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G. Wu and C.C. Zhang
plantation, the repopulation percentage of WT LT-HSC donors gradually increased to above 35%, whereas the percentage decreased to below 2% for WT ST-HSC plus MPP (Figure 4A). This indicated that LT-HSC play the major role in regeneration and that ST-HSC and MPP are quickly exhausted.
When WT and CAR cKO donors were compared, WT LT-HSC repopulated about twice as efficiently as CAR cKO cells in the first 6 weeks (Figure 4A). There was only a small difference for ST-HSC plus MPP between WT and CAR cKO cells on days 21 and 23 in Mac1+ population. In the B220+ population there were significant differences in LT-HSC, but not in ST-HSC plus MPP, between WT and CAR cKO donors (Figure 4A). This suggests that CAR cKO impairs the function of LT-HSC but not of ST-HSC and MPP. At 170 days after transplantation, the repopula- tion percentages donor LSKFC of WT and CAR cKO LT- HSC groups did not differ significantly (Figure 4B), indi- cating that CAR deficiency does not alter LT-HSC activity during regeneration. These results indicate that CAR stimulates regeneration mainly by affecting LT-HSC.
In order to evaluate whether lack of CAR is detrimental after bone marrow transplantation, we conducted anoth- er repopulation assay (Figure 4C). Here WT or CAR cKO BM cells were mixed with an equal number of CD45.1 BM cells and were injected into lethally irradiated recipi- ent mice. WT donor cells showed greater repopulation ability in both myeloid and lymphoid cells than CAR cKO donor cells in first 6 weeks after transplantation. After 6 weeks, the difference between WT and CAR cKO repopulation gradually diminished. At 16 weeks after BM transplantation, the repopulation percentage of the donor
BM cells from CAR cKO mice was not significantly differ- ent from that in mice that received WT cells (Figure 4C). In the second transplantation, CAR cKO donor cells had no defects in long-term repopulation ability (Figure 4C). This serial transplantation analysis indicates that the self- renewal of LT-HSC was not affected by CAR. In addition, CAR did not affect homing ability of HSC (Online Supplementary Figure 6). The limiting dilution analysis indicates that CAR cKO did not change the frequency of HSC in BM (Online Supplementary Table1). This result sug- gests that CAR enhances the speed of hematopoietic repopulation after BM transplantation but does not change HSC activity over the long term. Together, these results demonstrate that CAR supports hematopoietic regeneration after stress. Furthermore, the donor BM cells from Scl-CreERT/CARloxp/loxp mice in which CAR was specifically cKO in hematopoietic cells also had defects in initial repopulation after BM transplantation (Online Supplementary Figure 7), indicating that CAR on the hematopoietic cells plays a major role in regenera- tion. In repopulation assay with CAR cKO recipient mice, CAR cKO in the donor hematopoietic cells still resulted in defects in repopulation (Online Supplementary Figure 8), suggesting that CAR in the BM microenvironment is not essential for the function of CAR in regeneration.
Next, we assessed phenotypical LT-HSC (LSKFC), stem cells, and multiple progenitors in WT and CAR cKO mice after 5-FU treatment. None of these populations altered once the mice recovered from 5-FU injury (Figure 4D). In order to test whether the functional LT-HSC were affected by CAR after injury, BM transplantation was conducted with the donor cells collected from mice 1 month after 5-
AB
Figure 3. CAR stimulates progenitor production after 5-fluorouacil treatment and bone marrow transplantation. (A) One month after tamoxifen treatment, bone marrow (BM) cells from wild-type (WT) or CAR conditional knockout (cKO) mice were isolated before and at indicated days after 5-fluorouracil (5-FU) treatment (150 mg/kg) for colony forming units assays. (B) CFU assays of BM cells after transplantation of 1x106 total BM cells into lethally irradiated WT. *P<0.05; **P<0.001.
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haematologica | 2021; 106(8)