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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Presented here is a protocol that combines an in vitro neural-endothelial co-culture system and metabolic incorporation of sialoglycan with bioorthogonal functional groups to expand primary neural stem and progenitor cells and label their surface sialoglycoproteins for imaging or mass-spectrometry analysis of cell surface markers.

Abstract

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized niches in vivo and can be isolated and expanded in vitro, serving as an important resource for cell transplantation to repair brain damage. However, NSPCs are heterogeneous and not clearly defined at the molecular level or purified due to a lack of specific cell surface markers. The protocol presented, which has been previously reported, combines a neural-endothelial co-culture system with a metabolic glycan labeling method to identify the surface sialoglycoproteome of primary NSPCs. The NSPC-endothelial co-culture system allows self-renewal and expansion of primary NSPCs in vitro, generating a sufficient number of NSPCs. Sialoglycans in cultured NSPCs are labeled using an unnatural sialic acid metabolic reporter with bioorthogonal functional groups. By comparing the sialoglycoproteome from self-renewing NSPCs expanded in an endothelial co-culture with differentiating neural culture, we identify a list of membrane proteins that are enriched in NSPCs. In detail, the protocol involves: 1) set-up of an NSPC-endothelial co-culture and NSPC differentiating culture; 2) labeling with azidosugar per-O-acetylated N-azidoacetylmannosamine (Ac4ManNAz); and 3) biotin conjugation to modified sialoglycan for imaging after fixation of neural culture or protein extraction from neural culture for mass spectrometry analysis. Then, the NSPC-enriched surface marker candidates are selected by comparative analysis of mass spectrometry data from both the expanded NSPC and differentiated neural cultures. This protocol is highly sensitive for identifying membrane proteins of low abundance in the starting materials, and it can be applied to marker discovery in other systems with appropriate modifications

Introduction

Neural stem cells are defined as a multipotent cell population that can self-renew to maintain a stem cell pool and differentiate into neurons and glia. They are the major cell types in the nervous system and may offer great therapeutic potential in regenerative medicine through cell transplantation into diseased and injured brains1,2. As development proceeds, the neural stem cell population becomes heterogenous3,4, and the proportion of neural stem cells in the brain gradually decreases5. Generally speaking, embryonic neur....

Protocol

​All animal protocols used in this study were approved by the IACUC (Institutional Animal Care and Use Committee) of Tsinghua University and performed in accordance with guidelines of the IACUC. The laboratory animal facility at Tsinghua University has been accredited by the AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care International). For staging of embryos, mid-day of the vaginal plug identified was calculated as embryonic day 0.5 (E0.5).

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Representative Results

The whole procedure for in vitro expansion and metabolic labeling of primary embryonic NSPCs takes 6 days (Figure 1A). Quality of the BEND3 cell line and freshly isolated primary NSPCs are key to a successful experiment. BEND3 cells are the source of soluble factors that stimulate self-renewal and proliferation of NSPCs. It should be ensured that the BEND3 cells are free of any contamination and divide actively with minimal cell death before co-culturing with.......

Discussion

Surface markers are commonly used to label and purify specific cell types in vitro and in vivo17,18. Discovery of surface markers contributes greatly to regenerative medicine and stem cell researches by providing molecular tools to selectively enrich a stem cell population from normal or pathological tissues and culture dishes, offering a purified cell resource for clinical use or study of biological properties. However, progress in developing surface markers for.......

Acknowledgements

Figure 1B, 1C, 1E and 1F are reproduced from Bai et al.10 with permission from the Royal Society of Chemistry. We thank Yi Hao in X. C.'s lab for figure editing. This work is supported by the National Natural Science Foundation of China (No. 91753206 to Q. S. and X. C., No. 31371093 to Q. S., and Nos. 21425204 and 21672013 to X. C.).

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Materials

NameCompanyCatalog NumberComments
BEND3ATCCCRL-229
DMEMGibco11960044
L-glutamineGibco250300811%
Sodium pyruvateSigmaP52801%
N2 supplementGibco175020481 to 100
N-acetyl-L-cysteineSigmaA72501 mM
PapainWorthingtonLS00372610 U/mL
B27 supplementGibco175040441 to 50
Poly-L-lysineSigmaP4707
basic Fibroblast growth factorGibcoPHG026110 ng/mL
Penicillin-StreptomycinGibco151401221%
Fetal bovine serumGibco1009914110%
HBSSGibco14175095
Tripsin-EDTA, 0.25%Gibco25200056
DPBSGibco14190094
TranswellCorning3450
ParaformaldehydeSigma1581274%
SucroseSangonA100335
DAPIGibco62248
RIPA bufferThermo Scientific89900
SDS-PAGE loading buffer 2XSolarbioP1018
6-well plateCorning3335
Tris-Glycine protein gelinvitrogenxp00100box
mouse monoclonal anti-NestinDevelopmental Study Hybridoma BankRat-4011 to 20
mouse monoclonal anti-beta-tubulin IIISigmaT88601 to 1000
Alexa Fluor 488 goat anti-mouse IgG1invitrogenA-211211 to 1000
Alexa Fluor 546 goat anti-mouse IgG2binvitrogenA-211431 to 1000
Albumin Bovine VAmresco0332
Triton X-100Amresco0694
BCA assay kitThermo Scientific23225
dimethyl sulfoxideSigmaD2650
Brij97AladdinB129088
CuSO4Sigma209198
alkyne-biotinClick Chemistry ToolsTA105
BTTAAClick Chemistry Tools1236
Ac4ManNAzClick Chemistry Tools1084100 µM
9AzSiasynthesized in lab
sodium ascorbateSigmaA4034
MethanolSigma34860
EDTASangonA100322
NaClSangonA100241
SDSSangonA100227
Alexa Flour 647-conjugated streptavidininvitrogenS213741 to 1000
TriethanolamineSigmaV900257
Dynabeads M-280 Streptavidin invitrogen60210
ammonium bicarbonateSigma9830
Coomassie Brilliant Blue R-250Thermo Scientific20278
IsofluraneRWD Life Science Co.970-00026-00
DNase ISigmaDN2512 µg/mL
ureaSigmaU5378

References

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Neural Stem CellsNeural Progenitor CellsCell Surface MarkersMetabolic LabelingSialoglycanEndothelial Cell Co cultureAc4ManAzMAPK ERK PathwayPrimary Cortical CellsCell PurificationRegenerative Medicine

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