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Results: 7

1.
Figure 2

Figure 2. From: Stress hematopoiesis reveals abnormal control of self-renewal, lineage bias, and myeloid differentiation in Mll partial tandem duplication (Mll-PTD) hematopoietic stem/progenitor cells.

Expansion of MllPTD/WTmice HSPCs in CFU-spleen and replating assays. (A) Representative image of CFU-spleen assay. (B) Number of colonies was counted 8 or 12 days after transplantation (12 mice per group; 1 × 105 cells per mouse). *P < .05. **P < .01. (C) Diameter of each colony was measured under inverted microscope. (D) Frequency of CFU-Cs in the BM of WT and MllPTD/WT during serial replating on methylcellulose in vitro. (E) Proportion of CFU-Cs in serial replating. (F) Representative image of colonies from WT and MllPTD/WT BM cells in the second round of replating.

Yue Zhang, et al. Blood. 2012 August 2;120(5):1118-1129.
2.
Figure 5

Figure 5. From: Stress hematopoiesis reveals abnormal control of self-renewal, lineage bias, and myeloid differentiation in Mll partial tandem duplication (Mll-PTD) hematopoietic stem/progenitor cells.

Phenotypic ST-HSCs, MPP, and GMP from MllPTD/WT mice repopulate LT-HSCs. Representative FACS contour diagram shows the repopulation of LSK, LT-HSC/ST-HSC/MPP, and CMP/GMP/MEP in recipients transplanted with MllPTD/WT different fractions, MllPTD/WT LT-HSCs (A), ST-HSCs (B), MPP (C), and GMP (D). (E) Mean ± SD percentage of donor-derived MllPTD/WT cells (CD45.2+) in recipient BM. (F) Frequency of donor-derived lineage-repopulation myeloid or B or T cells compared with competitor (CD45.1) derived lineage-repopulation present in recipient BM. Data are at 12 weeks after transplantation (n = 4). (G) Secondary transplantation was performed 16 weeks after primary transplantation with sorted fractions from MllPTD/WT MPP transplanted recipients (n = 4). Data are mean ± SD percentage of donor-derived MllPTD/WT cells (CD45.2+) in recipient PB. (H) Frequency of donor-derived lineage-repopulation myeloid or B or T cells compared with competitor (CD45.1) derived lineage-repopulation present in secondary recipient PB 12 weeks after transplantation.

Yue Zhang, et al. Blood. 2012 August 2;120(5):1118-1129.
3.
Figure 6

Figure 6. From: Stress hematopoiesis reveals abnormal control of self-renewal, lineage bias, and myeloid differentiation in Mll partial tandem duplication (Mll-PTD) hematopoietic stem/progenitor cells.

Increased repopulating activity of HSPCs from MllPTD/WT mice correlates with acquisition of an intrinsic self-renewal program. (A) A single-cell culture was performed in the presence of cytokines for 2 weeks. (B) Cytospin slides were prepared from individual clones and stained with Camco Stain Pak. Black arrow indicates megakaryocyte; red arrow, neutrophil; green arrow, monocyte; and blue arrow, poly-erythrocyte. (C) Frequency of lineage formation of LSK/SLAM+ or LSK/SLAM or GMP in WT or MllPTD/WT are shown in Table 1. (D) Frequency of CFU-Cs in the GMP population sorted from WT or MllPTD/WT during serial replating on methylcellulose in vitro. For the first-round plating, 1 × 103 GMP cells were seeded per milliliter of hematopoietic methylcellulose colony-forming media. A total of 1 × 104 cells were planted for next-round replating. The cells are assayed in triplicate dishes of 1 mL. *P < .05. (E) Proportion of CFU-Cs in serial replating. (F) Representative image of colonies from WT and MllPTD/WT GMP cells in third-round replating.

Yue Zhang, et al. Blood. 2012 August 2;120(5):1118-1129.
4.
Figure 3

Figure 3. From: Stress hematopoiesis reveals abnormal control of self-renewal, lineage bias, and myeloid differentiation in Mll partial tandem duplication (Mll-PTD) hematopoietic stem/progenitor cells.

Increased competitive repopulating LT-HSCs in MllPTD/WT BM cells. (A) Experimental setup. Lethally irradiated groups of CD45.1+/CD45.2+ WT recipient mice are intravenously injected with 1.5 × 106 BM-MNCs from WT or MllPTD/WT (CD45.2+) mice together with an equal number of CD45.1+ competitor cells. PB is collected from recipients monthly and analyzed by FACS for the presence of CD45.2+ donor-derived cells. Secondary competitive BMT is performed 16 weeks after transplantation, with purified CD45.2+ BM cells from primary recipients together with CD45.1+ competitor cells at a 1:1 ratio. Chimerisms in primary BMT (B) and secondary competitive BMT (C) were assessed monthly. Data shown are the mean percentage ± SD of donor-derived cells (CD45.2+) in PB (n = 8). *P < .05. **P < .01. (D) The competitive repopulation unit (CRU) was calculated 16 weeks after BMT according to the formula: donor RU = % donor × competitor cell number/(100 − % donor), and compared between MllPTD/WT with WT. (E) WT or MllPTD/WT (CD45.2+) donor cells in a serial diluted dose were transplanted into lethally irradiated CD45.1+/CD45.2+ WT recipient mice along with 1 × 105 WT (CD45.1+) helper cells, and the donor engraftment in recipient blood was determined monthly (n = 4).

Yue Zhang, et al. Blood. 2012 August 2;120(5):1118-1129.
5.
Figure 4

Figure 4. From: Stress hematopoiesis reveals abnormal control of self-renewal, lineage bias, and myeloid differentiation in Mll partial tandem duplication (Mll-PTD) hematopoietic stem/progenitor cells.

Increased repopulating activity of HSPCs from MllPTD/WT mice is not restricted to phenotypically identified HSCs but also comes from ST-HSCs and myeloid progenitors. The fractions of WT or MllPTD/WT (CD45.2+) BM cells were transplanted into lethally irradiated CD45.1+/CD45.2+ WT recipient mice along with 1 × 105 WT (CD45.1+) helper cells. Engraftment was assessed 16 weeks after transplantation. Shown is the mean ± SD percentage of donor-derived WT or MllPTD/WT cells (CD45.2+) in recipient PB. (A) Equal number of LSK/SLAM+ (3.5 × 102) or LSK/SLAM (3.5 × 103) fractions from WT or MllPTD/WT BM cells were transplanted into CD45.1+/CD45.2+ WT recipient mice (2 experiments, n = 8). (B) WT or MllPTD/WT (CD45.2+) LT-HSCs (8 × 102) or ST-HSCs (1.2 × 103) or MPP (3.5 × 103) were transplanted into CD45.1+/CD45.2+ WT recipient mice (2 experiments, n = 8). (C) WT or MllPTD/WT (CD45.2+) GMP population cells (3.5 × 103) were transplanted into CD45.1+/CD45.2+ WT recipient mice (2 experiments, n = 8). (D) Frequency of lineage-repopulation myeloid (CD45.2+CD11b+) or B (CD45.2+B220+) or T (CD45.2+CD5+) cells compared with competitor (CD45.1) derived lineage-repopulation present in CD45.1+/CD45.2+ WT recipient mice. *P < .05. **P < .01. (E) MllPTD/WT (CD45.2+) GMP population cells in serial diluted dose (1.5 × 103, 5 × 103, 1 × 104, and 5 × 104) were transplanted into CD45.1+/CD45.2+ WT recipient mice (n = 4). Engraftment was assessed at 6, 12, and 20 weeks after transplantation (n = 4). (F) Frequency of donor-derived lineage-repopulation myeloid or B or T cells compared with competitor (CD45.1) derived lineage-repopulation present in CD45.1+/CD45.2+ WT recipient mice. Data are shown at 12 weeks after transplantation.

Yue Zhang, et al. Blood. 2012 August 2;120(5):1118-1129.
6.
Figure 7

Figure 7. From: Stress hematopoiesis reveals abnormal control of self-renewal, lineage bias, and myeloid differentiation in Mll partial tandem duplication (Mll-PTD) hematopoietic stem/progenitor cells.

Rapid expansion and reduced apoptosis of MllPTD/WT LSK/SLAM+ cells under stresses. (A) A single dose (150 mg/kg) of 5-FU was administered intraperitoneally into MllPTD/WT or WT (6-8 mice per group). BM cells were collected at the mentioned time point. Absolute number of LSK (A) and LSK/SLAM+ (B) in WT and MllPTD/WT expressed as mean ± SD per million BM cells. *P < .05. (C) Fold change of LSK or LSK/SLAM+ between day 14 and day 0 in WT and MllPTD/WT mice. (D) Apoptosis was checked by annexin V staining. Data shown are the mean percentage ± SD of annexin V+/7 AAD and annexin V+/7 AAD+ (n = 4). *P < .05. (E) Cell-cycle analysis was performed with BrdU flow kit. Percentage of cycling cells (G0/G1 and S/G2/M) are shown for LSK fraction at 10 days after 5-FU administration (2 experiments, n = 4). **P < .01. (F) Expression of Bcl-2 family protein in LSK fractions. BM cells were harvested 24 hours after 5-FU (150 mg/kg) intraperitoneal injection (F) or collected from primary 1:1 ratio competitive BMT recipients 2 months after transplantation (G). LSK cells were selected by using autoMACS. Western blots were done using the indicated antibodies (anti-Mcl1, anti-Bcl2, anti–Bcl-XL, anti-Bax, and antitubulin).

Yue Zhang, et al. Blood. 2012 August 2;120(5):1118-1129.
7.
Figure 1

Figure 1. From: Stress hematopoiesis reveals abnormal control of self-renewal, lineage bias, and myeloid differentiation in Mll partial tandem duplication (Mll-PTD) hematopoietic stem/progenitor cells.

BM content of HSPCs is reduced in number in MllPTD/WTmice. (A) BM of age- and sex-matched littermate WT controls and MllPTD/WT mice were analyzed at 2, 4, and 12 months of age, based on the immunophenotype analysis. Representative flow cytometry (FACS) contour diagram shows the frequency of LSK and LSK/SLAM+ BM cells of WT and MllPTD/WT mice. Shown is a representative of 8 experiments with similar results. (B) Absolute number of LSK cells of WT and MllPTD/WT mice. MllPTD/WT LSK population is reduced in absolute number during aging (n = 8). The difference between control and MllPTD/WT was significant at 8 and 12 months. *P < .05. (C) Absolute number of LSK/SLAM+ cells of WT and MllPTD/WT mice. MllPTD/WT LSK/SLAM+ populations are reduced in absolute number during aging (n = 8). The difference between control and MllPTD/WT was significant at 4, 8, and 12 months. *P < .05. **P < .01. (D) Absolute number of progenitors HPC, GMP, common myeloid progenitor cells (Linc-Kit+Sca1CD34+CD16/32mid; CMPs), and MEP of WT and MllPTD/WT mice. MllPTD/WT mice GMP population is increased at the expense of the MEP population (2 experiments, n = 8; 4-month-old mice were used). The differences of GMP and MEP between control and MllPTD/WT were significant (P < .05). (E) Apoptosis was checked by annexin V staining. Data are the mean percentage ± SD of annexin V+/7 AAD and annexin V+/7 AAD+. *P < .05. **P < .01. Experiments were performed in duplicate groups for 4 mice at 4-month-old per genotype repeated in 3 separate experiments. (F) Cell-cycle analysis was performed with a BrdU flow kit. Percentage of cycling cells: G0/G1 and S/G2/M are shown for LK (LinKit+) and LSK fractions (2 experiments, n = 4; 4-month-old mice were used). *P < .05. (G) H3K4 methylation in LSK fractions. BM cells were harvested and LSK cells were selected using autoMACS. Western blots were done using indicated antibodies (anti-H3K4me3, anti-H3K4me2, and anti-H3). Representative data were from 3 independent experiments.

Yue Zhang, et al. Blood. 2012 August 2;120(5):1118-1129.

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