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Updated: Oct 23, 2025
Immunophenotyping and Cell Sorting of Human MKs from Human Primary Sources or Differentiated In Vitro from Hematopoietic Progenitors
Published on: August 7, 2021
Immunophenotyping and Cell Sorting of Human MKs from Human Primary Sources or Differentiated In Vitro from
Andrea Acebes-Huerta1, Patricia Martínez-Botía1, Cristina Martín Martín2
1Platelet Research Lab, Instituto de Investigación Sanitaria del Principado de Asturias (ISPA).
Abstract:
Megakaryocyte (MK) differentiation encompasses a number of endomitotic cycles that result in a highly polyploid (reaching even >64N) and extremely large cell (40-60 µm). As opposed to the fast-increasing knowledge in megakaryopoiesis at the cell biology and molecular level, 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. Here, we present an immunophenotyping strategy that allows the identification of successive MK differentiation stages, with increasing ploidy status, in human primary sources or in vitro cultures with a panel integrating MK specific and non-specific surface markers. 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. This approach facilitates multi-Omics studies, with the aim to better understand the complexity of megakaryopoiesis and platelet production in humans. A better characterization of megakaryopoiesis may pose fundamental in the diagnosis or prognosis of lineage-related pathologies and malignancy.
Insights
This study introduces a novel flow cytometry method to identify megakaryocyte (MK) differentiation stages. This technique enhances understanding of megakaryopoiesis and aids in diagnosing related diseases.
Area of Science:
- Hematology
- Cell Biology
- Immunophenotyping
Background:
- Megakaryocyte (MK) differentiation involves endomitotic cycles, leading to large, polyploid cells.
- Current flow cytometry methods primarily identify mature MKs, leaving earlier stages unexplored.
- Understanding megakaryopoiesis is crucial for diagnosing blood-related pathologies.
Purpose of the Study:
- To develop an immunophenotyping strategy for identifying successive MK differentiation stages.
- To enable characterization of MKs across various ploidy levels.
- To facilitate multi-Omics studies for a deeper understanding of human megakaryopoiesis.
Main Methods:
- Developed an immunophenotyping panel with MK-specific and non-specific surface markers.
- Utilized fluorescence-activated cell sorting (FACS) under optimized pressure and nozzle conditions.
- Applied the strategy to human primary sources and in vitro cultures.
Main Results:
- Successfully identified successive MK differentiation stages with increasing ploidy.
- Demonstrated the feasibility of immunophenotyping large and fragile MKs.
- Established a method for enriching MKs for further analysis.
Conclusions:
- The developed strategy allows comprehensive characterization of megakaryopoiesis.
- This approach can significantly advance research into platelet production.
- Improved megakaryopoiesis characterization may aid in diagnosing and prognosing hematologic malignancies.
