Detection of human cells in human/sheep chimeric lambs with in vitro human stroma-forming potential

In utero transplantation of preimmune fetal sheep with human hematopoietic stem cells results in stable long-term hematopoietic chimerism. To clarify the mechanisms of support of human stem cells in chimeric sheep, we established stromas from bone marrow of 30 sheep transplanted in utero with human hematopoietic stem cells from adult bone marrow adult peripheral blood, or fetal liver. We examined the stromas for the presence or absence of human stromal elements in vitro. Human stromal elements were detected in 12 sheep as assessed by polymerase chain reaction (PCR) using human HLA-DQalpha-specific primers. The human origin of the PCR product was confirmed by Southern blotting using an HLA-DQalpha-specific probe. However, none of these stromal cells were positive for CD45 or CD14 as determined by fluorescence-activated cell sorting (FACS) analysis or by message expression using reverse transcriptase (RT)-PCR. In an attempt to further characterize these cells fibroblasts were isolated by panning, and DNA analysis confirmed the human origin of these cells in the same lambs. Of the fetuses injected with the highly enriched cells from adult human bone marrow 36% were found to harbor cells capable of forming human stromal elements in vitro in their marrow. Of those injected with human fetal liver and peripheral blood stem cells, 42 and 40%, respectively, exhibited in vitro human stromal cell-forming ability. These results indicate the long-term persistence of cells capable of giving rise to components of human marrow stroma in vitro in the human/sheep xenograft model.