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Rapid and efficient surface marker screening

A major challenge facing stem cell biology is the heterogeneous nature of cultures and differentiations. To sort viable cells to purify for use in later experiments, one must know the cell surface signature for the particular cell of interest. Since different types of related cells may share markers, researchers must find a unique multimarker signature for each, while other markers may be useful in distinguishing subpopulations of a particular type of cell.


BD Lyoplate™ cell surface marker screening panels provide a comprehensive and efficient solution for profiling stem cells and their derivatives for hundreds of human or mouse cell surface markers by flow cytometry or cellular imaging. Deciphering the cell surface proteome enables researchers to define strategies for the analysis and isolation of targeted cells from heterogeneous populations for functional studies, drug screening, in vivo animal studies, and cell therapy research.

The hundreds of monoclonal antibodies in each panel constitute one of the most cost-effective screening tools available for cellular analysis. To simplify the transition to more targeted, larger-scale experiments, all antibodies included in the screening panels are available in the BD Biosciences catalog.

Both the human (Cat. No. 560747) and mouse (Cat. No. 562208) panels contain three plates. Each well contains lyophilized, purified antibody to one cell surface marker or isotype control. Following reconstitution, the cellular samples are stained with purified antibodies, and detection reagents included with the panel are added. Finally, samples are analyzed by flow cytometry or imaging.

To provide flexibility while simplifying workflow, open wells allow the panel to be expanded to include additional markers. Powerful BD Biosciences analysis tools facilitate data mining and heatmap generation.

Considerations for flow cytometry vs image screening with BD Lyoplate panels

Property of sample Flow cytometry Imaging
Suspension cells X  
Rare cell populations X  
Subpopulation analysis
Co-staining with multiple markers
X  
Reporter lines X X
Specific morphology changes   X
Limited number of cells   X
Using surface marker screening to characterize and enrich neural stem cells and neurons
Using surface marker screening to characterize and enrich neural stem cells and neurons
A. H9 hESCs were induced using the SFEB method,1 and the BD Lyoplate™ human cell surface marker screening panel (Cat. No. 560747) was used to screen the resulting heterogeneous neural cultures on a BD™ LSR II flow cytometer to identify a surface marker signature for NSCs. After identifying potential hits, NSC cell surface phenotypes were verified and a CD184+CD44 CD271CD24+CD15mid sorting profile was used on a BD FACSAria™ II cell sorter to obtain a near-pure subpopulation of NSCs.
Using surface marker screening to characterize and enrich neural stem cells and neurons
Using surface marker screening to characterize and enrich neural stem cells and neurons
B. The purified NSCs were differentiated into a mixed culture of neuronal and glial cell populations, which was screened using the same panel to identify a surface marker signature for neurons. An imaging screen was chosen due to the unique morphology of neurons and the ability to co-stain with a neuron-specific marker. Potential hits were verified by flow cytometry, and a CD44 CD184CD24+CD15low sorting profile was used to purify neurons.1

 

Screening of neural populations

Neural cell populations derived from pluripotent stem cells are important for studying human disease and development. Pluripotent stem cells can be differentiated into self-renewing NSCs, which can be further differentiated into heterogeneous populations of neurons and glia.1 A key to further research is to identify surface marker signatures for each of these cell types.

In the example, the BD Lyoplate human cell surface marker screening panel (Cat. No. 560747) was used to identify cell surface phenotypes for NSCs and neurons. In panel A, heterogeneous neural induction cultures were screened by flow cytometry, and potential NSC markers were identified on a heatmap. A resulting NSC cell surface phenotype of CD184+CD44CD271CD24+CD15mid was verified using intracellular NSC markers. The surface phenotype was used to sort a near-pure subpopulation of NSCs, the ability of which to differentiate both in vivo and in vitro was later confirmed.

In panel B, the purified NSCs were differentiated into neuronal and glial cell populations, which were screened by imaging using the same panel to identify surface markers for isolating neurons. An imaging screen was chosen due to the unique morphology of neurons and the ability to co-stain with a neuronal-specific marker. A potential neuronal surface phenotype of CD44CD184CD24+CD15low was verified by flow cytometry and used to purify neurons. In addition to neural cells, the BD Lyoplate human cell surface marker screening panel has also been used to identify cell surface markers of cardiomyocytes derived from pluripotent stem cells.2 Most recently, this powerful methodology was used to develop a human stem cell model of Alzheimer’s disease.3


References

1. Yuan SH, Martin J, Elia J, et al. Cell surface marker signatures for the isolation of neural stem cells, glia and neurons derived from human pluripotent stem cells. PLoS One. 2011;6:e17540.

2. Uosaki H, Fukushima H, Takeuchi A, et al. Efficient and scalable purification of cardiomyocytes from human embryonic and induced pluripotent stem cells by VCAM1 surface expression. PLoS One. 2011;6:e23657.

3. Israel MA, Yuan SH, Bardy C, et al. Probing sporadic and familial Alzheimer’s disease using induced pluripotent stem cells. Nature. 2012;482:216-220.