In this study, we have isolated the CD13+
subset of cells from human bone marrow CD133+
stem/progenitor cells for both myeloid and erythroid lineage commitment. CD13 is a unique myeloid antigen expressed as early as the blast stage of lineage development and at high levels on myeloblasts and promyelocytes [19
]. CD36 has been used as an early erythroid marker [21
], although it is also expressed on late megakaryocytic and monocytic cells [23
]. Indeed, a high proportion of CD36+
progenitors only generated erythroid burst-forming unit (BFU-E) and erythroid colony-forming units (CFU-E) [23
]. In a recent paper, Singh et al.
] also showed that purified subpopulations of CD34+
cells from baboon bone marrow generated mostly BFU-E and CFU-E colonies. To date, few published studies have directly examined CD13+
subset cells in a steady state or under culture conditions. Our data showed a 23% of CD133+
cells at D0 (without EPO or G-CSF cytokine stimulation) were CD13+
. This value agrees with the studies by Oertel et al.
] and by Thoma et al.
], in which, the expression of CD13 was 33±15% and 70±13%, and the expression of CD36 was 22±8% and 6±3% on human bone marrow and cord blood, respectively. Our present study and above work [26
] showed that cells expressing CD13 and CD36 committed to myeloid and erythroid colonies, respectively, but no lymphocytes were detected in these colonies. In our study, in the presence of cytokines, CD133+
cells co-expressing CD13 and CD36 increased from 26% at D0 to 72% after culture for 14 days in EPO (E14), but decreased to 13% after culture for 14 days in G-CSF (G14). Meanwhile, in both E14 and G14 cultures, CD133+
cells decreased to less than 1% from their original >97% at D0, which is consistent with a previous report [28
]. It is unlikely that the minimal number of CD133+
cells at D14 can give rise to abundant erythroid or myeloid lineage cells at this point [14
]. However, some of the CD13+
cells may still express a low intensity of known stem cell markers (such as CD133 and CD34) or other unexpected marker(s). The CD13+
cells may represent an early branch of precursor cells, and have at least bi-differentiation potential for erythroid and myeloid development. It is notable that the increment of the CD13+
subset observed in G28 cells (up to 37% from 13% at G14) may largely reflect the expression of CD36 on late monocytes in this population of cells.
The observation of CD13+
cells in E14 (72%) and G14 (13%) confirms that cells co-expressing both myeloid and erythroid lineage-specific markers do exist. The colony-forming experiments further demonstrate this bi-directional differentiation potential. Co-expression of CD13 and CD36 antigens might reflect the intracellular overlap or balance of transcription factors, such as C/EBPa [7
], GATA-1 and PU.1 [29
], or other lineage switch-related genes, such as Myb LZR [30
] and glia maturation factor gamma [15
] although the molecular mechanisms controlling erythroid and myeloid commitment are largely unknown. These transcription factors or genes may function as a base on these cells of possessing bi-potential differentiation for their fate conversion (switching) or lineage plasticity in normal hematopoiesis.
In our studies, granulocyte commitment was always accompanied by monocyte colonies when CD13+
cells were induced for erythroid or myeloid cell differentiation, whereas myeloid lineage was not detected in CD13−
cells stimulated with EPO and/or G-CSF. These results indicate a sequence of hematopoietic lineage commitment, in which progenitors first generate myeloid lineage cells, followed by monocytes, and then erythroid cells under certain conditions . The results are consistent with a previous report on the order of myeloid and erythroid development from HSCs [31
Figure 4 Schematic representation of lineage-specific antigen expression of erythroid and myeloid development and cytokine stimulation in vitro. Lineage-specific markers of erythroid (CD36, in red), myeloid (CD13, in green), and progenitor (CD133, in yellow) cells (more ...)
In some hematopoietic disorders, such as the transient myeloproliferative disorder (TMD), acute myeloid leukemia (AML), and myelodysplastic syndromes (MDS), abnormal cells occur in more than one lineage. TMD is an abnormal proliferation of myeloid blasts in the blood that rapidly undergoes spontaneous remission without therapeutic intervention [32
]. A high percentage of TMD patients will develop AML [33
], so it is also considered as a pre-leukemic condition. CD36 antigen has been reported being expressed in all TMD patients [34
]. A subpopulation of immature blast cells expressing cross-lineage antigens, including CD13 and CD36, are observed in TMD, indicating the erythroid development of TMD even in the absence of CD34+
]. Erythroid differentiation in blast cells of TMD is further demonstrated by erythroid-specific gene expression [36
]. AML is a heterogeneous group of leukemias in which myeloid maturation is blocked at different differentiation stages, resulting in single- or mixed-cell lineage leukemic blasts. Thus, almost all AML cells express CD13, CD33, and CD117 [37
], of which most co-express CD36 [34
]. Furthermore, the expression of CD36 antigen on AML cells is related to a higher risk of relapse and a lower leukemia-free survival, and has been suggested as a key marker for an adverse prognosis irrespective of genetic abnormalities [37
]. MDS are also heterogeneous diseases of bone marrow cell precursors, with five categories including RAEB (refractory anemia with excess of blasts) [38
]. High expression of CD36, which was not associated with the expression of CD34 or CD71 in RAEB [39
], is an indicator of poor prognosis in patients with MDS.
These observations indicate that precursors expressing CD36 and CD13 could be involved in spontaneous recoveries or/and leukemic transformation processes in hematopoietic disorders. Such a mechanism would suggest that cells co-expressing CD36 and CD13 may represent a unique subgroup, which is independent of CD34 or CD71 in blast cells of these diseases [35
]. CD71 has been described as an erythroid antigen. The relationship between CD71 expressed levels on CD34 cells and erythroid and myeloid development has been reported by Mayani et al, showing that CD71 antigen was expressed at high level on CD34+ cells committing to the erythroid development, but at low level on CD34+ cells differentiating into the myeloid lineage [40
]. It is possible that CD34 or/and CD71 are expressed at a low levels on some CD13+CD36+ cells of bi-potential precursors. Thus, some conditions affecting exclusively one cell type with some abnormalities in the other cell lineage, such as Shwachman-Diamond neutropenia, may possibly spontaneously recover by employing these unique bi-directional cells. In malignant hematopoiesis, on the other hand, leukemic cells may be blocked at such stages and thus lose their ability for further differentiation. The special phenotype of cancer cells may in part be derived from the intrinsic properties of such bi-potential hematopoietic precursor cells. This hypothesis regarding the role of bi-potential precursors is supported by a recent report of a new erythroleukemic cell line, ERY-1, which possesses both the myeloid and erythroid markers CD13, CD33, CD36, and glycophorin A [6
EPO and G-CSF are major growth factors for inducing the proliferation and differentiation of erythroid and myeloid progenitor cells, respectively. Our data showed that the proliferation and differentiation of erythroid and myeloid phenotypes could be induced from the same source of CD133+
cells, which is consistent with the effects of these cytokines on hematopoiesis [41
]. Our observation of semi-solid cultures allows us to directly monitor the effect of EPO and G-CSF on the myeloid and erythroid lineage branching-point. The analysis of sorted CD13+
, or CD13−
cells from EPO- and G-CSF-treated CD133+
cells (E14, G14) confirms that EPO supports both erythroid [41
] and myeloid [44
] differentiation. This conclusion is drawn from the special setting in our experiments, in which fetal bovine serum and other cytokines (SCF, IL-3, IL-6, and GM-CSF) were included. This bi-effect of EPO on erythroid and myeloid differentiation provides for the possibility that inter-conversion or switching of erythroid and myeloid lineages during their development could occur. In contrast, G-CSF had no significant effect on erythroid progenitors in G14, consistent with previous data [45
]. The differential effects of EPO and G-CSF on erythroid and myeloid development may in part explain the observations in switch-culture experiments done previously [13
], in which E14 cells contain a number of CD13+
cells, rather than being exclusively committed to erythroid lineage, while G14 cells treated with EPO for another 14 days instead of G-CSF still retained some myeloid progenitor cells.
In conclusion, this is the first report on a subset of cells co-expressing CD13 and CD36 from human bone marrow. The CD13+
cells retain at least ‘bi-potential’ capacity of committing to erythroid and myeloid lineages in the presence of EPO or G-CSF. Thus, the identification of this specific subpopulation confirms the hypothesis of the existence of bi-potential cells in a relatively late committed lineage and provides a physiological basis for the phenomena of cross-lineage marker expression observed in hematopoietic malignancies [5
]. It also indicates that co-expression of erythroid- and myeloid-specific markers (e.g., CD36 and CD13) may be an intrinsic feature of normal hematopoiesis, rather than simply a hallmark of hematopoietic malignancies. More importantly, the subpopulation of CD13+
precursor cells, which are not transformed, may be used for further studies of the regulatory mechanisms controlling erythroid and myeloid development pathways underlying normal and leukemic hematopoiesis.