1. Volume 3, Issue 5, Pages 1539-1552 (May 2013)
Revision of the Human Hematopoietic Tree: Granulocyte Subtypes Derive from Distinct Hematopoietic Lineages 
André Görgens, Stefan Radtke, Michael Möllmann, Michael Cross, Jan Dürig, Peter A. Horn, Bernd Giebel 
Cell Reports 
Volume 3, Issue 5, Pages (May 2013)
DOI: /j.celrep
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2. Cell Reports 2013 3, 1539-1552DOI: (10.1016/j.celrep.2013.04.025)
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3. Figure 1
Progenitors with Long-Term Hematopoietic Potentials Are Highly Concentrated within CD133+CD34+ Cell Fractions
(A) Upper panels: CD133 expression on freshly enriched, UCB-derived CD34+ cells. Lower panels: relationship between CD133 and CD38 expression compared to isotype control on freshly enriched CD34+ cells.
(B) CD133 and CD38 expression compared to isotype controls on cultured (50–60 hr) CD34+ cells.
(C) CD133 and CD34 expression on cultured cells before sorting and following sorting for CD34+, CD34+CD133+, and CD34+CD133low.
(D) LTC-IC and NK-IC frequencies within sorted fractions of culture-derived (50–60 hr) cells are given as mean ± SD (*p < 0.05, **p < 0.01, ***p < 0.001).
(E) Quantification of human cell engraftment in NOD/SCID mice, given as a percentage of viable human CD45+ cells within the BM of NOD/SCID mice 8 weeks postintravenous transplantation. NOD/SCID mice were transplanted with 5 × 104 sorted CD133+CD34+ (CD133+) cells or CD133lowCD34+ (CD133low) cells. Identical symbols represent animals that received cells from the same UCB samples.
(F) Multilineage analyses of a CD133+CD34+-transplanted mouse. BM cells expressing human CD45+ cells were further analyzed for their expression of lymphoid and myeloid antigens.
(G) Analysis of human hematopoietic progenitor markers within the BM of transplanted NOD/SCID mice.
See also Figure S1.
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4. Figure 2
CD133+CD34+ and CD133lowCD34+ Cell Fractions Contain Different Qualities of Myeloid/Erythroid Progenitors
(A) Myeloid/erythroid colony formation of culture-derived CD133+CD34+ and CD133lowCD34+ cells. Colony numbers are indicated per 100 seeded cells (mean ± SD; **p < 0.01, ***p < 0.001).
(B) Flow cytometric analyses of dissociated colony cells. Erythroid cells (E) were discriminated from nonerythroid cells (NE) by anti-CD45 and anti-GPA staining. Erythroid progenitors were identified as CD71+GPAlow cells.
(C) Myeloid/erythroid colony formation from freshly isolated CD133+CD34+ and CD133lowCD34+ cells.
(D) Analyses of the cell surface phenotype of colony-derived cells originating from freshly isolated CD34+ cells.
(E) Analyses of the cell surface phenotype of cells derived from CD133+ (day 3) CFU-GM and CD133low (day 3) CFU-MIX colonies. A total of 12 individual colonies of each type was harvested from four different UCBs, stained, and analyzed by flow cytometry. Granulocytes were identified as CD45+ cells (CD45+), expressing the cell surface antigens CD15 and CD66b (GRAN). CD16 and CD49d expression on granulocytes was analyzed separately.
(F) For analyses of day 0 colony-derived cells, 12 individual CD133+ (day 0) CFU-G colonies and 19 CD133+ (day 0) CFU-MIX colonies were harvested from four different UCB samples and stained. Of the 19 CFU-MIX colonies tested, 4 contained CD16+/CD49d− granulocytes.
See also Figure S2.
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5. Figure 3
CD133+CD34+ and CD133lowCD34+ Generate Different Granulocyte Types
(A) Modified Wright’s staining of colonies derived from cultured cells. Neutrophils (Neutro) were detected exclusively in CD133+CD34+ CFU-GM colonies. In contrast, erythroid cells (Ery), MK, eosinophils (Eos), and basophils (Bas) as well as eosinophil/basophil hybrid cells (Hybrid) were found exclusively in CD133lowCD34+ CFU-MIX colonies. Macrophages (Mac) were identified in both colony types. Scale bars, 10 μm (n = 3 UCBs).
(B) Modified Wright’s staining of colonies derived from fresh cells. Day 0 CD133+CD34+ CFU-G colonies contained neutrophilic cells, ranging from immature to band neutrophils. CD133+CD34+ CFU-MIX colonies mainly contained immature cells, erythroid cells, and some macrophages. CD133lowCD34+ CFU-G colonies contained eosinophils, basophils, and a large proportion of leukocytes containing both eosinophil and basophil granules, but no neutrophils. CD133lowCD34+ CFU-MIX colonies included macrophages, erythroid cells, eosinophils, basophils, hybrid cells, a small proportion of MKs, and only few immature cells, but never neutrophils. Scale bars, 10 μm.
(C) Day 0 CD133+CD34+ and CD133lowCD34+ CFU-MIX colonies were harvested after 2 weeks (16 and 10 colonies, respectively, originating from two different UCBs) and replated in CFC assays. Secondary CFC potential was documented after 12 days. Cells of CD133+CD34+ CFU-MIX colonies revealed pronounced myeloid colony-formation potential (CFU-G/-M/-GM) with rare erythroid or CFU-MIX colonies. In contrast, cells from CD133lowCD34+ CFU-MIX colonies had no secondary colony-formation potential. Scale bars, 200 μm.
(D) The pool of secondary colony cells derived from day 0 CD133+CD34+ CFU-MIX colonies was analyzed flow cytometrically. Eight out of eight fractions contained CD16+ granulocytes.
(E) All pools of secondary day 0 CD133+CD34+ CFU-MIX colony cells (n = 16) contained mature, segmented neutrophils as well as eosinophils, basophils, macrophages, erythroid cells, and few MKs. Scale bars, 10 μm.
(F) Summary of the lineage analyses of day 0 colonies based on their histology.
(G) RT-PCR analyses of neutrophil-specific MPO, eosinophil-specific EPX, and basophil-specific HDC expression in day 3 colony-derived cells. In each of the three independent experiments, RNA was extracted from pools of five individually harvested CD133+CD34+ CFU-GM or CD133lowCD34+ CFU-MIX colonies.
(H) RT-PCR analyses of MPO, EPX, and HDC in day 0 colony-derived cells, derived either from CD133+CD34+ or CD133lowCD34+ CFU-G or CFU-MIX colonies. In three independent experiments for each colony type, RNA was extracted from pools of five individually harvested colonies.
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6. Figure 4
CFU-MIX, Erythroid, and MK Potentials Are Gradually Lost from CD34+CD133+ Cell Fractions upon Cultivation
(A) Sorting and cultivation strategy to analyze the erythro-myeloid lineage potential of CD133+CD34+ and CD133lowCD34+ subpopulations. Freshly enriched CD34+ cells were initially purified at day 0 as CD133+CD34+ (+) and CD133low/−CD34+ (−) cells. The descendants of these cells were sorted again on day 3 as ‘++, +−, or −− cells, and their descendants in turn on day 6 as +++, ++-, +−−, or −−−’ cells. At each time point, aliquots of each sorted cell fraction were analyzed in CFC assays (B), erythroid differentiation assays (C), and MK differentiation assays (D) with the remainder being returned to culture in the presence of SCF, TPO, and FLT3L.
(B) Analyses of the erythro-myeloid colony-formation potential of CD133+CD34+ and CD133lowCD34+ cells (n = 4). Colony numbers are indicated per 100 originally seeded cells (mean ± SD).
(C) Analyses of the erythroid differentiation potential of purified CD133+CD34+ and CD133lowCD34+ cells in erythroid differentiation assays.
(D) Analyses of the MK differentiation potential of purified CD133+CD34+ and CD133lowCD34+ cells in MK differentiation assays.
Numbers of erythroid (C) or MKs (D) generated were normalized to 1,000 originally seeded cells and are given as mean ± SD. Numbers above bars indicate numbers of independently performed experiments. Brackets indicate comparison analyses with p values.
(E) Quantitative PCR analysis of Gata-1 and Gata-2 expression, normalized to their expression in CD34+CD133+ (day 0) cells (n = 3; mean ± SEM).
See also Figure S3.
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7. Figure 5
Comparison of the In Vivo Differentiation Potential of Day 3 CD133+CD34+ and Day 9 CD133+CD34+ Cells
(A) Purification strategy for CD133+CD34+ cells after 3 or 9 days of culture. A total of 150,000 sorted day 3 cells or 2 × 106 sorted day 9 CD133+CD34+ cells were transplanted into sublethally irradiated NSG mice. BM of transplanted mice was harvested 8 weeks posttransplantation and analyzed for multilineage engraftment (Figure S4A) as well as for the content of CD133+CD34+ and CD133lowCD34+ cells within the human CD34+ cell fraction, given as mean ± SD.
(B) CD133+CD34+ (CD133+) and CD133lowCD34+ (CD133low) cells within the mouse BM were sorted by flow cytometry (Figure S4B) and transferred into CFC assays. The average colony-forming activity of these cells is given for each colony type or in total per 100 sort-purified CD133+CD34+ or CD133lowCD34+ cells as mean ± SD. Brackets indicate comparison analyses with p values: *p < 0.05, **p < 0.01, ***p <
See also Figure S4.
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8. Figure 6 Expression of CD133 on Defined Progenitor Subpopulations
(A) Expression of CD133 on immunophenotypically defined progenitor populations of freshly isolated UCB CD34+ cells using a CD45RA/CD135-based gating strategy similar to Doulatov et al. (2010).
(B) Expression of CD135 on defined CD34+ progenitors using a CD45RA/CD133-based gating strategy on the same data file presented in (A).
See also Figure S5.
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9. Figure 7
Cell Fate Analyses of Individual Progenitors Define a Revised Model of Early Hematopoiesis
(A) Sorting strategy to enrich for MPPs, LMPPs, MLPs, and GMPs.
(B) Representative examples of the flow cytometric characterization of the CD45+ progeny fraction of individually deposited progenitors raised for 4 weeks in the MS5 assay (Doulatov et al., 2010).
(C) Quantification of the myeloid, lymphoid, and lympho-myeloid lineage output of individually deposited cells of the MPP-, LMPP-, MLP-, and GMP-enriched cell fractions (mean ± SD).
(D) CFC assay of MPP-, LMPP-, MLP-, and GMP-enriched cell fractions (mean ± SD).
(E) Wright’s staining of cells isolated from CFC assays indicates the presence of basophils, eosinophils (asterisk), neutrophils (arrowhead), and macrophages (arrow) in MPP-derived colonies. In contrast, no basophils and eosinophils were detected in LMPP- and GMP-derived colonies (scale bars, 10 μm). Myeloid cell types detected within bulk colonies are given.
(F) Quantitative PCR analysis of Gata-1, Gata-2, C/EBPα, PU.1, and Notch1 expression in MPP-, LMPP-, MLP-, and GMP-enriched cell fractions as well as in erythro-myeloid CD133lowCD34+CD38+CD45RA− (CD133lowCD45RA−) cells, normalized to their expression within the MPP-enriched fraction (n = 3; mean ± SEM).
(G) Graphical overview of the classical (Reya et al., 2001), composite (Adolfsson et al., 2005), and revised model of hematopoiesis proposed here.
See also Figure S6 and Table S1.
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10. Figure S1
Hematopoietic Long-Term Potentials in CD34+ Cells, Related to Figure 1
(A) LTC-IC and NK-IC frequencies within freshly isolated, sort purified CD34+, CD133+CD34+ and CD133lowCD34+ cell fractions estimated in 7 independent experiments. Average frequencies are shown in percentages (mean ± SD) with p values: *p < 0.05, **p < 0.01, ***p <
(B) Quantification of human cell engraftment in NSG mice, given as percentage of viable human CD45+ cells within the BM of mice 2 or 4 weeks post intra-venous transplantation. In a series of 5 independent experiments a total of 19 mice were transplanted with 5 × 104 or 1,5 × 105 sort-purified CD133low (d3) cells.
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11. Figure S2
Analyses of Individual Colonies Obtained in CFC Assays, Related to Figure 2
(A) As shown at the example of a BFU-E colony individual colonies were harvested with a pipette under microscopic observation (scale bars 200 μm). To avoid contamination between neighboring colonies within the CFC-assays, purified cells were originally seeded at very low densities (≤100 cells/mL). To dissociate transferred colonies into single cell suspensions and to remove residual methyl-cellulose, colonies were washed several times and subsequently stained for flow-cytometric analyses.
(B) To correlate the colony appearance with the cell surface phenotype, colonies of each type were harvested and analyzed by flow cytometry (first row; scale bars 200 μm). non-erythroid [NE] cells were discriminated from erythroid [E] cells by anti-GPA/anti-CD45 staining (second row). non-erythroid cells were tested for their expression of distinct combinations of lineage markers; macrophages and monocytes were identified as CD14+CD15-CD66b- cells [Mac] and granulocytes as CD14-CD15+CD66b+ cells [Gran]. Progeny fractions of CFU-GMs and CFU-MIX colonies contained granulocytes and macrophages or granulocytes, macrophages and erythroid cells, respectively.
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12. Figure S3
Flow Cytometric Analyses of Cells Raised within Expansion Cultures and the Erythroid and MK Differentiation Assays, related to Figure 4
(A) Flow cytometric analyses of day 0 sorted cells and their progeny fractions after 3 days of cultivation. Day 0 CD133+CD34+ cells give rise to a population of cells maintaining the CD133+CD34+ cell surface phenotype (++) and a population with a reduced CD133 cell surface expression (+-). Progeny of CD133low/-CD34+ cells remained CD133 negative (−−−).
(B) Cells obtained within the erythroid differentiation assay were analyzed for their CD45, CD71 and GPA cell surface expression. Cells with a CD45lowCD71+GPA+ cell surface phenotype were judged to be erythroid cells. These analyses revealed that ++ fractions hardly contained any cells with an erythroid cell surface phenotype. In contrast, and −− cell fractions displayed strong erythroid potentials.
(C) Cells obtained within the megakaryocytic differentiation assay were analyzed for their CD45, CD41 and CD61 cell surface expression. Cells with a CD45+CD41+CD61+ cell surface phenotype were judged to be megakaryocytic cells. Only a few descendants of the ++ cells displayed the megakaryocyte phenotype. In contrast, cells as well as −− cells gave rise to well-defined fractions of megakaryocytic cells.
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13. Figure S4
Analyses of Multilineage Engraftment and Purification of CD133 Subpopulations from Mouse Bone Marrow, Related to Figure 5
(A) Multi-lineage analyses of engrafted d3 and a d9 CD133+CD34+ transplanted NSG mice.
(B) The applied gating strategy to purify human CD34+ cell subpopulations from BM of engrafted NSG mice including re-analyses of purified cells. Sorted cells were analyzed within the CFC-assays.
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14. Figure S5
Analyses of Lineage Antigens and CD135 in CD34+ Populations, Related to Figure 6
(A) Flow cytometric analysis of antigens commonly used for the depletion of lineage positive CD34+ cells, either gated on CD34+ cells (first row) or on CD34+CD133+ cells (second row). UCB-derived mononuclear cells were used as positive controls for antigens which did not bind to any CD34+ cells (third row).
(B) Comparison of CD133 and CD135 expression on total CD34+ cells and on CD34+CD38+ or CD34+CD38low cells, respectively. The relevant gating strategy is depicted in Figure 6.
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15. Figure S6
Granulocytic and Monocytic Potential of Distinct Progeny Fractions within the MS5 Assay, Related to Figure 7
In contrast to cells from the MPP-, LMPP- and GMP-enriched cell fractions, cells of the MLP-enriched fractions hardly showed any granulocytic potential (n = 4; mean ± SD).
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