Multilineage differentiation of human nonadherent CD34⁻ cells in the bone marrow of NOD/SCID mice.

(A) Bone marrow cells from a mouse given 10 000 NA CD34⁻cells were incubated with antibodies to various human antigens and analyzed by flow cytometry. Cells with low, medium, and high forward scatter (Ai, region R1) were gated and further analyzed. Panel Aii shows a histogram of CD45 (panleukocyte marker) expression. Panels Aiii and Aiv show expression of CD133 and CD34 (hematopoietic stem cell markers) and CD38 (a differentiation marker) within the CD45⁺ cell population. Most cells had a primitive CD133⁺CD34⁻CD38⁻ phenotype. (B) RT-PCR analysis of human CD133 and CD34 mRNA in bone marrow cells of NOD/SCID mice that received NA CD34⁻ cells. Lane 1 contains DNA molecular-weight markers. We used human CD34-specific primers to generate cDNA from bone marrow cells of a representative mouse recipient (lane 2), from highly enriched human CD34⁺cells from mobilized peripheral blood (positive control; lane 3), and from bone marrow cells of a representative mouse that received an injection of only PBS (lane 4). Lane 5 contains a sample of distilled water that was used in RT-PCR. We used human CD133-specific primers to generate cDNA from bone marrow cells of a representative mouse that received an injection of only PBS (lane 6); from highly enriched, mobilized CD34⁺ cells from human peripheral blood (lane 7); and from bone marrow cells of a representative mouse recipient (lane 8). (C) The human cells were tested for expression of the lineage-specific markers CD19 (a human pan–B-cell marker; Ci), CD56 (an NK-cell marker; Cvi), and CD3 (a human pan–T-cell marker; Ci-ii,Civ). Subpopulations of human cells with T-cell phenotype were analyzed on the basis of expression of CD2, CD4, CD5, and CD7 (Cv-vi) on CD3⁺ cells within gate R3 (R1+R2; Civ), CD33 and CD15 (myeloid markers; Avii), and glycophorin-A (an erythroid cell marker; Cviii).