Adult human megakaryocyte-erythroid progenitors are in the CD34+CD38mid fraction

Abstract

Bipotent megakaryocyte/erythroid progenitors (MEPs) give rise to progeny limited to the megakaryocyte (Mk) and erythroid (E) lineages. We developed a novel dual-detection functional in vitro colony-forming unit (CFU) assay for single cells that differentiates down both the Mk and E lineages (CFU-Mk/E), which allowed development and validation of a novel purification strategy for the identification and quantitation of primary functional human MEPs from granulocyte colony-stimulating factor-mobilized peripheral blood and bone marrow. Applying this assay to fluorescence-activated cell sorter-sorted cell populations, we found that the Lin(-)CD34(+)CD38(mid)CD45RA(-)FLT3(-)MPL(+)CD36(-)CD41(-) population is much more highly enriched for bipotent MEPs than any previously reported subpopulations. We also developed purification strategies for primary human lineage-committed Mk and E progenitors identified as CFU-Mk and burst forming unit-E. Comparative expression analyses in MEP, MkP, and ErP populations revealed differential expression of MYB We tested whether alterations in MYB concentration affect the Mk-E fate decision at the single cell level in MEPs and found that short hairpin RNA-mediated MYB knockdown promoted commitment of MEPs to the Mk lineage, further defining its role in MEP lineage fate. There are numerous applications for these novel enrichment strategies, including facilitating mechanistic studies of MEP lineage commitment, improving approaches for in vitro expansion of Mk and E cells, and developing improved therapies for benign and malignant hematologic disease.

Figure 1

Use of the dual CFU-Mk/E assay to quantify bipotent MEPs. (A) Schematic of the dual CFU-Mk/E assay to determine Mk/E potential of FACS-sorted populations. Candidate MEPs are plated in a collagen-based colony assay with EPO, TPO, SCF, IL-3, and IL-6 to promote growth of megakaryocyte and erythroid colonies. After 13 to 15 days of culture, colonies are fixed and stained for CD41a (Mk specific) and GlyA (E specific). (B) Representative images of different colony types are shown. (i) Typical BFU-E stains for GlyA (purple) only. (ii) CFU-Mk stains for CD41a (red) only. (iii-iv) Two representative CFU-Mk/E colonies show prominent CD41a⁺ Mk within a burst of GlyA⁺ cells. Scale bar, 100 µm.

Figure 2

Comparison of 3 different MEP purification strategies for enrichment of CFU-Mk/E. Human MPB mononuclear cells were fractionated using 3 different approaches and analyzed for colony formation in the dual Mk/E assay and methylcellulose as described. (A) Starting from previously gated Lin⁻ cells, CD34⁺CD38⁺ cells (*) were equivalently selected using each of 3 MEP purification strategies. (B-D) The candidate MEP population (orange) is CD45RA⁻ (x-axis) and either IL-3Ra⁻ (CD123, strategy 1), FLT3⁻ (CD135, strategy 2), or MPL⁺ (CD110, strategy 3). The corresponding CMP (blue) and GMP (purple) populations are highlighted for each strategy. CMPs were also sorted for comparison. Percentages of total CD34⁺Lin⁻ cells appear with gates. (E) FMO control used for strict gating of PE negative (bottom left gate) vs positive events shown in B-D. (F) For the dual Mk/E assay, colonies were enumerated based on dual immunohistochemistry for CD41a and GlyA. (G) For methylcellulose, colonies were enumerated based on typical morphology and validated with Wright-Geimsa–stained cytospins of selected colonies (data not shown). Results presented as an average for each colony type + standard deviation (SD) for ≥4 independent experiments except for strategy 1 (n = 3 for dual Mk/E and n = 2 for methylcellulose assays).

Figure 3

Improvement in MEP purification by combining FLT3 and MPL selection. (A) Schematic of FACS gating for further purifying FLT3⁻ MPB cells into MPL^hi and MPL^lo/− fractions. Representative flow plots with associated FMO controls used to gate for positive and negative populations. (B) Results of dual Mk/E assay showing average colony counts per 100 seeded cells from 3 experiments. The FLT3^-MPL^hi population has the highest Mk/E potential (blue). (C) Methylcellulose assay results showing average CFU-G/M + CFU-GEMM (gray) and BFU-E (red) colony counts per 100 seeded cells for different subpopulations. CFU-G/M + CFU-GEMM (gray) colonies likely arise from CMP contamination in the sorted MEP-enriched population. The FLT3⁻MPL^hi population has the lowest G/M contamination (P = .02 in comparison with FLT3⁻ population).

Figure 4

Majority of MEPs are in the CD38^mid fraction. (A) Sorting strategy used to evaluate MPL⁺ and MPL⁻ cells within CD38 negative, low, mid, and high subsets of Lin⁻CD34⁺ MPB cells followed by exclusion of FLT3⁺ and CD45RA⁺ cells. (B) Dual Mk/E and (C) methylcellulose colony assay results. Results are presented as average + SD from 3 independent experiments.

Figure 5

Improved strategies for isolating MEPs, MkPs, and ErPs in MPB and BM. (A) Improved strategy to enrich for MEPs (Lin⁻CD34⁺CD38^midCD45RA⁻FLT3⁻MPL⁺CD36⁻CD41⁻), MkPs (Lin⁻CD34⁺CD38^midFLT3⁻CD45RA⁻MPL⁺CD36⁻CD41⁺), and ErPs (Lin⁻CD34⁺CD38^hiFLT3⁻CD45RA⁻MPL⁻). Dual Mk/E assay results for (B) MPB and (C) BM indicate similar enrichment levels in MPB and BM. Methylcellulose assay from the same populations in (D) MPB and (E) BM showing low contamination with other myeloid colonies (CFU-G/M/GM).

Figure 6

MYB expression controls the MEP fate decision. MYB is differentially expressed in MEPs, MkPs, and ErPs, and MYB downregulation in MEPs promotes megakaryocytic commitment. (A) Quantitative reverse transcriptase-PCR analysis of multiple genes in purified MEP, MkP, and ErP subsets (GAPDH used as an internal control). Knockdown of MYB in CD34⁺ cells showing decreased (B) mRNA and (C) protein expression 24 hours after transduction. (D) Dual Mk/E assay results show increased CFU-Mk as the expense of both CFU-Mk/E and BFU-E when MYB is knocked down (P < .05). Results are presented as average + SD from 3 independent experiments.

Figure 7

Proposed model for human hematopoiesis. The classical model of hematopoiesis was based in hierarchical and sequential differentiation from hematopoietic stem cells to mature cells. However, recent studies provide evidence that mature cells such as megakaryocytes could be derived directly from HSCs. We propose a model that there is more than one origin of megakaryocytes, likely dependent on need. We observed that the heterogeneous mixture of cells within the Lin⁻CD34⁺CD38⁻ population includes cells that can give rise to megakaryocytes directly. This is in addition to a strictly bipotent MEP population that gives rise to megakaryocytes and erythrocytes. Solid arrows, validated in the manuscript.