Detecting Stem Cells with Flow-Cytometry
Flow cytometry is widely applied in immunology, hematology, cancer research, stem cell biology, and infectious disease diagnostics, providing an unparalleled level of detail in cellular analysis. In clinical settings, it is extensively used for immunophenotyping, leukemia and lymphoma diagnostics, and monitoring immune responses in diseases such as HIV/AIDS. In research, it serves as a critical tool for studying cellular heterogeneity, signal transduction pathways, and the effects of drugs on cellular populations. The ability to rapidly analyze thousands to millions of individual cells within minutes makes flow cytometry one of the most powerful techniques in modern biomedical science, with ongoing innovations continuing to enhance its resolution, sensitivity, and efficiency.
Fluidics in Flow Cytometry
Fluidic-based flow cytometry analysis is a sophisticated technique that allows for the high-throughput evaluation of physical, chemical, and fluorescent properties of individual cells or microscopic particles in suspension. The fluidic system is fundamental to the accuracy and reproducibility of flow cytometry, as it ensures that cells are introduced into the interrogation region in a precise and controlled manner. This process begins with the injection of the sample into a flow chamber, where it is enveloped by a sheath fluid—typically a phosphate-buffered saline or other isotonic solution—that hydrodynamically focuses the sample stream into a narrow core. The sheath fluid creates a laminar flow pattern, allowing cells to travel in a single file and ensuring that each particle passes individually through the laser interrogation point. This single-cell resolution is critical to minimizing coincidence events, in which multiple cells pass through simultaneously, potentially leading to inaccurate data interpretation.
Measuring with Laser Beams
As the cells or particles pass through the focused laser beam, they interact with the light, resulting in two key types of light scatter: forward scatter (FSC) and side scatter (SSC). Forward scatter is collected along the same axis as the laser beam and provides information about cell size, while side scatter is collected at a 90-degree angle and reflects the internal complexity or granularity of the cell. These scattered signals are detected by photodiodes and photomultiplier tubes (PMTs), which convert the light into electrical signals for quantification.
Fluorescent Markers
In addition to scatter measurements, flow cytometry is widely used to analyze fluorescence properties, which are crucial for identifying specific cell populations or molecular markers. Fluorescent dyes, conjugated antibodies, or genetically encoded fluorescent proteins can be used to label specific cell surface markers, intracellular proteins, nucleic acids, or other biomolecules. When excited by specific laser wavelengths, these fluorophores emit light at characteristic wavelengths, which are captured by an array of optical filters and detectors designed to isolate and measure fluorescence intensity from multiple fluorophores simultaneously.
Finding the Cells in Question
The detected scatter and fluorescence signals are then amplified, digitized, and processed by sophisticated software that translates them into visual representations such as histograms, scatter plots, and density plots. Using these plots, researchers can employ gating strategies to distinguish between different cell types, analyze subpopulations, and evaluate cellular functions such as apoptosis (cell death), cell cycle status, or cytokine production.
Stem Cell Detection Using Flow Cytometry
The detection of mesenchymal stem cells (MSCs), hematopoietic stem cells (HSCs), multilineage differentiating stress-enduring (MUSE) cells, and very small embryonic-like stem cells (VSELs) using fluidic-based flow cytometry relies on the precise identification of surface and intracellular markers that define each stem cell population. Flow cytometry is a crucial tool for stem cell research, as it allows for the high-throughput characterization of heterogeneous cell populations based on light scattering properties and fluorescence-labeled antibodies that bind to specific cellular markers.
Mesenchymal stem cells (MSCs) are typically isolated from bone marrow, adipose tissue, umbilical cord, or dental pulp. Their identification using flow cytometry relies on a combination of positive and negative surface markers. Freshly harvested cells often express markers known as CD73 and CD271. MSCs that have been cultured and expanded often change their marker expression and display markers such as CD90 and CD105. Regardless, MSCs lack hematopoietic (blood-creating) markers such as CD34, CD45, and HLA-DR.
Hematopoietic stem cells (HSCs) are characterized by their ability to differentiate into all blood cell lineages and are primarily found in the bone marrow and peripheral blood. Flow cytometry detection of HSCs is primarily based on the expression of CD34, a well-established marker for hematopoietic progenitors, while the absence of lineage-specific markers such as CD3, CD19, and CD14 helps to exclude differentiated hematopoietic cells. In addition, the primitive HSC population is often identified by the expression of CD38, CD90, and CD133
Multilineage differentiating stress-enduring (MUSE) cells represent a unique subset of stem cells with inherent ability to differentiate into many cell types (pluripotency), found in mesenchymal tissue and peripheral blood. These cells are characterized by the expression of stage-specific embryonic antigen-3 (SSEA-3), along with mesenchymal markers such as CD105 and CD90. Since MUSE cells naturally exhibit stress resistance and can survive under extreme conditions, their detection via flow cytometry often involves selective gating based on SSEA-3 positivity while excluding lineage-committed cells.
Very small embryonic-like stem cells (VSELs) are a highly primitive and rare population of stem cells with embryonic-like features, present in the bone marrow and peripheral blood. Their detection is challenging due to their small size and low abundance, but flow cytometry enables their identification using a distinct marker profile. VSELs express markers such as CD133, CXCR4, and SSEA-4, while lacking CD45, which differentiates them from hematopoietic cells. Because VSELs are extremely small, their forward scatter properties place them in the low FSC region, often overlapping with debris or apoptotic bodies.
Preparing Samples for Measurement
When analyzing MSCs, the sample is first prepared washing and enzymatic digestion to remove as many additional cells as possible. Following this, the remaining cells are stained by antibodies that interact with the CD Markers mentioned above. These antibodies have a fluorescent component that can be detected by the cytometer. The fluidic system ensures that each labeled cell passes through the laser interrogation point individually, where it emits fluorescence that is detected and processed. MSCs are distinguished based on their scatter profile, which typically reflects a medium-sized, relatively homogeneous population, and their distinct marker expression, which is visualized using multi-parametric gating strategies.
In all cases, the success of fluidic-based flow cytometry in detecting these stem cell populations depends on precise sample preparation, careful antibody panel design, and proper instrument calibration to optimize sensitivity and specificity. The hydrodynamic focusing of cells ensures that each stem cell is analyzed independently, while fluorescence compensation and gating strategies allow for the separation of target populations from background signals. The ability to analyze multiple parameters simultaneously makes flow cytometry an essential technique for identifying and characterizing stem cells, facilitating research in regenerative medicine, hematology, and developmental biology.
