CD271: An Important Marker for Mesenchymal Stem Cells (MSCs)?
Flow cytometry is a sophisticated technology that enables the analysis of individual cells as they flow in a stream through a laser beam.
This technique provides a wealth of information about each cell, including:
Size and granularity: Forward scatter (FSC) and side scatter (SSC) measurements reveal the size and internal complexity of a cell
Presence and abundance of specific molecules: By labeling cells with fluorescent antibodies that bind to specific molecules, flow cytometry can detect the presence and quantify the amount of those molecules on each cell. This allows researchers to identify and isolate specific cell populations based on their unique surface markers
Mesenchymal Stem Cells and Their Importance
Mesenchymal stem cells are multipotent stromal cells that reside within various tissues, including bone marrow, adipose tissue, umbilical cord blood, and dental pulp. These cells possess the remarkable ability to differentiate into a variety of cell types, including bone, cartilage, and fat cells. This makes them attractive candidates for regenerative therapies aimed at repairing damaged or diseased tissues.
Identifying MSCs: The Challenge of Heterogeneity
One of the main challenges in working with MSCs lies in their inherent heterogeneity. This means that even within a single tissue source, MSCs exhibit a diverse range of surface markers and functional properties. To date, no single marker has been universally accepted to definitively identify MSCs before culture. Instead, researchers often rely on a combination of markers and functional assays to characterize these cells.
The International Society for Cellular Therapy (ISCT) has proposed minimal criteria for defining MSCs.
These criteria include:
Plastic adherence: MSCs readily attach to plastic surfaces when cultured
Specific surface marker expression: They express CD73, CD90, and CD105 while lacking hematopoietic markers such as CD34 and CD45.
Multi-lineage differentiation potential: MSCs can differentiate into bone, cartilage, and fat cells under appropriate culture conditions.
While these criteria provide a framework for MSC characterization, the ongoing search for more specific markers remains a priority in the field.
CD271: A Promising Marker for MSC Identification
CD271, also known as the low-affinity nerve growth factor receptor (LNGFR) or p75, has emerged as a promising marker for identifying MSCs, particularly those derived from bone marrow and adipose tissue.
Several studies have demonstrated the specificity of CD271 for identifying MSCs from bone marrow.
Some key findings include:
The CD271+ fraction of bone marrow mononuclear cells (BM-MNC) contains all the colony-forming unit-fibroblast (CFU-F) activity, a key characteristic of MSCs
CD271-selected BM-MSCs display greater proliferative capacity than MSCs isolated solely by plastic adherence
CD271 remains one of the most selective markers for enriching MSC progenitors from bone marrow
Further investigations have confirmed the usefulness of CD271 in combination with other markers, such as CD45, for isolating fresh BM-MSCs6.... Research suggests that using CD271 in combination with CD45 allows for the quantification of the MSC population in bone marrow samples obtained for cell therapy27. Additionally, no colony-forming cells are present in the CD271-negative fraction of bone marrow cells, suggesting that bone marrow colony-forming activity is confined to CD45lowCD271high cells.
In adipose tissue, CD271+ MSCs have also shown enhanced proliferative capacity and multi-lineage differentiation potential12.
Studies have revealed that:
CD271+ adipose-derived MSCs (AD-MSCs) express higher levels of angiogenic genes and lower levels of inflammatory genes compared to CD271- AD-MSCs.
They possess a greater proportion of typical stem cell surface markers and are more likely to promote neoangiogenesis than their CD271- counterparts.
CD271+ AD-MSCs have demonstrated enhanced cartilage repair capabilities and reduced angiogenic activity compared to plastic-adherent MSCs.
While promising, it's important to note that CD271 expression can vary between individuals and tissue sources. Moreover, its expression can decline during cell cultures. These factors underscore the need for continued research to optimize CD271-based MSC isolation strategies and explore its potential in combination with other markers.
Flow Cytometry in Action: Identifying CD271+ MSCs
Flow cytometry provides a rapid and precise method for identifying and isolating CD271+ MSCs. Researchers typically employ a multi-marker approach, labeling cells with fluorescent antibodies against CD271 and other relevant markers, such as CD45 and CD90. By analyzing the fluorescence signals emitted by individual cells, flow cytometers can distinguish different cell populations and sort them based on their marker expression.
Here's a simplified overview of the process:
Sample preparation: Bone marrow aspirates, adipose tissue, or other sources are processed to obtain a single-cell suspension.
Antibody labeling: Cells are incubated with fluorescent antibodies targeting CD271, CD45, and other markers of interest.
Flow cytometry analysis: The labeled cells are passed through a flow cytometer, where they are illuminated by a laser beam. The emitted fluorescence signals are detected and analyzed, allowing for the identification and quantification of different cell populations.
Cell sorting (optional): If desired, specific cell populations, such as CD271+/CD45- MSCs, can be physically separated from the rest of the sample using a cell sorter.
The Advantages of Flow Cytometry for MSC Research
Flow cytometry offers several advantages for MSC research, including:
High sensitivity and specificity: It can detect rare cell populations, such as MSCs, even at very low frequencies.
Multi-parameter analysis: Flow cytometry allows for the simultaneous analysis of multiple markers on each cell, providing a comprehensive picture of cell heterogeneity and enabling the identification of distinct subpopulations.
Quantitative data: It provides quantitative data on marker expression, allowing for precise comparisons between different samples and experimental conditions.
Cell sorting capabilities: Flow cytometers equipped with cell sorters can physically isolate specific cell populations for further analysis or therapeutic use.
Looking Ahead: Future Directions and Challenges
Flow cytometry will undoubtedly continue to play a central role in advancing MSC research and clinical translation. Some exciting future directions include:
Developing more refined marker combinations: Combining CD271 with other markers, such as CD146, STRO-1, and SSEA-4, may further enhance MSC identification and isolation.
Exploring functional flow cytometry: Integrating functional assays with flow cytometry could provide insights into the biological properties of different MSC subpopulations.
Optimizing cell sorting strategies: Refinements in cell sorting techniques could improve the purity and yield of isolated MSCs for therapeutic applications.
However, challenges remain:
Standardization of protocols: Variations in flow cytometry protocols and data analysis methods can hinder comparisons between studies.
Cost and accessibility: Flow cytometry remains a relatively expensive technology, limiting its accessibility for some researchers.
The ongoing need for well-defined MSC markers: Despite promising progress, the search for definitive MSC markers remains a priority.
Conclusion
Flow cytometry stands as an indispensable tool in the quest to understand and harness the therapeutic potential of MSCs. While CD271 holds promise as a specific marker, ongoing research is crucial to refine its use and explore its potential in combination with other markers. As our knowledge of MSC heterogeneity deepens, flow cytometry will undoubtedly remain at the forefront of efforts to unlock the full potential of these remarkable cells for regenerative medicine.
