JCI - Hematopoietic stem cells proliferate until after birth and show a reversible phase-specific engraftment defect

All HSCs in the 14.5-dpc FL are rapidly proliferating. To measure the proportion of HSCs that are in cycle in the 14.5-dpc FL, we used 3 complementary strategies. In the first, we injected pregnant mice at 13.5 dpc with 100 mg/kg of 5-fluorouracil (5-FU) and then removed the fetuses 16 hours later, prepared cell suspensions from the FLs, and measured the number of HSCs present using a limiting dilution transplantation assay for long-term (16-week) competitive repopulating units (CRUs) (1). In these experiments, we detected very few CRUs in the FLs of the 5-FU–treated embryos (Figure 1A). A comparison of the CRU yield from the 5-FU–treated FLs with the control FLs from pregnant mice injected 13.5 dpc with PBS indicated that the 5-FU treatment had reduced the expected CRU population in vivo by more than 1,000-fold.

We then assessed the cycling status of HSCs in the 14.5-dpc FL by measuring the proportion of CRUs that survived a 16-hour exposure to high–specific activity ³H-thymidine (³H-Tdr) (30). Sixteen hours was anticipated to be sufficient to allow all cycling HSCs to enter S-phase, which we later confirmed (see below), with minimal exit of any quiescent cells from G₀ (31), as demonstrated for adult BM HSCs, most of which were in G₀ (Figure 1A). For these experiments, the Ter119⁺ (erythroid) cells were first removed from the FL cells to give a 10-fold enrichment in HSC content, and cells were then incubated in a serum-free medium supplemented with 50 ng/ml SF only. This growth factor condition was chosen based on other data demonstrating that freshly isolated 14.5-dpc FL CRUs are maintained at the same numbers for 16 hours under these conditions (M.B. Bowie and C.J. Eaves, unpublished observations). The results of the ³H-Tdr suicide experiments showed that this treatment reduced the number of CRUs in the suspensions of 14.5-dpc FL cells more than 100-fold (P < 0.001; Figure 1A), whereas the same treatment had no significant effect on the recovery of CRUs in similarly treated lineage marker–negative (lin^–) BM cells from young adult (10-week-old) mice compared with either control cells incubated without ³H-Tdr (P = 0.17) or starting values (data not shown).

We then assessed the distribution of CRUs between the G₀ and G₁/S/G₂/M fractions of Ter119^– 14.5-dpc FL cells. These subsets were isolated by fluorescence-activated cell sorting (FACS) on the basis of their staining with Hst and Pyronin Y (Py) (32). A representative FACS profile of the Hst- and Py-stained cells is shown in Figure 2A. The combined results of in vivo assays of 14.5-dpc sorted cells from 4 independent experiments are shown in Figure 3 and indicate that all transplantable CRU activity was confined to the G₁/S/G₂/M fraction. Based on the total number of G₀ cells assayed, the proportion of quiescent HSCs was estimated to be less than 0.02%.

HSCs undergo a complete and abrupt change in cycling activity between 3 and 4 weeks after birth. Since HSCs are known to be present in the BM of fetal mice at later times of gestation, it was of interest to investigate whether HSCs first become quiescent in the fetus at that site. To address this question, we used the 16-hour ³H-Tdr suicide assay to determine the cycling status of the CRUs present in the BM of mice at 18.5 dpc. For comparison, we also evaluated the cycling status of CRUs in the 18.5-dpc FL. The frequency of CRUs in these 2 tissues was 1 per 10⁵ and 1 per 7 × 10⁴ total nucleated cells (Supplemental Table 1; supplemental material available online with this article; doi:10.1172/JCI28310DS1); i.e., approximately 5- and 3.5-fold lower, respectively, than in adult BM (1 per 2 × 10⁴ total nucleated cells; ref. 33) and 6- and 4-fold lower, respectively, than in the 14.5-dpc FL (1 per 1.7 × 10⁴ total nucleated cells; ref. 33). After overnight exposure to high–specific activity ³H-Tdr, no CRUs were detected in the suspensions of either the 18.5-dpc fetal BM cells or the 18.5-dpc FL cells, in contrast to the control cells incubated in the same medium without ³H-Tdr (Figure 1B). Thus all HSCs in the fetus, regardless of their location, appear to be rapidly proliferating.

To further investigate the pace and timing of the transition of HSCs into a largely quiescent population, we analyzed the cycling status of CRUs in lin^– BM cell suspensions from 3- and 4-week-old (weanling) mice (lin^– 3- and 4-wk BM, respectively). In initial experiments, the CRU frequencies in the lin^– 3- and 4-wk BM cells were found to be the same (1 per 6.5 × 10³ and 1 per 6.3 × 10³ lin^– cells, respectively; Supplemental Table 2), approximately 2-fold lower than in the lin^– 10-wk BM cells (young adult mice; 1 per 2.9 × 10³ lin^– cells). We then fractionated 3- and 4-wk BM cells by FACS into their component G₀ and G₁/S/G₂/M subsets based on the gates shown in the left panels of Figure 2, B and C, and the sorted G₀ and G₁/S/G₂/M cells were assayed separately for CRU activity. Reanalysis of the sorted G₀ and G₁/S/G₂/M fractions after staining for Ki67 confirmed that the cells expressing this proliferation marker were confined to those we had designated as G₁/S/G₂/M (Figure 2B). Remarkably, the results of the in vivo assays showed that all of the CRUs detected in the 3-wk BM were also confined to the G₁/S/G₂/M fraction, whereas more than 98% of the CRUs in the 4-wk BM were found in the G₀ fraction (Figure 3). Thus, there was a rapid downregulation of CRU proliferative activity in the BM of mice between 3 and 4 weeks of age with little change in CRU number.

HSCs in S/G₂/M show a specific and reversible engraftment defect regardless of their developmental origin or route of injection into assay recipients. Given the previously reported engraftment defect of adult HSCs stimulated to enter S/G₂/M (19), it was of interest to determine whether the number of proliferating HSCs present early in development might be routinely underestimated due an inability to detect those in S/G₂/M. To investigate this possibility, the G₁/S/G₂/M population of Ter119^– 14.5-dpc FL cells was subdivided into its component G₁ and S/G₂/M fractions, and each of these 2 subsets was assayed separately for CRUs. In this case, the gate settings chosen to separate the G₁ (2n DNA) and S-phase cells (>2n DNA) were validated by the profiles obtained when the sorted cells were stained with propidium iodide (PI) and reanalyzed by FACS (Figure 4A).

All CRU activity detectable in the G₁/S/G₂/M fraction of Ter119^– 14.5-dpc FL cells was confined to the G₁ subset (Figure 4B and Figure 5A). Similar experiments performed with lin^– 3-wk BM cells showed that the CRUs in the G₁/S/G₂/M population from this source were likewise confined to the G₁ fraction (Figure 5A and data not shown). It is of interest to note that the S/G₂/M defect was specific to repopulating cells able to produce progeny in all lineages for at least 16 weeks. In contrast, cells with short-term repopulating activity (8 weeks) were readily detected in the S/G₂/M fraction as well as in the G₁ fraction, thus confirming the restriction of this cell cycle–dependent engrafting defect to cells with sustained multilineage repopulating activity (34, 35).

To determine whether the apparent engraftment defect of proliferating CRUs was reversible, we first assayed the CRU content of aliquots of the same isolated G₁ and S/G₂/M cells after they had been incubated for 6 hours at 37°C in serum-free medium containing 50 ng/ml SF. During this time, many of the G₁ cells progressed into S/G₂/M and many of the S/G₂/M cells moved into G₁, as seen by their altered PI (Figure 4A) and Hst (data not shown) staining profiles. In vivo assays showed that CRU activity reappeared when the S/G₂/M cells were cultured for 6 hours, whereas the CRU activity originally present in the G₁ cells was partially lost (Figure 4B).

We next asked whether the inability of intravenously transplanted CRUs in S/G₂/M to engraft recipient mice might be overcome by injecting the cells directly into the femoral BM space. However, we did not detect any CRUs after intrafemoral injection in this subset of Ter119^– 14.5-dpc FL cells (Figure 4B), even though the CRU numbers measured in the corresponding G₁ 14.5-dpc FL cells after intrafemoral injection were the same as after intravenous transplantation (1 per 3.6 × 10³ and 1 per 3.8 × 10³ cells, respectively).

The S/G₂/M engraftment defect of HSCs is overcome by pretreatment of the host with a CXCL12 antagonist. Previous reports have shown that CXCL12 can promote both the mobilization (36) and the homing (37–39) of HSCs. However, the mobilization of primitive hematopoietic cells can also be stimulated by blocking CXCL12/CXCR4 signaling, as achieved by in vivo administration of AMD3100, a CXCL12 antagonist (40). In addition, it has recently been shown that in vivo administration of AMD3100 can increase the competitive engrafting ability of transplanted BM cells in unirradiated hosts (41). These findings suggested that targeting the CXCL12/CXCR4 pathway might also affect the variable engraftment properties of cycling HSCs by influencing either the HSCs themselves or the transplanted host. To investigate these possibilities, we first asked whether pretreating either the cycling HSCs to be transplanted or their hosts with a specific antagonist of CXCL12 might alter the level of repopulation obtained 16 weeks later. The CXCL12 antagonist used in these experiments was SDF-1G2 (also called P2G because it is identical to stromal cell–derived factor 1 except that the proline at position 2 has been converted to glycine; ref. 42). SDF-1G2 is thus structurally quite different from AMD3100 but similar in its ability to block CXCL12 from binding to CXCR4 without activating CXCR4 (42, 43). SDF-1G2 and AMD3100 also share an ability to elicit effects on primitive hematopoietic cells both in vitro and in vivo (44). Mice were injected with 10 μg of SDF-1G2 or PBS 2 hours prior to the transplantation of FACS-sorted G₁ or S/G₂/M cells and analyzed for the presence of donor-derived blood cells 16 weeks later, and the results for 14.5-dpc FL and 3-wk BM cells were similar (Figure 5A). Treatment of recipients with SDF-1G2 had no effect on the repopulating activity of CRUs in G₁. In contrast, SDF-1G2 pretreatment of recipients of S/G₂/M cells enabled long-term multilineage repopulation to be readily detected (in 7 and 4 mice of 10 transplanted with 14.5-dpc FL and 3-wk BM S/G₂/M cells, respectively, versus 0 of 10 in PBS-injected controls for each). Moreover, the SDF-1G2–pretreated hosts showed levels of repopulation by both sources of S/G₂/M cells that were indistinguishable from those seen in mice transplanted with G₁ cells (Figure 6). On the other hand, when SDF-1G2 was applied directly to the cells to be transplanted for 30 minutes before injection, no difference in the engrafting activity of the transplanted G₁ or S/G₂/M cells was seen compared with untreated controls over a wide range of SDF-1G2 and CXCL12 concentrations tested, either with or without added SF (Figure 5B).

HSCs in S/G₂/M express higher levels of CXCL12 transcripts than do HSCs in G₁. To begin to understand the mechanism behind the observed HSC S/G₂/M engraftment defect and how it might be overcome by SDF-1G2 pretreatment of the host, we isolated highly purified populations of HSCs from 14.5-dpc FLs and from 3-wk BM (lin^–Sca-1⁺CD43⁺Mac1⁺ cells representing approximately 20% pure HSCs; M.B. Bowie and C.J. Eaves, unpublished observations) and sorted these into their corresponding G₀/G₁ and S/G₂/M fractions by Hst staining. Aliquots of approximately 200–800 cells were collected from each fraction in 3 independent sorting experiments, and transcript levels for Gapdh, c-Kit, c-mpl, CD44, α4-integrin (α4int), VCAM1, CXCR4, and CXCL12 were measured by quantitative real-time analysis of the cDNAs prepared from the isolated RNA extracts as described in Methods. Transcripts for all these genes were consistently detected in both the G₀/G₁ and S/G₂/M fractions of the highly purified suspensions of HSCs from 14.5-dpc FL and 3-wk BM, including CXCL12, which had not previously been shown to be expressed by HSCs (Figure 7). Interestingly, CXCL12 was also the only one of the genes assessed that was found to be expressed at significantly different levels in G₀/G₁ and S/G₂/M HSCs (9-fold higher in S/G₂/M HSCs; P < 0.05).