Background: Megakaryocyte (MK) differentiation encompasses a number of endomitotic cycles that result in a highly polyploid (>64N) and large (40-60 µm) cell. The characterization of megakaryopoiesis by flow cytometry is limited to the identification of mature MKs using lineage-specific surface markers, while earlier MK differentiation stages remain unexplored.

Aims: To develop an immunophenotyping strategy for the staging of megakaryopoiesis.

Methods: Surface protein expression was evaluated by flow cytometry on human peripheral blood mononuclear cells (PBMC), bone marrow samples and in vitro PBMC-derived megakaryocyte cultures using 7-color panel mixes.

Results: Here, we present an immunophenotyping strategy that allows the identification of successive MK differentiation stages with a panel integrating MK specific and non-specific surface markers (Table 1). We have seen that the combination of CD31/CD71 allows to set a number of gates which correspond to different stages towards MK differentiation. Further back-gating with MK-specific markers allows the separation of mature and immature MKs. Furthermore, in fresh samples, back-gating to verify the presence of other markers used, or to place the populations in the Forward/Side Scatter axes, refines the assessment of MK differentiation stages and allows to discard other cell types that could be present on the same populations. These populations distribute distinctly in different hematological pathologies (example in Figure 1A), which will be discussed in detail. Ploidy analysis of these populations shows a positive correlation of ploidy status and maturation of MKs (Figure 1B). Despite its size and fragility, MKs can be immunophenotyped using the above-mentioned panel and enriched by fluorescence-activated cell sorting under specific conditions of pressure and nozzle diameter (Figure 1C) for further studies (i.e. multi-Omics).

Conclusion(s): A better characterization of megakaryopoiesis by flow cytometry may pose fundamental in the diagnosis or prognosis of lineage-related pathologies and malignancy and we believe our strategy eases that path.

Table

Table 1. Rationale of the surface markers that are combined in the panels used and presented in the representative results section for flow cytometry phenotypical analysis of MK subpopulations.

image

Figure 1. A. Immunophenotyìng of megakaryopoiesis in human bone marrow samples from patients with different pathologies. B. Ploidy analysis of the populations selected based on CD31/CD71 expression, in human BM sample from an AML patient. C. Megakaryocyte cell sorting: representative gating strategy of a sample of MK in vitro culture at day 10 of differentiation.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/immunophenotyping-and-cell-sorting-of-human-mks-from-human-primary-sources-or-differentiated-in-vitro-from-hematopoietic-progenitors/