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A novel method for isolating Schwann cells using the extracellular domain of Necl1.

ABSTRACT: Myelinating cocultures of Schwann cells and dorsal root ganglion neurons are a powerful experimental system for probing the molecular mechanisms of axon-Schwann cell interaction. The isolation of a pure population of myelination-competent Schwann cells is a prerequisite for this experimental system. We describe here a protocol for a FACS-based isolation of Schwann cells utilizing a specific affinity reagent (Necl1-Fc) and the use of these isolated cells in myelinating cocultures. An advantage of the myelinating coculture system is that Schwann cells and the neurons can be genetically manipulated before they are cocultured. We further show that our method allows the isolation of virally transduced Schwann cells in a single purification step. This protocol for the FACS-based isolation of myelination-competent Schwann cells by Necl1-Fc and the use of these cells in myelinating cocultures should significantly facilitate future studies aimed at delineation of the molecular mechanisms of axon-Schwann cell interactions and myelination.

SUBMITTER: Spiegel I

PROVIDER: S-EPMC2760645 | biostudies-literature | 2009 Nov

REPOSITORIES: biostudies-literature

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A novel method for isolating Schwann cells using the extracellular domain of Necl1.

Spiegel Ivo I Peles Elior E

Journal of neuroscience research 20091101 15

Myelinating cocultures of Schwann cells and dorsal root ganglion neurons are a powerful experimental system for probing the molecular mechanisms of axon-Schwann cell interaction. The isolation of a pure population of myelination-competent Schwann cells is a prerequisite for this experimental system. We describe here a protocol for a FACS-based isolation of Schwann cells utilizing a specific affinity reagent (Necl1-Fc) and the use of these isolated cells in myelinating cocultures. An advantage of ...[more]

PMID: 19125407

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Project description:Extracellular vesicles that are derived from stem cells play an important role in the treatment of disease. To obtain high-quality small extracellular vesicles (sEVs), we optimized the culture conditions of human induced pluripotent stem cells (hiPSCs), the supernatant collection time, and sEVs extraction methods. Firstly, hiPSCs were cultured in extracellular vesicles-production medium (EVs-PM) containing different concentrations (0%, 0.25%, 0.5%, 2%, 5%, and 20%) of extracellular vesicle-depleted knockout serum replacement (ED-KSR), and the culture supernatants were collected continuously for 5 days. Then, the sEVs were isolated, followed by an evaluation of their characteristics. The survival rates of the hiPSCs lines that were cultured in EVs-PM containing 0.5% to 20% ED-KSR were not significantly different (P > 0.05). The survival rates of the hiPSCs in 0.5% ED-KSR after the culture supernatants were continuously collected for day 1, day 3, and day 5 were not statistically significant (P > 0.05). After 5 days of continuous collection of the supernatant, the hiPSCs expressed some pluripotent markers, while SSEA4 and TRA-1-60 expression changed gradually. The sEVs that were extracted by the two methods were all 50-200 nm, double-layered and oval or round cellular vesicles and expressed the marker proteins CD63, TSG101, and HSP70. The characteristics of sEVs extracted on day 1, day 3, and day 5 were almost identical on morphology, size and the relative quantity of annexin V-positive subpopulations. The PKH67 staining showed that the sEVs could be endocytosed by HepG2 cells and aggregated in the cytoplasm. The proliferation experiments showed that the sEVs can promote cell proliferation. In Conclusion, the 0.5% ED-KSR is the optimal concentration, and that the hiPSCs culture supernatant can be continuously collected for 5 days while maintaining high cell viability and some pluripotent characteristics. Both of the methods extraction can be used to obtain biologically active sEVs.

Dataset Information

A novel method for isolating Schwann cells using the extracellular domain of Necl1.

Publications

A novel method for isolating Schwann cells using the extracellular domain of Necl1.

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