Characterization of primary human fetal dissociated central nervous system cultures with an emphasis on microglia

S. C. Lee, W. Liu, C. F. Brosnan, Dennis W Dickson

Research output: Contribution to journalArticle

136 Citations (Scopus)

Abstract

BACKGROUND: We undertook a systematic examination of human fetal central nervous system dissociated cell cultures, particularly with respect to microglia and their dynamic interactions with other central nervous system cell components. EXPERIMENTAL DESIGN: The growth and differentiation of astrocytes, microglia, and small immature neuronal cells in mixed and single cell type-enriched cultures were followed by phase contrast microscopy, immunocytochemistry, flow cytometry, electron microscopy and immunoelectron microscopy, in regard to their phenotype change, proliferation and class II major histocompatibility complex antigen expression. RESULTS: Both astrocytes and microglia expressed marked phenotype heterogeneity, but they were consistently positive for glial fibrillary acidic protein and CD68, respectively. Highly enriched astrocyte and microglial cultures suitable for biochemical or molecular biologic studies were obtained. Using double immunocytochemical labeling techniques with the proliferating cell nuclear antigen and cell-type specific markers, astrocytes grown in serum-containing media showed a high labeling index, whereas microglia seldom exhibited a similar degree of proliferative activity. Microglia, however, proliferated in response to certain growth factors, such as granulocyte macrophage colony- stimulating factor. Both microglia and astrocytes expressed high basal levels of class II MHC antigens, which further increased with addition of interferon-γ. The microglia in dissociated cultures existed in two forms, ameboid and ramified. Microglial ramification was induced when ameboid microglia were co-cultured with a monolayer of astrocytes, suggesting a role for astrocytes in microglial differentiation. In addition, lipopolysaccharide in nanogram amounts consistently enhanced microglial survival and morphologic differentiation. The small bipolar cells, the most frequent cell type in mixed culture, were glial fibrillary acidic protein (-) and CD68 (-), and their size and shape remained unchanged under our culture conditions. A subpopulation of small bipolar cells was stained positive with an antibody to surface ganglioside, A2B5. Electron microscopic examination showed that their processes contained parallel microtubules bearing side arms consistent with immature neurons. CONCLUSIONS: Highly purified astrocyte and microglial cultures are obtained using the currently described technique. The current culture system is a valuable tool in studying human central nervous system biology and disease.

Original languageEnglish (US)
Pages (from-to)465-476
Number of pages12
JournalLaboratory Investigation
Volume67
Issue number4
StatePublished - 1992
Externally publishedYes

Fingerprint

Microglia
Astrocytes
Central Nervous System
Glial Fibrillary Acidic Protein
Histocompatibility Antigens Class II
Cell Culture Techniques
Phenotype
Phase-Contrast Microscopy
Systems Biology
Immunoelectron Microscopy
Central Nervous System Diseases
Proliferating Cell Nuclear Antigen
Cellular Structures
Granulocyte-Macrophage Colony-Stimulating Factor
Major Histocompatibility Complex
Microtubules
Interferons
Lipopolysaccharides
Intercellular Signaling Peptides and Proteins
Electron Microscopy

Keywords

  • Astrocytes
  • Class II major histocompatibility complex antigen
  • Human culture
  • Lipopolysaccharide
  • Microglia
  • Proliferation
  • Ramified microglia

ASJC Scopus subject areas

  • Pathology and Forensic Medicine

Cite this

Characterization of primary human fetal dissociated central nervous system cultures with an emphasis on microglia. / Lee, S. C.; Liu, W.; Brosnan, C. F.; Dickson, Dennis W.

In: Laboratory Investigation, Vol. 67, No. 4, 1992, p. 465-476.

Research output: Contribution to journalArticle

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N2 - BACKGROUND: We undertook a systematic examination of human fetal central nervous system dissociated cell cultures, particularly with respect to microglia and their dynamic interactions with other central nervous system cell components. EXPERIMENTAL DESIGN: The growth and differentiation of astrocytes, microglia, and small immature neuronal cells in mixed and single cell type-enriched cultures were followed by phase contrast microscopy, immunocytochemistry, flow cytometry, electron microscopy and immunoelectron microscopy, in regard to their phenotype change, proliferation and class II major histocompatibility complex antigen expression. RESULTS: Both astrocytes and microglia expressed marked phenotype heterogeneity, but they were consistently positive for glial fibrillary acidic protein and CD68, respectively. Highly enriched astrocyte and microglial cultures suitable for biochemical or molecular biologic studies were obtained. Using double immunocytochemical labeling techniques with the proliferating cell nuclear antigen and cell-type specific markers, astrocytes grown in serum-containing media showed a high labeling index, whereas microglia seldom exhibited a similar degree of proliferative activity. Microglia, however, proliferated in response to certain growth factors, such as granulocyte macrophage colony- stimulating factor. Both microglia and astrocytes expressed high basal levels of class II MHC antigens, which further increased with addition of interferon-γ. The microglia in dissociated cultures existed in two forms, ameboid and ramified. Microglial ramification was induced when ameboid microglia were co-cultured with a monolayer of astrocytes, suggesting a role for astrocytes in microglial differentiation. In addition, lipopolysaccharide in nanogram amounts consistently enhanced microglial survival and morphologic differentiation. The small bipolar cells, the most frequent cell type in mixed culture, were glial fibrillary acidic protein (-) and CD68 (-), and their size and shape remained unchanged under our culture conditions. A subpopulation of small bipolar cells was stained positive with an antibody to surface ganglioside, A2B5. Electron microscopic examination showed that their processes contained parallel microtubules bearing side arms consistent with immature neurons. CONCLUSIONS: Highly purified astrocyte and microglial cultures are obtained using the currently described technique. The current culture system is a valuable tool in studying human central nervous system biology and disease.

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