Cytokines: The Code to Unlock the Fate of Neural Stem Cells

What are Neural Stem Cells?

Neural Stem Cells (NSCs) are a type of stem cells present in the nervous system, possessing self-renewal capability and the potential to differentiate into neural cells (neurons) and glial cells (e.g., astrocytes and oligodendrocytes), which are primarily responsible for the development, repair, and functional maintenance of the nervous system.

Sources of Neural Stem Cells

Neural stem cells have diverse sources and can be categorized into two major types based on the acquisition scenario (embryonic/adult), tissue locations, and technical methods: endogenous natural sources and exogenous induced/cultured sources. Different sources of neural stem cells exhibit significant differences in acquisition difficulty, ethical controversy, and application scenarios.

Figure 1. Sources of neural stem cells

Differentiation of Neural Stem Cells

The differentiation of neural stem cells refers to the process by which neural stem cells gradually transform into functionally specialized terminal neural cells (e.g., neurons, astrocytes, and oligodendrocytes) under the regulation of specific internal and external factors. This process serves as the foundation for brain development and repair.

Neural stem cells primarily differentiate into the following three cell types:

1. Neurons: Responsible for receiving, integrating, and transmitting neural signals, forming the core of the nervous system functions.

2. Astrocytes: Provide structural support, maintain homeostasis of the neural microenvironment, and regulate synaptic functions.

3. Oligodendrocytes: Wrap axons to form myelin sheaths, insulating nerve fibers and accelerating electrical signal conduction.

Figure 2. Activation and differentiation of NSCs processes are controlled by multiple signalling molecules. ^[1]

Applications of Neural Stem Cells in Disease Treatment

Leveraging their self-renewal capacity and multipotent differentiation potential, neural stem cells achieve "repair and replacement" and "environmental regulation" for nervous system injuries or degenerative diseases. Currently, there are clear application directions and research progress in multiple disease areas.

I. Central Nervous System Degenerative Diseases

a) Alzheimer's Disease (AD):

• Mechanism: Transplanted NSCs differentiate into functional neurons, replenishing cells and reconstructing circuits; NSCs-secreted BDNF/NGF, inhibits Aβ deposition and tau phosphorylation, thereby alleviating neuroinflammation.
• Animal Experiments: In APP/PS1 mouse models, transplanted human-derived NSCs, the cells survive and differentiate into neurons, which improve the learning and memory abilities of the mice.

b) Parkinson's Disease (PD):

• Key Strategy: Overexpression of transcription factors such as Nurr1 and Lmx1a increases the differentiation efficiency of NSCs into dopaminergic neurons to over 50%.
• Animal Experiments: Transplantation into PD rats improved rotational behavior, increased dopamine levels, with no significant tumorigenic risk observed.

II. Central Nervous System Injuries

a) Spinal Cord Injury (SCI):

• Mechanism: NSCs differentiate into oligodendrocytes to restoring neural signal conduction; secreted IL-10/TGF-β inhibit excessive inflammatory responses after injury and reduce scar tissue formation.
• Animal Experiments: Post-transplantation promoted the recovery of hindlimb motor function in rats, increased axonal regeneration, with cell survival exceeding 6 months.

b) Cerebral Infarction:

• Approach: Supplement exogenous NSCs or activate "dormant" endogenous NSCs around the lesion.
• Animal Experiments: Within 1-2 weeks post-transplantation, rats showed improved limb movement and spatial cognition, reduced infarct volume, and no significant immune rejection.

III. Mental Disorders and Rare Diseases

a) Depression:

• Association: Reduced neurogenesis in the hippocampal region is associated with depressive behaviors.
• Animal Experiments: Injection of bFGF in CUMS mice activated endogenous NSCs, increased sucrose preference rate, shortened immobility time, and enhanced hippocampal neurogenesis.

b) Spinal Muscular Atrophy (SMA):

• Strategy: NSCs combined with gene therapy (transfection with viral vectors containing the SMN1 gene).
• Animal Experiments: Transplanted cells differentiated into motor neurons, expressed SMN protein, prolonged survival time, and improved motor function in mice.

Culture of Neural Stem Cells

I. Culture Methods of Neural Stem Cells

The culture methods for neural stem cells mainly include two categories: adherent culture and suspension neurosphere culture. The core principle is to simulate the in vivo neurogenic microenvironment to maintain their self-renewal capacity and differentiation potential.

Adherent Culture

Core Principle	Operational Points	Advantages	Disadvantages
Coat the culture dishs with substrates (e.g., polylysine, laminin) to make cells adhere, facilitating observation.	1. Culture dishes need to be pre-coated with substrate and incubated for 1-2 hours. 2. Medium composition is similar to suspension method; growth factor concentration can be appropriately reduced. 3. When the cell confluency reaches 70%-80%, trypsin is used for digestion and passage.	1. Uniform cell growth state, convenient for microscopic observation and manipulation. 2. Suitable for experiments like immunofluorescence staining and drug treatment. 3. Reduces differentiation heterogeneity caused by cell aggregation.	1. Slower expansion speed compared to suspension method. 2. Coating steps increase operational complexity and time. 3. Long-term culture may lead to partial cell differentiation.

Suspension Culture

Core Principle	Key Operational Points	Advantages	Disadvantages
Cells aggregate via autocrine factors to form spherical structures (neurospheres), maintaining stemness.	1. Medium requires addition of cytokines like EGF, bFGF. 2. No need to coat culture dishes; requires regular gentle pipetting to prevent attachment. 3. Passage every 3-5 days at a 1:3 ratio to avoid central necrosis due to oversized neurospheres.	1. Expand cell numbers rapidly. 2. Closer to the aggregated growth state of NSCs in vivo. 3. Relatively simple operation.	1. Uneven nutrient distribution inside neurospheres can lead to differentiation differences. 2. Difficult to perform experiments at the single-cell level (e.g., transfection).

Key Universal Operational Steps (Taking Suspension Culture as Example)

1. Cell Source Acquisition: The hippocampus and cortex of embryonic mice/rats, or the subventricular zone (SVZ) and hippocampal dentate gyrus (DG) of adult animals are commonly used. After isolating the tissues, trypsin or papain is used to digest them into single - cell suspensions.

2. Medium Preparation: Prepare serum-free DMEM/F12 medium supplemented with 2% B27 (to maintain neuronal viability), 20 ng/mL EGF, 20 ng/mL bFGF (to maintain stemness), and 1% penicillin-streptomycin (to prevent contamination).

3. Seeding and Culture: Seed the single-cell suspension at a density of 1x10⁵ - 5x10⁵ cells/mL into low-adhesion culture dishes. Incubate at 37°C in a 5% CO₂ incubator, observing neurosphere formation daily.

4. Passaging: When neurospheres reach 100-150 μm in diameter, gently pipette them into single cells or small clusters. Resuspend in fresh medium after centrifugation and pass at the appropriate ratio.

5. Identification and Detection: Detect stemness markers (such as Nestin and Sox2) through immunofluorescence staining, or detect the markers of neurons (β - III Tubulin) and astrocytes (GFAP) after induced differentiation to verify the stemness of the cells.

II. Induced Differentiation of Neural Stem Cells

The core of neural stem cell differentiation induction involves modulating the culture environment (e.g., removing growth factors, adding inducers) to direct cells from a stem state toward specialized differentiation into neurons, astrocytes, or oligodendrocytes. The differentiation pathway is primarily determined by the type of inducer and the culture system. Below is a comparison of induction protocols for the three major cell types:

Differentiation Directions	Key Induction Conditions	Induction Period	Identification Markers (Positive)
Neurons	1. Remove EGF/bFGF. 2. Add 10 μM Retinoic Acid (RA). 3. Use Poly-L-lysine coated plates.	7-10 days	β-III Tubulin (Tuj1), MAP2
Astrocytes	1. Add 10 ng/mL LIF to base medium. 2. Maintain 37°C, 5% CO₂ environment. 3. Refresh medium every 3 days.	14-21 days	Glial Fibrillary Acidic Protein (GFAP)
Oligodendrocytes	1. Culture with 10 ng/mL PDGF-AA for 7 days first. 2. Then switch to 50 ng/mL T3 medium. 3. Use low-adhesion culture plates throughout.	21-28 days	O4 antibody, Myelin Basic Protein (MBP)

 Key Cytokines in Neural Stem Cell Culture

Cytokines serve as the core regulatory signals in neural stem cell culture, directly determining whether cells maintain pluripotency, achieve proliferation, or initiate directed differentiation. They function as the "switch" and "fuel" of the entire culture system.

I. Core Functions: Providing Key Instructions

a) Maintaining Stemness and Promoting Proliferation

This is the most fundamental and crucial function of cytokines – keeping stem cells in a "young" state and enabling continuous division and expansion.

• Key Factors:

o Basic Fibroblast Growth Factor (bFGF/FGF-2): A key factor maintaining NSCs proliferation and self-renewal. It activates signaling pathways like MAPK, promotes cell cycle progression, and prevents premature differentiation.

o Epidermal Growth Factor (EGF): Binds to EGFR receptors on the cell surface, activating core signaling pathways PI3K-AKT and RAS-MAPK. Maintains multipotent differentiation capacity and promotes cell division.

· Mechanism of Action: These cytokines bind to the receptors on the cell surface, activate downstream signal pathways, transmit the instruction of "continue to divide, do not differentiate" to the nucleus, and initiate the expression of genes related to proliferation.

b) Guiding Directed Differentiation

When it is necessary for NSCs to differentiate into specific functional cells, factors maintaining stemness must be withdrawn, and new "differentiation commands" must be added.

• Differentiation into Neurons:

o Brain-Derived Neurotrophic Factor (BDNF): Binds to TrkB receptors, activating pathways that promote neuronal survival, maturation, synaptogenesis, and neurotransmitter expression. It is the key to obtaining functional neurons.

o Neurotrophin-3 (NT-3): Supports the survival and differentiation of specific neuronal subpopulations.

• Differentiation into Oligodendrocyte:

o Platelet-Derived Growth Factor-AA (PDGF-AA): Binds to PDGFRα receptors on oligodendrocyte precursor cells (OPCs), activating PI3K-AKT and PLCγ pathways, promoting the proliferation and migration of OPCs.

o Triiodothyronine (T3): A key hormone driving the maturation of OPCs into myelinating mature oligodendrocytes.

• Differentiation into Astrocytes:

o Leukemia Inhibitory Factor (LIF): Binds to the LIFR/gp130 receptor complex, activating the JAK-STAT3 signaling pathway, effectively inducing astrocyte differentiation.

o Serum (e.g., Fetal Bovine Serum, FBS): Contains various unknown inducing factors that can strongly induce astrocyte differentiation. However, due to its undefined composition, it is rarely used in precise research.

c) Promoting Cell Survival

NSCs are relatively fragile in vitro and prone to apoptosis. Many cytokines also have significant anti-apoptotic and pro-survival effects.

• Example: BDNF, IGF-1 (Insulin-like Growth Factor-1), etc., can significantly improve cell survival rates.

II. Application Strategies: Synergy and Timing

A successful culture protocol is not merely the simple addition of a single factor, but rather an art of multi-factor synergy and temporal control.

a) Synergistic Effects:

• bFGF + EGF: Synergistically promote NSCs expansion, with effects superior to single factors.
• BDNF + NT-3: Combined action enhances the efficiency and maturity of neuronal differentiation.

b) Temporal Regulation:

A typical culture and differentiation process is as follows (taking neuronal differentiation as example):

• Proliferation Phase: Use bFGF + EGF to extensively expand NSCs while maintaining stemness.
• Differentiation Induction Phase: Remove all mitogens and switch to BDNF to initiate the neuronal differentiation program.
• Maturation Phase: Continue BDNF treatment and optionally add cAMP to promote neuronal network maturation and functional synapse formation.

Cytokines serve as the "instructional language" governing neural stem cell fate. Deeply understanding the functions of core factors like bFGF, EGF, and BDNF, and mastering their "application syntax" in synergistic use and temporal regulation, is fundamental to successfully achieving NSCs culture, expansion, and directed differentiation in vitro. This ultimately enables their application in disease modeling, drug screening, and cell therapy.

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. In the process of NSCs culture, multiple key cytokines, such as bFGF, LIF, EGF, and TGF-β, are usually required to promote their proliferation and differentiation. Yeasen Biotech's high-quality cytokines can support NSCs research.

Product Data

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

Figure 3. 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 EGF

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. Fully biologically active when compared to standard.

Bioactivity of human IL-6

Figure 5. The ED₅₀ as determined by a cell proliferation assay using human TF-1 cells is 0.1-0.5 ng/mL.

Ordering Information

Name	Cat.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 FGF-10/KGF-2 Protein	91306ES	2μg/10μg/50μg/100μg/500μg/1mg
Recombinant Mouse KGF-2/FGF-10	91319ES	5μg/25μg/50μg/100μg/500μg
Recombinant Human FGF8b Protein	95132ES	25μg/100μg/500μg
Recombinant Mouse FGF-8 Protein	91317ES	5μg/100μg/500μg
Recombinant Human IGF-1 Protein	92211ES	10μg/100μg/500μg/1mg
Recombinant Mouse IGF-1	92208ES	10μg/100μg/500μg
Recombinant Human PDGF-AA Protein	91601ES	2μg/10μg/50μg/100μg/250μg/500μg
Recombinant Rat PDGF-AA	91604ES	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 Human/Mouse/Rat BDNF Protein	92129ES	25μg/100μg/500μg/1mg
Recombinant Human Neurotrophin 3/NT-3	92128ES	20μg/100μg
Recombinant Human NT-4 Protein	92114ES	2μg/10μg/50μg/100μg
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 LIF Protein	92111ES	5μg/50μg/100μg/500μg
Recombinant Mouse LIF Protein	92256ES	5μg/50μg/100μg/500μg
Recombinant Human CNTF Protein	92123ES	5μg/20μg/100μg/500μg
Recombinant Mouse CNTF	92124ES	5μg/50μg/100μg/500μg
Recombinant Human GDNF Protein	92102ES	2μg/10μg/50μg/100μg
Recombinant Mouse GDNF Protein	92103ES	2μg/10μg/50μg/100μg
Recombinant Human SHH	92566ES	5μg/25μg/50μg/100μg
Recombinant Mouse Sonic Hedgehog/SHH Protein	92589ES	5μg/25μg/100μg

 

1. Kaminska A, Radoszkiewicz K, Rybkowska P, Wedzinska A, Sarnowska A. Interaction of Neural Stem Cells (NSCs) and Mesenchymal Stem Cells (MSCs) as a Promising Approach in Brain Study and Nerve Regeneration. Cells 2022; 11(9).