Haploinsufficiency of GATA-2 perturbs adult hematopoietic stem-cell homeostasis by Neil P. Rodrigues, Viktor Janzen, Randolf Forkert, David M. Dombkowski, Ashleigh S. Boyd, Stuart H. Orkin, Tariq Enver, Paresh Vyas, and David T. Scadden Blood Volume 106(2):477-484 July 15, 2005 ©2005 by American Society of Hematology

Reduced expression of GATA-2 within GATA-2+/– bone marrow. Reduced expression of GATA-2 within GATA-2+/– bone marrow. RNA prepared from the total bone marrow (A) and Lin–c-kit+Sca-1+ cells (B) of each genotype was subjected to reverse transcriptase reaction and real-time PCR for GATA-2 and normalized to GAPDH. For each Lin–c-kit+Sca-1+ experiment, marrow cells from 2 to 3 animals of each genotype were pooled for cell sorting. Triplicates were used for each PCR reaction and the figures represent n = 2 experiments plus or minus SEM. Neil P. Rodrigues et al. Blood 2005;106:477-484 ©2005 by American Society of Hematology

Reduced numbers of primitive hematopoietic cells within GATA-2+/– bone marrow. Reduced numbers of primitive hematopoietic cells within GATA-2+/– bone marrow. Bone marrow nucleated cells from each genotype were assessed for CAFC ability using limiting dilution long-term culture assays. Five to 6 dilutions were employed for each genotype in individual experiments. The absolute number of CAFCs per harvest was measured at week 2 (A) (P = .019, n = 5), week 3 (B) (P = .001, n = 6), and week 5 (C) (P = .001, n = 6). Horizontal bars depict the mean of cumulative data. GATA-2+/– bone marrow displays a decrease in Lin–c-kit+Sca-1+CD34– hematopoietic stem-cells. Using flow cytometry, a 2-fold reduction of LKSCD34– cells in GATA-2+/– marrow compared with wild-type marrow is evident in the single experiment shown here (D). Numbers in R3 gates indicate % of LKS34– cells in marrow. The cumulative data of multiple independent experiments are depicted in panel E (P < .001, n = 8). To enumerate the size of the GATA-2+/– functional stem-cell pool in Lin–c-kit+Sca-1+ or Lin–c-kit+Sca-1+CD34– cells, each primitive cell population was sorted and cultured in limiting dilution long-term culture assays and CAFCs were scored at week 5. The cumulative data of multiple experiments are depicted in panel F (LKS: P = .003, n = 7; LKSCD34–: P = .005, n = 7). Error bars indicate the standard error of the mean. Statistical analysis was performed using the Student t test. Neil P. Rodrigues et al. Blood 2005;106:477-484 ©2005 by American Society of Hematology

Competitive transplantation reveals diminished hematopoietic repopulating ability of GATA-2+/– bone marrow. Competitive transplantation reveals diminished hematopoietic repopulating ability of GATA-2+/– bone marrow. Male bone marrow nucleated cells from each genotype were admixed in a 1:1 ratio and injected into the lateral veins of warmed female wild-type recipients that had received 10 Gy whole body irradiation. After 7 weeks, nucleated marrow cells from recipients were analyzed in long-term culture initiating cell (LTC-IC) and CFC assays. Discrete colonies derived from week-6 LTC-IC (stem-cells) and day-10 CFC (progenitor) assays were microisolated, lysed, and analyzed by genotyping PCR for the GATA-2 mutant and GATA-2 wild-type alleles. Nucleated marrow cells from recipients were similarly analyzed by CFC assays at 4 and 8 months after transplantation. There were 10 to 15 LTC-IC or CFC colonies screened per transplant recipient. Representative GATA-2 mutant allele PCR from cell lysates of LTC-ICs and CFCs from one recipient animal is shown (A). The mean percentage distribution of stem-cells (LTC-ICs) and progenitor cells (CFCs) from each genotype at 7 weeks after transplantation (LTC-ICs: P < .05, SEM = ± 9.3% for each genotype, n = 6 recipients; CFCs: P < .05, SEM = ± 10.5% for each genotype, n = 6 recipients) (B) and of progenitor cells at 4 and 8 months after transplantation (4 months: P < .05, SEM = ± 4.9% for each genotype, n = 6 recipients; 8 months: P < .05, SEM = ± 5.6% for each genotype n = 6 recipients) (C) are shown, with □ representing wild-type and ▪ the GATA-2+/– genotypes, respectively. Statistical analysis was performed using the Student t test. Neil P. Rodrigues et al. Blood 2005;106:477-484 ©2005 by American Society of Hematology

Altered proportion of quiescent primitive cells in GATA-2+/– bone marrow. Altered proportion of quiescent primitive cells in GATA-2+/– bone marrow. Bone marrow cells were stained with stem-cell markers (Sca-1 and c-kit) and lineage antibodies to separate stem-cells. Simultaneous staining with the DNA dye Hoechst 33 342 (Hst) was used to determine the percentage of S/G2/M in the Lin– c-kit+Sca-1+ compartment. Data from multiple experiments are summarized in panel A (P = .931; n = 6). To determine the fraction of G0 cells in the stem-cell compartment, the RNA dye Pyronin Y (PY) and Hst were used to stain Lin–c-kit+Sca-1+ cells and flow cytometry performed to determine the percentage of G0 (PYlo) in the G0/G1 fraction (Hstlow). Representative flow cytometry plots are shown (B) and data from multiple experiments are summarized in panel C (P = .004; n = 7). To functionally corroborate altered quiescence from GATA-2+/– animals, mice from GATA-2+/– and GATA-2+/+ groups were treated with 5-FU in vivo. One day after a single intraperitoneal injection of 5-FU at a dose of 150 mg/kg, nucleated cells were obtained and plated in limiting dilution long-term culture and CAFCs were scored at week 5 (D) (P = .048; n = 5). Error bars depict the standard error of the mean. All statistical analyses were performed using the Student t test. Neil P. Rodrigues et al. Blood 2005;106:477-484 ©2005 by American Society of Hematology

Freshly isolated primitive cells from GATA-2+/– bone marrow have an increased susceptibility for apoptosis. Freshly isolated primitive cells from GATA-2+/– bone marrow have an increased susceptibility for apoptosis. Bone marrow nucleated cells were stained with annexin-V, DNA dye DAPI, Sca-1, c-kit, and lineage antibodies, and Sca1+ckit+Lin– cells were analyzed for annexin-V/DAPI activity by flow cytometry. Cells excluding DAPI and binding annexin-V were considered apoptotic (A) (P = .011; n = 6). To examine intracellular Bcl-xL expression, bone marrow nucleated cells were stained with Sca-1, c-kit, and lineage antibodies, and Bcl-xL and the Lin–c-kit+Sca-1+ population was analyzed by flow cytometry. Representative flow cytometry plots for Bcl-xL expression are depicted in panel B; the Bcl-xL–positive fraction was defined relative to the isotype control staining (data not shown). M2 represents the % of Bcl-xL–positive cells. Multiple experiments are summarized in panel C (P = .050; n = 5). Error bars depict the standard error of the mean. All statistical analyses were performed using the Student t test. Neil P. Rodrigues et al. Blood 2005;106:477-484 ©2005 by American Society of Hematology

Bone marrow stem-cells from GATA-2+/+ and GATA-2+/– animals display similar self-renewal potential. Bone marrow stem-cells from GATA-2+/+ and GATA-2+/– animals display similar self-renewal potential. Two million bone marrow nucleated cells from male mice of each genotype were injected into female wild-type recipient mice that had been subjected to lethal whole body irradiation. Recipient mice were monitored daily for survival for 2 months. Y-chromosome PCR was used to assess donor contribution in transplantations (data not shown). Irradiated mice injected with PBS alone died within 10 to 14 days of irradiation (data not shown). After 2 months, marrow suspensions were prepared and 2 million nucleated cells were injected into new female irradiated animals. Another 3 transplantations were performed sequentially at 2-month intervals. For each serial transplantation, 10 recipients were injected with each genotypic group. The cumulative survival from the tertiary transplantation onwards is displayed (A). Limiting dilution long-term culture was performed on donor cells prior to transplantation (BMT 0) and after each cycle of transplantation (B) (n = 3 for each transplant, P > .05 for each transplantation cycle). The BMT 0 sample was prepared by pooling cells from the 3 donor mice that initially underwent transplantation. Error bars depict the standard error of the mean. Statistical analysis was performed using the Student t test. Neil P. Rodrigues et al. Blood 2005;106:477-484 ©2005 by American Society of Hematology