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. 2008 Feb 15;111(4):2444-51.
doi: 10.1182/blood-2007-09-115006. Epub 2007 Nov 30.

CD150- side population cells represent a functionally distinct population of long-term hematopoietic stem cells

David C Weksberg  1 , Stuart M Chambers, Nathan C Boles, Margaret A Goodell
Affiliations

CD150- side population cells represent a functionally distinct population of long-term hematopoietic stem cells

David C Weksberg et al. Blood. .

Abstract

Hematopoietic stem cells (HSCs) are a self-renewing population of bone marrow cells that replenish the cellular elements of blood throughout life. HSCs represent a paradigm for the study of stem-cell biology, because robust methods for prospective isolation of HSCs have facilitated rigorous characterization of these cells. Recently, a new isolation method was reported, using the SLAM family of cell-surface markers, including CD150 (SlamF1), to offer potential advantages over established protocols. We examined the overlap between SLAM family member expression with an established isolation scheme based on Hoechst dye efflux (side population; SP) in conjunction with canonical HSC cell-surface markers (Sca-1, c-Kit, and lineage markers). Importantly, we find that stringent gating of SLAM markers is essential to achieving purity in HSC isolation and that the inclusion of canonical HSC markers in the SLAM scheme can greatly augment HSC purity. Furthermore, we observe that both CD150(+) and CD150(-) cells can be found within the SP population and that both populations can contribute to long-term multilineage reconstitution. Thus, using SLAM family markers to isolate HSCs excludes a substantial fraction of the marrow HSC compartment. Interestingly, these 2 subpopulations are functionally distinct, with respect to lineage output as well as proliferative status.

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Figures

Figure 1
Figure 1
High correspondence of SLAM cells with the side population is dependent on stringent CD150 gating. (A) Whole bone marrow isotype controls for SLAM family members (CD48, CD41, and CD150). (B) When using SLAM family members to define HSCs, based on isotype controls, 45% of WBM cells are CD150+ and 0.4% are CD150+CD48−CD41− (termed SLAM cells). Only 23% of these cells are c-Kit and Sca-1 positive, and only 20% are side population (SP) cells. (C) When the gating for CD150 is more stringent (asterisks), reducing the number of SLAM cells to 0.01%, then both the percentage of c-Kit+ Sca-1+ and SP cells within the SLAM population increases to 86% and 84%, respectively. Percentages shown correspond to the gated proportion of the plot.
Figure 2
Figure 2
CD150 divides the SP into 2 distinct fractions. (A) From mice at 10 weeks of age, bone marrow SP cells that are c-Kit+, Sca-1+, Lin− (SPKLS) were examined for expression of SLAM family members CD41, CD48, and CD150. (B) At 10 weeks of age, all SPKLS cells express little or no CD41 and CD48, and only one-third of SPKLS cells express CD150. Percentages shown correspond to the gated proportion of the plot.
Figure 3
Figure 3
CD150 is heterogeneous with respect to dye efflux and changes with age within the SP. (A) The analysis scheme divides the SP into upper and lower portions, both of which are heterogeneous with respect to CD150. These relations are quantified in subsequent panels. Percentages shown correspond to the gated proportion of the plot. (B) The proportion of CD150+ cells increases with age within the SP. (C) When the shift in CD150+ SP cells is quantified, there is a significant change with age, which varies between upper and lower SP. Error bars represent SEM.
Figure 4
Figure 4
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.
Figure 5
Figure 5
CD150− and CD150+ HSCs exhibit differences in contribution to various hematopoietic compartments 24 weeks after transplantation. (A) A significant decrease in donor-derived whole bone marrow (WBM) and thymus engraftment, measured by flow cytometry, was observed for CD150− SPKLS HSCs compared with CD150+ or whole SPKLS HSCs. No statistical difference was observed for donor-derived peripheral blood and spleen chimerism. (B) All 3 populations of SPKLS cells self-renew to give rise to comparable levels of SPKLS, although CD150− SPKLS generate lower levels of KLS, MP, and CLP compartments as measured by flow cytometry. (C) WBM was examined for the presence of donor-derived CD150+ and CD150− SPKLS within transplant recipients that received CD150+ or CD150− SPKLS cells. Both donor populations were able to produce CD150+ and CD150− SPKLS cells, indicating no clear-cut hierarchy between the CD150+ and CD150− SPKLS cells. In addition, CD150+ SPKLS donor cells exhibited a trend toward generating a higher proportion of CD150+ cells in the recipient SP. This result, although not statistically significant, suggests a preference for the CD150+ SPKLS to preferentially produce CD150+ SPKLS on transplantation. Error bars represent SEM.
Figure 6
Figure 6
CD150 marks a population of more quiescent HSCs. SPKLS cells fractionated based on position within the SP (Hoechst Blue fluorescence) and CD150 expression (gates i-iv) were isolated from mice treated with the nucleotide analog BrdU for 3 days and examined by flow cytometry for BrdU incorporation, an indicator of transition through the cell cycle. CD150− SPKLS cells were substantially more proliferative in vivo than their CD150+ SPKLS counterparts, with the lower SP being generally less in cell cycle than the upper SP. Percentages shown correspond to the gated proportion of the plot.
Figure 7
Figure 7
SPKLS cells and SLAM cells are functionally overlapping populations. Non-SP cells can be found in significant numbers within the SLAM isolation scheme (particularly when using isotype controls to define the population), and these non-SP cells are not functional HSCs. Further, CD150− cells can be found within the SPKLS sort scheme. The gray area represents populations containing LT-HSC activity, with the darker gray central area representing the less proliferative myeloid-biased LT-HSCs. The area within the Venn diagram is roughly representative of the degree of overlap, but not precisely to scale.
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