Identification and Hematopoietic Potential of CD45– Clonal Cells with Very Immature Phenotype (CD45–CD34–CD38–Lin–) in Patients with Myelody
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《干细胞学杂志》
a Division of Hematology, Third Department of Internal Medicine, and
b Department of Bioregulation, Nippon Medical School, Tokyo, Japan;
c Department of Molecular-Targeting Cancer Prevention, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan;
d Department of Hygiene, Kansai Medical University, Osaka, Japan;
e Department of Industrial Science and Technology, Tokyo University of Science, Chiba, Japan
Key Words. Myelodysplastic syndromes ? CD45 ? Hematopoietic stem cells
Correspondence: Kiyoyuki Ogata, M.D., Division of Hematology, Third Department of Internal Medicine, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8603, Japan. Telephone: 81-3-3822-2131; Fax: 81-3-5685-1793; e-mail: ogata@nms.ac.jp
ABSTRACT
Hematopoietic stem cells (HSCs) are rare cell populations that are capable of self-renewal and blood cell production . Therefore, they maintain hematopoiesis throughout life. Further, HSCs might be able to differentiate into cells of other tissues such as muscle cells . Cells that express CD45 and CD34 but lack CD38 and lineage antigens (CD45+CD34+CD38–Lin–) are a well-documented HSC population . Recent data from multiple groups have indicated that cells which express CD45 but lack expression of CD34, CD38, and lineage antigens (CD45+CD34–CD38–Lin–) are probably less mature HSCs than are CD45+CD34+CD38–Lin– HSCs . CD45 is a hematopoietic lineage-restricted cell-surface marker that is expressed on all hematopoietic cells, from HSCs to mature blood cells, except for erythroid cells, platelets, and plasma cells, which lose this antigen during maturation .
Myelodysplastic syndromes (MDS) are hematological neoplasms in which neoplastic myeloid cells (i.e., neutrophilic, monocytic, megakaryocytic, and erythroid cells) proliferate in the bone marrow (BM). There is a hypothesis that the MDS malignant transformation occurs at the committed myeloid progenitor cell level , but recent data suggest that the transformation occurs in CD45+CD34+CD38– HSCs in MDS . The neoplastic hematopoietic cells in MDS have various degrees of defective differentiation capability in each patient, and thus the percentage of immature blast cells in the BM differs among patients. MDS is classified into several subgroups, based mainly on the blast percentages in the BM and peripheral blood (PB) . During the clinical course, patients with MDS often show a transition from the original MDS subtype to another subtype with a higher blast percentage and, finally, to secondary acute myeloid leukemia (AML) . Recently, based on the findings that immature cells are lighter than mature cells , a new density-centrifugation method for enriching blastoid immature cells from PB and BM samples was developed . In a prior study, we used this method to prepare blast-rich MDS specimens for immunophenotyping and found that MDS blasts have the immunophenotype of committed myeloid precursors (CD45+CD34+CD38+CD13+CD33+) . Further, we showed that, in accordance with disease progression (increase in blast percentage), the phenotype of MDS blasts became more immature (e.g., gain of CD7 and c-kit expression), at least in some patients .
Here we report detection of CD45–CD34–CD38–Lin– blastoid cells having a chromosomal aberration in the PB and BM samples of MDS. These cells were detected in the advanced disease stages of MDS. The freshly isolated CD45–CD34–CD38–Lin– cells did not form any hematopoietic colonies but differentiated into hematopoietic colony-forming cells and fully mature myeloid cells when cultured together with murine stroma cells. This newly identified cell population has the most immature immunophenotype so far identified in the hematopoietic lineage and is involved in the MDS clone.
MATERIALS AND METHODS
Detection of CD45–CD34–CD38–Lin– and CD45–CD34+CD38–Lin– Clonal Cells in MDS
Figure 1 shows a representative example of flow cytometric analysis of a sample after BR density centrifugation. In the CD45 versus side scatter (SSC) display, we detected a cell cluster that lacked CD45 expression and had low SSC (R3 in Fig. 1B). The CD45– was confirmed by using another antibody that recognizes a different epitope of the CD45 molecule. The forward scatter (FSC) showed that the size of cells in R3 ranged from lymphocyte size to myeloblast size, but cells smaller than myeloblasts were predominant (Fig. 1D). Immunophenotyping of the cells in R3 revealed that, in addition to the CD45–, the majority of cells were negative for hematopoietic lineage antigens (CD2, CD3, CD10, CD11b, CD13, CD15, CD16, CD19, CD20, CD33, CD41a, CD56, and GPA) and stem cell–related antigens (CD34, CD38, CD117, and CD133). They were negative for HLA-DR and KDR but positive for HLA-class I antigen and CD44. Only minor subpopulations of the cells in R3 were weakly positive for CD7, CD34, CD38, and CD133. Therefore, in this case the dominant cells in R3 were CD45–CD34–CD38–Lin–. Part of the flow cytometric immunophenotyping for CD45– cells as well as myeloblasts is shown in Figure 1E (CD45– cells, blue dots; myeloblasts , gray dots). The cardinal data of the antigen profiles for these two cell populations are presented in Table 1 (patient 1) and clearly differ considerably between CD45– cells and myeloblasts. Figure 2 shows the plots for another patient, and it is seen that one third of the CD45– cells (cells in R3, Fig. 2B) expressed CD34 and a few expressed CD38 and myeloid antigens (blue dots in Fig. 2C). Expression of other hematopoietic lineage antigens was sparse. Therefore, for this patient the dominant cells in R3 were CD45–CD34+CD38–Lin–. Again, the antigen profile differs considerably between the CD45– cells and myeloblasts (gray dots in Fig. 2C) in this patient (Table 1, patient 8).
Figure 1. Representative example of CD45–CD34–CD38–Lin– cell detection in a sample of myelodysplastic syndromes. (A): Forward scatter (FSC) versus side scatter (SSC) display of bone marrow cells after blastretriever density centrifugation (patient 1 of Table 1). (B): CD45 versus SSC display of the cells gated by R1 in panel A. The bold vertical line, the left side of which shows CD45–, was obtained from panel C. R2, R3, and R4 indicate myeloblasts, CD45– cells, and lymphocytes, respectively (the immunophenotype data for myeloblasts and CD45– cells are shown in Table 1). (C): The cells were stained with isotype-matched control immunoglobulin G (IgG) conjugated with peridin chlorophyll (PerCP). (D): Cell size of the cells gated by R3 in panel B (FSC versus SSC display). Similar results were obtained when CD7+ or CD38+ cells were excluded from the analysis. (E): Part of antigen-expression analysis of myeloblasts (gray dots) and CD45– cells (blue dots). Abbreviations: FITC, fluorescein isothiocyanate; GPA, glycophorin A; PE, phycoerythrin.
Table 1. Phenotypic characteristics of myeloblasts and CD45– cells
Figure 2. Representative example of CD45–CD34+CD38–Lin– cell detection in a sample of myelodysplastic syndromes. (A): Forward scatter (FSC) versus side scatter (SSC) display of bone marrow cells after blastretriever density centrifugation (patient 8 in Table 1). (B): CD45 versus SSC display of the cells gated by R1 in panel A. The left side of the bold vertical line shows CD45–. R2 and R3 indicate myeloblasts and CD45– cells, respectively. (C): Part of antigen-expression analysis of myeloblasts (gray dots) and CD45– cells (blue dots). Abbreviations: FITC, fluorescein isothiocyanate; PE, phycoerythrin; PerCP, peridin chlorophyll.
In total, we examined BR-treated samples from 60 patients by FCM. The BR treatment enriches immature blastoid cells and depletes other cells. However, when original samples contain large numbers of erythroblasts (Ebls) that are CD45–, the BR treatment often cannot deplete Ebls to a level that does not interfere with CD45–CD38–Lin– cell detection. For this reason, we omitted nine of the 60 patients from the analyses. In eight of the remaining 51 patients, we detected CD45–CD38–Lin– cells in the CD45– cell clusters, as exemplified in Figures 1 and 2. Tables 1 and 2 show the characteristics of these eight patients and their phenotypic data for CD45– cells and myeloblasts. In all patients, CD45– cells almost completely lacked expression of myeloid antigens (CD11b, CD13, CD15, and CD33) and lymphoid-specific antigens (CD2, CD3, CD10, and CD19). Compared with the myeloblasts in each patient, CD45– cells had much lower expression of stem cell–related antigens (CD34, CD38, CD117, and CD133), but CD34 expression on CD45– cells varied greatly among the patients. MDS myeloblasts often express CD7 and/or CD56 aberrantly. The expression of these antigens was reduced on CD45– cells in all patients whose myeloblasts expressed them, but in three patients (patients 1, 4, and 7) more than 10% of CD45– cells expressed CD7 weakly. CD45–CD38–Lin– cells were generally smaller than myeloblasts in all patients. The ratio of the cell number between CD45– cells and myeloblasts, which was determined by FCM (/ x 100), ranged from 3.1%–26.1% in these eight patients (median 6.5%). Interestingly, CD45–CD38–Lin– cells were detected not only in BM samples but also in PB samples (Table 1). For patients 1 and 6, we obtained both BM and PB samples and detected CD45–CD38–Lin– cells in both samples. As shown in Table 3, a CD45– cell cluster that contained CD45–CD38–Lin– cells was detected only in RAEB-t and AL-MDS patients, and not in any of the RAEB patients, even though the percentages of enriched blastoid cells and contaminating Ebls in the analyzed samples were similar in all three disease groups.
Table 2. Characteristics of patients in whose samples CD45–CD38–Lin– cells were detected
Table 3. Relationships between disease subtypes and CD45–CD38–Lin– cell detection
Next, we isolated the CD45–CD34–CD38–Lin– cells from six of the eight patients and CD45–CD34+CD38–Lin– cells from two of the eight patients by successive application of BR density centrifugation, MACS, and FACS. CD45+CD34+ cells expressing myeloid antigens (CD34+ myeloblasts) were similarly isolated from the same patients as control cells. The purity of the isolated cells was at least 98% when assessed by FCM (Fig. 3A). The isolated CD45–CD34–CD38–Lin– and CD45–CD34+CD38–Lin– cells were blastoid cells that had scanty cytoplasm with no granules (Fig. 3B). Based on the findings that CD45–CD38–Lin– blastoid cells appeared after the disease stage of MDS had progressed (RAEB-t and AL-MDS) and were present not only in BM but also in PB, it is highly probable that these cells were clonal in origin. We performed FISH analysis of purified CD45–CD38–Lin– cells and CD34+ myeloblasts in four of the eight patients and confirmed that CD45–CD34–CD38–Lin– and CD45–CD34+CD38–Lin– cells had the same chromosomal aberration as the myeloblasts in each patient (Fig. 3C and Table 4).
Figure 3. Isolation of CD45–CD34–CD38–Lin– cells and their morphological and cytogenetic analyses. (A): The top panel shows the CD34 versus CD45 display of myelodysplastic syndromes (MDS) cells after blastretriever (BR) density centrifugation. The middle two panels show the cells after magnetic cell sorting (MACS) treatment. The rectangles are gates for fluorescence-activated cell sorting (FACS). The bottom two panels show the isolated CD34+ myeloblasts (right) and CD45–CD34–CD38–Lin– cells (left) after FACS. In each MDS patient, the antibody-coated colloids used for MACS and the antibodies used for FACS were selected based on the immunophenotypes of CD45– cells. (B): Isolated CD45–CD34–CD38–Lin– cells (Wright-Giemsa stain). (C): Isolated CD45–CD34–CD38–Lin– cells from patient 4 were subjected to Giemsa staining (left) and then to fluorescence in situ hybridization (right). The red spot and green spots show X- and Y-chromosome signals. Abbreviations: FITC, fluorescein isothiocyanate; PE, phycoerythrin; PerCP, peridin chlorophyll.
Table 4. Results of fluorescence in situ hybridization analysis
Proliferation and Differentiation of CD45–CD34–CD38–Lin– Cells Cultured with HESS-5 Stroma Cells
We previously established a murine BM stromal cell line, HESS-5 , and showed that in combination with cytokines it dramatically supported the proliferation and differentiation of normal human HSCs, including CD45+CD34+38–Lin– cells and more immature CD45+CD34–CD38–Lin– cells . Thus, we examined whether HESS-5 and cytokines could expand and/or differentiate the newly identified CD45–CD34–CD38–Lin– cells of MDS. First, we cultured CD45–CD34–CD38–Lin– cells and CD34+ myeloblasts from one patient (patient 4) under various conditions. We confirmed that cytokines alone were much inferior to HESS-5 plus cytokines in expanding and/or differentiating these cells (data not shown). Among the examined cytokine combinations, a combination of IL-3, TPO, SCF, and Flk2 ligand was the most suitable for CD45–CD34–CD38–Lin– cell culture (Fig. 4A). Therefore, using this cytokine combination and HESS-5 cells, we cultured CD45–CD34–CD38–Lin– cells and CD34+ myeloblasts, which had been isolated from six patients, for up to 5 weeks and examined the time-course changes in the number, morphology, and immunophenotypes of the cells in the cultures. When normal CD34+ myeloblasts obtained from granulocyte colony-stimulating factor (G-CSF)–mobilized PB (normal control) were cultured under the present culture conditions, the cell number had increased 370-fold (mean of four experiments) on day 7 of the culture. In contrast, when CD34+ myeloblasts from six MDS patients were cultured, the increase in cell number was much less. In two patients (patients 2 and 3), almost all the cultured cells died during the first 7–10 days of culture. The data for the other four MDS patients are shown in Figure 4B. Among these four patients, the highest increase in cell number on day 7 was 50-fold (patient 4; the solid circles in Fig. 4B). These inferior results in MDS were expected because we and others had documented that the expansion and differentiation abilities of HSCs and hematopoietic progenitors were often markedly reduced in MDS patients compared with normal subjects . Moreover, the fold increase in the cell number of cultured CD45–CD34–CD38–Lin– MDS cells was much less compared with the cultured CD34+ myeloblasts in MDS. In addition to patients 2 and 3, in one other case (patient 5, the open star in Fig. 4B) almost all the cells in CD45–CD34–CD38–Lin– cell culture died during the first 10 days of culture. For the remaining three patients, the cell number decreased during the first 7–10 days, while small clusters of cells (aggregates of two to four cells) began to appear on days 7–10 of culture. These cell clusters later produced more cells. This is in contrast to the CD34+ myeloblast culture, in which cell clusters began to appear on day 3 of culture. The fold increase in cell number was always less in the CD45–CD34–CD38–Lin– cell cultures compared with CD34+ myeloblast cultures for each patient (Fig. 4B). Time-course photographs of the cultured cells from a representative patient are shown in Figure 4C.
Figure 4. Proliferation and differentiation of CD45–CD34–CD38–Lin– cells cocultured with HESS-5 stroma cells. (A): CD34+ myeloblasts (solid symbols) and CD45–CD34–CD38–Lin– cells (open symbols) obtained from patient 4 were cocultured with HESS-5 cells in the presence of various combinations of cytokines (circles: IL-3, TPO, SCF, and Flk2 ligand; rectangles: IL-3, TPO, SCF, Flk2 ligand, and vascular endothelial growth factor; triangles: TPO, SCF, and Flk2 ligand). (B): CD34+ myeloblasts (solid symbols) and CD45–CD34–CD38–Lin– cells (open symbols) obtained from four patients (shown as circles, rectangles, triangles, and stars) were cocultured with HESS-5 cells in the presence of IL-3, TPO, SCF, and Flk2 ligand. (C): Time-course photographs of the cultured cells from patient 4. On day 7 of culture, a marked increase in cells was observed in the myeloblast culture but not in the CD45–CD34–CD38–Lin– cell culture. Abbreviations: IL-3, interleukin-3; SCF, stem cell factor; TPO, thrombopoietin.
In two patients from whom relatively large numbers of cells were obtained for culture, we examined the immunophenotypes of the cultured cells at various time points. Representative results are shown in Figure 5. Regarding CD34+ myeloblast culture, the percentage of CD34+ cells in the culture decreased with time and became 0% on day 35 of culture (Fig. 5A, lower panel). Throughout the culture period, almost all cells expressed myeloid antigens (data not shown). On day 7 of CD45–CD34–CD38–Lin– cell culture, the CD45–CD34–CD38–Lin– cell percentage had decreased markedly, and CD45+CD34+ cells and CD45+CD34– cells expressing myeloid antigens had appeared (Fig. 5A, upper panel). These data, together with the finding that the cell number in CD45–CD34–CD38–Lin– cell cultures decreased during the first 7–10 days while small clusters of cells began to appear on days 7–10, indicate that most CD45–CD34–CD38–Lin– MDS cells could not survive in our culture system, and a minor subpopulation of CD45–CD34–CD38–Lin– cells contributed to the production of CD45+CD34+ cells and CD45+CD34– cells expressing myeloid antigens. On days 15 and 35 of the CD45–CD34–CD38–Lin– cell culture, the percentage of CD45+CD34+ cells had decreased, but a small percentage of CD45+CD34+ cells still existed on day 35. Throughout the culture period, almost all CD45+ cells expressed myeloid antigens (Fig. 5B). The findings for Wright-Giemsa–stained cytospin preparations at various time points were consistent with the FCM data and confirmed that both cultures produced myeloid cells in various stages of maturation (Fig. 5C). FISH analyses also confirmed that these cells were clonal in origin (data not shown).
Figure 5. Time courses of immunological and morphological characteristics of cultured CD45–CD34–CD38–Lin– cells. CD45–CD34–CD38–Lin– cells and CD34+ myeloblasts were cocultured with HESS-5 cells in the presence of interleukin-3, thrombopoietin, stem cell factor, and Flk2 ligand. (A): Expression of CD45 and CD34 on the cultured cells at various time points. The number in the upper right corner of each dot plot indicates the percentage of CD45+CD34+ cells. (B): Staining with lineage-specific antibodies on day 15 of CD45–CD34–CD38–Lin– cell culture showed most cells expressed myeloid antigens. (C): A Wright Giemsa–stained cytospin on day 15 of CD45–CD34–CD38–Lin– cell culture showed myeloid cells in various stages of maturation. The arrowhead and arrow indicate a neutrophil and a blast, respectively. Abbreviations: FITC, fluorescein isothiocyanate; GPA, glycophorin A; PE, phycoerythrin; PerCP, peridin chlorophyll.
Colony-Forming Activity and LTC-IC Activity of CD45–CD34–CD38–Lin– Cells
The results are shown in Figure 6. It has been reported that colony-forming activity is often defective in MDS . In the experiments reported here, we observed clear colony formation from freshly isolated CD34+ myeloblasts in only two of the five MDS patients examined. In contrast, freshly isolated CD45–CD34–CD38–Lin– cells did not form any colonies in any of the five MDS patients. Conversely, when these two cell populations from the five MDS patients were cultured with HESS-5 cells for 5 weeks and then examined, LTC-IC activity was detected in the cultured CD45–CD34–CD38–Lin– cells (clear activity in two patients) but not in the cultured CD34+ myeloblasts.
Figure 6. Colony-forming activity and long-term culture-initiating cell (LTC-IC) activity of CD45–CD34–CD38–Lin– cells. (A): Freshly isolated CD45–CD34–CD38–Lin– cells (light columns) and CD34+ myeloblasts (dark columns) were analyzed for colony-forming activity. (B): CD45–CD34–CD38–Lin– cells (light columns) and CD34+ myeloblasts (dark columns) were cocultured with HESS-5 stroma cells for 5 weeks and then analyzed for LTC-IC activity. Data in panels A and B are the mean ± SD of triplicate cultures. Forty colonies from the freshly isolated CD34+ myeloblasts of patient 4 (panel A) were from BFU-E. Other colonies in panels A and B were from granulocyte-macrophage colony-forming units.
In Vitro Hematopoietic Potential of CD45–CD34+CD38–Lin– Cells
For patient 4, we compared the in vitro hematopoietic potential among CD45–CD34–CD38–Lin– cells, CD45–CD34+CD38–Lin– cells, and CD34+ myeloblasts. When freshly isolated populations of these cells were cocultured with HESS-5 under the above-mentioned conditions, the proliferation and differentiation kinetics of CD45–CD34+CD38–Lin– cells were similar to those of CD34+ myeloblasts. That is, cell clusters began to appear on day 3 of culture, and the cell number had increased significantly by day 7 of culture (Fig. 7A). Further, freshly isolated CD45–CD34+CD38–Lin– cells had clear colony-forming potential (Fig. 7B).
Figure 7. In vitro hematopoietic potential of CD45–CD34+CD38–Lin– cells. (A): CD45–CD34+CD38–Lin– cells (gray circles), CD34+ myeloblasts (black circles), and CD45–CD34–CD38–Lin– cells (white circles) obtained from patient 4 were cocultured with HESS-5 cells in the presence of interleukin-3, thrombopoietin, stem cell factor, and Flk2 ligand. (B): Freshly isolated CD45–CD34+CD38–Lin– cells (middle column labeled CD45–CD34+), CD34+ myeloblasts (left column labeled Mbl), and CD45–CD34–CD38–Lin– cells (right column labeled CD45–CD34–) from patient 4 were analyzed for colony-forming activity. Data are the mean ± SD of duplicate cultures. Fifty-three and 19 colonies from CD34+ myeloblasts and CD45–CD34+CD38–Lin– cells, respectively, were from BFU-E. Other colonies were from granulocyte-macrophage colony-forming units.
NOD/SCID Repopulating Activity of CD45–CD34–CD38–Lin– Cells
Demonstration of human hematopoiesis in NOD/SCID mice has been used to confirm HSC activity of human cell samples . However, the published data indicate that MDS stem cells seldom or never establish hematopoiesis in NOD/SCID mice . Therefore, in this study we used IBMI of MDS cells to NOD/SCID mice. The IBMI technique detects HSC activity more sensitively compared with the conventional method—intravenous injection of HSCs to NOD/SCID. That is, IBMI, but not the conventional method, can detect HSC activity in CD34– HSCs, and it needs fewer HSCs to reconstitute human hematopoiesis than does the conventional method . Moreover, we treated some NOD/SCID mice with anti-asialo GM1 antiserum to facilitate HSC engraftment. Despite these approaches, we have so far been unable to detect any NOD/SCID repopulation activity in CD45–CD34–CD38–Lin– MDS cells (Table 5).
Table 5. Intrabone marrow injection of CD45–CD34–CD38–Lin– cells to nonobese diabetic/severe combined immunodeficient mice
DISCUSSION
This work was supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (No. 14571002) and a research fund from Kirin Brewery Co., Ltd. to K.O.
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b Department of Bioregulation, Nippon Medical School, Tokyo, Japan;
c Department of Molecular-Targeting Cancer Prevention, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan;
d Department of Hygiene, Kansai Medical University, Osaka, Japan;
e Department of Industrial Science and Technology, Tokyo University of Science, Chiba, Japan
Key Words. Myelodysplastic syndromes ? CD45 ? Hematopoietic stem cells
Correspondence: Kiyoyuki Ogata, M.D., Division of Hematology, Third Department of Internal Medicine, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8603, Japan. Telephone: 81-3-3822-2131; Fax: 81-3-5685-1793; e-mail: ogata@nms.ac.jp
ABSTRACT
Hematopoietic stem cells (HSCs) are rare cell populations that are capable of self-renewal and blood cell production . Therefore, they maintain hematopoiesis throughout life. Further, HSCs might be able to differentiate into cells of other tissues such as muscle cells . Cells that express CD45 and CD34 but lack CD38 and lineage antigens (CD45+CD34+CD38–Lin–) are a well-documented HSC population . Recent data from multiple groups have indicated that cells which express CD45 but lack expression of CD34, CD38, and lineage antigens (CD45+CD34–CD38–Lin–) are probably less mature HSCs than are CD45+CD34+CD38–Lin– HSCs . CD45 is a hematopoietic lineage-restricted cell-surface marker that is expressed on all hematopoietic cells, from HSCs to mature blood cells, except for erythroid cells, platelets, and plasma cells, which lose this antigen during maturation .
Myelodysplastic syndromes (MDS) are hematological neoplasms in which neoplastic myeloid cells (i.e., neutrophilic, monocytic, megakaryocytic, and erythroid cells) proliferate in the bone marrow (BM). There is a hypothesis that the MDS malignant transformation occurs at the committed myeloid progenitor cell level , but recent data suggest that the transformation occurs in CD45+CD34+CD38– HSCs in MDS . The neoplastic hematopoietic cells in MDS have various degrees of defective differentiation capability in each patient, and thus the percentage of immature blast cells in the BM differs among patients. MDS is classified into several subgroups, based mainly on the blast percentages in the BM and peripheral blood (PB) . During the clinical course, patients with MDS often show a transition from the original MDS subtype to another subtype with a higher blast percentage and, finally, to secondary acute myeloid leukemia (AML) . Recently, based on the findings that immature cells are lighter than mature cells , a new density-centrifugation method for enriching blastoid immature cells from PB and BM samples was developed . In a prior study, we used this method to prepare blast-rich MDS specimens for immunophenotyping and found that MDS blasts have the immunophenotype of committed myeloid precursors (CD45+CD34+CD38+CD13+CD33+) . Further, we showed that, in accordance with disease progression (increase in blast percentage), the phenotype of MDS blasts became more immature (e.g., gain of CD7 and c-kit expression), at least in some patients .
Here we report detection of CD45–CD34–CD38–Lin– blastoid cells having a chromosomal aberration in the PB and BM samples of MDS. These cells were detected in the advanced disease stages of MDS. The freshly isolated CD45–CD34–CD38–Lin– cells did not form any hematopoietic colonies but differentiated into hematopoietic colony-forming cells and fully mature myeloid cells when cultured together with murine stroma cells. This newly identified cell population has the most immature immunophenotype so far identified in the hematopoietic lineage and is involved in the MDS clone.
MATERIALS AND METHODS
Detection of CD45–CD34–CD38–Lin– and CD45–CD34+CD38–Lin– Clonal Cells in MDS
Figure 1 shows a representative example of flow cytometric analysis of a sample after BR density centrifugation. In the CD45 versus side scatter (SSC) display, we detected a cell cluster that lacked CD45 expression and had low SSC (R3 in Fig. 1B). The CD45– was confirmed by using another antibody that recognizes a different epitope of the CD45 molecule. The forward scatter (FSC) showed that the size of cells in R3 ranged from lymphocyte size to myeloblast size, but cells smaller than myeloblasts were predominant (Fig. 1D). Immunophenotyping of the cells in R3 revealed that, in addition to the CD45–, the majority of cells were negative for hematopoietic lineage antigens (CD2, CD3, CD10, CD11b, CD13, CD15, CD16, CD19, CD20, CD33, CD41a, CD56, and GPA) and stem cell–related antigens (CD34, CD38, CD117, and CD133). They were negative for HLA-DR and KDR but positive for HLA-class I antigen and CD44. Only minor subpopulations of the cells in R3 were weakly positive for CD7, CD34, CD38, and CD133. Therefore, in this case the dominant cells in R3 were CD45–CD34–CD38–Lin–. Part of the flow cytometric immunophenotyping for CD45– cells as well as myeloblasts is shown in Figure 1E (CD45– cells, blue dots; myeloblasts , gray dots). The cardinal data of the antigen profiles for these two cell populations are presented in Table 1 (patient 1) and clearly differ considerably between CD45– cells and myeloblasts. Figure 2 shows the plots for another patient, and it is seen that one third of the CD45– cells (cells in R3, Fig. 2B) expressed CD34 and a few expressed CD38 and myeloid antigens (blue dots in Fig. 2C). Expression of other hematopoietic lineage antigens was sparse. Therefore, for this patient the dominant cells in R3 were CD45–CD34+CD38–Lin–. Again, the antigen profile differs considerably between the CD45– cells and myeloblasts (gray dots in Fig. 2C) in this patient (Table 1, patient 8).
Figure 1. Representative example of CD45–CD34–CD38–Lin– cell detection in a sample of myelodysplastic syndromes. (A): Forward scatter (FSC) versus side scatter (SSC) display of bone marrow cells after blastretriever density centrifugation (patient 1 of Table 1). (B): CD45 versus SSC display of the cells gated by R1 in panel A. The bold vertical line, the left side of which shows CD45–, was obtained from panel C. R2, R3, and R4 indicate myeloblasts, CD45– cells, and lymphocytes, respectively (the immunophenotype data for myeloblasts and CD45– cells are shown in Table 1). (C): The cells were stained with isotype-matched control immunoglobulin G (IgG) conjugated with peridin chlorophyll (PerCP). (D): Cell size of the cells gated by R3 in panel B (FSC versus SSC display). Similar results were obtained when CD7+ or CD38+ cells were excluded from the analysis. (E): Part of antigen-expression analysis of myeloblasts (gray dots) and CD45– cells (blue dots). Abbreviations: FITC, fluorescein isothiocyanate; GPA, glycophorin A; PE, phycoerythrin.
Table 1. Phenotypic characteristics of myeloblasts and CD45– cells
Figure 2. Representative example of CD45–CD34+CD38–Lin– cell detection in a sample of myelodysplastic syndromes. (A): Forward scatter (FSC) versus side scatter (SSC) display of bone marrow cells after blastretriever density centrifugation (patient 8 in Table 1). (B): CD45 versus SSC display of the cells gated by R1 in panel A. The left side of the bold vertical line shows CD45–. R2 and R3 indicate myeloblasts and CD45– cells, respectively. (C): Part of antigen-expression analysis of myeloblasts (gray dots) and CD45– cells (blue dots). Abbreviations: FITC, fluorescein isothiocyanate; PE, phycoerythrin; PerCP, peridin chlorophyll.
In total, we examined BR-treated samples from 60 patients by FCM. The BR treatment enriches immature blastoid cells and depletes other cells. However, when original samples contain large numbers of erythroblasts (Ebls) that are CD45–, the BR treatment often cannot deplete Ebls to a level that does not interfere with CD45–CD38–Lin– cell detection. For this reason, we omitted nine of the 60 patients from the analyses. In eight of the remaining 51 patients, we detected CD45–CD38–Lin– cells in the CD45– cell clusters, as exemplified in Figures 1 and 2. Tables 1 and 2 show the characteristics of these eight patients and their phenotypic data for CD45– cells and myeloblasts. In all patients, CD45– cells almost completely lacked expression of myeloid antigens (CD11b, CD13, CD15, and CD33) and lymphoid-specific antigens (CD2, CD3, CD10, and CD19). Compared with the myeloblasts in each patient, CD45– cells had much lower expression of stem cell–related antigens (CD34, CD38, CD117, and CD133), but CD34 expression on CD45– cells varied greatly among the patients. MDS myeloblasts often express CD7 and/or CD56 aberrantly. The expression of these antigens was reduced on CD45– cells in all patients whose myeloblasts expressed them, but in three patients (patients 1, 4, and 7) more than 10% of CD45– cells expressed CD7 weakly. CD45–CD38–Lin– cells were generally smaller than myeloblasts in all patients. The ratio of the cell number between CD45– cells and myeloblasts, which was determined by FCM (/ x 100), ranged from 3.1%–26.1% in these eight patients (median 6.5%). Interestingly, CD45–CD38–Lin– cells were detected not only in BM samples but also in PB samples (Table 1). For patients 1 and 6, we obtained both BM and PB samples and detected CD45–CD38–Lin– cells in both samples. As shown in Table 3, a CD45– cell cluster that contained CD45–CD38–Lin– cells was detected only in RAEB-t and AL-MDS patients, and not in any of the RAEB patients, even though the percentages of enriched blastoid cells and contaminating Ebls in the analyzed samples were similar in all three disease groups.
Table 2. Characteristics of patients in whose samples CD45–CD38–Lin– cells were detected
Table 3. Relationships between disease subtypes and CD45–CD38–Lin– cell detection
Next, we isolated the CD45–CD34–CD38–Lin– cells from six of the eight patients and CD45–CD34+CD38–Lin– cells from two of the eight patients by successive application of BR density centrifugation, MACS, and FACS. CD45+CD34+ cells expressing myeloid antigens (CD34+ myeloblasts) were similarly isolated from the same patients as control cells. The purity of the isolated cells was at least 98% when assessed by FCM (Fig. 3A). The isolated CD45–CD34–CD38–Lin– and CD45–CD34+CD38–Lin– cells were blastoid cells that had scanty cytoplasm with no granules (Fig. 3B). Based on the findings that CD45–CD38–Lin– blastoid cells appeared after the disease stage of MDS had progressed (RAEB-t and AL-MDS) and were present not only in BM but also in PB, it is highly probable that these cells were clonal in origin. We performed FISH analysis of purified CD45–CD38–Lin– cells and CD34+ myeloblasts in four of the eight patients and confirmed that CD45–CD34–CD38–Lin– and CD45–CD34+CD38–Lin– cells had the same chromosomal aberration as the myeloblasts in each patient (Fig. 3C and Table 4).
Figure 3. Isolation of CD45–CD34–CD38–Lin– cells and their morphological and cytogenetic analyses. (A): The top panel shows the CD34 versus CD45 display of myelodysplastic syndromes (MDS) cells after blastretriever (BR) density centrifugation. The middle two panels show the cells after magnetic cell sorting (MACS) treatment. The rectangles are gates for fluorescence-activated cell sorting (FACS). The bottom two panels show the isolated CD34+ myeloblasts (right) and CD45–CD34–CD38–Lin– cells (left) after FACS. In each MDS patient, the antibody-coated colloids used for MACS and the antibodies used for FACS were selected based on the immunophenotypes of CD45– cells. (B): Isolated CD45–CD34–CD38–Lin– cells (Wright-Giemsa stain). (C): Isolated CD45–CD34–CD38–Lin– cells from patient 4 were subjected to Giemsa staining (left) and then to fluorescence in situ hybridization (right). The red spot and green spots show X- and Y-chromosome signals. Abbreviations: FITC, fluorescein isothiocyanate; PE, phycoerythrin; PerCP, peridin chlorophyll.
Table 4. Results of fluorescence in situ hybridization analysis
Proliferation and Differentiation of CD45–CD34–CD38–Lin– Cells Cultured with HESS-5 Stroma Cells
We previously established a murine BM stromal cell line, HESS-5 , and showed that in combination with cytokines it dramatically supported the proliferation and differentiation of normal human HSCs, including CD45+CD34+38–Lin– cells and more immature CD45+CD34–CD38–Lin– cells . Thus, we examined whether HESS-5 and cytokines could expand and/or differentiate the newly identified CD45–CD34–CD38–Lin– cells of MDS. First, we cultured CD45–CD34–CD38–Lin– cells and CD34+ myeloblasts from one patient (patient 4) under various conditions. We confirmed that cytokines alone were much inferior to HESS-5 plus cytokines in expanding and/or differentiating these cells (data not shown). Among the examined cytokine combinations, a combination of IL-3, TPO, SCF, and Flk2 ligand was the most suitable for CD45–CD34–CD38–Lin– cell culture (Fig. 4A). Therefore, using this cytokine combination and HESS-5 cells, we cultured CD45–CD34–CD38–Lin– cells and CD34+ myeloblasts, which had been isolated from six patients, for up to 5 weeks and examined the time-course changes in the number, morphology, and immunophenotypes of the cells in the cultures. When normal CD34+ myeloblasts obtained from granulocyte colony-stimulating factor (G-CSF)–mobilized PB (normal control) were cultured under the present culture conditions, the cell number had increased 370-fold (mean of four experiments) on day 7 of the culture. In contrast, when CD34+ myeloblasts from six MDS patients were cultured, the increase in cell number was much less. In two patients (patients 2 and 3), almost all the cultured cells died during the first 7–10 days of culture. The data for the other four MDS patients are shown in Figure 4B. Among these four patients, the highest increase in cell number on day 7 was 50-fold (patient 4; the solid circles in Fig. 4B). These inferior results in MDS were expected because we and others had documented that the expansion and differentiation abilities of HSCs and hematopoietic progenitors were often markedly reduced in MDS patients compared with normal subjects . Moreover, the fold increase in the cell number of cultured CD45–CD34–CD38–Lin– MDS cells was much less compared with the cultured CD34+ myeloblasts in MDS. In addition to patients 2 and 3, in one other case (patient 5, the open star in Fig. 4B) almost all the cells in CD45–CD34–CD38–Lin– cell culture died during the first 10 days of culture. For the remaining three patients, the cell number decreased during the first 7–10 days, while small clusters of cells (aggregates of two to four cells) began to appear on days 7–10 of culture. These cell clusters later produced more cells. This is in contrast to the CD34+ myeloblast culture, in which cell clusters began to appear on day 3 of culture. The fold increase in cell number was always less in the CD45–CD34–CD38–Lin– cell cultures compared with CD34+ myeloblast cultures for each patient (Fig. 4B). Time-course photographs of the cultured cells from a representative patient are shown in Figure 4C.
Figure 4. Proliferation and differentiation of CD45–CD34–CD38–Lin– cells cocultured with HESS-5 stroma cells. (A): CD34+ myeloblasts (solid symbols) and CD45–CD34–CD38–Lin– cells (open symbols) obtained from patient 4 were cocultured with HESS-5 cells in the presence of various combinations of cytokines (circles: IL-3, TPO, SCF, and Flk2 ligand; rectangles: IL-3, TPO, SCF, Flk2 ligand, and vascular endothelial growth factor; triangles: TPO, SCF, and Flk2 ligand). (B): CD34+ myeloblasts (solid symbols) and CD45–CD34–CD38–Lin– cells (open symbols) obtained from four patients (shown as circles, rectangles, triangles, and stars) were cocultured with HESS-5 cells in the presence of IL-3, TPO, SCF, and Flk2 ligand. (C): Time-course photographs of the cultured cells from patient 4. On day 7 of culture, a marked increase in cells was observed in the myeloblast culture but not in the CD45–CD34–CD38–Lin– cell culture. Abbreviations: IL-3, interleukin-3; SCF, stem cell factor; TPO, thrombopoietin.
In two patients from whom relatively large numbers of cells were obtained for culture, we examined the immunophenotypes of the cultured cells at various time points. Representative results are shown in Figure 5. Regarding CD34+ myeloblast culture, the percentage of CD34+ cells in the culture decreased with time and became 0% on day 35 of culture (Fig. 5A, lower panel). Throughout the culture period, almost all cells expressed myeloid antigens (data not shown). On day 7 of CD45–CD34–CD38–Lin– cell culture, the CD45–CD34–CD38–Lin– cell percentage had decreased markedly, and CD45+CD34+ cells and CD45+CD34– cells expressing myeloid antigens had appeared (Fig. 5A, upper panel). These data, together with the finding that the cell number in CD45–CD34–CD38–Lin– cell cultures decreased during the first 7–10 days while small clusters of cells began to appear on days 7–10, indicate that most CD45–CD34–CD38–Lin– MDS cells could not survive in our culture system, and a minor subpopulation of CD45–CD34–CD38–Lin– cells contributed to the production of CD45+CD34+ cells and CD45+CD34– cells expressing myeloid antigens. On days 15 and 35 of the CD45–CD34–CD38–Lin– cell culture, the percentage of CD45+CD34+ cells had decreased, but a small percentage of CD45+CD34+ cells still existed on day 35. Throughout the culture period, almost all CD45+ cells expressed myeloid antigens (Fig. 5B). The findings for Wright-Giemsa–stained cytospin preparations at various time points were consistent with the FCM data and confirmed that both cultures produced myeloid cells in various stages of maturation (Fig. 5C). FISH analyses also confirmed that these cells were clonal in origin (data not shown).
Figure 5. Time courses of immunological and morphological characteristics of cultured CD45–CD34–CD38–Lin– cells. CD45–CD34–CD38–Lin– cells and CD34+ myeloblasts were cocultured with HESS-5 cells in the presence of interleukin-3, thrombopoietin, stem cell factor, and Flk2 ligand. (A): Expression of CD45 and CD34 on the cultured cells at various time points. The number in the upper right corner of each dot plot indicates the percentage of CD45+CD34+ cells. (B): Staining with lineage-specific antibodies on day 15 of CD45–CD34–CD38–Lin– cell culture showed most cells expressed myeloid antigens. (C): A Wright Giemsa–stained cytospin on day 15 of CD45–CD34–CD38–Lin– cell culture showed myeloid cells in various stages of maturation. The arrowhead and arrow indicate a neutrophil and a blast, respectively. Abbreviations: FITC, fluorescein isothiocyanate; GPA, glycophorin A; PE, phycoerythrin; PerCP, peridin chlorophyll.
Colony-Forming Activity and LTC-IC Activity of CD45–CD34–CD38–Lin– Cells
The results are shown in Figure 6. It has been reported that colony-forming activity is often defective in MDS . In the experiments reported here, we observed clear colony formation from freshly isolated CD34+ myeloblasts in only two of the five MDS patients examined. In contrast, freshly isolated CD45–CD34–CD38–Lin– cells did not form any colonies in any of the five MDS patients. Conversely, when these two cell populations from the five MDS patients were cultured with HESS-5 cells for 5 weeks and then examined, LTC-IC activity was detected in the cultured CD45–CD34–CD38–Lin– cells (clear activity in two patients) but not in the cultured CD34+ myeloblasts.
Figure 6. Colony-forming activity and long-term culture-initiating cell (LTC-IC) activity of CD45–CD34–CD38–Lin– cells. (A): Freshly isolated CD45–CD34–CD38–Lin– cells (light columns) and CD34+ myeloblasts (dark columns) were analyzed for colony-forming activity. (B): CD45–CD34–CD38–Lin– cells (light columns) and CD34+ myeloblasts (dark columns) were cocultured with HESS-5 stroma cells for 5 weeks and then analyzed for LTC-IC activity. Data in panels A and B are the mean ± SD of triplicate cultures. Forty colonies from the freshly isolated CD34+ myeloblasts of patient 4 (panel A) were from BFU-E. Other colonies in panels A and B were from granulocyte-macrophage colony-forming units.
In Vitro Hematopoietic Potential of CD45–CD34+CD38–Lin– Cells
For patient 4, we compared the in vitro hematopoietic potential among CD45–CD34–CD38–Lin– cells, CD45–CD34+CD38–Lin– cells, and CD34+ myeloblasts. When freshly isolated populations of these cells were cocultured with HESS-5 under the above-mentioned conditions, the proliferation and differentiation kinetics of CD45–CD34+CD38–Lin– cells were similar to those of CD34+ myeloblasts. That is, cell clusters began to appear on day 3 of culture, and the cell number had increased significantly by day 7 of culture (Fig. 7A). Further, freshly isolated CD45–CD34+CD38–Lin– cells had clear colony-forming potential (Fig. 7B).
Figure 7. In vitro hematopoietic potential of CD45–CD34+CD38–Lin– cells. (A): CD45–CD34+CD38–Lin– cells (gray circles), CD34+ myeloblasts (black circles), and CD45–CD34–CD38–Lin– cells (white circles) obtained from patient 4 were cocultured with HESS-5 cells in the presence of interleukin-3, thrombopoietin, stem cell factor, and Flk2 ligand. (B): Freshly isolated CD45–CD34+CD38–Lin– cells (middle column labeled CD45–CD34+), CD34+ myeloblasts (left column labeled Mbl), and CD45–CD34–CD38–Lin– cells (right column labeled CD45–CD34–) from patient 4 were analyzed for colony-forming activity. Data are the mean ± SD of duplicate cultures. Fifty-three and 19 colonies from CD34+ myeloblasts and CD45–CD34+CD38–Lin– cells, respectively, were from BFU-E. Other colonies were from granulocyte-macrophage colony-forming units.
NOD/SCID Repopulating Activity of CD45–CD34–CD38–Lin– Cells
Demonstration of human hematopoiesis in NOD/SCID mice has been used to confirm HSC activity of human cell samples . However, the published data indicate that MDS stem cells seldom or never establish hematopoiesis in NOD/SCID mice . Therefore, in this study we used IBMI of MDS cells to NOD/SCID mice. The IBMI technique detects HSC activity more sensitively compared with the conventional method—intravenous injection of HSCs to NOD/SCID. That is, IBMI, but not the conventional method, can detect HSC activity in CD34– HSCs, and it needs fewer HSCs to reconstitute human hematopoiesis than does the conventional method . Moreover, we treated some NOD/SCID mice with anti-asialo GM1 antiserum to facilitate HSC engraftment. Despite these approaches, we have so far been unable to detect any NOD/SCID repopulation activity in CD45–CD34–CD38–Lin– MDS cells (Table 5).
Table 5. Intrabone marrow injection of CD45–CD34–CD38–Lin– cells to nonobese diabetic/severe combined immunodeficient mice
DISCUSSION
This work was supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (No. 14571002) and a research fund from Kirin Brewery Co., Ltd. to K.O.
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