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Stem Cells, Vol. 16, No. 1, 38-48, January 1998
© 1998 AlphaMed Press

Characterization of a Population of Cells in the Bone Marrow that Phenotypically Mimics Hematopoietic Stem Cells: Resting Stem Cells or Mystery Population?

Troy D. Randalla, Irving L. Weissman

Department of Pathology, Stanford University Medical Center, Stanford, California, USA;
a Trudeau Institute, Inc., Saranac Lake, New York, USA

Key Words. Hematopoietic stem cells • c-kit • CD34 • CD38 • Sca-1 • 5-fluorouracil

Dr. Troy Randall, Trudeau Institute, Inc., P.O. Box 59, Saranac Lake, NY 12983, USA.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
We have identified a population of cells in murine bone marrow that has many of the phenotypic characteristics attributed to resting hematopoietic stem cells but does not reconstitute irradiated mice. These cells express high levels of Sca-1, H-2K and CD38 and low levels of Thy-1.1, but do not express CD34 nor any of the lineage markers including CD3, CD4, CD5, CD8 NK1.1, I-A, B220, Ig(MGA), CD40, kappa, Mac-1, Gr-1 or Ter119. In addition, this population can be found at normal frequency in nu/nu as well as rag-1–/– mice. These cells incorporate only low levels of Rh123, are resistant to the cytotoxic effects of 5-fluorouracil and, consistent with their resting phenotype, less than 2% of these cells are in the S/G2/M phases of the cell cycle. The only phenotypic characteristic that distinguishes these cells from the lineage– Sca-1+, Thy-1.1low long-term reconstituting hematopoietic stem cell population is their lack of c-kit expression. Here we have explored the possibility that these cells represent a truly resting population of hematopoietic stem cells. We found that the lineage–, Sca-1+, c-kit– cells do not respond to hematopoietic growth factors in vitro, either alone or in combination with stromal layers. Furthermore, these cells do not form in vivo spleen colonies nor do they have the ability to reconstitute irradiated mice. Thus, this population may represent either a population of resting stem cells for which we lack the appropriate activating stimulus, or simply represent a "mystery population" that phenotypically mimics most of the physical properties of resting stem cells. Given the close phenotypic similarity of the c-kit– mystery population cells to the c-kit+ long-term reconstituting stem cells, investigators must be rigorous to exclude their effects from other stem cell assays.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
Hematopoietic stem cells have been defined in the mouse as clonogenic multipotent precursors to the lymphoid, myeloid and erythroid lineages that can also self-renew [1]. The pool of multipotent hematopoietic cells in mice includes three subsets of clonogenic progenitors based on phenotype, productive life span and degree of self-renewal: long-term stem cells that self-renew indefinitely, short-term stem cells that self-renew for three to six weeks and multipotent progenitors that do not self-renew extensively at limited dilution in vivo [1]. Thus, only long-term stem cells can produce both mature cells as well as more stem cells for the life of the animal. The best functional assay for stem cells is the transfer of genetically distinct cells into an irradiated (or otherwise compromised) congenic host and to test for the donor-derived repopulation of all blood lineages [2]. This is the only assay that measures both reconstitution of all the lineages (particularly the T cells) and that also measures the self-renewing potential of the stem cells. Prolonged donor-derived myeloid reconstitution is the best readout of continuous stem cell activity since monocytes and granulocytes are short-lived.

The fact that hematopoietic stem cells can easily be transferred in congenic systems has allowed the physical and functional characterization of purified populations of stem cells from hematopoietic tissues. Attempts to purify hematopoietic stem cells have focused on the physical characteristics of the cells [2, 3], as well as on the proteins that they express on their surface [4-7]. Murine long-term reconstituting hematopoietic stem cells have been shown to express characteristically high levels of H2-K [6, 8] and Sca-1 [4, 5], low levels of Thy-1.1 [4, 9] and undetectable levels of the lineage markers Mac-1, Gr-1, Ter119, B220, CD3, CD4 and CD8 [1, 9]. The most primitive resting stem cells are also thought to incorporate low levels of Rh123 due to a combination of relatively few mitochondria [10-12] and the effects of the mdr gene product [13].

Many (if not most) hematopoietic stem cells in adult animals are thought to be quiescent, or at least noncycling [14, 15]. In fact, highly purified populations of long-term reconstituting stem cells such as the Lineage–, Sca-1+, Thy-1.1low, c-kit+ cells of the bone marrow have been shown to have only around 4% of their cells in the S/G2/M phases of the cell cycle [1]. In addition, the most efficient long-term reconstituting stem cells incorporate very low levels of Rh123, consistent again with the idea that the most primitive stem cells are resting [10, 11]. The most compelling evidence supporting the idea of resting stem cells, however, is that the most primitive hematopoietic stem cells are resistant to the effects of single doses of cytotoxic agents such as 5-fluorouracil [16-18], hydroxyurea [19, 20] or cyclophosphamide [21]. Thus, stem cells must either be in a nondividing state or are resistant to these drugs in other ways. Taken together, these experiments suggest that there is a population of stem cells that may be quiescent and awaiting an activating stimulus. Combined with studies showing that genetically marked clones of stem cells can become active after long periods of dormancy [22-24], some investigators have proposed that the hematopoietic system is controlled at least in part by clonal succession, in which a large pool of hematopoietic stem cells is inactive and is slowly recruited into the active (cycling) pool [25, 26].

Here we have looked for a candidate population of quiescent hematopoietic stem cells and have shown that by using many of the conventional criteria for purifying murine hematopoietic stem cells, we can also copurify a quiescent population of cells that appears phenotypically very similar to long-term reconstituting stem cells, yet has no detectable hematopoietic activity. We have been unable to assign this population of cells to any known hematopoietic lineage, nor have we detected any functional activity of this population. Given the physical similarity of this population to populations of "true" stem cells in the bone marrow, it is tempting to speculate that the two populations are directly related. Regardless of the lack of functional data for these cells, investigators should remain aware that this population significantly overlaps with many "purified" stem cell preparations and can significantly bias any experimental observations.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Conclusion
 References
 
Mice
All mice were bred and maintained in the Stanford Research Animal Facility (Stanford, CA). C57BL/Ka-Thy-1.1 (Ly5.2) mice were used as bone marrow donors and as sources of purified stem cells. C57BL/Ka-Thy-1.1-Ly5.1 mice were used as irradiated recipient animals at 9-12 weeks of age.

Antibodies
Monoclonal antibodies against the following surface molecules were used: fluorescein isothiocyanate (FITC)- labeled anti-Thy-1.1 (19XE5), FITC, phycoerythrin (PE)- or biotin-labeled anti-Sca-1 (E13), FITC-labeled anti-c-kit (3C11; Pharmingen; San Diego, CA), FITC-labeled anti-CD3 (145-2C11), biotin-labeled anti-B220 (RA3-6B2), biotin-labeled anti-CD34 (RAM34; Pharmingen), PE-labeled anti-Mac-1 (M1/70), PE-labeled anti-CD40 (1C10), PE-labeled anti-NK1.1 (PK136; Pharmingen), PE-labeled anti-H-2Kb (AF6-88.5), PE-labeled anti-I-Ab (AF6-120.1; Pharmingen), FITC-labeled anti-GR1 (8C5) allophycocyanin (APC)-labeled anti-Ly5.1 (A20), APC-labeled anti-CD38 (NIM-R5) and APC-labeled anti-Ly5.2 (AL1-4A2) were used as directly conjugated fluorochromes. Anti-CD4 (GK1.5), anti-erythroid antigen (Ter119), anti-CD5 (53-7.3), and anti-CD8 (53-6.7) were used as unlabeled primary antibodies. Secondary reagents included Texas red-labeled goat anti-rat IgG, (Caltag; South San Francisco, CA), Texas red-labeled avidin (Cappel; Durham, NC) and goat anti-rat or streptavidin magnetic beads (Miltenyi Biotec; Auburn, CA).

Preparation and Staining of Bone Marrow
Bone marrow was obtained by flushing the femurs and tibias with phosphate-buffered saline (PBS) containing 2% calf serum. Erythrocytes were eliminated by lysis with NH4Cl. The remaining cells were filtered through nylon mesh and then stained with the indicated combinations of antibodies. Cells were stained in PBS with 2% calf serum for 15 min on ice, washed in PBS with 2% serum and centrifuged through a serum layer. After the final round of staining and washing, the cells were resuspended in PBS 2% calf serum containing 1 mg/ml propidium iodide.

MACS Depletion of Lineage Positive Cells
For cell sorting, bone marrow was initially depleted of lineage positive cells using MACS (Miltenyi Biotec). Cells were stained with unconjugated or biotinylated anti-lineage antibodies and then allowed to bind either goat anti-rat or streptavidin magnetic beads. One hundred ml of beads were used per 108 cells. Cells were then applied to a C-type MACS column and the nonadherent (lineage negative) cells were stained with the subsequent antibodies as indicated in Results.

Cell Cycle Analysis
The DNA content of cells was determined by lysing the cell populations in 0.1% Triton X-100, 1 mg/ml RNase, 10 mg/ml propidium iodide in PBS with 1% bovine serum albumin and measuring the resulting fluorescence in the phycoerythrin channel. All cytometry was performed on a dual-laser FACS (Becton Dickinson; Mountain View, CA) and made available through the shared user group at Stanford University.

Rhodamine 123 Uptake
Bone marrow was resuspended at 5 x 106 cells/ml in RPMI 5% fetal calf serum containing 0.1 mg/ml Rh123 (Molecular Probes; Eugene, OR). Cells were incubated in the dark for 30 min at 37°C. Cells were washed once and resuspended in fresh RPMI 5% fetal calf serum for an additional 30 min at 37°C [13]. Cells were subsequently washed and stained as described above and were maintained on ice or at 4°C until analyzed by FACS.

Treatment with 5-FU
Mice were treated with single doses of 5-fluorouracil (5-FU) (150 mg 5-FU/kg body weight) from a stock solution of 10 mg/ml in PBS. Bone marrow was recovered at the times indicated and stained as described above.

Competitive Long-Term Reconstitution Assays
Irradiated recipients received 950 rads from an x-ray source operated at 200 kV delivering 85 rad/min. Mice were irradiated in a split dose administered four to five h apart. After irradiation, mice were maintained on antibiotic water (neomycin sulfate 1.1 g/l and polymyxin B sulfate 106 U/l) for at least six weeks. Progenitor cells sorted from Ly5.2 donors were injected retro-orbitally into the irradiated recipients along with 2 x 105 whole bone marrow (WBM) cells from normal Ly5.1 mice as a source of radioprotective cells. For the analysis of reconstitution, mice were bled from the tail and assayed for the presence of Ly5.2 positive cells in each of the lineages. Blood was collected in PBS with 10 mmol/l EDTA and 1% dextran. Erythrocytes were allowed to sediment at 37°C for 45 min, and the leukocytes in the supernatant were collected and stained for lineage markers and for Ly5.2. Anti-B220 was used to identify B cells, anti-CD3 for T cells and the combination of anti-Mac-1 and anti-GR-1 was used to identify myeloid cells.

Colony Forming Unit-Spleen (CFU-S) Assays
CFU-S assays were performed as described [27]. Sorted cells were retro-orbitally injected into lethally irradiated animals. After 12 days, mice were sacrificed and spleens were removed and fixed in 70% ethanol, 5% formaldehyde and 5% glacial acetic acid. Macroscopic colonies were counted by inspection.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Conclusion
 References
 
Most procedures for the isolation of hematopoietic stem cells include the elimination of the mature cells of the bone marrow using a panel of antibodies to lineage markers [4, 5]. In these experiments, the lineage cocktail included antibodies to T cells (CD3, CD4, CD5, CD8), B cells (B220, IgH, kappa), myeloid cells (Mac-1, Gr-1), erythroid cells (Ter119), natural killer (NK) cells (NK1.1) and APCs (I-A, CD40). When the lineage positive cells are eliminated from a FACS analysis of adult bone marrow ( Fig. 1A), one can see that only a minority of lineage– cells are Sca-1+. These lineage–, Sca-1+ cells can be further divided into two distinct populations based on the expression of c-kit ( Fig. 1B). The c-kit– population has levels of Thy-1.1 that range from low to intermediate ( Fig. 1B) although this expression is lower than that found on T cells (not shown). In contrast, the c-kit+ cells within the lineage–, Sca-1+ gate consist of cells that express low levels of Thy-1.1 and cells that are Thy-1.1– ( Fig. 1B). Since previous work has shown that all the stem cells in the bone marrow of this strain of mouse are found within the lineage–, Sca-1+ Thy-1.1low population [1, 9], it was reasonable to expect that the stem cells fell into one or both of the boxes in Figure 1B. When these cells were examined for characteristics of cell size using the forward and side scatter parameters, it appeared that the c-kit– cells ( Fig. 1B, box a) had scatter profiles consistent with small resting cells ( Fig. 1C), while the c-kit+ cells ( Fig. 1B, box B) were slightly larger and more granular ( Fig. 1D).



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  		Figure 1. c-kit defines two distinct subpopulations within the lineage–, Sca-1+, Thy-1.1low fraction of bone marrow. Panel A: expression of Sca-1 and c-kit on lineage– cells from normal bone marrow. Cells above the horizontal line are Sca-1+. Panel B: the expression of Thy-1.1 and c-kit on lineage–, Sca-1+ cells as indicated in A. Panel C: forward and side scatter properties of lineage–, Sca-1+, Thy-1.1low, c-kit– cells from box a. Panel D: forward and side scatter properties of lineage–, Sca-1+, Thy-1.1low, c-kit+ cells from box b.

 
Since we had previously observed that long-term reconstituting stem cells in C57BL/6-Thy-1.1 mice were found in the CD38high fraction of the lineage–, Sca-1+ bone marrow population [28], we examined the expression of CD38 relative to c-kit on lineage–, Sca-1+ cells. In Figure 2 we show that the lineage–, Sca-1+, c-kit+ cells have a wide range of CD38 expression ( Fig. 2, boxes B and C) as previously described [28], while the lineage–, Sca-1+ c-kit– cells ( Fig. 2, box A) uniformly express high levels of CD38, consistent with the phenotype of the long-term reconstituting stem cells. In order to examine the cell cycle characteristics of these populations, the lineage–, Sca-1+ cells were initially sorted into a c-kit–, CD38high population ( Fig. 2, box A) and a lineage–, Sca-1+, c-kit+ population, including both CD38high and CD38low cells ( Fig. 2, boxes B and C together). The c-kit–, CD38high cells were then sorted a second time as a single population ( Fig. 2, box A), while the c-kit+ cells were sorted into two populations: the CD38high cells ( Fig. 2, box B) and the CD38low/– cells ( Fig. 2, box C). These three populations were then permeablized in the presence of propidium iodide and analyzed for DNA content ( Fig. 2). The c-kit–, CD38high population had only 2% of its cells in the S/G2/M phases of the cell cycle as seen in Figure 2a, consistent with a population that is predominantly quiescent. The c-kit+, CD38high population was relatively more active with 11% of cells in the S/G2/M phases of the cell cycle ( Fig. 2b), while the c-kit+, CD38low/– population was the most active with 16% of cells in S/G2/M ( Fig. 2c). As a comparison, 17% of the cells in unfractionated whole bone marrow are in S/G2/M ( Fig. 2 WBM).



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  		Figure 2. Cell cycle analysis of subpopulations within the lineage–, Sca-1+ fraction of adult bone marrow. Lineage–, Sca-1+ cells were sorted into A) CD38high, c-kit– cells; B) CD38high, c-kit+ cells; and C) CD38low/–, c-kit+ cells. Each population was sorted twice to ensure purity. Sorted cells were permeablized in the presence of propidium iodide and examined for DNA content. Histogram a: DNA content of cells from box A. Histogram b: DNA content of cells from box B. Histogram c: DNA content of cells from box C. Histogram WBM: DNA content of cells from unfractionated whole bone marrow. Numbers beside the histograms refer to the percent of cells in the S/G2/M phases of the cell cycle.

 
Since the majority of the cells in the lineage–, Sca-1+ Thy-1.1low, CD38high, c-kit– population appeared to be in a resting state, we examined their ability to incorporate the vital dye Rh123. Rh123 is incorporated into mitochondrial membranes [12] and can serve as an indicator of the relative "activity" of a cell, although this interpretation may be complicated by the fact that Rh123 can also be effluxed from cells by the mdr gene product [13]. In Figure 3A the relative incorporation of Rh123 is shown for unfractionated bone marrow. This has been arbitrarily divided into low, intermediate and high levels of Rh123 as indicated by the vertical lines. Interestingly, the lineage–, Sca-1+ c-kit– population, as a whole, incorporated extremely low levels of Rh123, with 44% falling into the Rh123low gate and 51% falling into the Rh123intermediate gate while only 5% of these cells were in the Rh123high gate ( Fig. 3B, shaded histogram). In sharp contrast, only a small minority (4%) of lineage–, Sca-1+, c-kit+ cells were included in the Rh123low gate ( Fig. 3B, open histogram). Furthermore, only 29% of the c-kit+ cells were in the Rh123intermediate gate, while 67% of the c-kit+ cells were in the Rh123high gate ( Fig. 3B, open histogram). Unfortunately, due to the limitation of having only four colors on the cell sorter, the inclusion of Rh123 in the FITC channel necessitated the elimination of either CD38 or Thy-1.1. This meant that while the c-kit– population was essentially pure, the c-kit+ population was a combination of both CD38high and CD38low/– cells, as in Figure 2, boxes B and C, or both Thy-1.1low and Thy-1.1– cells as in Figure 1B. Regardless, the lineage–, Sca-1+, c-kit– population retains very low levels of Rh123, consistent with the observed phenotype of the most primitive hematopoietic stem cells [7, 12, 13].



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  		Figure 3. Incorporation of Rh123 by c-kit+ or c-kit– cells in the lineage–, Sca-1+ fraction of bone marrow. Erythrocyte-depleted whole bone marrow cells were incubated at 5 x 107 cells/ml in RPMI containing 5% fetal calf serum and 0.1 mg/ml Rh123 for 30 min at 37°C to allow Rh123 uptake by the cells. Cells were washed once and resuspended in RPMI, 5% fetal calf serum and incubated for an additional 30 min at 37°C. All subsequent steps were performed on ice or at 4°C. Cells were then stained with antibodies to lineage markers, Sca-1 and c-kit, and analyzed by FACS. Panel A: incorporation of Rh123 by whole bone marrow. Panel B: incorporation of Rh123 by lineage–, Sca-1+, c-kit– cells (shaded histogram) and by lineage–, Sca-1+, c-kit+ cells (open histogram). Plots have been divided into regions corresponding to low (L), intermediate (M) and high (H) incorporation of Rh123. In the lineage–, Sca-1+, c-kit– population 44% are Rh123low, 51% Rh123intermediate and 5% are Rh123high. In the lineage–, Sca-1+, c-kit+ population 4% are Rh123low, 29% Rh123intermediate and 67% are Rh123high

 
The idea that most hematopoietic stem cells exist in a resting state has its origins in experiments showing that stem cells are resistant to the effects of cytotoxic agents such as 5-FU [17, 18, 20, 29]. When mice are treated with a single dose of 5-FU, the population of long-term reconstituting stem cells is relatively undiminished [17, 30] while other hematopoietic precursors such as colony forming unit-spleen (CFU-S) are virtually eliminated [20]. We treated mice with a single dose of 5-FU and examined the cells within the lineage–, Sca-1+ population to test whether a particular subpopulation was resistant to the effects of 5-FU over a time course of 18 days post-5-FU treatment ( Fig. 4). As seen in the left column of Figure 4, the population of lineage–, Sca-1+, c-kit+ cells in untreated mice ( Fig. 4, day 0, box b), which has been previously shown to include long-term reconstituting stem cells, is found at a frequency similar to the lineage–, Sca-1+, c-kit– population of resting cells that we have identified here ( Fig. 4, day 0, box a). Both of these populations are mostly contained within the lineage–, Sca-1+, Thy-1low population ( Fig. 4, day 0, box c), although there are some Thy-1.1– cells in the c-kit+ population as seen in Figure 1. Between two and six days after treatment with 5-FU the overall lineage–, c-kit+ population has virtually disappeared (around a 1,000-fold decrease; see Fig. 4, day 2-day 6 and Fig. 5A), consistent with the idea that the majority of hematopoietic progenitors that are not long-term reconstituting stem cells are rapidly cycling, and thus killed by cytotoxic agents. By 18 days post-5-FU, however, the c-kit+ population has been re-established ( Fig. 4, day 18, box b). In contrast, the absolute numbers of the lineage–, Sca-1+, Thy-1low, c-kit– population appeared to be unaffected by 5-FU treatment ( Fig. 5B) and thus was selectively enriched, especially on day 6 in the lineage–, Sca-1+, Thy-1.1low population ( Fig. 4, day 6).



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  		Figure 4. Disappearance of c-kit+ populations in the bone marrow after 5-FU treatment. Mice were treated with a single dose of 5-FU (150 mg/kg) and bone marrow was collected from untreated mice (labeled as 0) and mice that had been treated 2, 4, 6 and 18 days previously (labeled as 2, 4, 6 and 18, respectively). Cells were stained with antibodies to lineage markers, Sca-1, c-kit and Thy-1.1, and the expression of Sca-1 and c-kit on lineage– cells is shown in the left column, while the expression of Sca-1 and Thy-1.1 on lineage– cells is shown in the right column. Of particular note are the populations of lineage–, Sca-1+, c-kit– cells (box a), lineage–, Sca-1+, c-kit+ cells (box b) and the lineage–, Sca-1+, Thy-1.1low cells (box c) for the full time course.

 


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  		Figure 5. Cellularity of the bone marrow in mice treated with 5-FU. Bone marrow was collected from groups of five mice treated two, four and six days previously with a single dose of 5-FU as well as from untreated mice. The bone marrow samples were made into single cell suspensions, counted, and stained as described in Figure 4. The frequencies of each population were determined by integration using FACSDesk software. Frequencies were multiplied by the total number of cells recovered from both femurs and tibias to obtain absolute numbers of cells for the indicated populations.

 
The absolute number of cells in each population was calculated based on the total number of cells recovered from two femurs and tibias per mouse and the frequency of the cells as determined by FACS ( Fig. 5). The cellularity of the bone marrow decreased about 20-fold over the first six days after 5-FU treatment ( Fig. 5A) consistent with other reports [19, 31-33]. However, the cellularity of the lineage–, c-kit+ population decreased about 1,000-fold within the first two days as seen in Fig. 5A. In other experiments we have observed that this drop actually occurs within the first 24 h (not shown). The lineage–, Sca-1+ Thy-1low, c-kit+ population drops around 100-fold within this time period ( Fig. 5B), but the lineage–, Sca-1+ Thy-1low, c-kit– population remains relatively constant ( Fig. 5B), as is predicted for a resting stem cell population based on functional studies [17]. Although the absolute numbers of the lineage–, Sca-1+, c-kit– population stayed the same, the frequency of this population was dramatically increased as seen in the FACS plots of Figure 4 and consistent with other functional studies [17, 34]. This was particularly true on day 6 post-5-FU ( Fig. 4, day 6) since the cellularity of the bone marrow was lowest at this time ( Fig. 5).

Although the physical properties of the lineage–, Sca-1+, Thy-1low, c-kit– population are consistent with the predicted characteristics of a resting stem cell population, we wanted to evaluate the hematopoietic properties of these cells functionally. Initially we compared the frequency of day 12 CFU-S within lineage–, Sca-1+, Thy-1.1low, c-kit– to that of the c-kit+ population. We injected one group of lethally irradiated recipients with 100 sorted lineage–, Sca-1+, Thy-1.1low, c-kit– cells per animal and another group with 100 sorted lineage–, Sca-1+, Thy-1.1low, c-kit+ cells. At day 12, the 10 mice receiving c-kit+ cells had on average 7.6 colonies per spleen for a frequency of 1/13. In contrast, there were no colonies present in the 8 survivors of 10 injected mice that had received the c-kit– cells (0/800).

Since it has been proposed by several groups that a subpopulation of long-term reconstituting stem cells lacks CFU-S activity [2, 14, 35], we examined the reconstituting potential of the c-kit– and c-kit+ cell populations in competitive reconstitution assays. Lineage–, Sca-1+ CD38high cells were sorted into c-kit+ and c-kit– populations from Ly5.2 expressing donors. Three hundred cells of each population were injected into irradiated Ly5.1 recipients in a competitive reconstitution assay. Reconstitution was measured by determining the percentage of Ly5.2-expressing cells in both myeloid and lymphoid lineages in peripheral blood. As seen in Figure 6, while the c-kit+ cells gave robust, multilineage reconstitution in each of the mice at the 16-week time point, mice reconstituted with c-kit– cells showed no evidence of donor type reconstitution in any lineage at 16 weeks ( Fig. 6) or at any other time measured. In fact, within the context of bone marrow transplantation, these cells appeared to be hematopoietically inert as they produced no spleen colonies in irradiated mice, had no colony forming ability in the presence of cytokines (combinations of stem cell factor, interleukin 3 [IL-3] and IL-6) or stromal cells, and were not radioprotective (not shown).



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  		Figure 6. Competitive long-term reconstitution potential of lineage–, Sca-1+, CD38high, c-kit– and lineage–, Sca-1+, CD38high, c-kit+ cells. The lineage–, Sca-1+, CD38high, c-kit– and lineage–, Sca-1+, CD38high, c-kit+ populations were sorted from the bone marrow of adult Ly5.2 mice as shown in Figures 2A and B Three hundred cells of each population were injected into lethally irradiated Ly5.1 recipients along with 2x 105 whole bone marrow cells of the Ly5.1 host genotype. Repopulation was assayed by measuring the percentage of donor-derived cells in the blood that expressed the lineage markers CD3 (T cells), B220 (B cells), Mac-1 and GR-1 (myeloid cells). The table represents the percentage of donor-derived cells in each lineage at 16 weeks post reconstitution.

 
To test if the lineage–, Sca-1+, c-kit– cells could be derived from c-kit+ stem cells, we transferred 1,000 lineage–, Sca-1+, c-kit+ cells from Ly5.2, Thy-1.1 donors into irradiated Ly5.1 (Thy-1.2) recipients. The recipients were allowed to repopulate their hematopoietic systems for 14 weeks, and bone marrow was subsequently harvested and analyzed for lineage–, Sca-1+, Thy-1.1low, c-kit– cells that were negative for the host marker Ly5.1. Figure 7 shows the expression of Thy-1.1 and c-kit on lineage–, Sca-1+, Ly5.1– cells from the bone marrow of one of the reconstituted animals. The box indicates the donor-derived lineage–, Sca-1+, Thy-1.1low, c-kit– cells. These cells are not only Ly5.1–, but are also Thy-1.1+, indicating their donor origin. Thus, it is clear that the lineage–, Sca-1+, c-kit– population can originate from c-kit+ stem cell populations since donor-derived cells of this population can be observed after the transfer of c-kit+ stem cells. Figure 7 is representative of data obtained from five recipient mice.



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  		Figure 7. Reconstitution of lineage–, Sca-1+, c-kit– cells by transfer of lineage–, Sca-1+, c-kit+ cells. The lineage–, Sca-1+, CD38high, c-kit+ population of cells that is known to contain long-term reconstituting stem cells was sorted from Ly5.2 mice. One thousand sorted cells of this population were injected into irradiated Ly5.1 recipients. Fourteen weeks post reconstitution, bone marrow was recovered from recipients and stained with antibodies to lineage markers, Sca-1, Thy-1.1, c-kit and Ly5.1 The expression of Thy-1.1 and c-kit on the lineage–, Sca-1+ fraction of Ly5.1– cells (donor type) is shown. The box indicates the repopulated lineage–, Thy-1.1low, c-kit– cells.

 
The apparent phenotypic similarity of the c-kit– population to long-term reconstituting hematopoietic stem cells in the absence of a functional readout was somewhat mystifying. We attempted to identify a hematopoietic lineage to which the lineage–, Sca-1+, c-kit– cells (mystery population) could be assigned. Since the mystery population cells physically looked like small resting lymphocytes, and CD38, Sca-1 and Thy-1 can all be markers for lymphoid cells, we examined both nu/nu and rag-1–/– mice for the presence of the mystery population in the bone marrow. Both of these strains of mice contained the lineage–, Sca-1+, Thy-1.1low, c-kit– population at normal frequency (not shown), demonstrating that these cells did not belong to either the T or B lineage subsequent to antigen receptor rearrangement. In addition, we found no evidence of the expression of CD3, Ig(M,G,A), kappa or NK1.1 which might have been consistent with cells of the lymphoid lineages. Furthermore, the c-kit– cells did not respond to IL-2, nor exhibit any killing activity towards NK target cells that would indicate an NK-like origin (not shown).

Since it appeared unlikely that the lineage–, Sca-1+, c-kit– cells were actually stem cells, we examined other lympho/hematopoietic tissues such as spleen, thymus and lymph nodes for the presence of c-kit– cells and compared their frequency with the lineage–, Sca-1+, c-kit+ cells in the same tissues. Table 1 shows that the c-kit– population is found in spleen, thymus and lymph nodes, although its frequency is highest in bone marrow. This is in contrast to the stem cell-containing c-kit+ population which is found predominantly in bone marrow, to a lesser extent in spleen and is almost undetectable in both the thymus and lymph nodes. Thus, while the c-kit– population is more widespread in various lymphoid tissues, its overall frequency is still extremely low and very similar to that of the lineage–, Sca-1+, c-kit+ population.


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  		Table 1.
 
We next examined the expression of other stem cell associated markers on these cells in the hope that with some marker other than c-kit we could at least eliminate this population from stem cell sorting procedures. One phenotypic characteristic that has been used to identify stem cell-containing populations from humans is the expression of CD34 [7]. We examined the expression of CD34 on both kit+ and kit– cells within the lineage–, Sca-1+ population and found that while the lineage– population was split evenly between CD34+ and CD34– cells ( Fig. 8B), the c-kit– mystery population was essentially devoid of CD34+ cells, and the c-kit+ stem cell-containing population was almost completely negative for CD34 ( Fig. 8). While this is contradictory to data that suggest that human progenitors are CD34+ [31], murine stem cells have been found to lack the expression of the CD34 antigenic epitope that is recognized by the RAM34 antibody that we used in this study [32].



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  		Figure 8. Expression of CD34 on lineage–, Sca-1+, c-kit– and lineage–, Sca-1+, c-kit+ cells. BM cells were stained with the lineage cocktail of antibodies and as seen in A, the majority of cells were lineage+. The lineage– cells to the left of the gate in A were analyzed for CD34 expression in B. Lineage– cells were also analyzed for the expression of c-kit and Sca-1 and were divided into CD34– and CD34+ fractions as shown by the vertical line in B. The lineage–, Sca-1+, c-kit– cells are contained within box a while the lineage–, Sca-1+, c-kit+ cells are contained within box b.

 
Another marker used to identify stem cell-containing populations from murine adult bone marrow is the high level of H2-K expression [6]. We compared the expression of H2-K on lineage–, Sca-1+, Thy-1.1low, c-kit+ with that on cells of the mystery population ( Fig. 9). The expression of H2-Kb on WBM is shown in Figure 9, panel A. While most cell are positive, they express relatively low levels of H2-Kb. In contrast, both the lineage–, Sca-1+, c-kit+ (open histogram) as well as the lineage–, Sca-1+, c-kit– populations (shaded histogram) express equivalently high levels of H2-Kb, consistent with the phenotype of stem cells as reported by others [5, 6, 8]. In addition, both populations express equivalently high levels of CD45 (Ly5), as expected for hematopoietic cells in general (not shown).



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  		Figure 9. Expression of H-2Kbon lineage–, Sca-1+, c-kit– and lineage–, Sca-1+, c-kit+ cells. H-2Kbexpression on unfractionated bone marrow is shown in panel A. H-2Kb expression on lineage–, Sca-1+, c-kit– cells is shown in panel B (shaded histogram) and the expression of H-2Kbon lineage–, Sca-1+, c-kit+ cells is shown in panel B (open histogram).

 
In early experiments we had observed that the relative frequency of the c-kit– mystery population appeared to be somewhat variable relative to the frequency of the lineage–, Sca-1+, c-kit+ cells in the bone marrow. Even though cells were typically examined from the bone marrow of mice within the relatively narrow age window of four to eight weeks, the variation appeared to correlate with the age of the mice. When we examined the frequencies of these two populations as a function of age ( Fig. 10), we found that very young mice (one to three weeks) had a slightly increased frequency of lineage–, Sca-1+, Thy-1.1low, c-kit+ cells and a very low frequency of the c-kit– mystery population. At five to six weeks of age the frequencies of the two populations are very similar at around 0.02%. Then, during the next several months, the lineage–, Sca-1+, Thy-1.1low, c-kit+ population levels off at around 0.01% while the c-kit– mystery population continues to increase in frequency ( Fig. 10).



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  		Figure 10. Changes in the frequency of the lineage–, Sca-1+, Thy-1.1low, c-kit– and the lineage–, Sca-1+, Thy-1.1low, c-kit+ populations over time. Bone marrow was obtained from mice at 1, 3, 8 and 20 weeks of age and stained with antibodies for lineage markers, Sca-1, Thy-1.1 and c-kit. Frequencies of the lineage–, Sca-1+, Thy-1.1low, c-kit– and the lineage–, Sca-1+, Thy-1.1low, c-kit+ populations were obtained by integration using FACSDesk.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Conclusion
 References
 
In this report we describe a rare population of cells from murine bone marrow that phenotypically mimics many of the characteristics attributed to hematopoietic stem cells. The lineage–, Sca-1+, Thy-1.1low, CD38high, CD34–, H2-Khigh, c-kit– population of cells found in normal bone marrow is strikingly similar to that observed on functionally characterized hematopoietic stem cells, with the exception of the lack of c-kit expression [33, 36, 37]. In fact, almost all of the procedures for isolating stem cells from the murine bone marrow that do not involve the expression of c-kit will assuredly copurify these cells as well [4-6, 11]. The cells of the mystery population morphologically look like resting lymphocytes but have no markers or functional properties associated with either B, T or NK cells. The fact that this population is found in normal frequency and numbers in rag-1–/– mice formally eliminates the possibility that it is actually any form of antigen receptor bearing B or T cells. Furthermore, these cells have no markers of NK cells, nor do they exhibit any form of killing activity either with or without IL-2. The possibility that these cells are precursors to lymphocytes prior to antigen receptor rearrangement is unlikely as well, since these cells are both small and noncycling in contrast to most early precursors. These cells have absolutely no physical characteristics of any myeloid cells, and yet they are certainly of the hematopoietic lineage since they express the leukocyte common antigen, CD45 (Ly5) and can be repopulated by c-kit+ hematopoietic stem cells. Obviously we would like to know if there is a direct relationship between the c-kit– mystery population and c-kit+ "true" stem cells. Although mystery population cells can clearly be derived from c-kit+ stem cells, it remains unclear if they are actually a population of resting stem cells that is in some sort of equilibrium with the "active" c-kit+ stem cells or if they are simply members of another unidentified hematopoietic lineage downstream of the stem cell.

Is it possible that there are populations of stem cells that have no functional assay at this point? To date, the most stringent assay for hematopoietic stem cells is the competitive long-term reconstitution assay [38]. Although it is now taken for granted that hematopoietic stem cells can be easily transferred in syngeneic systems and are triggered to restore the hematopoietic system, it is possible that there is an additional class of resting stem cells (such as the mystery population) that cannot be assayed by transplantation in the absence of the appropriate activating signal. Treatment of mice with 5-FU would be an obvious candidate for such an activating stimulus, since 5-FU would eliminate cycling progenitors [14, 30, 39, 40], and any quiescent stem cells would then be forced into cycle in order to replace the depleted hematopoietic system [41]. Furthermore, as seen in Figures 4 and 5GoGo, the vast majority of c-kit+ cells disappear after 5-FU treatment while the c-kit– mystery population cells remain. Interestingly, we and others have found that many long-term reconstituting stem cells can be found in the c-kitlow/– fraction of the bone marrow after 5-FU treatment [32, 41, 42]. While it is possible that these cells represent resting c-kit– mystery population cells that have been activated to replace the hematopoietic system, other evidence suggests that the c-kit+ stem cells can temporarily lose expression of c-kit under these conditions and become indistinguishable from the mystery population cells [30, 36, 43, 44]. It is becoming increasingly clear that stem cells do not have a static cell surface phenotype and that events which perturb the hematopoietic system, such as treatment with 5-FU or mobilization protocols, change both the physical as well as the functional properties of stem cells [30, 40, 42]. Thus, one must be careful not to equate the presence or absence of a particular phenotypically defined population with the presence or absence of stem cell activity.

It is interesting that the mystery population not only mimics the phenotype of hematopoietic stem cells, but that it is found at a frequency similar to that of stem cells in the bone marrow and at a similarly low frequency in spleen, thymus and lymph nodes ( Table 1). Since there are relatively few mystery population cells in young mice where the hematopoietic system is just being established and their frequency increases with age, one could imagine that once the hematopoietic system is well established a few weeks after birth many of the "active" c-kit+ stem cells are put into storage for future use. This would also explain the fact that the c-kit+ "active" stem cells can produce the c-kit– mystery population cells. The mystery population cells might represent senescent stem cells that are simply no longer functional and for some reason these cells accumulate with age or after transplantation. More likely, however, these represent cells totally unrelated to early hematopoietic progenitors, and have a function that we have failed to assay.


   Conclusion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
References
 
Without a functional assay, the c-kit– mystery population will have to remain an enigma. However, in a practical sense, investigators analyzing "purified" stem cell populations should remain aware that almost all sorting or enrichment procedures for hematopoietic stem cells that do not involve the selection of c-kit+ cells will copurify the c-kit– mystery population and, depending on the age of the mice, could dramatically bias their experimental results. This is particularly true after 5-FU treatment, when even c-kit expression fails to separate the mystery population from functional stem cells [30, 43, 44].


   Acknowledgments
 
The authors would like to thank Dr. Frances Lund for critical review of the manuscript prior to submission, Nobuko Uchida for help with the RH123 analysis and Tim Knaack for cell sorting.

Supported in part by National Institutes of Health grant CA 42251 and by a grant to Irving L. Weissman from Systemix/Novartis. Troy D. Randall was a recipient of a Helen Hay Whitney Foundation Fellowship during the course of this work.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 References
 

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accepted for publication November 14, 1997.



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