1. Volume 23, Issue 11, Pages 3223-3235 (June 2018)
Variable SATB1 Levels Regulate Hematopoietic Stem Cell Heterogeneity with Distinct Lineage Fate 
Yukiko Doi, Takafumi Yokota, Yusuke Satoh, Daisuke Okuzaki, Masahiro Tokunaga, Tomohiko Ishibashi, Takao Sudo, Tomoaki Ueda, Yasuhiro Shingai, Michiko Ichii, Akira Tanimura, Sachiko Ezoe, Hirohiko Shibayama, Terumi Kohwi-Shigematsu, Junji Takeda, Kenji Oritani, Yuzuru Kanakura 
Cell Reports 
Volume 23, Issue 11, Pages (June 2018)
DOI: /j.celrep
Copyright © 2018 The Authors Terms and Conditions

2. Cell Reports 2018 23, 3223-3235DOI: (10.1016/j.celrep.2018.05.042)
Copyright © 2018 The Authors Terms and Conditions

3. Figure 1
Functional Significance of SATB1 in HSC Self-Renewal and Differentiation
(A) The number of LSK CD150+ Flt3− cells/femur in Tie2-Cre+ Satb1+/+ or Satb1flox/flox mice (n = 4; mean ± SE).
(B) Long-term reconstituting capacity of HSCs obtained from Tie2-Cre+ Satb1+/+ or Satb1f/f mice. The chimerism of CD45.2+ MNCs in recipient BM is shown (n = 7–8).
(C–E) Transplantation results using Mx1-Cre Satb1-cKO mice.
(C) Long-term reconstituting capacity of HSCs obtained from CD45.2+ Mx1-Cre+ Satb1+/+ or Satb1flox/flox mice in WT recipients (n = 3 or 7, respectively; left). HSCs obtained from CD45.1+ WT mice were inversely transplanted into Mx1-Cre+ Satb1+/flox or Satb1flox/flox mice (n = 10 or 13, respectively; right). The chimerisms of donor-type MNCs in recipient PB are shown.
(D and E) Reconstituted lineage proportion in primary recipients with (D) Mx1-Cre+ Satb1-cKO HSCs or (E) in primary recipients in inverse transplantation. Percentages of Mac1+ and/or Gr1+ myeloid cells and Mac1− Gr1− B220+ or CD3ε+ lymphoid cells in CD45.2+ MNCs are shown.
(F) Differentiation capacity of HSCs from Tie2-Cre+ Satb1+/+ or Satb1flox/flox mice. Results of co-cultures with MS5 stromal cells are shown. Co-cultures were started with 100 LSK CD150+ Flt3− cells/well on day 0, and analyses were performed on day 10. B-lymphocyte formation rates in CD45+ Mac1− Gr1− cells are shown as contour plots. The bar graph shows the absolute number of B lymphocytes/well grown from Tie2-Cre+ Satb1+/+ or Satb1flox/flox HSCs (n = 4).
(G and H) Growth capacity of Mx1-Cre+ Satb1+/+ or Satb1flox/flox HSCs in co-cultures with OP9 or OP9-DL1 cells. On day 0, cultures were started with 100 cells/well.
(G) The absolute number of B lymphocytes/well was counted on days 4, 7, and 10 (n = 4). The absolute number of CD45+ CD4− CD8− double-negative (DN) T lymphocytes/well were counted on day 14 (n = 4).
(H) The number of DN CD44+ CD25− (DN1) cells and DN CD44+ CD25+ (DN2) cells are shown (n = 4).
∗p < 0.05 (see also Figure S1).
∗p < 0.05; ∗∗p < 0.01.
Cell Reports  , DOI: ( /j.celrep )
Copyright © 2018 The Authors Terms and Conditions

4. Figure 2 Establishment of SATB1-Reporter Mice
(A) Knockin strategy and construct of a targeting vector for SATB1-reporter mice. Margins for recombination are represented with dotted lines.
(B) Observation of BM MNCs in SATB1/Tomato-reporter mice. Each axis shows SATB1, c-Kit, and Flt3 levels. Gating strategies for each fraction in BM MNCs are shown: HSCs, LSK CD150+ CD48− Flt3−; LMPPs, LSK Flt3+ interleukin-7Ra (IL-7Ra)−; CLPs, Lin− c-Kitlow Sca-1−/low Flt3+ IL-7Ra+; GMPs, Lin− CD4− CD8− IgM− IL-7Ra− Sca-1− c-Kit+ FCγRHi CD34Hi; and MEPs, Lin− CD4− CD8− IgM− IL-7Ra− Sca-1− c-Kit+ FCγRLo CD34Lo.
(C) SATB1/Tomato expression in HSCs and progenitors. Red histograms show the fluorescent intensity of cells obtained from SATB1-reporter mice. Gray shadows indicate autofluorescence of the same fraction of WT littermates. Each panel represents HSCs, lymphoid-lineage progenitors (upper panels), and progenitors for myeloid and megakaryoid and/or erythroid lineages (lower panels).
(D) Expression levels of HSC-related antigens in SATB1− and SATB1+ CD150+ Flt3− LSK cells. Mean fluorescence intensity (MFI) was calculated and is shown as bar graphs (n = 4).
∗p < 0.05; ∗∗p < 0.01 (see also Figure S2).
Cell Reports  , DOI: ( /j.celrep )
Copyright © 2018 The Authors Terms and Conditions

5. Figure 3 Functional Assessment of SATB1− and SATB1+ HSCs In Vitro
(A) A sorting strategy for SATB1− and SATB1+ cells from the LSK CD150+ Flt3− HSC-enriched fraction.
(B) Evaluation of the intensities of SATB1/Tomato in sorted HSCs.
(C) Determination of Satb1 mRNA levels in sorted SATB1− and SATB1+ HSCs (n = 6).
(D) Colony formation in methylcellulose colony assays. Sorted SATB1− and SATB1+ LSK CD150+ Flt3− cells (n = 100) were seeded on day 0, and the number of each type of colony (CFU-Mix, CFU-GM/M/G, and BFU-E) were counted on day 10 (n = 3).
(E) Growth capacity of SATB1− and SATB1+ HSCs in co-cultures with MS5 cells. On day 0, cultures were started with 100 cells/well. The absolute numbers of CD45+ hematopoietic cells (bar graphs, left upper panel), myeloid cells (bar graphs, left lower panel), and B lymphocytes (line graph, right) per well were counted on days 4 and 10 (n = 4).
∗p < 0.05; ∗∗p < 0.01; ∗∗∗p <
Cell Reports  , DOI: ( /j.celrep )
Copyright © 2018 The Authors Terms and Conditions

6. Figure 4 Transcriptome Evaluation of SATB1− and SATB1+ HSCs
(A–C) Results of RNA-seq analysis of total RNA samples isolated from SATB1− and SATB1+ CD150+ Flt3− LSK cells to evaluate changes in genetic expression between HSC type (see also Figure S3).
(A) Volcano plot showing comprehensive changes in gene expression, with changes in individual gene expression compared between HSCs (p < 1; Fisher’s exact test). Red lines divide genes according to increase or decrease in expression level and p < 0.05 (corresponding to 1.3 on the −log10 scale).
(B) Top 10 downregulated and upregulated genes and their fold changes (FCs). Lymphoid-lineage-related genes are indicated (yellow; genes restricted to p < 0.05).
(C) Results of functional annotation analysis (genes restricted to p < 0.05). Several lymphoid-lineage-related pathways are shown in red.
(D–F) Expression levels of specific genes were evaluated, and data represent the expression levels in SATB1+ HSCs as compared with those in SATB1− HSCs. ∗p < 0.05.
(D) Lymphoid-lineage-related genes.
(E) Myeloid-lineage-related genes (left) and erythroid-lineage-related genes (right).
(F) Genes involved in maintaining HSC stemness (left) and lymphoid- or myeloid-biased HSCs (right).
∗p < 0.05; ∗∗p < 0.01.
Cell Reports  , DOI: ( /j.celrep )
Copyright © 2018 The Authors Terms and Conditions

7. Figure 5 Functional Assessment of SATB1− and SATB1+ HSCs In Vivo
(A–C) Results of primary BM transplantation. Donor SATB1− and SATB1+ LSK CD150+ Flt3− cells were sorted from CD45.2+ SATB1/Tomato-reporter mice and transplanted into lethally irradiated CD45.1+ WT mice (n = 8–9).
(A) Chimerism in primary recipients. Evaluation of percentages of CD45.2+ MNCs in total CD45+ MNCs in the BM of each recipient is shown.
(B) Lineage output in the BM of primary recipients (see also Figure S4). Frequencies of CLPs (in CD45.2+ Lin− cells) and myeloid progenitors (in CD45.2+ Lin− Sca-1− cells) in SATB1− and SATB1+ HSC-transplanted recipients are shown. The gating strategies for each fraction are shown: CLPs, Lin− Sca-1−/low c-Kitlow Flt3+ IL-7Rα+ and myeloid progenitors, Lin− Sca-1− c-KitHi IL-7Ra−.
(C) Reconstruction of the HSC fraction in the BM of primary recipients. Each panel is gated on CD45.2+ LSK cells.
(D) SATB1 intensity in reconstituted HSCs in primary recipients. Each histogram is gated on CD45.2+ LSK CD150+ Flt3− cells, and the gray shadowed line indicates autofluorescence in WT mice.
(E–H) Results of secondary transplantation. Whole-BM MNCs were collected from primary recipients and transplanted into lethally irradiated CD45.1+ WT mice. The gating strategies for each fraction were same as in (A)–(C).
(E) Chimerism in recipients of secondary transplantation. Evaluation of percentages of CD45.2+ MNCs in total CD45+ MNCs in the BM of each recipient (n = 4–6).
(F) Lineage output in the BM of secondary recipients (see also Figure S4). Frequencies of reconstituted CLPs and myeloid progenitors are shown (n = 5–8).
(G) Reconstruction of the original HSC fraction of secondary recipients.
(H) SATB1 intensity in reconstituted HSCs of secondary recipients.
∗p < 0.05; ∗∗p < See also Figure S4.
Cell Reports  , DOI: ( /j.celrep )
Copyright © 2018 The Authors Terms and Conditions

8. Figure 6
Examination of the Differentiation Potential of HSCs according to SATB1 Levels
(A) Transplantation strategy. LSK CD150+ Flt3− cells in SATB1/Tomato-reporter mice were subdivided into five groups according to SATB1/Tomato intensity. The borders of each group are defined by cell-number percentages (histogram).
(B) CD150 expression in single HSCs in each subfraction (group 1, 76 cells; group 2, 56 cells; group 3, 64 cells; group 4, 78 cells; and group 5, 79 cells). Medians are shown with bars.
(C–E) Ten cells (C and D) or single cells (E) for each fraction were transplanted into lethally irradiated CD45.1+ recipients, and analysis was performed after 4 months.
(C) Engraftment rates in recipients of each group. The frequencies of successfully engrafted recipients with at least 2% BM chimerism are shown. Data were summarized from three independent experiments (total analyzed recipients for groups 1, 2, 3, 4, and 5: n = 12, 15, 9, 13, and 13, respectively; successfully engrafted recipients for groups 1, 2, 3, 4, and 5: n = 3, 7, 6, 10, and 10, respectively).
(D) Percentages of reconstituted myeloid cells (upper panel) and lymphoid cells (lower panel) in CD45.2+ BM MNCs. The medians are shown with bars.
(E) SATB1/Tomato intensity of each single LSK CD150+ Flt3− HSC (plotted as a dot) reconstituted after single-cell HSC transplantation. Vertical axis indicates the distribution of recovered HSCs with respect to SATB1/Tomato intensity. Data from a representative recipient in each group are shown. MFIs are shown with bars. The borders of each group were drawn as dashed lines, which were defined as described in (A). Distribution data are summarized and shown as 100% in the stacked bar chart (in a dashed-line rectangle).
∗p < 0.05; ∗∗∗p <
Cell Reports  , DOI: ( /j.celrep )
Copyright © 2018 The Authors Terms and Conditions

9. Figure 7
Visualization of HSC Heterogeneity and Fluctuation according to SATB1 Expression
(A) Three-dimensional representation of the HSC fraction. SATB1/Tomato, CD86, and CD41 levels were estimated by flow cytometry. Three-dimensional scatterplot and 50% probability ellipsoid for total plots are drawn. Cells with the upper one-third of SATB1 intensity are drawn in red, and others are drawn in blue.
(B) Associations between SATB1 and CD86 (left) and between SATB1 and CD41 (right) in each single HSC. The linear approximations for each scatterplot are drawn in red and represented as mathematical formulas.
(C) A dynamic view of the cellular states of HSCs and their differentiation. Each axis represents the expression levels of SATB1 and other lineage-related genes. The coil-like trajectory represents time-related fluctuations in each gene-expression level. Lymphoid-biased HSCs are shown as red dots, whereas myeloid-biased HSCs are shown as blue dots.
Cell Reports  , DOI: ( /j.celrep )
Copyright © 2018 The Authors Terms and Conditions