The importance of MSC characterization

Sources and characteristics of mesenchymal stem cells

Mesenchymal stem cells are a heterogeneous population of progenitor cells that can be derived from various tissue sources, including the bone marrow, adipose tissue, and umbilical cord. MSCs show stem cell characteristics, such as self-renewal and multipotency (ability to differentiate into multiple cell types) (Fig. 1).^1,2

Investigators use different methods for cell isolation and expansion and take different approaches to characterize their MSCs. The heterogeneity of MSCs makes it challenging to determine whether the cells used in different studies are similar enough for the results and outcomes across various groups to be compared and contrasted.^3,4

Importance of mesenchymal stem cell characterization

Studies have shown that billions of dollars are spent each year in the United States alone on preclinical research that is not reproducible.⁵ Factors contributing to the lack of reproducibility in preclinical experiments include poor study design, use of different biological reagents, and inconsistent reference materials. The reproducibility of preclinical research can be improved by using validated materials and technologies and by adopting standardized practices for cell characterization.

Proper characterization of MSCs ensures their identity, purity, and functional integrity. Accurate characterization is essential for:

• Quality control: Robust characterization protocols are crucial for maintaining consistent and reproducible results in research and clinical applications.⁶
• Facilitating clinical translation: Accurate characterization is a prerequisite for the successful translation of MSC-based therapies from bench to bedside.⁷
• Comparability: Standardized characterization methods allow for the comparison of results across different studies and laboratories, promoting collaboration and advancing scientific knowledge.⁸
• Regulatory compliance: As MSC-based therapies progress toward clinical trials and eventual commercialization, rigorous characterization protocols are essential for meeting regulatory requirements and ensuring product quality and safety.⁹

MSC characterization guidelines

To provide a common set of standard criteria for MSC research, the International Society for Cell and Gene Therapy (ISCT)* has defined three minimum criteria to ensure accurate identification of human MSCs (Fig. 2)¹⁰:

• Plastic adherence under standard culture conditions.
• Expression of specific surface markers (positive for CD105, CD73, and CD90; negative for CD45, CD34, CD14/CD11b, CD79α/CD19, and HLA-DR).
• Potential for differentiation into adipocytes, osteoblasts, and chondrocytes under standard in vitro conditions.

Similarly, the International Federation for Adipose Therapeutics and Science (IFATS) guidelines provide recommendations specific to the characterization of adipose-derived MSCs (AD-MSCs), including the expression of MSC surface markers, the lack of expression of hematopoietic and endothelial markers, and differentiation potential into adipogenic, osteogenic, and chondrogenic.¹¹ These guidelines serve as a foundation for standardized MSC characterization, ensuring consistency and facilitating cross-study comparisons and collaborations.

* formerly known as International Society for Cellular Therapy

MSC characterization technologies

Various techniques are employed to validate the identity and function of MSCs. These include cell surface marker analysis, functional assays, and gene expression profiling.¹² The selection of the characterization method depends on the features of the MSCs under question.

MSC marker expression

MSCs are characterized by the expression of stem cell surface markers (e.g., CD90, CD105, CD73, CD44, and CD29) and lack of expression of hematopoietic and endothelial markers (CD45, CD31, CD34, and CD144).¹⁰ Flow cytometry is commonly used to analyze the expression of MSC-specific markers, enabling positive selection of cells expressing MSC markers and negative selection of cells expressing hematopoietic and endothelial markers.¹³ This process allows for the isolation of MSCs from a heterogeneous cell population.

Characterization of MSC surface markers by flow cytometry following isolation from the bone marrow using our MSC Growth Medium showed a defined MSC population according to the recommended ISCT markers for determining MSC identity (Fig. 3).

Self-renewal capacity

Like other stem cell populations, MSCs are characterized by self-renewal capacity. Colony-forming unit (CFU) assays are commonly used to evaluate the self-renewal and proliferative capacity of MSCs by quantifying their ability to form colonies from single cells seeded at low densities.¹⁴

Expansion of bone marrow-derived MSCs using our MSC Growth Medium XF resulted in a stable growth performance over several passages (Fig. 4). The MSCs used in this investigation demonstrated self-renewal capabilities, in accordance with the ISCT guidelines (see Fig. 2).

Multipotency

MSCs are characterized by tri-lineage differentiation potential as they can differentiate into osteoblasts, chondrocytes, and adipocytes.¹⁰ Therefore, in vitro differentiation assays are commonly used to evaluate the ability of MSCs to differentiate into osteogenic (bone), chondrogenic (cartilage), and adipogenic (fat) lineages under defined culture conditions.¹⁵

Differentiation of expanded bone marrow MSCs into adipocytes, chondrocytes, and osteoblasts, in accordance with ISCT criteria, was assayed in passage-3 cells using our MSC differentiation media (Fig. 5). MSCs successfully differentiated into all three cell types, demonstrating their multipotency. Adipogenic differentiation resulted in cells with intracellular lipid vacuoles, typical of mature adipocytes (Fig. 5A). Formation of cartilage spheroids confirmed the chondrogenic differentiation of MSCs (Fig. 5B), and Alizarin Red S staining of extracellular calcium deposits in mineralized cells confirmed MSC differentiation into mature osteoblasts (Fig. 5C).

Challenges in MSC characterization

Several challenges exist, including:

• Heterogeneity within MSC populations: MSCs isolated from different tissue sources or even within the same tissue can exhibit significant heterogeneity in their surface marker expression, differentiation potential, and functional characteristics.⁴
• Variations in isolation and culture methods: The methods used for MSC isolation and culture can influence their properties and behavior, making it challenging to standardize protocols across different laboratories.⁶
• Influence of tissue source and donor age on MSC characteristics: The tissue source and age of the donor can impact the characteristics and functionality of the isolated MSCs, introducing variability in the cell populations.¹⁶
• Lack of standardized protocols for functional assays: While differentiation assays are commonly used, there is a lack of standardized protocols and quantitative measures for assessing the functional capabilities of MSCs, hampering cross-study comparisons.¹⁷

Our solutions for standardized MSC culture and characterization

The MSC isolation method and growth medium used for culturing play a crucial role in obtaining a well-defined MSC population that is in agreement with the ISCT guidelines. Standardization is essential for ensuring consistency, reproducibility, and comparability of MSC research and clinical applications.

As cell culture experts, we provide high-quality MSCs and a comprehensive portfolio of specialized media for MSC culture, ensuring high-quality and reproducible results. These include:

• Mesenchymal Stem Cell Growth Medium XF: A serum-free, xeno-free medium optimized for MSC expansion and characterization.
• Mesenchymal Stem Cell Growth Medium 2: A low-serum medium tailored for MSC culture and differentiation.
• PromoExQ MSC Growth Medium XF: A chemically defined, xeno-free medium designed for MSC expansion in a GMP-regulated environment.

Conclusions and future prospects

Mesenchymal stem cell characterization is a critical step in unlocking the full potential of these versatile cells in regenerative medicine. By adhering to standardized protocols and employing robust characterization techniques, you can ensure the identity, purity, and functional integrity of MSCs, facilitating reliable and reproducible results.

As research in the MSC field continues to advance, the development of more comprehensive, inclusive, and harmonized characterization guidelines will be instrumental in accelerating clinical progress and in translating MSC-based therapies into real-world applications. Advancements in technology will likely lead to more robust and efficient methods for MSC characterization, such as single-cell analysis for deeper insights into cellular heterogeneity. Moreover, a deeper understanding of MSC biology will pave the way for strategies to improve their therapeutic potential, such as preconditioning them to enhance their homing and engraftment capabilities.¹⁸

References