CD150⁻ cells are transiently reconstituting multipotent progenitors with little or no stem cell activity

To the editor:

Goodell and colleagues recently published a paper in Blood (Weksberg et al¹) that confirmed many of our conclusions from prior studies demonstrating the utility of signaling lymphocytic activation molecule (SLAM) family markers to enhance the purification of mouse hematopoietic stem cells (HSCs).^2–5 However, they took issue with 2 conclusions. First, they argued that SLAM family members are not sufficient for the identification of HSCs and second that “a substantial fraction” of HSCs are CD150⁻, in contrast to our published data. However, the data in the Goodell paper do not support these conclusions.

Weksberg et al¹ argued that in contrast to our published data,^2–5 only a minority of cells expressing SLAM family markers (CD150⁺CD48⁻CD41⁻) expressed the HSC markers c-kit and Sca-1. However, Figure 1B in the Goodell paper shows that they used an inappropriately loose gate that included mainly CD150⁻ cells in the CD150⁺CD48⁻CD41⁻ population. Because the gate included 1% of cells in the isotype control and 0.4% of cells in the experimental sample, the authors did not attempt to sort the CD150⁺CD48⁻CD41⁻ population characterized in prior studies, which represents only 0.007% of cells.³ The gate used by Weksberg et al¹ is different from that illustrated in our papers (compare Figure 2A from Kiel et al² to Figure 1B in Weksberg et al¹). Obviously, SLAM markers will render poorer purity than reported in prior studies if gates are set to include mainly CD150⁻ cells.

The only experimental support offered by Weksberg et al¹ for the existence of CD150⁻ HSCs was an experiment in which 100 CD150⁻ c-Kit⁺Lineage⁻Sca-1⁺ (KLS) side population (SP^KLS) cells were injected into irradiated mice.¹ They observed significantly less reconstitution than that observed from CD150⁺ SP^KLS cells but nonetheless concluded that CD150⁻ cells represent “a substantial fraction” of HSCs. However, to test this conclusion it would be necessary to perform a more careful clonal analysis. What percentage of CD150⁻ cells actually give long-term multilineage reconstitution: 1%, 10%, 100%? Without such data it is impossible to know what percentage of HSCs are CD150⁻, whether such cells represent a significant fraction of HSCs, or whether the HSC activity could be explained by rare contamination from CD150⁺ cells. Weksberg et al did not even disclose what fraction of mice that received transplants of 100 CD150⁻ SP^KLS cells showed long-term multilineage reconstitution. If some did not, this would prove that less than 1% of CD150⁻ SP^KLS cells are HSCs, disproving the conclusions of this paper.

We have performed clonal analyses of the CD150⁻ fraction of c-kit⁺Sca-1⁺CD48⁻ (KS48) or c-kit⁺Thy-1^lowLineage⁻Sca-1⁺ (KTLS) cells. We found less than 5% of HSCs in the CD150⁻ fraction of whole bone marrow, KS48, or KTLS cells (Table 1). CD150⁻ cells give transient multilineage reconstitution of irradiated mice. These results have been independently replicated by other labs in papers^6,7 not cited by Weksberg et al.¹ Our conclusion that CD150⁻ KS48 cells are mainly transiently reconstituting multipotent progenitors is consistent with data in the Goodell paper that showed more rapid proliferation and reduced myeloid reconstitution from CD150⁻ SP^KLS cells compared with CD150⁺ SP^KLS cells. Goodell's conclusion that CD150⁻ cells represent a substantial population of rapidly dividing HSCs that can give rise to CD150⁺ HSCs but that have less reconstituting potential than CD150⁺ HSCs is internally inconsistent, inconsistent with more rigorous data from multiple labs, and inconsistent with decades of work demonstrating that HSCs are slowly dividing.

Table 1.

CD150⁺ cells consistently include all or almost all of the HSC activity in adult bone marrow while CD150⁻ cells include transiently reconstituting multipotent progenitors and little or no HSC activity

Source, cells	% of all cells	Donor cell reconstitution, % of all recipient mice (n/N)			HSC frequency	% of all HSCs in this population
		Long-term multilineage	Transient multilineage	Other (e.g., lymphoid)
Adult bone marrow
20,000 CD150+	8.8	93 (13/14)	7.1 (1/14)	0 (0/14)	1 in 7,600	97.6
180,000 CD150−	91	5.5 (1/18)	89 (16/18)	5.5 (1/18)	1 in 3,150,000	2.4
Adult bone marrow
5 KS CD150+CD48−	0.011	85 (11/13)	0 (0/13)	7.7 (1/13)	1 in 3.2	95.2
5 KS CD150−CD48−	0.024	3.6 (2/56)	66 (37/56)	20 (11/56)	1 in 138.0	4.8
10 KS CD150−CD48−	0.024	0 (0/7)	57 (4/7)	43 (3/7)	<1 in 70	0
5 KTLS CD150−	0.005	0 (0/10)	20 (2/10)	20 (2/10)	<1 in 50	0
Mobilized spleen
20,000 CD150+	13	89 (8/9)	11 (1/9)	0 (0/9)	1 in 9,100	100
180,000 CD150−	87	0 (0/10)	0 (0/10)	100 (10/10)	<1 in 1,800,000	0
Reconstituted bone marrow
20,000 CD150+	12	75 (3/4)	0 (0/4)	0 (0/4)	1 in 14,400	100
180,000 CD150−	88	0 (0/5)	0 (0/5)	40 (2/5)	<1 in 900,000	0

In rows where reconstitution categories do not add to 100%, the remaining mice were unreconstituted by donor cells. Some of these data were previously published.^3,4 Transient multilineage reconstitution means myeloid chimerism was lost within 16 weeks of transplantation.

n indicates number in group; N, total number.

Authorship

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Sean J. Morrison, 5435 Life Sciences Institute, 210 Washtenam Avenue, Howard Hughes Medical Institute and Center for Stem Cell Biology, University of Michigan, Ann Arbor, MI 48109-2216; e-mail: seanjm@umich.edu.