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* These authors contributed equally
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.
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
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....
​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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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.......
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.......
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.).
....Name | Company | Catalog Number | Comments |
BEND3 | ATCC | CRL-229 | |
DMEM | Gibco | 11960044 | |
L-glutamine | Gibco | 25030081 | 1% |
Sodium pyruvate | Sigma | P5280 | 1% |
N2 supplement | Gibco | 17502048 | 1 to 100 |
N-acetyl-L-cysteine | Sigma | A7250 | 1 mM |
Papain | Worthington | LS003726 | 10 U/mL |
B27 supplement | Gibco | 17504044 | 1 to 50 |
Poly-L-lysine | Sigma | P4707 | |
basic Fibroblast growth factor | Gibco | PHG0261 | 10 ng/mL |
Penicillin-Streptomycin | Gibco | 15140122 | 1% |
Fetal bovine serum | Gibco | 10099141 | 10% |
HBSS | Gibco | 14175095 | |
Tripsin-EDTA, 0.25% | Gibco | 25200056 | |
DPBS | Gibco | 14190094 | |
Transwell | Corning | 3450 | |
Paraformaldehyde | Sigma | 158127 | 4% |
Sucrose | Sangon | A100335 | |
DAPI | Gibco | 62248 | |
RIPA buffer | Thermo Scientific | 89900 | |
SDS-PAGE loading buffer 2X | Solarbio | P1018 | |
6-well plate | Corning | 3335 | |
Tris-Glycine protein gel | invitrogen | xp00100box | |
mouse monoclonal anti-Nestin | Developmental Study Hybridoma Bank | Rat-401 | 1 to 20 |
mouse monoclonal anti-beta-tubulin III | Sigma | T8860 | 1 to 1000 |
Alexa Fluor 488 goat anti-mouse IgG1 | invitrogen | A-21121 | 1 to 1000 |
Alexa Fluor 546 goat anti-mouse IgG2b | invitrogen | A-21143 | 1 to 1000 |
Albumin Bovine V | Amresco | 0332 | |
Triton X-100 | Amresco | 0694 | |
BCA assay kit | Thermo Scientific | 23225 | |
dimethyl sulfoxide | Sigma | D2650 | |
Brij97 | Aladdin | B129088 | |
CuSO4 | Sigma | 209198 | |
alkyne-biotin | Click Chemistry Tools | TA105 | |
BTTAA | Click Chemistry Tools | 1236 | |
Ac4ManNAz | Click Chemistry Tools | 1084 | 100 µM |
9AzSia | synthesized in lab | ||
sodium ascorbate | Sigma | A4034 | |
Methanol | Sigma | 34860 | |
EDTA | Sangon | A100322 | |
NaCl | Sangon | A100241 | |
SDS | Sangon | A100227 | |
Alexa Flour 647-conjugated streptavidin | invitrogen | S21374 | 1 to 1000 |
Triethanolamine | Sigma | V900257 | |
Dynabeads M-280 Streptavidin | invitrogen | 60210 | |
ammonium bicarbonate | Sigma | 9830 | |
Coomassie Brilliant Blue R-250 | Thermo Scientific | 20278 | |
Isoflurane | RWD Life Science Co. | 970-00026-00 | |
DNase I | Sigma | DN25 | 12 µg/mL |
urea | Sigma | U5378 |
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