What are stem cells?

Stem cells are a kind of cells with self-renewal ability and multipotent differentiation potential. Stem cells can maintain the stability of their own cell population through division, while differentiating into cells with many different functions, thus participating in tissue repair, growth and development and other life activities.

Figure 1. Classification of stem cells

What are mesenchymal stem cells?

Mesenchymal Stem Cells (MSCs) are a type of adult stem cells with multipotent differentiation potential and immunomodulatory functions, which were also called bone marrow mesenchymal stem cells in the early stage because they were initially discovered in the bone marrow. Subsequent, research revealed their presence in various tissues such as adipose, umbilical cord, placenta, dental pulp, and skeletal muscle.

1. Core characteristics of MSCs

Mesenchymal stem cells possess three core properties, which also serve as the identification criteria defined by the International Society for Cell & gene Therapy (ISCT):

a) Adherent growth: Adherent growth in plastic dishes under standard culture conditions.

b) Specific surface marker expression:

Positive expression (≥ 95%): CD105, CD73, CD90.

Negative expression (≤ 2%): CD45, CD34, CD14 or CD11b, CD79α or CD19, HLA-DR.

c) Multipotent differentiation potential: Capable of differentiating into osteoblasts, chondrocytes and adipocytes under in vitro induction conditions.

2. Source of MSCs

Mesenchymal stem cells are derived from various sources, each exhibiting distinct characteristics in terms of proliferative capacity, differentiation tendency, and clinical application.

3. Differentiation of MSCs

The differentiation capacity of mesenchymal stem cells is one of their core biological properties and refers to the potential to differentiate into many different types of cells under specific induction conditions (e.g., cytokines, chemicals, or microenvironment signaling regulation). The differentiation capacity of mesenchymal stem cells makes them "universal" repair cells, which directly replenish functional cells and promote tissue regeneration by directionally inducing differentiation into cell types corresponding to damaged tissues, avoiding the limitations of "replacement therapy" in traditional treatments (such as insufficient organ transplant donors, immune rejection). At present, related research has entered the clinical stage in the fields of bone repair, cartilage regeneration, myocardial repair, etc., demonstrating significant therapeutic potential.

Figure 2. Applications of mesenchymal stem cells with multiple differentiation potential for repair of various tissues. ^[1]

4. Functions and applications of MSCs

In vitro culture of mesenchymal stem cells

In vitro culture of mesenchymal stem cells is the core method to obtain sufficient and high-quality cells to meet the needs of research and clinical demands. It also serves as the foundation for exploring their biological characteristics and realizing standardized applications.

Figure 3. Typical extraction process of adipose-derived mesenchymal stem cells from adipose tissue of mouse. ^[1]

In vitro culture of MSCs involves four core stages: isolation and acquisition, primary culture, passage and expansion, and functional induction.

a) Isolation and acquisition: MSCs are typically isolated from bone marrow, adipose, umbilical cord and other tissues, and single cell suspensions are obtained by density gradient centrifugation or enzymatic digestion. During this stage, penicillin/streptomycin should be added to the basal medium (e.g., α-MEM, DMEM) to suppress contamination, while avoiding premature addition of cytokines to interfere with the initial state of the cell.

b) Primary culture: Inoculated cells should be cultured quietly in a 5% CO₂ incubator at 37℃, avoiding frequent medium changes for the first 3-4 days to facilitate cell adhesion. When confluence reaches 80%-90%, digest cells with 0.25% trypsin for passage. Targeted cytokine supplementation may commence at this stage.

c) Passage and expansion: Post-passage cells proliferate rapidly. Regularly monitor cell morphology (typically spindle-shaped or fibroblast-like) and adjust cytokine combinations to maintain stemness and proliferative activity. MSCs typically passaged 3–5 times are suitable for subsequent experiments, avoiding senescence or phenotypic alterations in high-passage cells.

d) Functional induction: Based on research requirements, directed differentiation (e.g., osteogenic, adipogenic, chondrogenic) is achieved by switching to induction media (containing specific cytokines). The induction period typically lasts 1–3 weeks, during which differentiation outcomes are validated via staining (e.g., alizarin red, Oil Red O) or molecular marker detection.

Common cytokines in MSCs culture and their mechanism of action

In vitro culture of mesenchymal stem cells (MSCs) is a critical step for both basic research and clinical applications. Cytokines, as the core molecules to regulate the biological behavior of MSCs, run through the whole process of culture.

Common cytokines and their mechanisms of action

1. Adhesion and survival related cytokines

• Fibroblast growth factor (FGF): FGF-2, as a "foundational factor" for MSCs culture, activates FGFR1 receptor, initiates MAPK/ERK and PI3K/Akt signaling pathways, enhances cell adhesion to extracellular matrix, increases primary cell adhesion rate by 30% -50%, while inhibiting early apoptosis.
• Transforming growth factor-β (TGF-β): Through Smad2/3 pathway, induce cells to secrete matrix components such as fibronectin and collagen to construct a microenvironment conducive to cell survival. Combined with FGF-2 significantly reduces cell mortality in primary cultures.

2. Proliferation and stemness maintenance cytokines

• Platelet-derived growth factor (PDGF): PDGF-BB shortens the G1 phase of the cell cycle through the PI3K/Akt pathway, dose-dependently promote MSCs proliferation at concentrations ranging from 5 to 20 ng/mL, and maintain high expression of cell surface markers (CD29, CD44, CD90) and inhibit spontaneous differentiation.
• Fibroblast growth factor (FGF): FGF-2 is a potent pro-proliferative factor that promotes self-renewal and inhibits premature differentiation of MSCs. It is commonly used to improve cell viability in primary culture and passage.
• Insulin-like growth factor-1 (IGF-1): Synergizes with PDGF to promote proliferation by activating the MAPK/ERK pathway, reduces the population doubling time of MSCs to 20-24 hours at 10-50 ng/mL, with a karyotype abnormality rate below 1% during passaging.
• Epidermal growth factor (EGF): Synergizes with FGF and PDGF to promote MSCs proliferation, exhibiting particularly potent effects on adipose-derived MSCs.
• Stem cell factor (SCF): Maintains the stemness and proliferation ability of MSCs, commonly employed in bone marrow MSCs culture.

3. Directed differentiation inducing cytokines

a) Osteogenic differentiation:

• Bone morphogenetic proteins (BMPs): BMP-2 is the core inducer, activating the key osteogenic transcription factor Runx2 via the Smad1/5/8 pathway. Combined with sodium β-glycerophosphate and vitamin C, it induces high expression of osteocalcin and alkaline phosphatase within approximately 14 days.
• Transforming growth factor-β (TGF-β): Synergizes with BMPs to enhance osteogenic differentiation while also promoting chondrogenic differentiation.
• Insulin-like growth factor-1 (IGF-1): Promotes osteoblast maturation and mineralization.

b) Adipogenic differentiation:

Insulin and dexamethasone form the foundational combination. Supplementing with interleukin-6 (IL-6) activates the PPARγ pathway, promoting lipodendrite formation. Oil Red O staining yields strong positivity around day 21.

c) Chondrogenic differentiation:

• Transforming growth factor-β (TGF-β1, TGF-β3): TGF-β3 induces cartilage-specific type II collagen synthesis by regulating Sox9 transcription factor and needs to be used in pellet culture system with ascorbic acid to form cartilage-like tissue in 4 weeks.
• Bone morphogenetic proteins (BMPs): Synergize with TGF-β to enhance cartilage differentiation, especially promote cartilage matrix synthesis.

4. Other Function-Related cytokines

• Tumor necrosis factor-α (TNF-α): Low concentrations of TNF-α induces MSCs to secrete anti-inflammatory factors such as IL-6 and IL-10, enhancing their immunosuppressive function, and are commonly used to prepare MSCs culture medium with immunoregulatory activity.
• Interferon-γ (IFN-γ): Promoting indoleamine 2,3-dioxygenase (IDO) expression in MSCs by activating the JAK-STAT pathway significantly improves their ability to inhibit T cell proliferation. This make IFN-γ a key factor in optimizing the immunoregulatory function of MSCs.
• Vascular endothelial growth factor (VEGF): It promotes the angiogenic ability of MSCs and is commonly used in combination to induce vascularization tissue engineering constructs.

Mastering the action patterns and application techniques of cytokines is the core prerequisite for achieving efficient culture and functional regulation of MSCs, laying the foundation for their application in cell therapy, tissue engineering, and other fields. With the development of culture technology, combined with biomaterial loading, gene editing and other means to optimize the delivery and effect of cytokines, the application boundary of MSCs will be further expanded.

Yeasen HiActi^® Cytokines

Yeasen has developed a series of HiActi® cytokines specifically designed for cell culture. Strict quality control and validation of cellular functions have ensured that the products have high activity, high purity, high stability, and low endotoxin levels. There are a wide variety of products, of which FGF-2, BMP-2, IGF-1, IL-6, SCF, PDGF, VEGF and other cytokines are suitable for the culture and functional study of mesenchymal stem cells.

Product Data

Bioactivity of human bFGF/FGF-2/FGF-basic

Figure 4. The ED₅₀ as determined by a cell proliferation assay using murine balb/c 3T3 cells is less than 1 ng/mL, corresponding to a specific activity of > 1.0 × 10⁶ IU/mg.

Bioactivity of Human/Mouse/Rat TGF-beta 1

Figure 5. Measured by the (CAGA) 12-luciferase reporter assay. The EC₅₀ for this effect is 1.092 ng/mL.

Bioactivity of human BMP-2

Figure 6. The ED₅₀ as determined by alkaline phosphatase inducing production of murine ATDC5 cells is 200 ng/mL, corresponding to a specific activity of > 5.0 × 10³ IU/mg.

Ordering Information

Product Name	CAS.NO	Size
Recombinant Human bFGF/FGF-2 Protein	91330ES	10μg/100μg/500μg/1mg
Recombinant Mouse bFGF/FGF-2 Protein	91315ES	10μg/50μg/100μg/500μg
Recombinant Human EGF Protein	92708ES	100μg/500μg/1mg
Recombinant Mouse EGF Protein	92703ES	100μg/500μg/1mg
Recombinant Human FGF-4 Protein	91303ES	5μg/50μg/100μg/500μg
Recombinant Mouse FGF-4 Protein	91331ES	5μg/25μg/50μg/100μg/500μg
Recombinant Human IGF-1 Protein	92211ES	10μg/100μg/500μg/1mg
Recombinant Mouse IGF-1	92208ES	10μg/100μg/500μg
Recombinant Human PDGF-BB Protein	91605ES	2μg/10μg/50μg/100μg/500μg
Recombinant Mouse PDGF-BB	91602ES	10μg/50μg/100μg/500μg
Recombinant Human/Mouse/Rat TGF-beta 1/TGF-β1 Protein	91701ES	2μg/10μg/100μg
Recombinant Human TGF-β2 Protein (CHO)	91709ES	10μg/50μg/1mg
Recombinant Human TGF-beta 3 Protein	91705ES	10μg/50μg/100μg
Recombinant Rat VEGF164	91512ES	10μg/100μg/500μg
Recombinant Human VEGF165 Protein	91517ES	2μg/10μg/50μg/100μg/1mg
Recombinant Human IL-6 Protein	90107ES	5μg/20μg/50μg/100μg/1mg
Recombinant Mouse IL-6 Protein	90146ES	2μg/10μg/50μg/100μg/500μg
Recombinant Human SCF Protein	92251ES	2μg/10μg/50μg/100μg/500μg/1mg
Recombinant Mouse SCF Protein	92260ES	2μg/10μg/50μg/100μg/500μg
Recombinant Human BMP-2 Protein, His Tag	92060ES	10μg/50μg/250μg/500μg
Recombinant Human/Rhesus macaque/Mouse/Rat/Canine BMP2 Protein	95644ES	25μg/100μg/500μg
Recombinant Human BMP-4 Protein, His Tag	92070ES	2μg/10μg/50μg/250μg/500μg
Recombinant Mouse BMP-4 Protein, His Tag	92056ES	5μg/20μg/100μg/500μg/1mg
Recombinant Human TNF-alpha Protein, His tag	90601ES	10μg/100μg/500μg
Recombinant Mouse TNF-α/TNFSF2	90621ES	5μg/20μg/50μg/100μg/1mg
Recombinant Human IFN-γ Protein	91207ES	20μg/50μg/100μg/500μg
Recombinant Mouse IFN-gamma Protein	91212ES	5μg/50μg/100μg/500μg

 

1. Han Y, Li X, Zhang Y, Han Y, Chang F, Ding J. Mesenchymal Stem Cells for Regenerative Medicine. Cells 2019; 8(8).