MS4A3 Marks Early Myeloid Differentiation in Human Hematopoiesis

Understanding lineage specific markers contributes to investigation into lineage commitment processes in hematopoiesis. Particularly in the human study, information about hematopoietic lineage divergence is essential to refine hematopoietic lineage tree. Lineage markers are also potentially useful for therapeutic target, such as CD20 in B-cell lymphoma, and CD33 in acute myeloid leukemia. We have recently reported that special AT-rich sequence-binding protein 1 (SATB1), a global chromatin organizer, promotes lymphocyte production from hematopoietic stem cells (HSCs) (Immunity 38;1105, 2013). Expression level of SATB1 increases with early lymphoid differentiation, whereas it is shut off in committed myeloid progenitors. To search a novel cell surface molecule that marks the point of branching lineage along early myeloid and lymphoid differentiation, we performed microarray analyses comparing SATB1-overexpressed HSCs with mock-transduced HSCs. The results drew our attention to membrane-spanning 4-domains, subfamily A, member 3 (MS4A3). MS4A3, also called hematopoietic cell-specific transmembrane 4 (HTm4), is a member of the MS4A family. CD20, encoded by MS4A1 gene, belongs to the same family. We observed that expression level of MS4A3 in SATB1-overexpressed HSCs was decreased almost one tenth of that of mock HSCs.

To confirm the relationship of SATB1 and MS4A3 in human hematopoietic cells, we first used chronic myeloid leukemia cell line K562, which was found to clearly express MS4A3 on their cell surface. While SATB1 expression was undetectable in original K562 cells, the exogenous expression of SATB1 significantly reduced their MS4A3 expression level, suggesting that SATB1 negatively regulates MS4A3 expression in human cells.

Next, we analyzed MS4A3 expression pattern in primary human hematopoietic stem/progenitor cells. Bone marrow (BM) cells were obtained from healthy donors or patients with acute myeloid leukemia. The Institutional Review Board of Osaka University School of Medicine approved all of protocols, and written informed consents were obtained from all participants. Mononuclear cells were separated from the BM samples by density gradient centrifugation, and subsequently applied to cell sorting for Lineage marker-negative (Lin^-) CD34⁺ CD38^- HSCs, Lin^- CD34⁺ CD38⁺ IL-3 receptor α (IL-3Rα)⁺ CD45RA^- common myeloid progenitors (CMPs), Lin^- CD34⁺ CD38⁺ IL3-Rα⁺ CD45RA⁺ granulocyte-macrophage progenitors (GMPs) and Lin^- CD34⁺ CD38⁺ IL-3Rα^- CD45RA^-megakaryocyte-erythroid progenitors (MEPs). MS4A3 expression levels of the sorted cells were analyzed with real-time RT-PCR. We detected more than 10-fold amount of MS4A3 transcripts in CMPs than HSCs. Furthermore, its expression level continuously increased along myeloid lineage differentiation to GMP. On the other hand, megakaryocyte-erythroid lineage differentiation was not accompanied by MS4A3 expression and the amount of MS4A3 transcripts in MEPs was minimum as in HSCs.

Flow cytometry analyses confirmed that HSCs and MEPs do not express MS4A3 on their cell surface whereas the MS4A3 expression on CMPs and GMPs is detectable. Further, the Lin^- CD34⁺ CD38⁺ CD33⁺ cells could be fractionated according to the intensity of cell surface MS4A3 expression. To investigate the significance of cell surface MS4A3 expression for functional analyses of myeloid progenitor cells, we performed methylcellulose colony-forming assays. We found that MS4A3⁺ cells in Lin^- CD34⁺ CD38⁺ CD33⁺ fraction only produced granulocyte/macrophage colonies, losing erythroid colony- and mixed colony-forming capacity. These results suggest that cell surface expression of MS4A3 is useful to distinguish granulocyte/macrophage lineage-committed progenitors from other lineage-related ones in early human hematopoiesis. We also analyzed MS4A3 expression in BM cells obtained from patients with acute leukemia. Flow cytometry analyses revealed that leukemia cells of some patients expressed substantial amount of cell surface MS4A3.

In conclusion, MS4A3 is useful to monitor early stage of myeloid differentiation in human hematopoiesis. In addition, our findings of MS4A3 expression on myeloid leukemia cells, while no expression on normal HSCs, imply that MS4A3 might be a therapeutic target molecule in myelogenous leukemia. Further studies would clarify the application of MS4A3 to anti-leukemia therapy.