Prospective isolation of human clonogenic common myeloid progenitors

Abstract

The hierarchical development from hematopoietic stem cells to mature cells of the hematolymphoid system involves progressive loss of self-renewal capacity, proliferation ability, and lineage potentials. Here we show the prospective isolation of early developmental intermediates, the human clonogenic common myeloid progenitors and their downstream progeny, the granulocyte/macrophage and megakaryocyte/erythrocyte progenitors. All three populations reside in the lineage-negative (lin(-)) CD34(+)CD38(+) fraction of adult bone marrow as well as in cord blood. They are distinguishable by the expression of the IL-3R alpha chain, the receptor of an early-acting hematopoietic cytokine, and CD45RA, an isoform of a phosphotyrosine phosphatase involved in negative regulation of cytokine signaling. Multipotent progenitors, early lymphoid progenitors, and the here-defined myeloid progenitors express distinct profiles of hematopoiesis-affiliated genes. The isolation of highly purified hematopoietic intermediates provides tools to better understand developmental programs underlying normal and leukemic hematopoiesis.

Figure 1

Prospective isolation of myeloid progenitors in adult human bone marrow. (a) The lin⁻CD34⁺CD38⁺ fraction was subdivided according to IL-3Rα and CD45RA expression (a–c). (b) Reanalysis of the sorted populations. Percentages relative to mononuclear bone marrow cells are shown. (c) Additional antigen expression profile on CD34⁺CD38⁻ (brown), IL-3Rα^loCD45RA⁻ (red), IL-3Rα^loCD45RA⁺ (green), and IL-3Rα⁻CD45RA⁻ (blue) populations. Isotype control gated on total lin⁻CD34⁺CD38⁺ cells (black) and marker expression on CD34⁻ bone marrow cells (dashed line) are shown.

Figure 2

Colony formation in methylcellulose. (a) Morphology of day-14 colonies derived from sorted progenitors (inverted light microscope, ×4). (Right Insert) CFU-megakaryocyte/erythroid (×10). (b) Cellular morphology form single-picked colonies: CFU-granulocyte/erythroid/macrophage/megakaryocyte (GEMM, Left), CFU-granulocyte/macrophage (Center), burst-forming units/erythroid (Right), and CFU-megakaryocyte (Right Insert) (Giemsa, ×100 oil). (c) Clonogenic myeloid colony formation in methylcellulose. Scored were 288 wells receiving lin⁻CD34⁺CD38⁻ cells (three donors), 336 wells receiving lin⁻CD34⁺CD38⁺IL-3Rα^loCD45RA⁻ cells, and 384 wells each receiving lin⁻CD34⁺CD38⁺IL-3Rα^loCD45RA⁺ and lin⁻CD34⁺CD38⁺IL-3Rα⁻CD45RA⁻ cells (four donors each). CFU-MegE, CFU-megakaryocyte/erythroid; CFU-GM, CFU-granulocyte/macrophage; BFU-E, burst-forming units/erythroid; CFU-Meg, CFU-megakaryocyte; CFU-M, CFU-macrophage; CFU-G, CFU-granulocyte.

Figure 3

Lin⁻CD34⁺CD38⁺IL-3Rα^loCD45RA⁻ cells are progenitors of IL-3Rα^loCD45RA⁺ and IL-3Rα⁻CD45RA⁻ cells and have clonal bipotent granulocyte/macrophage and erythrocyte/megakaryocyte potential. (a) IL-3Rα^loCD45RA⁻ cells give rise to both IL-3Rα^loCD45RA⁺ and IL-3Rα⁻CD45RA⁻ cells after 72-h culture on Sys-1stromal cells in the presence of SCF, IL-11, FL, Epo, and Tpo. (b) Colony readout of sorted cells from primary cultures (gates in a) in methylcellulose. CFU-MegE, CFU-megakaryocyte/erythroid; CFU-Meg, CFU-megakaryocyte; BFU-E, burst-forming units erythroid; CFU-GM, CFU-granulocyte/macrophage; CFU-M, CFU-macrophage; CFU-G, CFU-granulocyte. (c) Myeloid differentiation potential of single IL-3Rα^loCD45RA⁻ cells. Single cells were expanded in primary cultures and then transferred to methylcellulose (see Materials and Methods).

Figure 4

IL-3Rα^loCD45RA⁻ cells contain no lymphoid-committed progenitor cells. (a) Development of CD19⁺ and CD56⁺ cells from 33 lin⁻ CD34⁺CD38⁺IL-7Rα⁺CD10⁺ cells after 4 weeks of culture. (b) No CD19⁺ or CD56⁺ cells develop from 5,000 IL-3Rα^loCD45RA⁻ cells under the same conditions. Plots show cell fractions gated on human CD45⁺ cells.

Figure 5

Differential mRNA expression of hematopoiesis-affiliated genes in primitive progenitors (CD34⁺CD38⁻), myeloid-committed progenitors, and lymphoid-committed progenitors. Total CD34⁺ bone marrow cells were used as controls. GM-CSFR, granulocyte/macrophage colony-stimulating factor receptor; G-CSFR, granulocyte colony-stimulating factor receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; vWG, von Willebrand factor; C/EBPɛ, CCAAT-enhancer-binding protein ɛ; MPO, myeloperoxidase; EpoR, erythropoietin receptor; TdT, terminal deoxynucleotidyl transferase.