Page 154 - 2021_06-Haematologica-web

G. Wu and C.C. Zhang
CAR stimulates progenitor production during regeneration
The Lin-Sca1+cKit+ (LSK) population of hematopoietic cells is primarily responsible for production of progeni- tors during regeneration.25 We compared the production of hematopoietic progenitors by WT and CAR cKO mice after dosing with 150 mg/kg 5-FU and after BM transplan- tation (Figure 3A). The CAR cKO mice produced progen- itors more slowly during recovery. Before 5-FU adminis- tration there were no differences in pre-B or myeloid colony forming units (CFU) in WT and CAR cKO mice. Four days after 5-FU injection, there were significantly more pre-B CFU and myeloid CFU but fewer granulocyte, erythrocyte, monocyte, megakaryocyte (GEMM) CFU in WT mice than in CAR cKO mice, suggesting a slower hematopoietic differentiation in CAR cKO mice upon 5- FU-induced injury. When the mice recovered from injury at 7 days after 5-FU injection, the CAR cKO mice had the same number of myeloid CFU and the difference in pre- B cell CFU between WT and CAR cKO mice was smaller than on day 4 (Figure 3A). We also injected WT or CAR
cKO BM cells into lethally irradiated recipient mice and conducted CFU assays for the BM cells. WT donor cells produced more pre-B and myeloid CFU than CAR cKO donor cells on days 9 and 11 post transplantation (Figure 3B). The difference disappeared at 2 months after trans- plantation, suggesting CAR cKO only took effect during regeneration. These results indicate that CAR stimulates production of differentiated progenitors.
CAR stimulates entry of “long-term”-hematopoietic stem cells into cell cycle
Although CAR promotes progenitor production during regeneration, it did not affect frequency of progenitors or stem cells during homeostasis (Online Supplementary Figure 5). During regeneration, HSC enter the cell cycle and expand.2 During expansion, HSC may self-renew or differentiate. In order to investigate whether CAR regu- lates the function of LT-HSC, ST-HSC, and MPP upon transplantation stress, we collected WT and cKO LT-HSC (LSKFC) and ST-HSC plus MPP (Lin-Sca1+cKit+CD34+) for competitive repopulation assays. From day 17 after trans-
AB
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E
Figure 1. CAR is expressed in differentiated hematopoietic cells, and its expression increases in hematopoietic stem cells during regeneration. (A) CAR expression (determined by real-time qantitative polymerase chain reaction [RT-qPCR] ) in hematopoietic cells, hematopoietic stem cells (HSC), and progenitors including Lin-, LK (Lin-cKit+Sca1-), LSK (Lin-cKit+Sca1+), LSKFC (Lin-Sca1+cKit+CD135-CD34-), CMP (LKCD34+FcrR-), MEP (LKCD34-FcrR-), granulocyte-monocyte progenitor (GMP) (LKCD34+FcrR+), and CLP (Lin-cKitlowSca1lowCD135+CD127+). (B) Right panel, CAR expression (determined by RT-qPCR) in LT-HSC (selected as LSKFC) at day 1.5 and day 7 after 5-fluorouracil (5-FU) treatment (5-FU-1.5d and 5-FU-7d, respectively) and at 9 and 24 days post bone marrow (BM) transplantation (BMT-9d and BMT- 24d, respectively); Left panel, BM cells from wild-type (WT) and CAR conditional knockout (cKO) for analysis CAR expression on LSKFC. (C) CAR surface expression on LK, LSK and Lin-cKit- population after 5-FU treatment. (D) Flow chart of repopulation experiment for LSKFC CAR- or CAR+. (E) Repopulation results of LSKFC CAR- or CAR+ (n=7-9). One and half days after treated with 5-FU, BM cells from mice were isolated for flow cytometry analysis (panel B (left) and C) or for isolating LSKFC cells (D and E). The mRNA levels were calculated based on non-treated LSKFC, and experiments were repeated three times. **P<0.001.
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haematologica | 2021; 106(8)