CD150− side population cells possess long-term, multilineage reconstitution potential. (A) Representative sort scheme for transplants that are CD150+ (n = 10), CD150− (n = 14), or both (total SPKLS; n = 9) is shown. Sca-1/c-Kit and Hoechst Blue/CD150 scatter plots contain more cells to emphasize the CD150+ and CD150− populations. Exceptionally stringent gates were used to define CD150+ and CD150− subpopulations, and a purity check of sorted cells (using an example from a CD150− sort) was used to verify sort quality. The SPKLS donor population includes all events shown in the Hoechst Blue/CD150 scatter plot. (B) One hundred cells from each donor population were isolated from CD45.2 mice and transplanted into CD45.1 lethally irradiated recipient mice, along with 250 000 WBM competitor cells from CD45.1 animals. Donor-derived engraftment (percentage of CD45.2) within the peripheral blood was measured by flow cytometry at 4, 8, 12, and 16 weeks after transplantation, indicating long-term engraftment (greater than 16 weeks) from all 3 populations. An example flow cytometric analysis of an individual animal from each cohort is shown. (C) Lineage contribution of donor-derived hematopoiesis was determined using a staining scheme (illustrated here) that separates the peripheral blood into myeloid cells, B cells, and T cells. (D) Donor- derived (percentage of CD45.2) multilineage reconstitution within the peripheral blood was measured by flow cytometry at 4, 8, 12, and 16 weeks after transplantation, indicating long-term multilineage engraftment (16 weeks) from all 3 donor populations. The proportion of the specified lineages within all donor-derived test cells is indicated. An example flow cytometric analysis of an individual animal from each cohort is shown. Data shown are from 2 separate transplantations and are representative of several experiments. Donor-derived T cells were not observed at 4 weeks after transplantation. Error bars represent SEM. Percentages shown correspond to the gated proportion of the plot.
