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Updated: Jun 12, 2026
Collection of Serum- and Feeder-free Mouse Embryonic Stem Cell-conditioned Medium for a Cell-free Approach
Published on: January 8, 2017
Novel approach for formation of platelet-like particles from mouse embryonic stem cells without using feeder cells
Kayoko Tsuji1, Masaaki Ohnuma, Stephanie M Jung
1Department of Protein Biochemistry, Institute of Life Science, Kurume University, Kurume, Japan. k-tsuji@1si.kurume-u.ac.jp
Abstract:
Megakaryocytes (MKs) and platelet-like particles (PLPs) have generally been obtained by culturing embryonic stem (ES) cells over feeder cells. However, using feeder cells need many labor-consuming processes and the MK and PLP fractions obtained are often contaminated by such cells and their fragments. Here we describe our new culture system for differentiating mouse ES cells to MKs and PLPs without using feeder cells. ES cells are differentiated to cells with MK-like morphology and properties, including proplatelet formation, high ploidy (>8N), and CD41 expression. The culture medium contained PLPs expressing platelet glycoproteins, CD41 and GPIb. Integrin alpha(IIb)beta(3) of PLPs can be activated by thrombin. Addition of the metalloproteinase inhibitor TAPI-2 to the culture increased the surface expression of GPIbalpha and augmented the adhesion of PLPs to immobilized von Willebrand factor through decreasing the shedding of GPIbalpha. Thus our mouse ES cells culture system is a suitable and efficient method for obtaining MKs and functional PLPs that obviates the need for feeder cells.
Insights
This study presents a new method to generate megakaryocytes (MKs) and platelet-like particles (PLPs) from mouse embryonic stem (ES) cells without feeder cells, improving efficiency and purity.
Area of Science:
- Hematology
- Stem Cell Biology
- Cell Culture Technology
Background:
- Megakaryocytes (MKs) and platelet-like particles (PLPs) are typically derived from embryonic stem (ES) cells cultured on feeder cells.
- Feeder cell co-cultures are labor-intensive and often result in contamination of MK and PLP fractions.
Purpose of the Study:
- To develop a feeder-cell-free culture system for efficient differentiation of mouse ES cells into MKs and functional PLPs.
- To characterize the resulting MKs and PLPs for key markers and functional properties.
Main Methods:
- Mouse ES cells were cultured in a novel system designed to induce differentiation without feeder cells.
- Characterization involved assessing morphology, proplatelet formation, ploidy, CD41 expression, and platelet glycoprotein expression on PLPs.
- Functional assays included thrombin-induced integrin activation and adhesion to von Willebrand factor, with and without TAPI-2 treatment.
Main Results:
- The feeder-free system successfully differentiated ES cells into MKs exhibiting proplatelet formation, high ploidy (>8N), and CD41 expression.
- Generated PLPs expressed platelet glycoproteins (CD41, GPIb), showed thrombin-activatable integrin alpha(IIb)beta(3), and adhered to von Willebrand factor.
- Inhibition of GPIb shedding with TAPI-2 enhanced PLP adhesion and GPIbalpha surface expression.
Conclusions:
- A novel, efficient, and feeder-cell-free culture system for generating MKs and functional PLPs from mouse ES cells has been established.
- This method overcomes the limitations of traditional feeder cell-based approaches, offering a cleaner and more streamlined production of these critical cell types.
