLO/FCS confocal microscope (Carl Zeiss BV) with 40×/1.3 NA water objective and Vectashield medium (Vector Laboratories). LSM image software was used (Carl Ziess BV). (H-I) Representative semiquantitative PCR analysis of hematopoietic tissue DNA 4 months after transplantation from (H upper panel) a primary recipient injected with 2 ee of E12 placenta CD41⁻ cells or (I upper panel) 0.1 ee of E14 FL. Representative semiquantitative PCR analysis of peripheral blood DNA from 6 secondary recipients injected with BM cells from the primary E12 placenta CD41⁻ recipient (H lower panel) and 5 recipients injected with BM cells from the primary E14 FL CD41⁻ recipient (I lower panel). Donor indicates the human β-globin PCR fragment, and Myo indicates the myogenin DNA normalization control PCR fragment. DNA dilution controls (0%-100%) were used to quantitate percentages of donor chimerism that are indicated below each lane. PB indicates peripheral blood; Th, thymus; LN, lymph node; Sp, spleen; M, myeloid (sorted from BM); E, erythroid (sorted from BM); L, lymphoid (sorted from BM); B, B lymphoid (sorted from spleen); and T, T lymphoid (sorted from spleen).