Volume 154, Issue 5, Pages 1112-1126 (August 2013) Clonal Analysis Unveils Self-Renewing Lineage-Restricted Progenitors Generated Directly from Hematopoietic Stem Cells  Ryo Yamamoto, Yohei Morita, Jun Ooehara, Sanae Hamanaka, Masafumi Onodera, Karl Lenhard Rudolph, Hideo Ema, Hiromitsu Nakauchi  Cell  Volume 154, Issue 5, Pages 1112-1126 (August 2013) DOI: 10.1016/j.cell.2013.08.007 Copyright © 2013 Elsevier Inc. Terms and Conditions

Cell 2013 154, 1112-1126DOI: (10.1016/j.cell.2013.08.007) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 1 Experimental Design for In Vivo Analysis of Single Hematopoietic Stem and Progenitor Cells (A) Gating strategy to purify single KuO+CD150+CD41−CD34−KSL (fraction I) cells, CD150+CD41+CD34−KSL (fraction II) cells, CD150−CD41−CD34−KSL (fraction III) cells, Flt3−CD34−KSL cells (MPPs), and Flt3+CD34+KSL cells (LMPPs) from Kusabira-Orange (KuO) mouse BM cells. BM cells were stained with antibodies and were gated as indicated. Doublets and dead cells were excluded before sorting. For single-cell sorting, the presence of one cell per well was verified under an inverted microscope. (B) Single fraction I, fraction II, or fraction III cells were sorted from BM cells of KuO mice and were individually transplanted with 2 × 105 competitor cells from Ly5.1/Ly5.2-F1 mice into lethally irradiated Ly5.2 mice. Chimerism of KuO+ neutrophils/monocytes, erythrocytes, platelets, B cells, and T cells in peripheral blood (PB) was analyzed 2, 3, 4, 8, 16, and 24 weeks after primary transplantation (in addition, some mice were also analyzed at 12 weeks, some were also analyzed at 20 weeks, and some were also analyzed at 12 and 20 weeks). Secondary transplantation was performed by transferring 1 × 107 whole BM cells from primary recipient mice. PB chimerism was analyzed 4, 12, 16, and 20 weeks after secondary transplantation. (C) Definition of repopulating cell: When all five types of mature blood cells in an individual mouse showed stable chimerism (0.1% or more) 20 weeks after secondary transplantation, the original single cell was retrospectively defined as a long-term HSC (LT-HSC). When five types of mature blood cells in an individual mouse showed chimerism (0.1% or more) 24 weeks after primary transplantation but at least one lineage disappeared in secondary recipient mice, the original single cell was defined as an intermediate-term HSC (IT-HSC). When, in a primary recipient mouse, mature blood cells of at least one lineage disappeared (that is, showed less than 0.1% chimerism) before 24 weeks after transplantation, the original single cell was defined as a short-term HSC (ST-HSC). When only neutrophils/monocytes, erythrocytes, and platelets, but not cells of B and T lineages, were detected in mice, the original single cell was defined as a common myeloid repopulating progenitor (CMRP). When only erythrocytes and platelets were detected in mice, the original single cell was defined as a megakaryocyte-erythroid repopulating progenitor (MERP). When only platelets were detected in mice, the original single cell was defined as a megakaryocyte repopulating progenitor (MkRP). See also Figures S1 and S2. Cell 2013 154, 1112-1126DOI: (10.1016/j.cell.2013.08.007) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 2 Myeloid-Restricted Progenitors with Long-Term Repopulating Ability Are Present in the Phenotypically Defined HSC Compartment Single fraction I, fraction II, or fraction III cells, 50 MPPs or 50 LMPPs with 2 × 105 competitor cells (Ly5.1/Ly5.2-F1) were transplanted into lethally irradiated Ly5.2 mice. PB chimerism was analyzed periodically in primary and secondary recipient mice. Based on reconstitution kinetics data, single test donor cells were classed as LT-HSCs, IT-HSCs, ST-HSCs, CMRPs, MERPs, MkRPs, or “other.” Chimerism kinetics of each lineage (neutrophil/monocyte, erythrocyte, platelet, B cell, and T cell) are shown for mice that had received a single LT-HSC (n = 13), IT-HSC (n = 18), ST-HSC (n = 26), CMRP (n = 37), MERP (n = 5), or MkRP (n = 22); multiple MPPs (n = 5); or multiple LMPPs (n = 4). See also Figure S2. Cell 2013 154, 1112-1126DOI: (10.1016/j.cell.2013.08.007) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 3 MERPs and MkRPs Are Present among Fraction I and II but Not among Fraction III Single fraction I, fraction II, or fraction III cells were transplanted with 2 × 105 competitor cells (Ly5.1/Ly5.2-F1) into lethally irradiated Ly5.2 mice. KuO+ blood cells were detected in 46 of 83 mice transplanted with single fraction I cells (55.4%), 39 of 88 mice transplanted with single fraction II cells (44.3%), and 45 of 88 mice transplanted with single fraction III cells (51.1%). Based on reconstitution kinetics data, single test donor cells were classed as LT-HSCs, IT-HSCs, ST-HSCs, CMRPs, MERPs, MkRPs, or “other.” (A) Average reconstitution kinetics of LT-HSCs, IT-HSCs, ST-HSCs, CMRPs, MERPs, and MkRPs in fraction I, fraction II, and fraction III are shown (platelet, Plt; neutrophil/monocyte, nm; erythrocyte, E; B cell, B; and T cell, T). Error bar, SEM; for numbers of mice, see (B). (B) All repopulating cells among fraction I, fraction II, and fraction III are classed as LT-HSCs, IT-HSCs, ST-HSCs, CMRPs, MERPs, MkRPs, or “other.” Single fraction I cells exhibited reconstitutive behavior of LT-HSCs (n = 13, 15.7%), IT-HSCs (n = 8, 9.6%), CMRPs (n = 11, 13.3%), MERPs (n = 3, 3.6%), or MkRPs (n = 11, 13.3%). Single fraction II cells showed reconstitutive behavior of IT-HSCs (n = 3, 3.4%), ST-HSCs (n = 1, 1.1%), CMRPs (n = 19, 21.6%), MERPs (n = 2, 2.3%), MkRPs (n = 11, 12.5%), or “other” (n = 3, 3.4%). “Other”-type reconstitution encompassed nmEPltB (nm, E, Plt, and B)-type (n = 1, 1.1%) and nmEPltT (nm, E, Plt, and T)-type (n = 2, 2.3%) reconstitution. Single fraction III cells exhibited reconstitutive behavior of IT-HSCs (n = 7, 8.0%), ST-HSCs (n = 25, 28.4%), CMRPs (n = 7, 8.0%), and others (n = 6, 6.8%). “Other”-type reconstitution encompassed nmBTE (nm, E, B, and T)-type (n = 2, 2.3%), nmB (nm and B)-type (n = 2, 2.3%), and nmEPltB (nm, E, Plt and B)-type (n = 2, 2.3%) reconstitution. See also Figure S3. Cell 2013 154, 1112-1126DOI: (10.1016/j.cell.2013.08.007) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 4 CD34+KSL Fraction Contains Myeloid-Restricted Progenitors with Lower Repopulating Capability than MyRPs Ten Flt3−CD34+KSL cells (MPPs; A), or ten Flt3+CD34+KSL cells (LMPPs; B) were transplanted with 2 × 105 competitor cells (Ly5.1/Ly5.2-F1) into each of 18 lethally irradiated Ly5.2 mice (two cohorts of nine mice each). Differentiation potentials of transplanted cells into mature blood lineages (platelet, neutrophil/monocyte, erythrocyte, B cell, and T cell) in each mouse are shown with colored boxes (yellow, black, red, blue, and green, respectively). Reconstitution kinetics data of all mice transplanted with MPPs (A) or LMPPs (B) are shown at right. See also Figure S5. Cell 2013 154, 1112-1126DOI: (10.1016/j.cell.2013.08.007) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 5 HSCs Asymmetrically Give Rise to HSCs and to MkRPs or CMRPs (A) Single fraction I cells from KuO mice were sorted into 96-well plates, one cell per well, and were incubated in the presence of SCF and TPO. After 36–40 hr, during which interval the individual cells had divided once, the two daughter cells resulting from division of each initially sorted cell were individually transplanted into 204 individual (102 paired) lethally irradiated Ly5.2 mice with 2 × 105 competitor cells from a Ly5.1/Ly5.2-F1 mouse. PB cells from the recipient mice were periodically analyzed after transplantation. The platelet, neutrophil/monocyte, erythrocyte, B cell, and T cell differentiation potentials in daughter cells were determined. The mother cells were considered to have had the combined differentiation potentials of the two daughter cells. In 27 out of 102 pairs, both daughter cells reconstituted hematopoiesis in recipient mice. (B–D) Representative reconstitution kinetics for HSC-HSC (B), HSC-MkRP (C), and HSC-CMRP, CMRP-CMRP, and CMRP-MkRP pairs (D) are shown. All data for repopulating cell type in daughter cells are shown in Table S1. See also Figure S6. Cell 2013 154, 1112-1126DOI: (10.1016/j.cell.2013.08.007) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 6 Myeloid Bypass Model in Hematopoietic Stem Cells HSCs self-renew and give rise to lineage-restricted progenitor cells. In the conventional hematopoietic differentiation model (right side), HSCs in the CD34−KSL population differentiate into multipotent progenitors (MPPs, Flt3−CD34+KSL) of reduced self-renewal potential and MPPs eventually produce lymphoid-primed multipotent progenitors (LMPPs, Flt3+CD34+KSL) or lineage-committed progenitor cells in a stepwise manner. All mature blood lineages are considered to pass through the MPP and/or LMPP stage in the CD34+KSL population. Subsets of HSCs (myeloid-biased, balanced, and lymphoid-biased HSCs; and α, β, γ, and δ cells) are reported by other groups (Müller-Sieburg et al., 2002; Dykstra et al., 2007). In contrast, in the myeloid bypass model (left side), the CD34−KSL cell population contains CMRPs, MERPs, and MkRPs in addition to HSCs. These MyRPs are produced by HSCs (LT-HSCs, IT-HSCs or ST-HSCs). MyRPs can clonally expand via self-renewal as in HSCs, B cells, and T cells. The CD34+KSL population, which is downstream from the CD34−KSL population, also contains lineage-committed progenitors including myeloid-restricted progenitors, whereas “true” MPPs and LMPPs are minor populations in the CD34+KSL population. Together, MyRPs, rather than “MPPs and LMPPs,” are considered to be the major suppliers of myeloid cells (platelets, erythrocytes, and neutrophils/monocytes) in the hematopoietic system at a single-cell level. LMPP fraction yields cells with various differentiation potentials (B, nm, nmB, nmET, nmEB, nmEPlt, and nmEPltB) and these progenitors might derive from ST-HSCs in the CD34−KSL population but not from MPPs in the CD34+KSL population. Because cells in the CD34+KSL fraction have oligopotent differentiation potentials rather than multipotent potentials, CD34+KSL cells are considered to be OPPs. Cell 2013 154, 1112-1126DOI: (10.1016/j.cell.2013.08.007) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure S1 Peripheral Blood Analysis after Transplantations, Related to Figure 1 Gating strategy to detect Kusabira-Orange (KuO)+cells among platelets, neutrophils/monocytes, erythrocytes, B cells, or T cells in peripheral blood. Nucleated blood cells (Ly5.1+Ly5.2−) were subdivided into Gr-1+Mac-1+B220−CD4−CD8− cells (neutrophils/monocytes), B220+Gr-1−Mac-1−CD4−CD8− cells (B cells), and CD4+CD8+Gr-1−Mac-1−B220− cells (T cells). Erythrocytes and platelets were detected as FSChighTer-119+CD41− cells (erythrocytes) and FCSlowCD41+Ter-119− cells (platelets), respectively. Cell 2013 154, 1112-1126DOI: (10.1016/j.cell.2013.08.007) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure S2 Representative Flow Cytometric Data of Mice Transplanted with a Single LT-HSC, IT-HSC, ST-HSC, CMRP, MERP, or MkRP, Related to Figures 1 and 2 Chimerism kinetics data of an LT-HSC (A), IT-HSC (B), ST-HSC (C), CMRP (D), MERP (E) and MkRP (F) mouse are shown. The boxes show KuO+ cells among Ly5.1-positive cells, neutrophils/monocytes, B cells, T cells, erythrocytes, and platelets. In an LT-HSC mouse (A), chimerism was stable (0.1% or more) for all five types of KuO+ mature blood cells 20 weeks after secondary transplantation. In an IT-HSC mouse (B), chimerism (0.1% or more) for all five types of KuO+ mature blood cells 24 weeks after primary transplantation, but at least one lineage disappeared (that is, showed less than 0.1% chimerism) in secondary recipient mice. In a ST-HSC mouse (C), mature blood cells of at least one lineage among five lineages disappeared (that is, showed less than 0.1% chimerism) before 24 weeks after primary transplantation. In a CMRP mouse (D), only neutrophils/monocyte, erythrocyte, and platelet reconstitution, but not B and T lineage reconstitution, was detected in peripheral blood. In an MERP mouse (E), only erythrocyte and platelet reconstitution was detected in peripheral blood. In an MkRP mouse (F), only platelet reconstitution was detected in peripheral blood. Cell 2013 154, 1112-1126DOI: (10.1016/j.cell.2013.08.007) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure S3 Chimerism Kinetics of Mice with nmB-Type Reconstitution, Related to Figure 3 Two mice receiving single fraction III cells showed nmB-type reconstitution. Cell 2013 154, 1112-1126DOI: (10.1016/j.cell.2013.08.007) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure S4 Representative Flow Cytometric Data of BM Cells of LT-HSC-, IT-HSC-, ST-HSC-, CMRP- and MkRP-Recipient Mice, Related to Table 1 BM cells of LT-HSC- (A, n = 3), IT-HSC- (B, n = 4), ST-HSC- (C, n = 4), CMRP- (D, n = 3) and MkRP (E, n = 4)-recipient mice were analyzed after transplantation. BM cells were stained with antibodies against a lineage cocktail and CD150, CD41, CD34, c-Kit, Sca-1, and Flt3 for evaluation of phenotypically defined HSCs (myeloid-biased/balanced and lymphoid-biased HSCs), MPPs, LMPPs and MkPs; with antibodies against a lineage cocktail and CD34, c-Kit, Sca-1, and FcγR for evaluation of phenotypically defined myeloid progenitors (CMPs, GMPs, and MEPs), or with antibodies against a lineage cocktail and c-Kit, Sca-1, IL7Rα, and Flt3 for evaluation of phenotypically defined lymphoid progenitors (CLPs). Flow cytometric data of each KuO+ hematopoietic subset are shown. Cell 2013 154, 1112-1126DOI: (10.1016/j.cell.2013.08.007) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure S5 MPP and LMPP Fraction Cells Are Lineage-Committed at a Single Cell Level, Related to Figure 4 Single MPPs or LMPPs were transplanted with competitor cells into 26 or 48 lethally irradiated Ly5.2 mice with competitor cells, respectively. In 10 of 26 (38.5%) and 16 of 48 mice (33.3%), KuO+mature blood lineages were detected. Differentiation potentials of transplanted cells into mature blood lineages (platelet, neutrophil/monocyte, erythrocyte, B cell, and T cell) in each mouse are shown with colored boxes (yellow, black, red, blue, and green, respectively). Cell 2013 154, 1112-1126DOI: (10.1016/j.cell.2013.08.007) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure S6 Megakaryocyte Colony Formation by Hematopoietic Stem Cells and Differentiation Potential in PDC Assay, Related to Figure 5 (A), Single CD150+CD34−KSL or CD150−CD34−KSL cells from wild-type mouse BM cells sorted into 96-well plates were cultured in serum-free medium with SCF and TPO. Representative colonies consisting of only large cells (large cell colony, top) or small cells (other colony, bottom) obtained on culture of CD150+CD34−KSL cells are shown (day 7). (B) Colonies with only large cells (large cell colonies) and colonies with small cells (other colonies) arose, respectively, from 5.0 ± 1.0 and 86.0 ± 1.0 (mean ± SD) cells in the CD150+CD34−KSL cell group and 0.0 ± 0.0 and 90 ± 1.6 per 96 cells (n = 3) in the CD150−CD34−KSL cell group at day 7. (C) Colonies consisting of only large cells were stained with FITC-conjugated anti-CD41 antibody or its isotype antibody. Of note is that large cells marked for CD41. (D) Sixty CD150+CD34−KSL cells from wild-type mouse BM were cultured in semisolid medium with SCF, TPO, IL-3, IL-6, and EPO for 12 days. Colonies were fixed and stained for acetylcholinesterase and with Harris’ hematoxylin. Shown are an acetylcholinesterase-expressing megakaryocyte colony (D, top) and a colony without acetylcholinesterase expression (D, bottom). (E) Megakaryocyte-restricted colonies arose from 5.3 ± 0.57 (mean ± SD) cells in the CD150+CD34−KSL cell group and 0.0 ± 0.0 cells in the CD150−CD34−KSL cell group per 60 cells (n = 3) cultured in semisolid medium as for (D), above. (F) Repopulating cell types in freshly isolated single fraction I cells (representing mother cells; data also shown in Figure 3B, four independent experiments) and fraction I cells allowed to divide once in short-term culture (representing daughter cells; data also shown in Table S1, three independent experiments) obtained in paired-daughter cell assays were defined as LT-HSC, IT-HSC, ST-HSC, CMRP, MERP, or MkRP according to their differentiation potentials as determined from results of primary and secondary transplantations. Proportions of CMRP, MERP, and MkRP did not significantly differ between freshly isolated cells and daughter cells (p > 0.1, Student’s t test). (G) Transplanted cells’ differentiation potentials along neutrophil/monocyte, erythrocyte, platelet, B cell, and T cell lineages were compared among freshly isolated single fraction I cells (representing mother cells; data also shown in Figure 3B), fraction I cells allowed to divide once in short-term culture (representing daughter cells; data also shown in Table S1 in paired daughter cell assays. Differentiation potentials did not significantly differ from one another (p > 0.1, Student’s t test). Error bar, SEM. Cell 2013 154, 1112-1126DOI: (10.1016/j.cell.2013.08.007) Copyright © 2013 Elsevier Inc. Terms and Conditions