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a Using surface markers that enable the identification of LT-HSC and MPP1-4 populations (Kit, Lineage Markers, Sca1, <t>Cd48,</t> Cd150, Flk2), flow cytometry analysis shows the frequencies for CT and TKO LT-HSC/MPP1-MPP4. CT , white bars; TKO , black bars. n=7. b 100 cells from each subpopulation were plated into standard methylcellulose. The number and identity of colonies were quantified after 8 days in culture (n=3). c 100 CT and TKO MPP1-MPP4 cells were co-cultured with OP9 cells and the number of B cells (Cd45 + , B220 + ) and Myeloid cells (Cd45 + , Mac1 + ) were quantified by flow cytometry after 10 days in culture (n=4). d CT / TKO MPP3 and MPP4 were isolated by flow cytometry and placed in liquid culture. After 5 days, surface expression of the pan-myeloid marker MacI was assessed in cultured cells to quantify their differentiation rate. e heatmap showing the expression of representative genes for different steps of hematopoiesis in CT and TKO LT-HSC, MPP1-4 subpopulations. f Individual sc-Seq datasets for CT and TKO LT-HSCs were merged for analysis and individually displayed to determine the distribution of clusters from the merged dataset per genotype. g UMAP display of sc-Seq analysis for CT (left) and TKO (right) MPP4. Cells surrounded by a dotted line represent a projected subcluster. h Trajectory analysis by Monocles-2D identifies a bipotential (myeloid/lymphoid) fate in CT MPP4 (left panel) while the lymphoid fate is obliterated in TKO MPP4 (right panel). Cells surrounded by a dotted circle represent a projected subcluster (i.e located at the end of the trajectory).
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a Using surface markers that enable the identification of LT-HSC and MPP1-4 populations (Kit, Lineage Markers, Sca1, <t>Cd48,</t> Cd150, Flk2), flow cytometry analysis shows the frequencies for CT and TKO LT-HSC/MPP1-MPP4. CT , white bars; TKO , black bars. n=7. b 100 cells from each subpopulation were plated into standard methylcellulose. The number and identity of colonies were quantified after 8 days in culture (n=3). c 100 CT and TKO MPP1-MPP4 cells were co-cultured with OP9 cells and the number of B cells (Cd45 + , B220 + ) and Myeloid cells (Cd45 + , Mac1 + ) were quantified by flow cytometry after 10 days in culture (n=4). d CT / TKO MPP3 and MPP4 were isolated by flow cytometry and placed in liquid culture. After 5 days, surface expression of the pan-myeloid marker MacI was assessed in cultured cells to quantify their differentiation rate. e heatmap showing the expression of representative genes for different steps of hematopoiesis in CT and TKO LT-HSC, MPP1-4 subpopulations. f Individual sc-Seq datasets for CT and TKO LT-HSCs were merged for analysis and individually displayed to determine the distribution of clusters from the merged dataset per genotype. g UMAP display of sc-Seq analysis for CT (left) and TKO (right) MPP4. Cells surrounded by a dotted line represent a projected subcluster. h Trajectory analysis by Monocles-2D identifies a bipotential (myeloid/lymphoid) fate in CT MPP4 (left panel) while the lymphoid fate is obliterated in TKO MPP4 (right panel). Cells surrounded by a dotted circle represent a projected subcluster (i.e located at the end of the trajectory).
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a Using surface markers that enable the identification of LT-HSC and MPP1-4 populations (Kit, Lineage Markers, Sca1, <t>Cd48,</t> Cd150, Flk2), flow cytometry analysis shows the frequencies for CT and TKO LT-HSC/MPP1-MPP4. CT , white bars; TKO , black bars. n=7. b 100 cells from each subpopulation were plated into standard methylcellulose. The number and identity of colonies were quantified after 8 days in culture (n=3). c 100 CT and TKO MPP1-MPP4 cells were co-cultured with OP9 cells and the number of B cells (Cd45 + , B220 + ) and Myeloid cells (Cd45 + , Mac1 + ) were quantified by flow cytometry after 10 days in culture (n=4). d CT / TKO MPP3 and MPP4 were isolated by flow cytometry and placed in liquid culture. After 5 days, surface expression of the pan-myeloid marker MacI was assessed in cultured cells to quantify their differentiation rate. e heatmap showing the expression of representative genes for different steps of hematopoiesis in CT and TKO LT-HSC, MPP1-4 subpopulations. f Individual sc-Seq datasets for CT and TKO LT-HSCs were merged for analysis and individually displayed to determine the distribution of clusters from the merged dataset per genotype. g UMAP display of sc-Seq analysis for CT (left) and TKO (right) MPP4. Cells surrounded by a dotted line represent a projected subcluster. h Trajectory analysis by Monocles-2D identifies a bipotential (myeloid/lymphoid) fate in CT MPP4 (left panel) while the lymphoid fate is obliterated in TKO MPP4 (right panel). Cells surrounded by a dotted circle represent a projected subcluster (i.e located at the end of the trajectory).
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a Using surface markers that enable the identification of LT-HSC and MPP1-4 populations (Kit, Lineage Markers, Sca1, Cd48, Cd150, Flk2), flow cytometry analysis shows the frequencies for CT and TKO LT-HSC/MPP1-MPP4. CT , white bars; TKO , black bars. n=7. b 100 cells from each subpopulation were plated into standard methylcellulose. The number and identity of colonies were quantified after 8 days in culture (n=3). c 100 CT and TKO MPP1-MPP4 cells were co-cultured with OP9 cells and the number of B cells (Cd45 + , B220 + ) and Myeloid cells (Cd45 + , Mac1 + ) were quantified by flow cytometry after 10 days in culture (n=4). d CT / TKO MPP3 and MPP4 were isolated by flow cytometry and placed in liquid culture. After 5 days, surface expression of the pan-myeloid marker MacI was assessed in cultured cells to quantify their differentiation rate. e heatmap showing the expression of representative genes for different steps of hematopoiesis in CT and TKO LT-HSC, MPP1-4 subpopulations. f Individual sc-Seq datasets for CT and TKO LT-HSCs were merged for analysis and individually displayed to determine the distribution of clusters from the merged dataset per genotype. g UMAP display of sc-Seq analysis for CT (left) and TKO (right) MPP4. Cells surrounded by a dotted line represent a projected subcluster. h Trajectory analysis by Monocles-2D identifies a bipotential (myeloid/lymphoid) fate in CT MPP4 (left panel) while the lymphoid fate is obliterated in TKO MPP4 (right panel). Cells surrounded by a dotted circle represent a projected subcluster (i.e located at the end of the trajectory).

Journal: bioRxiv

Article Title: E2f coordinates the cell cycle and cell fate of hematopoietic progenitors to drive stress myelopoiesis

doi: 10.1101/2025.06.16.659412

Figure Lengend Snippet: a Using surface markers that enable the identification of LT-HSC and MPP1-4 populations (Kit, Lineage Markers, Sca1, Cd48, Cd150, Flk2), flow cytometry analysis shows the frequencies for CT and TKO LT-HSC/MPP1-MPP4. CT , white bars; TKO , black bars. n=7. b 100 cells from each subpopulation were plated into standard methylcellulose. The number and identity of colonies were quantified after 8 days in culture (n=3). c 100 CT and TKO MPP1-MPP4 cells were co-cultured with OP9 cells and the number of B cells (Cd45 + , B220 + ) and Myeloid cells (Cd45 + , Mac1 + ) were quantified by flow cytometry after 10 days in culture (n=4). d CT / TKO MPP3 and MPP4 were isolated by flow cytometry and placed in liquid culture. After 5 days, surface expression of the pan-myeloid marker MacI was assessed in cultured cells to quantify their differentiation rate. e heatmap showing the expression of representative genes for different steps of hematopoiesis in CT and TKO LT-HSC, MPP1-4 subpopulations. f Individual sc-Seq datasets for CT and TKO LT-HSCs were merged for analysis and individually displayed to determine the distribution of clusters from the merged dataset per genotype. g UMAP display of sc-Seq analysis for CT (left) and TKO (right) MPP4. Cells surrounded by a dotted line represent a projected subcluster. h Trajectory analysis by Monocles-2D identifies a bipotential (myeloid/lymphoid) fate in CT MPP4 (left panel) while the lymphoid fate is obliterated in TKO MPP4 (right panel). Cells surrounded by a dotted circle represent a projected subcluster (i.e located at the end of the trajectory).

Article Snippet: The remaining white blood cells were stained with a cocktail of lineage antibodies (Cd3, Cd4, Cd8, B220, Ter119, Mac1 and Gr1) as well as antibodies against Kit, Sca1, Flk2/Flt3, Cd48, Cd150, FcgR, Cd34, Ly6c, Ly6g, Rank and Cd115 (all purchased from eBiosciences) and βchain receptor (Miltenyi).

Techniques: Flow Cytometry, Cell Culture, Isolation, Expressing, Marker