Prospective isolation and global gene expression analysis of the erythrocyte colony-forming unit (CFU-E)‏ by Grzegorz Terszowski, Claudia Waskow, Peter Conradt, Dido Lenze, Jessica Koenigsmann, Dirk Carstanjen, Ivan Horak, and Hans-Reimer Rodewald Blood Volume 105(5):1937-1945 March 1, 2005 ©2005 by American Society of Hematology

Phenotypic isolation, morphology, and potential of 6 distinct hematopoietic progenitor populations. Phenotypic isolation, morphology, and potential of 6 distinct hematopoietic progenitor populations. (A) Lin–IL-7Rα–Sca-1– BM cells were analyzed for expression of c-Kit versus IL-3Rα (i) or c-Kit versus antibody isotype control (ii). Cells shown in i correspond to 2% of all nucleated BM cells. Lin–Sca-+–IL-7Rα– cells were sorted into c-Kit+IL-3Rα– (green gate in i) and c-Kit+IL-3Rα+ (red gate in i) populations. Cells shown in iii and xi correspond to 0.52% and 0.68% of all nucleated BM cells, respectively. Purified c-Kit+IL-3Rα– cells (iii) were restained for expression CD71 versus CD41 (iv), and sorted into CD71+CD41– (v), CD71–CD41– (vii), or CD71–CD41+ (ix) cells. Sorted c-Kit+IL-3Rα+ cells (xi) were stained for expression of FcγR (CD16/CD32) versus CD41 (xii), and further separated into FcγRhiCD41– (xiii), FcγRloCD41– (xv), or FcγRloCD41+ (xvii) cells. Numbers in i, iv, and xii are relative percentages of the populations in the indicated regions. Numbers shown in v, vii, ix, xiii, xv, and xvii are percentages of each population per total nucleated BM cells. Cytospins from each population were stained with May-Grünwald-Giemsa stain (vi,viii,x,xiv,xvi,xviii). The scale bar (shown in xviii) corresponds to 15 μM and applies to panels vi, viii, x, xiv, xvi, and xviii. Based on their potential, the 6 progenitors were designated as erythrocyte progenitors (EPs; v), IL-3Rα– common myeloid progenitors (CMP IL-3Rα–; vii), IL-3Rα – megakaryocyte progenitors (MP1; ix), granulocyte-monocyte progenitors (GMPs; xiii), IL-3Rα+ common myeloid progenitors (CMP IL-3Rα+; xv), and IL-3Rα+ megakaryocyte progenitors (MP2; xvii). (B) Purified BM progenitors (as shown in panel A) were plated at 2000 cells/culture into methylcellulose assays promoting the generation of BFU-E (i), CFU-Meg (ii), or CFU-GM (iii) colonies. Colonies were counted after 8 days. BFU-E colonies were typed by benzidine staining for globin. CFU-Meg and CFU-GM colonies were identified by morphology. EPs lack potential for BFU-E, CFU-Meg, or CFU-GM colonies. Data shown are representative for one of 3 experiments. Formation of more mature CFU-Ss day 8 (iv), and more immature CFU-Ss day 12 (v) was analyzed by transfer of HSCs (2500 cells/mouse; number of recipients [n] = 2), CMP IL-3Rα– (2500 cells/mouse; n = 2), CMP IL-3Rα+ (2500 cells/mouse; n = 2), GMP (2500 cells/mouse; n = 2), EP (2500 cells/mouse; n = 2), MP1 (2500 cells/mouse; n = 2), or MP2 (2500 cells/mouse; n = 2). Injection of total BM (2 × 105 cells/mouse; n = 2) or PBS (n = 2) served as positive and negative controls, respectively. After 8 or 12 days macroscopic colonies were counted. CFU-Ss are restricted to HSC and CMP subpopulations (iv-v). Data shown are representative for one of 2 experiments. Grzegorz Terszowski et al. Blood 2005;105:1937-1945 ©2005 by American Society of Hematology

Parallel reduction in the frequencies of CFU-E colonies and EPs in mice lacking the transcription factor ICSBP. Lin–Sca-1–IL-7Rα– cells from ICSBP+/+ (A) and from ICSBP–/– (E) mice were analyzed for expression of c-Kit and IL-3Rα. Parallel reduction in the frequencies of CFU-E colonies and EPs in mice lacking the transcription factor ICSBP. Lin–Sca-1–IL-7Rα– cells from ICSBP+/+ (A) and from ICSBP–/– (E) mice were analyzed for expression of c-Kit and IL-3Rα. C-Kit+IL-3Rα– cells were further separated by staining for CD71 versus CD41 (B,F). Frequencies of EPs were strongly reduced in ICSBP–/– (G) compared to ICSBP+/+ (C) BM. This decline in the EP frequencies parallels the reduction in the CFU-E frequencies in total BM from ICSBP–/– (H) compared to ICSBP+/+ mice (D). Numbers in panels A, B, E, and F are relative percentages of the populations in the indicated regions. Data in shown panels C, D, G, and H are mean numbers from duplicate CFU-E cultures for each experiment (Exp.). Grzegorz Terszowski et al. Blood 2005;105:1937-1945 ©2005 by American Society of Hematology

“Cross-phenotyping” of IL-3Rα+ and IL-3Rα– BM progenitors for CD34 and FcγR expression. “Cross-phenotyping” of IL-3Rα+ and IL-3Rα– BM progenitors for CD34 and FcγR expression. Lin–c-Kit+Sca-1–IL-7Rα– cells (black gate in panel B) were stained for CD34 versus FcγR expression and resolved (E) into CD34+FcγRlo (CMP), CD34+FcγRhi (GMP), and CD34–FcγRlo (MEP).6 For comparison, c-Kit+lin–Sca-1–IL-7Rα– cells were sorted into IL-3Rα– (A) and IL-3Rα+ (C) subsets, and then restained for CD34 and FcγR expression. c-Kit+IL-3Rα– cells include MEP and CMP phenotypes (D), and c-Kit+IL-3Rα+ cells include GMP and CMP phenotypes (F). Grzegorz Terszowski et al. Blood 2005;105:1937-1945 ©2005 by American Society of Hematology

Erythrocyte reconstitution in vivo. Erythrocyte reconstitution in vivo. Host erythropoiesis was transiently suppressed by irradiation (850 rad; split dose). After irradiation, mice were given intravenous injections of PBS (number of recipients [n] = 2; A), total BM (2 × 105 cells/mouse; n = 2; B), HSC (2500 cells/mouse; n = 2; C), CMP IL-3Rα– cells (5000 cells/mouse; n = 2; D), EPs (5000 cells/mouse; n = 2; E), MP1 (5000 cells/mouse; n = 1; F), CMP IL-3Rα+ (5000 cells/mouse; n = 2; G), GMP (5000 cells/mouse; n = 2; H), or MP2 (5000 cells/mouse; n = 1; I). Percentages of reticulocytes in peripheral blood were determined at the indicated time points. Data shown are representative for one of 2 experiments. Grzegorz Terszowski et al. Blood 2005;105:1937-1945 ©2005 by American Society of Hematology

Differential gene expression profile comparing EPs and GMPs Differential gene expression profile comparing EPs and GMPs. RNAs from cell sorter-purified EPs and GMPs from normal mice were analyzed by microarray analyses for differences in global gene expression. Differential gene expression profile comparing EPs and GMPs. RNAs from cell sorter-purified EPs and GMPs from normal mice were analyzed by microarray analyses for differences in global gene expression. Panel A depicts a selection of genes that are highly and differentially expressed in either GMPs (top) or EPs (bottom). Depicted is the fold difference in GMP over EP (positive numbers) and EP over GMP (negative numbers) hybridization signals. Panel B shows the relative expression pattern of transcription factors in alphabetical order comparing EPs and GMPs. Red indicates increased expression; black indicates lack of expression. Repeats of the same gene name reflect distinct probes for that gene. The full list of expression data has been deposited at http://www.ncbi.nlm.nih.gov/geo. Grzegorz Terszowski et al. Blood 2005;105:1937-1945 ©2005 by American Society of Hematology

Phenotypic and functional map of myeloid pathways in mouse BM Phenotypic and functional map of myeloid pathways in mouse BM. IL-3Rα expression defines subsets of CMPs and progenitors with prospective potential for erythrocytes (EP), megakaryocytes (MP1 and MP2), or granulocytes and monocytes (GMP). Phenotypic and functional map of myeloid pathways in mouse BM. IL-3Rα expression defines subsets of CMPs and progenitors with prospective potential for erythrocytes (EP), megakaryocytes (MP1 and MP2), or granulocytes and monocytes (GMP). The c-Kit+IL-3Rα+ population includes the IL-3Rα+ CMP subpopulation, GMPs, and the IL-3Rα+ megakaryocyte progenitor termed MP2. The c-Kit+IL-3Rα– population includes the IL-3Rα– CMP subsets, the IL-3Rα– megakaryocyte progenitor termed MP1, and EPs. The relationship of stem cells and common myeloid and committed myeloid stages to CFU-S, BFU-E, and CFU-E is indicated for each compartment. Despite their major myeloid potential, we also detected a minor level of B-cell progenitor activity in mice given transplants of CMP 3Rα– or CMP 3Rα+ cells. The level of B-cell reconstitution from CMPs was about 10-fold lower when compared to CLPs and about 40-fold lower when compared to HSCs or BM. Phenotypic abbreviations are c-Kit (K), lin (L), and Sca-1 (S). Grzegorz Terszowski et al. Blood 2005;105:1937-1945 ©2005 by American Society of Hematology