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CAR promotes hematopoietic regeneration
FU treatment. There was no difference in the long-term repopulation ability between WT and CAR cKO donors in this experiment (Figure 4E). Together, our results indicated that self-renewal of LT-HSC was not affected by CAR nei- ther during homeostasis nor during regeneration.
In order to further understand how CAR affects LT-HSC, the population LSKFC cells were identified from mice on different days after 5-FU treatment and cell cycle states were analyzed (Figure 5). Before 5-FU injection, there was no difference between WT and CAR cKO cell cycle status. At 1.5 days after treatment, there was a significantly lower frequency of WT LSKFC in the G0 stage, and more WT LSKFC cells in apoptosis than prior to treatment because 5- FU kills cycling cells. After 3 days, more WT LSKFC than
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CAR cKO cells were in G1 stage and fewer were in G0 stage (Figure 5). These results suggest that CAR is needed for the transition into the cell cycle. On day 5 post 5-FU treatment, more than 90% of LSKFC cells were cycling and there were no differences between WT and CAR cKO pop- ulations. This implies that CAR is needed immediately after injury. Compared to CAR cKO mice, LSKFC of WT mice returned to homeostasis earlier, as more WT than CAR cKO LSKFC were at the G0 stage on day 6. In addi- tion, CAR+ LSKFC cells were positive for Ki67 staining and thus were cycling, whereas most CAR- LSKFC cells were Ki67 negative right after 5-FU treatment (Online Supplementary Figure 9). Thus, CAR appears to stimulate quiescent HSC to enter the cell cycle.
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Figure 4. CAR stimulates regeneration by forcing hematopoietic stem cells into the cell cycle but does not affects self-renewal. (A) Competitive repopulation assays were performed with LT-HSC (LSKFC, LSKCD34-CD135-) or “short-term” HSC (ST-HSC) plus multipotent hematopoietic progenitors (MPP) (LSKCD34+) from wild-type (WT) or CAR conditional knockout (cKO) donors (1 month after tamoxifen treatment) and CD45.1 WT competitor bone marrow (BM). Percentages of donor peripheral leukocytes in total peripheral blood (PB), the Mac1+ population, and the B220+ population after BM transplantation are plotted. (B) Percentages of donor LT-HSC (CD45.2) in total BM LT-HSC (CD45.1 plus CD45.2) 170 days after transplantation. (C) Competitive repopulation assays after initial BM transplantation were per- formed with a 1:1 ratio of donor (CD45.2) and CD45.1 WT competitor BM. Shown are the percentages of donor peripheral leukocytes (CD45.2) in total PB, the Mac1+ population, and the B220+ population. Data are means ± standard error of the mean, n=7-9 mice. (D) Number of LSK and LSKFC cells in BM before and after treat- ment with 150 mg/kg 5-FU (n=3-9 mice per group). (E) Donor mice (1 month after tamoxifen treatment) were injected with 150 mg/kg 5-FU, and 1 month later, com- petitive repopulation assays were performed with a 1:1 ratio of donor and CD45.1 WT competitor BM. Shown are the percentages of donor peripheral leukocytes in total PB, the Mac1+ population, and the B220+ population. Data are means ± standard error of the mean, n=9-10 mice. *P<0.05; **P<0.001. NS: no significant difference.
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