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Home › Learning centers › Stem cell research › Technical notes › Neural stem cells › RHB-A neural stem cell medium

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A fully defined, serum-free culture system for neural stem cell maintenance and differentiation

RHB-A cell culture medium

  • Improved maintenance and expansion of human neural stem cells
  • Differentiation of human pluripotent stem cells into neurons and astrocytes
Introduction Results Conclusions Methods

Introduction  

Differentiation into cells of neural lineage is a challenging and time-consuming process. Conventional differentiation media initially provided a baseline culture system for generating neural cells. However, these media were relatively inefficient, giving low percentages of neural cells, and cumbersome, in that the type of medium used for differentiation had to be changed completely before expansion of intermediate neural stem (NS) cells. Furthermore, these media were often specific only to mouse cells, necessitating the development of a medium compatible with human cells.

RHB-A medium is the next-generation formulation of N2B27-based medium for NS cell culture and neural differentiation. This optimized medium was developed to both improve the differentiation of human pluripotent stem cells into neurons, while also enabling the maintenance and expansion of NS cells upon supplementation with epidermal growth factor (EGF) and fibroblast growth factor-basic (FGF-basic).

In the following experiments, RHB-A medium was directly compared to different vendors' media for the expansion of human NS cells and was further tested for the differentiation of those expanded cells into both neurons and astrocytes.

Results  

Maintenance of linear growth

A human NS cell line (Human Neural Cortex Stem Cell Line Kit, Cat. # Y40050) was grown in either the RHB-A system or a system from one of two different vendors (Vendors A and B) for 35 days. The cells in the RHB-A system showed a linear growth curve until Day 28, whereas cells in Vendor A's system showed little growth during cultivation, and Vendor B's system was unable to maintain the NS cell culture.

Fold-expansion profiles of human NS cell lines cultured in different media

Figure 1. Fold-expansion profiles of human NS cell lines cultured in different media.

Differentiation into neurons and astrocytes

NS cells cultured for 28 days in the RHB-A system (as shown above) were then differentiated into neurons or astrocytes, as described in the Methods section. Following differentiation, cells were immunolabeled for microtubule-associated protein 2 (MAP2) and glial fibrillary acidic protein (GFAP), markers for neurons and astrocytes, respectively. Under conditions for neuron differentiation, most of the cells were positively labeled with an anti-MAP2 antibody but showed no labeling with the anti-GFAP antibody. On the other hand, under conditions for astrocyte differentiation, the culture showed mostly GFAP-positive cells and much lower number of cells showed positive staining for MAP2. Together, these results provide visual confirmation of the differentiation of NS cells into neurons and astrocytes while cultured in RHB-A medium.

Immunolabeling of neuron marker, MAP2, and astrocyte marker, GFAP

Figure 2. Immunolabeling of neuron marker, MAP2, and astrocyte marker, GFAP.

Conclusions  

The RHB-A system enables the stable proliferation of NS cell cultures while maintaining neuronal and astrocytic differentiation potential. Thus, this second-generation N2B27 medium can be widely used as a basal medium for the maintenance, expansion, and differentiation of NS cells. Flexibility is offered in the form of RHB-BASAL medium, which does not contain any growth factors or neuronal supplements, allowing it to be tailored to specific requirements depending on cell type.

Methods  

Cell culture

The human neural cortex stem cell line (Human Neural Cortex Stem Cell Line Kit) was cultured for four weeks in the RHB-A system, StemPro NSC SFM (Thermo Fisher Scientific), or Stemline Neural Stem Cell Expansion Medium (Sigma-Aldrich Corp.). The cells were cultured according to each manufacturer's recommendations. Tissue culture plates precoated with 10 µg/ml of laminin (Thermo Fisher Scientific) were used for the RHB-A system, with EGF (PeproTech) and FGF-basic (PeproTech) at final concentrations of 20 ng/ml each in the medium. The cells were seeded at 4 x 104 cells/cm2 and passaged once a week.

Differentiation into neurons or astrocytes

Human neural cortex system cells were cultured for four weeks in the RHB-A system. The resulting expanded cells were differentiated into neurons or astrocytes with the procedures described below.

For differentiation into neurons, cells were seeded at 25,000 cells/cm2 on plates precoated with poly-L-ornithine and laminin, and cultured in RHB-BASAL medium supplemented with 0.5% NDiff N2-AF, 1% B27 supplement (Thermo Fisher Scientific), and 10 ng/ml of FGF-basic (Day 0). On Day 7, the differentiation medium was switched to a 1:1 ratio of RHB-BASAL medium to Neurobasal medium, minus phenol red (Thermo Fisher Scientific), supplemented with 0.25% NDiff N2-AF, 1% B27 supplement, 10 ng/ml of FGF-basic, and 0.5% GlutaMAX (Thermo Fisher Scientific). On Day 14, the differentiation medium was switched to Neurobasal medium, minus phenol red, supplemented with 2% B27 supplement, 20 ng/ml BDNF (PeproTech), and 1% GlutaMAX. The cells were cultured for an additional 14 days. During cultivation, the differentiation medium was changed every other day.

For differentiation into astrocytes, the cells were seeded at 25,000 cells/cm2 on plates precoated with poly‑L‑ornithine and laminin, and cultured in RHB-A medium supplemented with 10 ng/ml of BMP-4 (R&D Systems) for 22 days. During cultivation, the differentiation medium was changed every other day.

After differentiation, GFAP and MAP2 expression levels were examined by immunolabeling.

Related Products

Cat. # Product Size Price License Quantity Details
Y40001 RHB-A® 500 mL USD $301.00

RHB-A, supplemented with Epidermal Growth Factor (EGF) and Fibroblast Growth Factor-2 (FGF-2), enables the maintenance and continual expansion of symmetrically-dividing NS cells in defined, serum-free adherent culture. In growth factor supplemented RHB-A, NS cells have been demonstrated to retain their neurogenic capacity for over 100 generations, with full maintenance of diploid karyotype. RHB-A in the presence of EGF and FGF-2 also supports the derivation of clonogenic NS cell lines. Culture of adherent NS cells in RHB-A with sequential growth factor withdrawal leads to differentiation into functional neurons. RHB-A supplemented with EGF and FGF-2 has recently been used for the propagation of glioblastoma stem cell lines.

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Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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Adherent human neural stem cells differentiated in RHB-A culture medium express neural lineage markers, including microtubule associate protein 2 (Panel B), neuron-specific class III beta-tubulin (Panel C), gamma amino butyric acid (Panel D), and glial fibrillary acidic protein (Panel E)

Adherent human neural stem cells differentiated in RHB-A culture medium express neural lineage markers, including microtubule associate protein 2 (Panel B), neuron-specific class III beta-tubulin (Panel C), gamma amino butyric acid (Panel D), and glial fibrillary acidic protein (Panel E)
Adherent human neural stem cells differentiated in RHB-A culture medium express neural lineage markers, including microtubule associate protein 2 (Panel B), neuron-specific class III beta-tubulin (Panel C), gamma amino butyric acid (Panel D), and glial fibrillary acidic protein (Panel E). A phase contrast image (Panel A) and a merge of neuron-specific class III beta-tubulin and glial fibrillary acidic protein images (F) are also shown.

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Adherent human neural stem cells cultured in RHB-A medium supplemented with epidermal growth factor and fibroblast growth factor-2 express neural stem cell markers

Adherent human neural stem cells cultured in RHB-A medium supplemented with epidermal growth factor and fibroblast growth factor-2 express neural stem cell markers
Adherent human neural stem cells cultured in RHB-A medium supplemented with epidermal growth factor and fibroblast growth factor-2 express neural stem cell markers. Expression of nestin (Panel A), vimentin (Panel B), 3CB2 (Panel C), and microtubule associated protein 2 (Panel D) were assessed by immunofluorescence.

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Adherent mouse neural stem cells cultured in RHB-A medium supplemented with epidermal growth factor and fibroblast growth factor-2 express sex determining region Y-box 2 (Sox2, red) and Nestin (green)

Adherent mouse neural stem cells cultured in RHB-A medium supplemented with epidermal growth factor and fibroblast growth factor-2 express sex determining region Y-box 2 (Sox2, red) and Nestin (green)
Adherent mouse neural stem cells cultured in RHB-A medium supplemented with epidermal growth factor and fibroblast growth factor-2 express sex determining region Y-box 2 (Sox2, red) and Nestin (green).

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Adherent mouse neural stem cells cultured in RHB-A medium supplemented with epidermal growth factor and fibroblast growth factor-2 express neural stem cell markers, including Nestin, Vimentin (3CB2), radial glial cell marker-2 (RC2), glial fibrillary acidic protein (GFAP), and microtubule associate protein (MAP)

Adherent mouse neural stem cells cultured in RHB-A medium supplemented with epidermal growth factor and fibroblast growth factor-2 express neural stem cell markers, including Nestin, Vimentin (3CB2), radial glial cell marker-2 (RC2), glial fibrillary acidic protein (GFAP), and microtubule associate protein (MAP)
Adherent mouse neural stem cells cultured in RHB-A medium supplemented with epidermal growth factor and fibroblast growth factor-2 express neural stem cell markers, including Nestin, Vimentin (3CB2), radial glial cell marker-2 (RC2), glial fibrillary acidic protein (GFAP), and microtubule associate protein (MAP).

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Human neural cortex stem cell lines were differentiated into either neurons (MAP2, red) or astrocytes (GFAP, green) using RHB-A

Human neural cortex stem cell lines were differentiated into either neurons (MAP2, red) or astrocytes (GFAP, green) using RHB-A
Human neural cortex stem cell lines were differentiated into either neurons (MAP2, red) or astrocytes (GFAP, green) using RHB-A.

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Fold expansion profiles of human neural stem cell lines cultured by different vendor’s systems

Fold expansion profiles of human neural stem cell lines cultured by different vendor’s systems
Fold expansion profiles of human neural stem cell lines cultured by different vendor’s systems. RHB-A exhibits robust growth and expansion of human neural stem cells.

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Y40001: RHB-A

Y40001: RHB-A
Y40000 RHB-BASAL™ 500 mL USD $191.00

RHB-Basal is a proprietary, defined, serum-free basal medium specifically formulated for the propagation and differentiation of adherent NS cells. RHB-Basal does not contain any growth factors or neuronal supplements. Therefore, customers can tailor RHB-Basal to suit the specific requirements of their cell type by the addition of preferred supplements.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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