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A protocol for isolation and enriched monolayer cultivation of neural precursor cells from mouse dentate gyrus.

Babu H, Claasen JH, Kannan S, Rünker AE, Palmer T, Kempermann G - Front Neurosci (2011)

Bottom Line: The cultures are expanded under serum-free conditions in Neurobasal A medium with addition of the mitogens Epidermal growth factor and Fibroblast growth factor 2 as well as the supplements Glutamax-1 and B27.Under differentiation conditions, the precursor cells reliably generate approximately 30% neurons with appropriate morphological, molecular, and electrophysiological characteristics that might reflect granule cell properties as their in vivo counterpart.We also highlight potential modifications to the protocol.

View Article: PubMed Central - PubMed

Affiliation: Institute for Stem Cell Biology and Regenerative Medicine, Stanford University Stanford, CA, USA.

ABSTRACT
In vitro assays are valuable tools to study the characteristics of adult neural precursor cells under controlled conditions with a defined set of parameters. We here present a detailed protocol based on our previous original publication (Babu et al., 2007) to isolate neural precursor cells from the hippocampus of adult mice and maintain and propagate them as adherent monolayer cultures. The strategy is based on the use of Percoll density gradient centrifugation to enrich precursor cells from the micro-dissected dentate gyrus. Based on the expression of Nestin and Sox2, a culture-purity of more than 98% can be achieved. The cultures are expanded under serum-free conditions in Neurobasal A medium with addition of the mitogens Epidermal growth factor and Fibroblast growth factor 2 as well as the supplements Glutamax-1 and B27. Under differentiation conditions, the precursor cells reliably generate approximately 30% neurons with appropriate morphological, molecular, and electrophysiological characteristics that might reflect granule cell properties as their in vivo counterpart. We also highlight potential modifications to the protocol.

No MeSH data available.


In cultures of dentate gyrus neural precursor cells virtually all cells express cellular marker for proliferation, Ki67 (A), and for progenitor cells of the nervous system, such as Nestin (A–C), Sox2 (C) or vimentin (D). Many cells express also the radial glia marker BLBP (B) and Id1 (F), a dominant-negative bHLH transcriptional regulator expressed in a fraction of adult neural stem cells important for their self-renewal capacity. Only few cells express β3-tubulin (E) indicating neuronally committed cells. No cells were positive for S100β (F), a marker that labels mature astrocytes. Cells were grown until about 80% confluent (3–5 days) before fixing and staining. Blue stain in (A–F) is the nucleic dye DAPI. Scale bar in (A): 50 μm, in (A′) and (B) for (A′–F): 20 μm.
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Figure 3: In cultures of dentate gyrus neural precursor cells virtually all cells express cellular marker for proliferation, Ki67 (A), and for progenitor cells of the nervous system, such as Nestin (A–C), Sox2 (C) or vimentin (D). Many cells express also the radial glia marker BLBP (B) and Id1 (F), a dominant-negative bHLH transcriptional regulator expressed in a fraction of adult neural stem cells important for their self-renewal capacity. Only few cells express β3-tubulin (E) indicating neuronally committed cells. No cells were positive for S100β (F), a marker that labels mature astrocytes. Cells were grown until about 80% confluent (3–5 days) before fixing and staining. Blue stain in (A–F) is the nucleic dye DAPI. Scale bar in (A): 50 μm, in (A′) and (B) for (A′–F): 20 μm.

Mentions: The isolated and expanded cells display the typical morphology of adult neural precursor cells Figures 2A–C and express the typical neural precursor cell markers Nestin Figure 3C, or vimentin Figure 3D. Many cells also express the radial glia marker BLBP Figure 3B or transcription factor Id1 involved in self-renewal Figure 3F. In contrast, only few cells appear further committed to the neuronal lineage (expressing βIII-tubulin Figure 3E), and there are no identifiable mature astrocytes (expressing S100β Figure 3F). Virtually all cells express Ki67, a cellular marker for proliferation Figure 3A. Similarly, incubation with BrdU, a thymidine analog that is incorporated during DNA synthesis, labels almost all cells, indicating robust proliferation of the cultured neural precursor cells (not shown here, but see figures in Babu et al., 2007). Under differentiation conditions, neural precursor cells gradually acquire the morphology of postmitotic neurons and astrocytes (Figures2 D–F).


A protocol for isolation and enriched monolayer cultivation of neural precursor cells from mouse dentate gyrus.

Babu H, Claasen JH, Kannan S, Rünker AE, Palmer T, Kempermann G - Front Neurosci (2011)

In cultures of dentate gyrus neural precursor cells virtually all cells express cellular marker for proliferation, Ki67 (A), and for progenitor cells of the nervous system, such as Nestin (A–C), Sox2 (C) or vimentin (D). Many cells express also the radial glia marker BLBP (B) and Id1 (F), a dominant-negative bHLH transcriptional regulator expressed in a fraction of adult neural stem cells important for their self-renewal capacity. Only few cells express β3-tubulin (E) indicating neuronally committed cells. No cells were positive for S100β (F), a marker that labels mature astrocytes. Cells were grown until about 80% confluent (3–5 days) before fixing and staining. Blue stain in (A–F) is the nucleic dye DAPI. Scale bar in (A): 50 μm, in (A′) and (B) for (A′–F): 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3140691&req=5

Figure 3: In cultures of dentate gyrus neural precursor cells virtually all cells express cellular marker for proliferation, Ki67 (A), and for progenitor cells of the nervous system, such as Nestin (A–C), Sox2 (C) or vimentin (D). Many cells express also the radial glia marker BLBP (B) and Id1 (F), a dominant-negative bHLH transcriptional regulator expressed in a fraction of adult neural stem cells important for their self-renewal capacity. Only few cells express β3-tubulin (E) indicating neuronally committed cells. No cells were positive for S100β (F), a marker that labels mature astrocytes. Cells were grown until about 80% confluent (3–5 days) before fixing and staining. Blue stain in (A–F) is the nucleic dye DAPI. Scale bar in (A): 50 μm, in (A′) and (B) for (A′–F): 20 μm.
Mentions: The isolated and expanded cells display the typical morphology of adult neural precursor cells Figures 2A–C and express the typical neural precursor cell markers Nestin Figure 3C, or vimentin Figure 3D. Many cells also express the radial glia marker BLBP Figure 3B or transcription factor Id1 involved in self-renewal Figure 3F. In contrast, only few cells appear further committed to the neuronal lineage (expressing βIII-tubulin Figure 3E), and there are no identifiable mature astrocytes (expressing S100β Figure 3F). Virtually all cells express Ki67, a cellular marker for proliferation Figure 3A. Similarly, incubation with BrdU, a thymidine analog that is incorporated during DNA synthesis, labels almost all cells, indicating robust proliferation of the cultured neural precursor cells (not shown here, but see figures in Babu et al., 2007). Under differentiation conditions, neural precursor cells gradually acquire the morphology of postmitotic neurons and astrocytes (Figures2 D–F).

Bottom Line: The cultures are expanded under serum-free conditions in Neurobasal A medium with addition of the mitogens Epidermal growth factor and Fibroblast growth factor 2 as well as the supplements Glutamax-1 and B27.Under differentiation conditions, the precursor cells reliably generate approximately 30% neurons with appropriate morphological, molecular, and electrophysiological characteristics that might reflect granule cell properties as their in vivo counterpart.We also highlight potential modifications to the protocol.

View Article: PubMed Central - PubMed

Affiliation: Institute for Stem Cell Biology and Regenerative Medicine, Stanford University Stanford, CA, USA.

ABSTRACT
In vitro assays are valuable tools to study the characteristics of adult neural precursor cells under controlled conditions with a defined set of parameters. We here present a detailed protocol based on our previous original publication (Babu et al., 2007) to isolate neural precursor cells from the hippocampus of adult mice and maintain and propagate them as adherent monolayer cultures. The strategy is based on the use of Percoll density gradient centrifugation to enrich precursor cells from the micro-dissected dentate gyrus. Based on the expression of Nestin and Sox2, a culture-purity of more than 98% can be achieved. The cultures are expanded under serum-free conditions in Neurobasal A medium with addition of the mitogens Epidermal growth factor and Fibroblast growth factor 2 as well as the supplements Glutamax-1 and B27. Under differentiation conditions, the precursor cells reliably generate approximately 30% neurons with appropriate morphological, molecular, and electrophysiological characteristics that might reflect granule cell properties as their in vivo counterpart. We also highlight potential modifications to the protocol.

No MeSH data available.