Human mPB-derived cFCs enhance repopulation of hematopoietic CD34⁺ progenitors in NOD/SCID mice in vivo and clonogenicity in methylcellulose in vitro. (A) The content of human CD45⁺ cells in NOD/SCID BM at 8 weeks after transplantation with CD34⁺ cells alone (n = 16) or together with CD8⁺ cells (n = 12), CD8⁺TCR⁻ cells (n = 3) or cFCs (n = 10), as indicated. Bar indicates mean repopulation efficiency. Cells are derived from 3 CB and 3 mPB donors. (B) Representative FACS analysis of human hematopoietic lineages in NOD/SCID BM. Percentages in FACS plots refer to lineage marker–positive cells of total cells in the BM. (C) CFU assay of CB CD34⁺ cells (250 cells) preincubated for 2 hours at 37°C alone or with CD8⁺ cells, CD8⁺TCR⁻ cells or cFCs (750 cells each) from 2 mPB donors. CFU-GM and CFU-GEMM, in triplicates or quadruplicates, were counted after 14 days. SEM and P values refer to total colony numbers. Specifically for the CD34⁺ plus cFC group, P values of CFU-GEMM were less than .001 (Donor 1) and less than .01 (Donor 2), and of CFU-GM were greater than 0.5. (D) Large GEMM-CFU–derived colonies formed after coculture with cFCs are shown. Micrographs were acquired by imaging methylcellulose cultures with a Leica microscope (Leica Microsystems, Wetzlar, Germany) fitted with 100× objective, DS-5M camera head, and camera control unit DS-L1 image acquisition software (Nikon, Tokyo, Japan). (E) CFU assay of CB CD34⁺ cells (250 cells) preincubated for 2 hours at 37°C with cFCs (750 cells), without and with anti-Flt3L (Flt3L-Ab) or control mAb (c-Ab), and CFU assay of cFCs or CD8⁺ cells alone, as indicated. Preincubation with Flt3L-Ab did not inhibit growth of CD34⁺ cells alone (not shown). CFU-GM and CFU-GEMM, in triplicates were counted after 14 days. SEM and P values refer to total colony numbers; n.s., not significant.