Mesenchymal Stem Cell Morphology – Structure, Growth Pattern, and Identification
Mesenchymal stem cells (MSCs) are multipotent adult stem cells capable of differentiating into various cell types, including osteoblasts, adipocytes, and chondrocytes. They are widely studied for their potential in regenerative medicine, tissue engineering, and immune modulation therapies.One of the earliest and most accessible ways to evaluate MSCs in culture is through their morphology—the visual appearance of the cells under a microscope. In vitro, mesenchymal stem cells display a characteristic spindle-shaped, fibroblast-like morphology that helps distinguish them from other cell types.
Understanding MSC morphology is crucial for identifying healthy cultures, detecting contamination or senescence, and monitoring the effects of media, passage number, or stress. While morphology alone does not confirm MSC identity, it serves as a valuable first-line tool for stem cell researchers to assess the overall health and quality of their cultures.
Initial assessment of MSC morphology under the microscope often includes noting cell shape, adherence, cytoplasmic granularity, and colony formation. These indicators can quickly inform whether the culture conditions are optimal or require adjustment.
Typical Morphology of MSCs in Culture
When cultured in vitro, mesenchymal stem cells (MSCs) display a well-defined and recognizable morphology. They are typically described as spindle-shaped or fibroblast-like, with elongated cytoplasm and tapered ends. This distinctive appearance makes them relatively easy to identify during early culture observation.
Under standard culture conditions, MSC size ranges between 15–30 µm, though this can vary depending on passage number and confluence. Each cell generally features an oval or elongated nucleus, centrally located and often clearly visible under phase-contrast microscopy.
One of the defining features of MSCs is their ability to adhere to plastic surfaces—a hallmark used during isolation. Within 24–48 hours of plating, MSCs begin to spread and align, forming swirled or whorled growth patterns, especially as cultures approach 70–80% confluence.
At low density, early MSC colonies may appear as discrete clusters of spindle-shaped cells—this is known as CFU-F morphology (Colony-Forming Unit–Fibroblast). These colonies indicate the clonogenic potential of the MSC population and are commonly used to evaluate stem cell proliferation capacity.
Consistent MSC appearance in culture, including shape, growth behavior, and colony formation, reflects the health and integrity of the cell population. Any deviation—such as increased size, loss of shape, or irregular alignment—may suggest culture stress or cellular aging.
Changes in MSC Morphology by Passage
Early-Passage MSCs (P2–P4)
At early passages, MSCs exhibit their classic spindle-shaped, fibroblast-like appearance.
They are:
These elongated cells typically show high differentiation potential and are ideal for downstream applications, including in vitro assays and clinical preps. Early-passage morphology reflects a healthy and functionally potent MSC population.
Late-Passage MSCs (>P5)
With increasing passages, especially beyond P5–P6, MSCs begin to show morphological changes associated with senescence:
These MSC senescence indicators often correlate with reduced stemness, compromised differentiation ability, and altered surface marker expression. Late-passage MSCs are less desirable for experiments or therapeutic use due to diminished potency.
Morphology and Differentiation Potential
There is a clear connection between MSC morphology and their differentiation capacity:
They are:
Thus, morphological assessment by passage number can help predict whether MSCs are still suitable for research or clinical protocols.
Observing MSC Morphology
Using Phase Contrast Microscopy and Live-Cell Imaging
Phase contrast microscopy is the standard, non-invasive method for examining MSC morphology under the microscope. It allows researchers to visualize live, unstained cells with clear contrast—ideal for observing:
For more dynamic monitoring, live-cell imaging systems can track MSC appearance in culture over time, offering insights into behavior such as migration, division, and stress responses in real-time.
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To explore the internal structure of MSCs, researchers commonly use immunofluorescence staining to highlight components of the cytoskeleton, including:
- Actin filaments (F-actin): Defines shape and motility
- Vimentin: A mesenchymal marker confirming MSC identity
Visualizing the MSC cytoskeleton helps evaluate structural integrity, especially during differentiation or when exposed to mechanical or chemical stress.
Monitoring Morphological Changes Due to Stress
Changes in morphology can signal problems in the culture, such as:
|
Visual Cue
|
Potential Cause
|
|---|---|
|
Rounded or floating cells |
Apoptosis, enzyme overexposure, pH shift |
|
Granularity or vacuolization |
Oxidative stress, late passage |
|
Loss of adherence |
Contamination, media issues |
|
Heterogeneous shapes |
Mixed population or early differentiation |
Factors Influencing MSC Morphology
While mesenchymal stem cells (MSCs) generally follow a typical fibroblast-like growth pattern, their morphology is highly sensitive to changes in the culture environment. Variations in medium composition, oxygen levels, and substrate stiffness can significantly affect MSC shape, size, and behavior.
Culture Medium Composition and Supplements
The formulation of the culture medium—whether it includes fetal bovine serum (FBS), human platelet lysate, or is fully xeno-free—can influence MSC morphology in different media.
- Rich, serum-based media promote faster spreading and elongation
- Serum-free or chemically defined media may cause delayed adherence or altered morphology
Additives such as growth factors or cytokines can also impact cytoskeletal tension and cellular shape.
Confluency Levels and Passaging Techniques
MSC culture confluency plays a major role in morphology:
- At low density, MSCs are well-spaced and fully spread
- At high confluency, cells align in swirls and begin overlapping
Delayed passaging can lead to stress-induced morphological changes, including flattening or granulation.
Oxygen Levels and Incubation Environment
MSC growth pattern can shift under different oxygen tensions.
- Normoxic conditions (21% O₂) are standard
- Hypoxic conditions (1–5% O₂) may maintain stemness and alter morphology
Temperature fluctuations, CO₂ imbalance, or poor humidity can also influence cell shape and health.
Substrate Stiffness and Coating
MSCs respond to the physical properties of their environment:
- Standard tissue culture plastic supports strong adhesion
- Coatings with ECM proteins like collagen or fibronectin can enhance elongation and mimic in vivo conditions
- Softer substrates may reduce spreading and induce rounder morphology
Together, these factors determine how MSCs grow, organize, and interact with their environment.
Morphology as an Indicator of Cell Health
The morphology of MSCs offers quick, visual clues about the viability and functional status of a culture. Trained researchers can often detect early warning signs of decline or transformation just by looking at the cells under a microscope.
Signs of a Healthy MSC Culture
These features indicate high viability, robust proliferation, and preservation of multipotency.
Morphological Signs of Stress or Senescence
Such changes are typical MSC senescence indicators and may signal reduced differentiation capacity.
When to Discard or Recover a Culture
Healthy MSC morphology is a cornerstone of reproducible research and therapeutic success. Regular assessment allows early intervention and ensures only potent, functional cells are used.