American Society of Hematology

Figure 2.

$Clonal LSK SLAM fetal liver cell in vivo transplantations reveal decreased engraftment levels and myeloid-lineage bias for Gpr56 deficient HSCs. (A) Experimental setup for in vivo transplantation of E13.5 FL-sorted LSK SLAM cells. (B-C) One FL equivalent of LSK SLAM sorted E13.5 cells from WT and VavCre:loxGpr56 cKO embryos (Ly5.2) were injected into adult irradiated recipients (Ly5.1). Donor cell chimerism in recipient PB at week 4 and 16 posttransplant (B) and hematopoietic tissues at week 18 posttransplant (C). Analysis is by Ly5.1/Ly5.2 flow cytometry. Mean ± SEM is shown. n = 6 per group. (D) Limiting dilution clonal HSC transplantation. Percentage of donor cell engraftment of individual adult irradiated recipient mice as measured by Ly5.1/Ly5.2 flow cytometry of PB at 4, 16, and 23 weeks postinjection of 3, 10, and 30 LSK SLAM E13.5 FL cells. WT controls (red squares) and VavCre:loxGpr56 knockout (cKO, blue empty squares). n = 8 per group. Horizontal line at 5% indicates cutoff for reconstitution. Horizontal lines indicate average percentage engraftment. Circled individual symbols indicate mice used for secondary transplantations. *P ≤ .05; **P ≤ .01. (E) Percentage of donor cell engraftment at 23 weeks postinjection as measured by Ly5.1/Ly5.2 flow cytometry of BM, Sp, LN, and Thy cells from 7 recipient mice injected with 30 LSK-SLAM FL cells. *P ≤ .05. (F) RT-PCR relative expression of Gpr56 normalized to β-actin in LSK BM cells from primary recipients injected with WT and VavCre:loxGpr56 cKO sorted E13.5 FL cells (n = 5). Mean ± SEM are shown. (G) Percentage of lymphoid and myeloid cell contribution in PB of 22 individual adult irradiated recipient mice as measured by flow cytometry of peripheral blood at 23 weeks postinjection of 10 and 30 LSK-SLAM WT control and VavCre:loxGpr56 cKO E13.5 FL cells. (H) Ratio of B-T lymphoid and G-M myeloid for cohort of recipients in panel G with indicators for limits of lymphoid-biased, balanced and myeloid-biased HSC outputs. B, balanced; Ly, lymphoid bias; My, myeloid bias. >87% B-T=Ly; 75% to 87% B-T=B; <75% B-T=My. (I) Percentages of lymphoid-biased, balanced and myeloid-biased HSC engrafted recipients from panel H. Fisher exact test determined statistically significant differences in the My-bias fraction. *P ≤ .05.$

Clonal LSK SLAM fetal liver cell in vivo transplantations reveal decreased engraftment levels and myeloid-lineage bias for Gpr56 deficient HSCs. (A) Experimental setup for in vivo transplantation of E13.5 FL-sorted LSK SLAM cells. (B-C) One FL equivalent of LSK SLAM sorted E13.5 cells from WT and VavCre:loxGpr56 cKO embryos (Ly5.2) were injected into adult irradiated recipients (Ly5.1). Donor cell chimerism in recipient PB at week 4 and 16 posttransplant (B) and hematopoietic tissues at week 18 posttransplant (C). Analysis is by Ly5.1/Ly5.2 flow cytometry. Mean ± SEM is shown. n = 6 per group. (D) Limiting dilution clonal HSC transplantation. Percentage of donor cell engraftment of individual adult irradiated recipient mice as measured by Ly5.1/Ly5.2 flow cytometry of PB at 4, 16, and 23 weeks postinjection of 3, 10, and 30 LSK SLAM E13.5 FL cells. WT controls (red squares) and VavCre:loxGpr56 knockout (cKO, blue empty squares). n = 8 per group. Horizontal line at 5% indicates cutoff for reconstitution. Horizontal lines indicate average percentage engraftment. Circled individual symbols indicate mice used for secondary transplantations. *P ≤ .05; **P ≤ .01. (E) Percentage of donor cell engraftment at 23 weeks postinjection as measured by Ly5.1/Ly5.2 flow cytometry of BM, Sp, LN, and Thy cells from 7 recipient mice injected with 30 LSK-SLAM FL cells. *P ≤ .05. (F) RT-PCR relative expression of Gpr56 normalized to β-actin in LSK BM cells from primary recipients injected with WT and VavCre:loxGpr56 cKO sorted E13.5 FL cells (n = 5). Mean ± SEM are shown. (G) Percentage of lymphoid and myeloid cell contribution in PB of 22 individual adult irradiated recipient mice as measured by flow cytometry of peripheral blood at 23 weeks postinjection of 10 and 30 LSK-SLAM WT control and VavCre:loxGpr56 cKO E13.5 FL cells. (H) Ratio of B-T lymphoid and G-M myeloid for cohort of recipients in panel G with indicators for limits of lymphoid-biased, balanced and myeloid-biased HSC outputs. B, balanced; Ly, lymphoid bias; My, myeloid bias. >87% B-T=Ly; 75% to 87% B-T=B; <75% B-T=My. (I) Percentages of lymphoid-biased, balanced and myeloid-biased HSC engrafted recipients from panel H. Fisher exact test determined statistically significant differences in the My-bias fraction. *P ≤ .05.

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