J Korean Soc Spine Surg.  2006 Dec;13(4):225-233.

Establishment of a Multipotent Neural Stem Cell Line from the Adult Mouse Cerebrum

• Kim KS
• Choi YS
• Seo SY
• Kim BH
• Kim YS
• Lee MC

Abstract

STUDY DESIGN: Establishment of a multipotent neural stem cell line from the adult mouse cerebrum.
OBJECTIVES
To establish a daughter cell line, B2A1, from B2 cells through the limiting dilution method, and to determine if the cells have the characteristics of neural stem cell (NSCs) using immunocytochemistry and RT-PCR methods. SUMMARY AND LITERATURE REVIEW: In the development of NSCs, differentiated organ or tissue-derived multipotent stem cells have attracted considerable interest because of the lack of ethical issues. Previously, a glial precursor cell line (B2 cells) was generated from the primary cultures of oligodendrocytes/ astrocytes in an adult BALB/c mouse brain. These cells exhibited the cell-type specific markers for immature neuroectodermal cells, astrocytes, and oligodendrocytes in serum-contained media.
MATERIALS AND METHODS
The primary cultures of oligodendrocytes/astrocytes were established from the whole brains of 12 to 16-week-old BALB/c mice from either gender. After 6 months with 25 serial passages, the culture consisted of a morphologically homogeneous cell population, which was designated as B2 cells. A subclone, B2A1, was isolated from B2 cells through two consecutive limiting dilutions.
RESULTS
More than 90% of B2A1 cells showed immunopositivity for nestin, a specific marker for NSC. The cells also showed immunopositivity for the neuronal, astrocytic and oligodendroglial markers. These cells expressed the genotypic mRNA messages for both neural progenitor cells and differentiated neuronoglial cells. These positive immunocytochemical reactions and mRNA messages for neuronoglial cells varied according to the extrinsic growth factors used. However, the treatment of extrinsic growth factors did not produce any significant differences in the nestin-immunopositive cells.
CONCLUSIONS
B2A1 cells have the immunocytochemical and cytogenetic properties of NSCs, and the capacity to differentiate into neuronoglial cells.

Keyword

Neural stem cell; B2A1 cell; Adult mouse cerebrum

MeSH Terms

Adult*
Animals
Astrocytes
Brain
Cell Line
Cerebrum*
Cytogenetics
Ethics
Humans
Immunohistochemistry
Intercellular Signaling Peptides and Proteins
Mice*
Multipotent Stem Cells
Nestin
Neural Plate
Neural Stem Cells*
Neurons
Nuclear Family
Oligodendroglia
RNA, Messenger
Serial Passage
Stem Cells
Intercellular Signaling Peptides and Proteins
RNA, Messenger

Figure

• 

  Fig. 1. Cytologic characteristics of B2A1 cells disclosed bi- or tripolar cells (A) phase contrast microscopy (B) and show immunopositivities for nestin (C) β tubulin III, (D) and GFAP.

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  Fig. 2. Multipotential differentiation of B2A1 cells by treatment of growth factors. Most of all cells were immunopositive for nestin in any culture condition (A). Immunopositive cells for βtubulin III significantly increased in PDGF-contained media (B), while GFAP (C) and galactocerebroside (D) positive cells markedly increased in c-AMP or PDGF-contained media.

• 

  Fig. 3. Gene expression of cell type specific markers studied by RT-PCR in B2A1 cells. The cells expressed both neural progenitor cell markers (nestin, Notch1, PDGFα) and differentiated cell markers for neurons (NF-L), astrocytes (GFAP), and oligodendrocytes (MBP). The expression levels were variable related to addition of growth factors.

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