Lineage marker expression on mouse hematopoietic stem cells

•Both Mac-1-negative and Mac-1-low HSCs exist in adult mouse bone marrow.•Both CD41-negative and CD41-low HSCs exist in adult mouse bone marrow.•No use of Mac-1 and CD41 as markers in HSC purification is recommended. Whether hematopoietic stem cells (HSCs) express lineage markers is controversial. In this study, we highly purified HSCs from the adult bone marrow of C57BL/6 mice and examined their gene expression and reconstitution potential. We first focused on the integrin family. Single-cell reverse transcription polymerase chain reaction revealed that the expression of ItgaM/Itgb2 (Mac-1) and Itga2b/Itgb3 (CD41/CD61) gradually increased along HSC differentiation, whereas Itga4, Itga5, Itga6, and ItgaV (CD51) together with Itgb1 were highly expressed in both HSCs and hematopoietic progenitor cells (HPCs). We next fractionated HSCs based on their expression of Mac-1, CD41, and CD51 by flow cytometry. We detected Mac-negative and Mac-low, but not Mac-high cells, in the HSC population. We also detected CD41-negative, -low, and -high cells in the HSC population. Competitive repopulation revealed that Mac-1-negative and -low HSCs were functionally similar, and CD41-negative and -low HSCs were functionally similar, at the single-cell level, but CD41-high HSCs were not detectable. We then found that the selection of Mac-1-negative HSCs or CD41-negative HSCs had no advantage in HSC purification. We moreover found that HSCs expressed more CD51 than CD41, and HPCs expressed more CD41 than CD51, suggesting that CD51 expression was gradually replaced by CD41 expression during megakaryocyte differentiation. We concluded that low levels of Mac-1 and CD41 expression are irrelevant to the self-renewal and differentiation potentials in HSCs. Whether hematopoietic stem cells (HSCs) express lineage markers is controversial. In this study, we highly purified HSCs from the adult bone marrow of C57BL/6 mice and examined their gene expression and reconstitution potential. We first focused on the integrin family. Single-cell reverse transcription polymerase chain reaction revealed that the expression of ItgaM/Itgb2 (Mac-1) and Itga2b/Itgb3 (CD41/CD61) gradually increased along HSC differentiation, whereas Itga4, Itga5, Itga6, and ItgaV (CD51) together with Itgb1 were highly expressed in both HSCs and hematopoietic progenitor cells (HPCs). We next fractionated HSCs based on their expression of Mac-1, CD41, and CD51 by flow cytometry. We detected Mac-negative and Mac-low, but not Mac-high cells, in the HSC population. We also detected CD41-negative, -low, and -high cells in the HSC population. Competitive repopulation revealed that Mac-1-negative and -low HSCs were functionally similar, and CD41-negative and -low HSCs were functionally similar, at the single-cell level, but CD41-high HSCs were not detectable. We then found that the selection of Mac-1-negative HSCs or CD41-negative HSCs had no advantage in HSC purification. We moreover found that HSCs expressed more CD51 than CD41, and HPCs expressed more CD41 than CD51, suggesting that CD51 expression was gradually replaced by CD41 expression during megakaryocyte differentiation. We concluded that low levels of Mac-1 and CD41 expression are irrelevant to the self-renewal and differentiation potentials in HSCs. Hematopoietic stem cells (HSCs) reside at the top of the hematopoietic hierarchy and differentiate into all blood cell lineages [1Wang Z Ema H Mechanisms of self-renewal in hematopoietic stem cells.Int J Hematol. 2016; 103: 498-509Crossref PubMed Scopus (21) Google Scholar]. It is generally accepted, based on an early study [2Spangrude GJ Heimfeld S Weissman IL Purification and characterization of mouse hematopoietic stem cells.Science. 1988; 241: 58-62Crossref PubMed Scopus (2181) Google Scholar], that HSCs do not express lineage markers. Many studies, including our own, have used anti-Mac-1 (ItgaM/Itgb2, CD11b/CD18) antibody as one of the lineage antibodies in a lineage cocktail to remove myeloid lineage-committed cells for HSC purification. Mac-1 is known to be expressed in HSCs at the fetal liver stage [3Morrison SJ Hemmati HD Wandycz AM Weissman IL The purification and characterization of fetal liver hematopoietic stem cells.Proc Natl Acad Sci USA. 1995; 92: 10302-10306Crossref PubMed Scopus (442) Google Scholar], but its expression is presumed to be lost in adult bone marrow (BM) HSCs. However, it has been reported that some adult BM HSCs express Mac-1 [4Morrison SJ Lagasse E Weissman IL Demonstration that Thy(lo) subsets of mouse bone marrow that express high levels of lineage markers are not significant hematopoietic progenitors.Blood. 1994; 83: 3480-3490Crossref PubMed Google Scholar, 5Morrison SJ Wandycz AM Hemmati HD Wright DE Weissman IL Identification of a lineage of multipotent hematopoietic progenitors.Development. 1997; 124: 1929-1939Crossref PubMed Google Scholar, 6Ishida A Zeng H Ogawa M Expression of lineage markers by CD34+ hematopoietic stem cells of adult mice.Exp Hematol. 2002; 30: 361-365Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar]. Itga2b/Intgb3 (CD41/CD61) is expressed in platelets and plays a role in their aggregation [7Phillips DR Charo IF Parise LV Fitzgerald LA The platelet membrane glycoprotein IIb-IIIa complex.Blood. 1988; 71: 831-843PubMed Google Scholar]. This complex is also expressed in megakaryocytes and their progenitor cells [8Pronk CJ Rossi DJ Mansson R et al.Elucidation of the phenotypic, functional, and molecular topography of a myeloerythroid progenitor cell hierarchy.Cell Stem Cell. 2007; 1: 428-442Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar, 9Pietras EM Reynaud D Kang YA et al.Functionally distinct subsets of lineage-biased multipotent progenitors control blood production in normal and regenerative conditions.Cell Stem Cell. 2015; 17: 35-46Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar, 10Nishikii H Kanazawa Y Umemoto T et al.Unipotent megakaryopoietic pathway bridging hematopoietic stem cells and mature megakaryocytes.Stem Cells. 2015; 33: 2196-2207Crossref PubMed Scopus (37) Google Scholar, 11Dumon S Heath VL Tomlinson MG Gottgens B Frampton J Differentiation of murine committed megakaryocytic progenitors isolated by a novel strategy reveals the complexity of GATA and Ets factor involvement in megakaryocytopoiesis and an unexpected potential role for GATA-6.Exp Hematol. 2006; 34: 654-663Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar]. Thus, CD41 can be used as one of the megakaryocyte lineage markers. It has been reported that some adult BM HSCs express CD41 [12Ferkowicz MJ Starr M Xie X et al.CD41 expression defines the onset of primitive and definitive hematopoiesis in the murine embryo.Development. 2003; 130: 4393-4403Crossref PubMed Scopus (256) Google Scholar]. CD41 expression may mark myeloid-biased HSCs [13Gekas C Graf T CD41 expression marks myeloid-biased adult hematopoietic stem cells and increases with age.Blood. 2013; 121: 4463-4472Crossref PubMed Scopus (186) Google Scholar]. Contrarily, it has also been reported that HSCs do not express CD41 in adult BM [14Kiel MJ Yilmaz OH Iwashita T Terhorst C Morrison SJ SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells.Cell. 2005; 121: 1109-1121Abstract Full Text Full Text PDF PubMed Scopus (2339) Google Scholar, 15Yamamoto R Morita Y Ooehara J et al.Clonal analysis unveils self-renewing lineage-restricted progenitors generated directly from hematopoietic stem cells.Cell. 2013; 154: 1112-1126Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar]. Whether CD41 is expressed on adult HSCs thus remains controversial. To address these issues, we examined Mac-1 and CD41 expression in highly purified mouse HSCs and found their low level of expression in a proportion of HSCs, which was functionally verified by transplantation assays. Here, we report the existence of Mac-1-negative or -low and CD41-negative or -low HSCs in adult BM. We further examine the relationship between CD41 (Intga2b) and CD51 (IntgaV) expression on HSCs because the expression of CD51/CD61 has been reported [16Umemoto T Yamato M Shiratsuchi Y et al.Expression of integrin beta3 is correlated to the properties of quiescent hemopoietic stem cells possessing the side population phenotype.J Immunol. 2006; 177: 7733-7739Crossref PubMed Scopus (32) Google Scholar], and CD41 and CD51 may share CD61 (Intgb3) as β-subunit integrin. From the practical point of view, we recommend not using the depletion of Mac-1+ cells or the selection of CD41− cells as a process of HSC purification. C57BL/6 (B6-CD45.2) mice were purchased from Beijing HFK Bioscience Company (Beijing, China). CD45.1 congenic B6 mice (B6-CD45.1) and GFP transgenic B6 mice were bred and maintained at the State Key Laboratory of Experimental Hematology. Eight- to 12-week-old female mice were used for all experiments. All experimental protocols using mice were approved by the Institutional Animal Care and Use Committee at the Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College. BM cells were obtained from the femurs, tibias and iliac crests of B6-CD45.1 or B6-GFP mice. Prior to antibody staining, c-Kit-positive cells were enriched with anti-c-Kit antibody-conjugated MACS beads (Miltenyi Biotec). Lineage depletion by MACS beads was not performed. Cells were stained with allophycocyanin (APC)/eFluor 780 (eF780)-conjugated anti-Mac-1 (M1/70), CD3 (SK7), CD4 (Gk1.5), CD8a (53-6.7), B220 (RA3-6B2), Gr-1 (RB6-8C5), Ter-119 (TER-119), or all seven antibodies. Cells were also stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD34 (RAM34), APC-conjugated anti-c-Kit (2B8), phycoerythrin-cyanine 7 (PE-Cy7)-conjugated anti Sca-1 (D7), Brilliant Violet (BV) 421-conjugated anti-CD48 (HM48-1), PE-conjugated anti-CD150 (TC15-12F12.2), and BV510 or PerCP/eF710-conjugated anti-CD41 (eBioMWReg30) antibodies. Cells were analyzed and sorted with a FACS Aria III (BD Biosciences). Data were exported with FCS 3.0 and analyzed by Flowjo. HSC1 was defined as CD150+CD41−CD34−Kit+Sca-1+Lineage− (CD150+41−34−KSL) cells. HSC2 was defined as CD150−41−34− KSL cells. HPC1 was defined as CD150+41+34−KSL cells. HPC2 was defined as CD150+41+34+ KSL cells. HPC3 was defined as CD150−41−34+ KSL cells (see the sorting gates for these populations in Figure 1A). A competitive repopulation assay was performed with 5 × 105 BM cells from B6-CD45.2 mice as competitor cells. Single or multiple cells were mixed with competitor cells and transplanted into B6-CD45.1 mice that had been irradiated twice at a dose of 4.75 Gy. Secondary transplantation was performed by transferring 2 × 107 BM cells of primary recipient mice into lethally irradiated B6-CD45.2 mice. Peripheral blood cells from the recipient mice were analyzed after the following procedures. Erythrocytes were lysed with buffer containing 0.15 mol/L NH4Cl, 10 mmol/L KHCO3, and 0.1 mmol/L EDTA in water (pH 7.2). Cells were stained with FITC-conjugated anti-CD45.1 (A20), PE-conjugated anti-CD45.2 (104), PE-Cy7-conjugated anti-CD4, APC-conjugated anti-CD8a, PerCP-Cy5.5-conjugated anti-B220, and APC/eF780-conjugated anti-Mac-1 and Gr-1 antibodies. Cells were analyzed on a Canto II (Becton Dickinson). The percentage of donor cells was calculated using the following formula: (% CD45.1+ cells) × 100/(% CD45.1+ test cells + % CD45.2+ cells). When the percentage of donor cells was ≥0.1, the hematopoietic system was considered to be reconstituted with donor cells (positive mice). Mac-1/Gr-1+ cells were considered to be of the myeloid lineage. B220+ cells were considered to be of the B-lymphoid lineage. CD4+ or CD8+ cells were considered to be of the T-lymphoid lineage. ST-HSCs were defined as HSCs with myeloid reconstitution potential for <6 months after transplantation and with lymphoid reconstitution potential. LT-HSCs were defined as HSCs with myeloid reconstitution potential for >6 months after transplantation. Single cells were sorted by a FACS Aria III into 96-well U-bottom plates, in which each well contained 200 µL α-MEM supplemented with 10% fetal bovine serum, 100 mg/mL penicillin/streptomycin, 50 ng/mL mouse stem cell factor, 50 ng/mL mouse thrombopoietin, 10 ng/mL mouse interleukin-3, and 1 IU/mL human erythropoietin, and cultured at 37°C in a humidified atmosphere with 5% CO2. On day 14 of culture, colonies were scored if a well contained ≥50 cells. Colonies were individually subjected to centrifugation onto glass slides by a Shandon Cytospin 4 (Thermo Scientific). Glass slides were stained with May–Giemsa staining solution. Cells were morphologically classified as neutrophils, macrophages, erythroblasts, and megakaryocytes. Megakaryocyte colonies were scored by in situ observation of four or more cells per well. Some megakaryocyte colonies were stained with anti-CD41 antibody to confirm CD41 expression. Single cells were sorted into 48 wells of a 96-well plate, where each well contained 10 μL of a reverse transcription and specific target amplification mixture consisting of 2.5 μL 0.2× primers containing 18 sets of α-integrin primers, 8 sets of β-integrin primers, and Gapdh primers, 5.0 μL 2× reaction mix, 0.5 μLIII, and 2.0 μL Tris–EDTA buffer. Reverse transcription (RT) was performed at 50°C for 15 min. The samples were incubated at 95°C for 2 min, followed by 22 cycles of 95°C for 15 sec and 60°C for 4 min. Five microliters from each of the samples was mixed with 20 μL of Tris– EDTA. Then, 2.7 μL of the diluted samples was mixed with 3.0 μL Taqman universal polymerase chain reaction PCR master mix (Applied Biosystems) and 0.3 μL sample loading buffer (for a total of 6.0 μL of sample loading mix). After 3.0 μL of 20× concentrations of each set of primers was mixed with 3.0 μL of assay loading reagent (a total of 6.0 μL of assay loading mix), 5 μL of the sample loading mix and 5 μL of the assay loading mix were applied to a 48-chip array, and 48 × 27 reactions were prepared by an integrated fluidics circuit controller. The chip was set on a Fluidigm BioMark and incubated at 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 60 sec. Data were analyzed with BioMark real-time PCR analysis software (Fluidigm). The PCR primers purchased from Thermo Fisher Scientific. When threshold cycle values (Ct) were <27.65, cells were considered to express the gene (positive cells). The Mann–Whitney test and Fisher's exact test were performed. We first examined the expression of all members of the integrin family [17Shimaoka M Takagi J Springer TA Conformational regulation of integrin structure and function.Annu Rev Biophys Biomol Struct. 2002; 31: 485-516Crossref PubMed Scopus (431) Google Scholar] in purified HSCs and HPCs by single-cell RT-PCR. We used two HSC populations: HSC1 and HSC2 (Figure 1A). HSC1 was enriched in long-term (LT >6 months) HSCs, and HSC2 was enriched in short-term HSCs (ST <6 months) [15Yamamoto R Morita Y Ooehara J et al.Clonal analysis unveils self-renewing lineage-restricted progenitors generated directly from hematopoietic stem cells.Cell. 2013; 154: 1112-1126Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar, 18Morita Y Ema H Nakauchi H Heterogeneity and hierarchy within the most primitive hematopoietic stem cell compartment.J Exp Med. 2010; 207: 1173-1182Crossref PubMed Scopus (299) Google Scholar, 19Ema H Morita Y Suda T Heterogeneity and hierarchy of hematopoietic stem cells.Exp Hematol. 2014; 42 (74–82.e72)Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar]. We used three HPC populations: HPC1, HPC2, and HPC3 (Figure 1A). HPC1 was enriched in myeloid HPCs. HPC2 was enriched in day 12 colony-forming cells in spleen, and HPC3 was enriched in lymphoid-primed multipotent progenitors [15Yamamoto R Morita Y Ooehara J et al.Clonal analysis unveils self-renewing lineage-restricted progenitors generated directly from hematopoietic stem cells.Cell. 2013; 154: 1112-1126Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar, 18Morita Y Ema H Nakauchi H Heterogeneity and hierarchy within the most primitive hematopoietic stem cell compartment.J Exp Med. 2010; 207: 1173-1182Crossref PubMed Scopus (299) Google Scholar, 20Adolfsson J Mansson R Buza-Vidas N et al.Identification of Flt3+ lympho-myeloid stem cells lacking erythro-megakaryocytic potential: a revised road map for adult blood lineage commitment.Cell. 2005; 121: 295-306Abstract Full Text Full Text PDF PubMed Scopus (886) Google Scholar, 21Dong F Bai H Wang X et al.Mouse acute leukemia develops independent of self-renewal and differentiation potentials in hematopoietic stem and progenitor cells.Blood Adv. 2019; 3: 419-431Crossref PubMed Scopus (9) Google Scholar]. The relationship between Trampp's classification and our classification has previously been reported [21Dong F Bai H Wang X et al.Mouse acute leukemia develops independent of self-renewal and differentiation potentials in hematopoietic stem and progenitor cells.Blood Adv. 2019; 3: 419-431Crossref PubMed Scopus (9) Google Scholar]. We used a lineage antibody cocktail consisting of seven lineage antibodies (see Methods) in this experiment. Mac-1+ cells were depleted from these purified HSCs and HPCs. Thus, the expression of integrin genes, particularly ItgaM/Ingb2, might be underestimated. Consistent with previous work [22Wagers AJ Allsopp RC Weissman IL Changes in integrin expression are associated with altered homing properties of Lin(–/lo)Thy1.1(lo)Sca-1(+)c-kit(+) hematopoietic stem cells following mobilization by cyclophosphamide/granulocyte colony-stimulating factor.Exp Hematol. 2002; 30: 176-185Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar], the expression of Itga4, Itga5, and Itga6, together with that of Itgb1, was detected in HSC1 and HSC2 (Figure 1B). Interestingly, these integrins were also expressed in HPC1, HPC2, and HPC3. ItgaM, and Itga2b-expressing cells were also detected at a low frequency in HSC1. The frequencies of these integrin-expressing cells, together with those of Itgb2- and Itgb3-expressing cells, gradually increased from HSCs to HPCs, suggesting that their expression is associated with cell differentiation. Of note was that the frequency of Itgb2-expressing cells in HSC2 remained very low. In addition, ItgaV-expressing cells were detected in both HSC and HPC populations. The frequencies of Itgb3- and Itgb5-expressing cells were very low in HSC1 and neither Itgb6- nor Itgb8-expressing cells were detected in HSC1 and HSC2. Thus, ItgaV/Itgb1 as a heterodimer might be expressed in HSCs. From these results, we decided to focus on integrins αM (Mac-1) and αIIb (CD41), and αV (CD51) for the further study. We examined the expression of seven lineage markers individually and simultaneously by flow cytometry on CD201+CD150+CD48−CD41−c-Kit+Sca-1+ (CD201+150+48−41− KS) cells, which are highly enriched in LT-HSCs (Figure 2A) [15Yamamoto R Morita Y Ooehara J et al.Clonal analysis unveils self-renewing lineage-restricted progenitors generated directly from hematopoietic stem cells.Cell. 2013; 154: 1112-1126Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar, 23Wang X Dong F Zhang S et al.TGF-beta1 negatively regulates the number and function of hematopoietic stem cells.Stem Cell Rep. 2018; 11: 274-287Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar]. A significant proportion of the cells expressed low levels of Mac-1 but not of CD3, CD4, CD8, B220, Gr-1, and Ter119 (Figure 2B). Notably, we never detected a high level of Mac-1 expression in CD201+150+48−41− KS cells. We next performed competitive repopulation using the sorting gates illustrated in Supplementary Figure E1 (online only, available at www.exphem.org). Transplantation of 20 Mac-1-negative or -low CD201+150+48−41− KS cells similarly revealed LT reconstitution activity in both populations (Figure 2C). After single-cell transplantation, two LT-HSCs and one ST-HSC were detected in the 26 recipients of Mac-1negCD201+150+48−41−KS cells. Four LT-HSCs and one ST-HSC were detected in the 29 recipients of Mac-1lowCD201+150+48−41−KS cells (Figure 2D). According to our definition, myeloid lineage reconstitution persisted <6 months after transplantation for ST-HSCs and >6 months after transplantation for LT-HSCs, respectively. ST-HSCs are almost equivalent to lymphoid-biased HSCs, and LT-HSCs are almost equivalent to myeloid-biased or balanced HSCs [19Ema H Morita Y Suda T Heterogeneity and hierarchy of hematopoietic stem cells.Exp Hematol. 2014; 42 (74–82.e72)Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar]. To evaluate whether depletion of Mac-1+ cells is effective for HSC purification, we chose B220, Ter-119, and Gr-1 as the major three lineage markers and compared the degree of HSC purification between three- and seven-lineage depletion (the three lineages were B220, Ter-119, and Gr-1; the seven lineages consisted of B220, Ter-119, Gr-1, Mac-1, CD4, CD8, and CD3). Competitive repopulation with 10 CD150+41−34− KSL3 cells(three-lineage depletion) and 10 CD150+41−34− KSL7 cells (seven-lineage depletion) resulted in similar LT reconstitution activity in both populations (Figure 3A). Single-cell transplantation was also performed with 30 mice in each group. Three LT-HSCs and three ST-HSCs were detected in the three-lineage depletion, and four LT-HSCs and two ST-HSCs were detected in the seven-lineage depletion (Figure 3B). These data suggested that regardless of whether three- or seven-lineage antibodies are used for lineage depletion, the quality of purified HSCs is similar, but a proportion of HSCs may be lost during the purification procedures when seven lineages, including Mac-1, are used. For this experiment, three-lineage depletion (L3) was used. We operationally separated CD150+34−KSL3 cells into CD41-negative (CD41neg), CD41-low (CD41low), and CD41-high (CD41high) fractions (Figure 4A). CD41neg, CD41low, and CD41high cells accounted for approximately 40%, 40%, and 20% of CD150+34−KSL3 cells, respectively. Competitive repopulation was performed on 30 CD41neg, CD41low, or CD41high CD150+34−KSL3 cells. LT reconstitution activity was similarly detected in CD41neg and CD41low CD150+34−KSL3 cells. However, only a low level of ST reconstitution activity was detected in CD41highCD150+34−KSL3 cells (Figure 4B). After single-cell transplantation, six LT-HSCs and two ST-HSCs were detected in the 19 recipients of CD41negCD150+34−KSL3 cells, and five LT-HSCs and one ST-HSC were detected in the 16 recipients of CD41lowCD150+34−KSL3 cells. In contrast, no HSCs were detected in the 19 recipients of CD41high CD150+34−KSL3 cells (Figure 4C). We examined CD41 and CD51 expression by flow cytometry on CD150+48−KL3 cells (Figure 5A). These cells expressed varying amounts of CD41 and CD51. We operationally fractionated CD150+48−KL3 cells into CD51+CD41−, CD51+CD41+, CD51−CD41−, and CD51−CD41+ cells (Figure 5A). Competitive repopulation with 20 cells revealed LT reconstitution activity in CD51+CD41−, CD51+CD41+, and CD51−CD41−CD150+48−KL3 cells, but not in CD51−CD41+CD150+48−KL3 cells (Figure 5B). To confirm this, we fractionated CD150+48−KL3 cells into CD51+ and CD51−CD41+ cells (Figure 5C) and performed competitive repopulation with 20 cells. LT reconstitution activity was again detected in CD51+CD150+48−KL3 cells but not in CD51−CD41+CD150+48−KL3 cells (Figure 5D), suggesting that CD51−CD41+CD150+48−KL3 cells were more differentiated cells. To characterize these cells, we performed a single-cell colony assay. In this assay 59.7 ± 21.0% (mean ± SD, n = 6) of CD51+CD150+48−KL3 cells formed colonies, of which the frequencies of multilineage colonies consisting of neutrophil/macrophage/erythroblast/megakaryocyte (nmEM), nmE, and nmM colonies were 32.5 ± 17.0% (n = 6) and that of megakaryocyte colonies was 10.3 ± 10.7% (n = 6). On the other hand, 61.1 ± 11.0% (mean ± SD, n = 6) of CD51−CD41+CD150+48− KL3 cells formed colonies, of which multilineage colonies accounted for 3.3 ± 6.6% (n = 6) and megakaryocyte colonies accounted for 56.1 ± 8.6% (n = 6) (Figure 5E). The frequency of multipotent colonies was significantly greater in CD51+CD150+48−KL3 cells than in CD51−CD41+CD150+48−KL3 cells (p = 0.0077), whereas the frequency of megakaryocyte colonies was significantly greater in CD51−CD41+CD150+48−KL3 cells than in CD51+CD150+48−KL3 cells (p = 0.0050). These data provide support that CD51−CD41+CD150+48−KL3 cells are enriched in megakaryocyte progenitors. We used anti-CD41 antibody (clone: MWReg30) in cell staining for flow cytometry sorting. It has been reported that this antibody has a neutralizing effect and reduces the reconstitution activity when HSCs are reacted with a certain amount of this antibody and transplanted [13Gekas C Graf T CD41 expression marks myeloid-biased adult hematopoietic stem cells and increases with age.Blood. 2013; 121: 4463-4472Crossref PubMed Scopus (186) Google Scholar]. To examine whether the amount of anti-CD41 antibody used for staining procedure reduces the reconstitution activity of HSCs in our experiments, 20 CD150+48−34−KSL3 cells were transplanted with or without anti-CD41 antibody staining. In this experiment, the CD41 gate was not used for sorting, regardless of whether anti-CD41 antibody was used (Figure 6A–D). There was no significant difference in reconstitution activity 6 months after transplantation between the groups in which antibody was added and the groups in which antibody was not added (Figure 6F).Supplementary Figure E1Sorting gates for Mac-1-negative or -low CD201+150+41−34− KS cells.Show full caption(A–D) Sequential gating for CD201+150+41−34− KS cells. (E) Sorting gates of Mac-1-negative and Mac-1-low CD201+150+41−34− KS cells. (F) CD3 was used as a negative control.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A–D) Sequential gating for CD201+150+41−34− KS cells. (E) Sorting gates of Mac-1-negative and Mac-1-low CD201+150+41−34− KS cells. (F) CD3 was used as a negative control. We finally examined how CD41 gating was effective for further purification of HSCs. Single CD150+48−34−KSL3 cells (Figure 6A–D) and CD41−CD150+48−34−KSL3 cells (Figure 6A–E) were sorted from the same stained sample and transplanted into a group of 20 lethally irradiated mice with competitor cells. No significant difference was found in the frequencies of repopulating cells between these two groups (Figure 6G). In this single-cell transplantation, the percentage of chimerism was not taken into account because the number of repopulating cells in competitor cells appeared to influence the results more than the reconstitution potential level in single test donor cells [24Ema H Uchinomiya K Morita Y Suda T Iwasa Y Repopulation dynamics of single haematopoietic stem cells in mouse transplantation experiments: importance of stem cell composition in competitor cells.J Theor Biol. 2016; 394: 57-67Crossref PubMed Scopus (3) Google Scholar]. As CD41high cells accounted for a small proportion of CD150+48−34−KSL3 cells, the selection of CD41-negative cells did not increase the purity of LT-HSCs. Over the last decades, flow cytometry instruments have been significantly improved mainly because of the development of new laser and fluorescent dyes. As a result, the stable sorting of very rare cells with high speed and efficiency has become possible. We were able to address the controversial issues with a small number of sorted cells or even at the single-cell level. We detected single Mac-1-negative and -low HSCs in adult BM, consistent with previous studies [4Morrison SJ Lagasse E Weissman IL Demonstration that Thy(lo) subsets of mouse bone marrow that express high levels of lineage markers are not significant hematopoietic progenitors.Blood. 1994; 83: 3480-3490Crossref PubMed Google Scholar, 5Morrison SJ Wandycz AM Hemmati HD Wright DE Weissman IL Identification of a lineage of multipotent hematopoietic progenitors.Development. 1997; 124: 1929-1939Crossref PubMed Google Scholar, 6Ishida A Zeng H Ogawa M Expression of lineage markers by CD34+ hematopoietic stem cells of adult mice.Exp Hematol. 2002; 30: 361-365Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar]. A low level of Mac-1 expression is detected in fetal liver HSCs [3Morrison SJ Hemmati HD Wandycz AM Weissman IL The purification and characterization of fetal liver hematopoietic stem cells.Proc Natl Acad Sci USA. 1995; 92: 10302-10306Crossref PubMed Scopus (442) Google Scholar]. Mac-1low HSCs in the adult BM might have arisen from the fetal liver. We detected single CD41-negative and -low HSCs, but not CD41-high HSCs, in adult BM. A low level of CD41 expression is detected in pre-HSCs at the aorta–gonad–mesonephros region [25Rybtsov S Sobiesiak M Taoudi S et al.Hierarchical organization and early hematopoietic specification of the developing HSC lineage in the AGM region.J Exp Med. 2011; 208: 1305-1315Crossref PubMed Scopus (158) Google Scholar, 26Zhou F Li X Wang W et al.Tracing haematopoietic stem cell formation at single-cell resolution.Nature. 2016; 533: 487-492Crossref PubMed Scopus (196) Google Scholar]. The proportion of CD41low HSCs in BM increases in aged mice [13Gekas C Graf T CD41 expression marks myeloid-biased adult hematopoietic stem cells and increases with age.Blood. 2013; 121: 4463-4472Crossref PubMed Scopus (186) Google Scholar, 27Bernitz JM Kim HS MacArthur B Sieburg H Moore K Hematopoietic stem cells count and remember self-renewal divisions.Cell. 2016; 167 (1296–1309.e1210)Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar]. These data suggest that the expression of these integrin molecules on HSCs is developmentally regulated. Hence, knowing their functional role in the regulation of HSCs is of great interest. In general, the percentage of chimerism is taken into account for transplantation of bulk cells, but not for single-cell transplantation. In the case of bulk cell transplantation, the percentage of chimerism depends on the numbers of repopulating cells in both test donor cells and competitor cells. To compare the reconstitution potentials in different test donor cells, we may be able to compare the means of chimerism if the numbers of repopulating cells in both test donor cells and competitor cells and the number of recipient mice are sufficient. However, in the case of single-cell transplantation, the percentage of chimerism depends only on the number of repopulating cells in competitor cells. Therefore, the frequency of positive mice is more meaningful than the level of reconstitution in positive mice [24Ema H Uchinomiya K Morita Y Suda T Iwasa Y Repopulation dynamics of single haematopoietic stem cells in mouse transplantation experiments: importance of stem cell composition in competitor cells.J Theor Biol. 2016; 394: 57-67Crossref PubMed Scopus (3) Google Scholar]. The time length and lineage contribution after transplantation are also important parameters in single-cell transplantation. Competitive repopulation has served as the most reliable assay for HSCs. However, it is often difficult to obtain the same percentages of chimerism from different experiments with this in vivo assay. It is likely that the sorting efficiency, the number of repopulating cells in test donor cells and competitor cells, and the conditions of donor and recipient mice may somewhat differ from experiment to experiment. Therefore, it is important to repeat experiments to have consistent data. In this study, we instead performed both bulk-cell transplantation and single-cell transplantation, providing complementary data to compare HSC activity in two or more samples. Both ST lymphoid-biased HSCs and LT myeloid-biased HSCs were detected in Mac-1-negative and -low fractions (Figure 2). Similarly, both ST lymphoid-biased HSCs and LT myeloid-biased HSCs were detected in CD41-negative and -low fractions (Figure 4). These data suggested that the expression of Mac-1 and CD41 is not associated with the self-renewal potential and lineage-biased differentiation potentials in HSCs. Platelet-biased HSCs have been reported [28Sanjuan-Pla A Macaulay IC Jensen CT et al.Platelet-biased stem cells reside at the apex of the haematopoietic stem-cell hierarchy.Nature. 2013; 502: 232-236Crossref PubMed Scopus (357) Google Scholar, 29Carrelha J Meng Y Kettyle LM et al.Hierarchically related lineage-restricted fates of multipotent haematopoietic stem cells.Nature. 2018; 554: 106-111Crossref PubMed Scopus (161) Google Scholar, 30Grover A Sanjuan-Pla A Thongjuea S et al.Single-cell RNA sequencing reveals molecular and functional platelet bias of aged haematopoietic stem cells.Nat Commun. 2016; 7: 11075Crossref PubMed Scopus (139) Google Scholar]. These HSCs were marked with von Willebrand factor (vWF) expression by using vWF-reporter transgenic mouse. vWF+ platelet-biased HSCs were detected in both CD41-negative and -low CD150+48+34−KSL cells [29Carrelha J Meng Y Kettyle LM et al.Hierarchically related lineage-restricted fates of multipotent haematopoietic stem cells.Nature. 2018; 554: 106-111Crossref PubMed Scopus (161) Google Scholar]. These data together with our data suggested that CD41 expression level is not related to the potentials of LT-HSCs and platelet-biased HSCs. CD150+201+48−34−KS cells (Supplementary Figure E2E, online only, available at www.exphem.org) are enriched in LT-HSCs, whereas CD150−201+48−34−KS cells (Supplementary Figure E2F) are enriched in ST-HSCs [18Morita Y Ema H Nakauchi H Heterogeneity and hierarchy within the most primitive hematopoietic stem cell compartment.J Exp Med. 2010; 207: 1173-1182Crossref PubMed Scopus (299) Google Scholar, 31Kent DG Copley MR Benz C et al.Prospective isolation and molecular characterization of hematopoietic stem cells with durable self-renewal potential.Blood. 2009; 113: 6342-6350Crossref PubMed Scopus (228) Google Scholar]. The proportion of Mac-1low cells increased and the proportion of CD41low cells decreased in the CD150− ST-HSC population compared with that of the CD150+LT-HSC population. It was most likely that CD51 was expressed with CD29 (Itgb1) in HSCs (Figure 1). The expression of CD51/CD29 and possibly CD51/CD61 also was gradually replaced by that of CD41/CD61 in the megakaryocyte differentiation pathway (Figure 5). CD41 expression increased while a high level of CD150 expression was maintained along the megakaryocyte differentiation pathway [8Pronk CJ Rossi DJ Mansson R et al.Elucidation of the phenotypic, functional, and molecular topography of a myeloerythroid progenitor cell hierarchy.Cell Stem Cell. 2007; 1: 428-442Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar, 9Pietras EM Reynaud D Kang YA et al.Functionally distinct subsets of lineage-biased multipotent progenitors control blood production in normal and regenerative conditions.Cell Stem Cell. 2015; 17: 35-46Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar, 10Nishikii H Kanazawa Y Umemoto T et al.Unipotent megakaryopoietic pathway bridging hematopoietic stem cells and mature megakaryocytes.Stem Cells. 2015; 33: 2196-2207Crossref PubMed Scopus (37) Google Scholar, 32Haas S Hansson J Klimmeck D et al.Inflammation-induced emergency megakaryopoiesis driven by hematopoietic stem cell-like megakaryocyte progenitors.Cell Stem Cell. 2015; 17: 422-434Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar]. We used anti-CD41 antibody, which has a neutralizing effect. However, we did not detect any neutralizing activity in this study, presumably because the amount of antibody we used for staining was insufficient to exert such an effect. In vitro megakaryocyte colony formation by CD41high CD150+48−KL cells was consistent with previous studies [8Pronk CJ Rossi DJ Mansson R et al.Elucidation of the phenotypic, functional, and molecular topography of a myeloerythroid progenitor cell hierarchy.Cell Stem Cell. 2007; 1: 428-442Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar, 9Pietras EM Reynaud D Kang YA et al.Functionally distinct subsets of lineage-biased multipotent progenitors control blood production in normal and regenerative conditions.Cell Stem Cell. 2015; 17: 35-46Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar, 10Nishikii H Kanazawa Y Umemoto T et al.Unipotent megakaryopoietic pathway bridging hematopoietic stem cells and mature megakaryocytes.Stem Cells. 2015; 33: 2196-2207Crossref PubMed Scopus (37) Google Scholar]. Because CD150+201+48−34−KSL3 cells uniformly expressed CD51 (data not shown), the use of CD51 as an additional marker appeared to be not useful for further purification of HSCs. The expression of Mac-1 and CD41 seemed almost mutually exclusive in HSC populations (Supplementary Figure E2). We speculated the existence of two distinct differentiation pathways. In one pathway, LT-HSCs give rise to ST-HSCs, and this differentiation pathway is at least in part marked by Mac-1 expression. The other pathway is the myeloid and megakaryocyte differentiation pathway, which is marked by CD41 expression. Because a low-level expression of Mac-1 and CD41 in HSC1 is not associated with HSC function, we might consider their expression as “footprints” of HSCs at this very early stage of hematopoiesis. Finally, concerning the HSC purification protocols, this study suggested that the use of anti-Mac-l antibody should be excluded from lineage depletion and that the use of anti-CD41 antibody has little advantage with respect to the degree of purification of HSCs. The authors declare no competing financial interests. This work was supported by grants from the National Key Research and Development Program of China Stem Cell and Translational Research (2017YFA0104900 and 2016YFA0100600), CAMS Innovation Fund for Medical Sciences (CIFMS; 2016-I2M-1-017 and 2017-I2M-1-015), Ministry of Science and Technology of China (2015CB964403, 2015CB964404), and National Natural Science Foundation of China (81670105, 81470279, 81421002, and 81500085).