Figure 1B, 1C, 1E and 1F are reproduced from Bai&#160;et al.10&#160;with permission from the Royal Society of Chemistry. We thank Yi Hao in X. C.&#39;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.).

&#8203;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).
NOTE: ...

<ol>
	<li>Weissman, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+Cells:+Units+of+Development,+Units+of+Regeneration,+and+Units+in+Evolution.">Stem Cells: Units of Development, Units of Regeneration, and Units in Evolution.</a> Cell. 100, 157-168 (2000).</li><li>Gage, F. H., Temple, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+Stem+Cells:+Generating+and+Regenerating+the+Brain.">Neural Stem Cells: Generating and Regenerating the Brain.</a> Neuron. 80, 588-601 (2013).</li><li>Gal, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+and+Morphological+Heterogeneity+of+Neural+Precursors+in+the+Mouse+Neocortical+Proliferative+Zones.">Molecular and Morphological Heterogeneity of Neural Precursors in the Mouse Neocortical Proliferative Zones.</a> Journal of Neuroscience. 26, 1045-1056 (2006).</li><li>Kawaguchi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+gene+profiling+defines+differential+progenitor+subclasses+in+mammalian+neurogenesis.">Single-cell gene profiling defines differential progenitor subclasses in mammalian neurogenesis.</a> Development. 135, 3113-3124 (2008).</li><li>Temple, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+neural+stem+cells.">The development of neural stem cells.</a> Nature. 414, 112-117 (2001).</li><li>Kwan, K. Y., Sestan, N., Anton, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+co-regulation+of+neuronal+migration+and+laminar+identity+in+the+neocortex.">Transcriptional co-regulation of neuronal migration and laminar identity in the neocortex.</a> Development. 139, 1535-1546 (2012).</li><li>Kriegstein, A., Alvarez-Buylla, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Glial+Nature+of+Embryonic+and+Adult+Neural+Stem+Cells.">The Glial Nature of Embryonic and Adult Neural Stem Cells.</a> Annual Review of Neuroscience. 32, 149-184 (2009).</li><li>Wang, X., Tsai, J. W., LaMonica, B., Kriegstein, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+subtype+of+progenitor+cell+in+the+mouse+embryonic+neocortex.">A new subtype of progenitor cell in the mouse embryonic neocortex.</a> Nature Neuroscience. 14, 555-561 (2011).</li><li>Taverna, E., G&#246;tz, M., Huttner, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Cell+Biology+of+Neurogenesis:+Toward+an+Understanding+of+the+Development+and+Evolution+of+the+Neocortex.">The Cell Biology of Neurogenesis: Toward an Understanding of the Development and Evolution of the Neocortex.</a> Annual Review of Cell and Developmental Biology. 30, 465-502 (2014).</li><li>Bai, Q. R., Dong, L., Hao, Y., Chen, X., Shen, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+glycan+labeling-assisted+discovery+of+cell-surface+markers+for+primary+neural+stem+and+progenitor+cells.">Metabolic glycan labeling-assisted discovery of cell-surface markers for primary neural stem and progenitor cells.</a> Chemical Communications. 54, 5486-5489 (2018).</li><li>Shen, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cells+stimulate+self-renewal+and+expand+neurogenesis+of+neural+stem+cells.">Endothelial cells stimulate self-renewal and expand neurogenesis of neural stem cells.</a> Science. 304, 1338-1340 (2004).</li><li>Qian, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Timing+of+CNS+cell+generation:+a+programmed+sequence+of+neuron+and+glial+cell+production+from+isolated+murine+cortical+stem+cells.">Timing of CNS cell generation: a programmed sequence of neuron and glial cell production from isolated murine cortical stem cells.</a> Neuron. 28, 69-80 (2000).</li><li>Varki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycan-based+interactions+involving+vertebrate+sialic-acid-recognizing+proteins.">Glycan-based interactions involving vertebrate sialic-acid-recognizing proteins.</a> Nature. 446, 1023-1029 (2007).</li><li>Cheng, B., Xie, R., Dong, L., Chen, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+Remodeling+of+Cell-Surface+Sialic+Acids:+Principles,+Applications,+and+Recent+Advances.">Metabolic Remodeling of Cell-Surface Sialic Acids: Principles, Applications, and Recent Advances.</a> ChemBioChem. 17, 11-27 (2016).</li><li>Lin, S. H., Guidotti, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+Membrane+Proteins.">Purification of Membrane Proteins.</a> Methods in Enzymology. 463, 619-629 (2009).</li><li>Schmidt, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pilot+Study+on+Mass+Spectrometry-Based+Analysis+of+the+Proteome+of+CD34+CD123++Progenitor+Cells+for+the+Identification+of+Potential+Targets+for+Immunotherapy+in+Acute+Myeloid+Leukemia.">Pilot Study on Mass Spectrometry-Based Analysis of the Proteome of CD34+CD123+ Progenitor Cells for the Identification of Potential Targets for Immunotherapy in Acute Myeloid Leukemia.</a> Proteomes. 6, (2018).</li><li>Crisan, M., Dzierzak, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+many+faces+of+hematopoietic+stem+cell+heterogeneity+Development.">The many faces of hematopoietic stem cell heterogeneity Development.</a> Development. 144, 4195-4195 (2017).</li><li>Uchida, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+isolation+of+human+central+nervous+system+stem+cells.">Direct isolation of human central nervous system stem cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 97, 14720-14725 (2000).</li><li>Qin, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artificial+Cysteine+S-Glycosylation+Induced+by+Per-O-Acetylated+Unnatural+Monosaccharides+during+Metabolic+Glycan+Labeling.">Artificial Cysteine S-Glycosylation Induced by Per-O-Acetylated Unnatural Monosaccharides during Metabolic Glycan Labeling.</a> Angewandte Chemie International Edition. , (2018).</li><li>Gry, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlations+between+RNA+and+protein+expression+profiles+in+23+human+cell+lines.">Correlations between RNA and protein expression profiles in 23 human cell lines.</a> BMC Genomics. 10, 365 (2009).</li><li>Hennen, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+LewisX+Glycoprotein+Screen+Identifies+the+Low+Density+Lipoprotein+Receptor-related+Protein+1+(LRP1)+as+a+Modulator+of+Oligodendrogenesis+in+Mice.">A LewisX Glycoprotein Screen Identifies the Low Density Lipoprotein Receptor-related Protein 1 (LRP1) as a Modulator of Oligodendrogenesis in Mice.</a> Journal of Biological Chemistry. 288, 16538-16545 (2013).</li><li>Seet, B. T., Dikic, I., Zhou, M. M., Pawson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reading+protein+modifications+with+interaction+domains.">Reading protein modifications with interaction domains.</a> Nature Reviews Molecular Cell Biology. 7, 473-483 (2006).</li><li>O&#8217;Brian, C. A., Chu, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ReviewPost-translational+disulfide+modifications+in+cell+signaling&#8212;role+of+inter-protein,+intra-protein,+S-glutathionyl,+and+S-cysteaminyl+disulfide+modifications+in+signal+transmission.">ReviewPost-translational disulfide modifications in cell signaling&#8212;role of inter-protein, intra-protein, S-glutathionyl, and S-cysteaminyl disulfide modifications in signal transmission.</a> Free Radical Research. 39, 471-480 (2005).</li><li>Williamson, A. J. K., Whetton, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+requirement+for+proteomics+to+unravel+stem+cell+regulatory+mechanisms.">The requirement for proteomics to unravel stem cell regulatory mechanisms.</a> Journal of Cellular Physiology. 226, 2478-2483 (2011).</li><li>Christensen, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Type-specific+Post-translational+Modifications+of+Mouse+Osteopontin+Are+Associated+with+Different+Adhesive+Properties.">Cell Type-specific Post-translational Modifications of Mouse Osteopontin Are Associated with Different Adhesive Properties.</a> Journal of Biological Chemistry. 282, 19463-19472 (2007).</li><li>Yanagisawa, M., Yu, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+expression+and+functions+of+glycoconjugates+in+neural+stem+cells.">The expression and functions of glycoconjugates in neural stem cells.</a> Glycobiology. 17, 57R-74R (2007).</li><li>Best, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Click+Chemistry+and+Bioorthogonal+Reactions:+Unprecedented+Selectivity+in+the+Labeling+of+Biological+Molecules.">Click Chemistry and Bioorthogonal Reactions: Unprecedented Selectivity in the Labeling of Biological Molecules.</a> Biochemistry. 48, 6571-6584 (2009).</li></ol>

The authors have nothing to disclosure.

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...

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&#160;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 neural stem cells and other neural progenitor cells, collectively called neural stem and progenitor cells (NSPCs), are located in the germinal zones, the ventricular zone, and the subventricular zone in mice6. In the embryonic brain, neural stem cells generate neurons directly or indirectly through intermediate progenitor cells (IPCs), and in some species through the outer subventricular zone progenitors (oRGs)7,8. The specific molecular signature, morphology, location in the stem cell niche, and differentiation potential all determine the role of each subtype in brain organogenesis and clinical applications9. However, the currently available cell surface markers cannot unequivocally discriminate and purify different subtypes of NSPCs, limiting the understanding and utilization of these subtypes.
The identification of primary NSPCs surface markers is limited by three major hurdles. The first one is the limited cell number of NSPCs in the tissue, making it difficult&#160;to prepare cell surface protein samples for common mass spectrometry analysis. The second limitation is the difficulty in producing pure cell subtypes for generating subtype-specific membrane protein data. Finally, the third challenge is the low ratio of cell surface proteins in whole cell proteins, which hampers their detection sensitivities by mass spectrometry analysis.
To overcome these problems, we developed a chemoproteomic approach to selectively enrich and identify cell surface proteins in primary NSPCs by metabolically&#160;labeling the sialoglycoproteins10. To generate a sufficient number of NSPCs, we took advantage of an established protocol to expand and maintain primary embryonic NSPCs in undifferentiated states in vitro, by co-culturing NSPCs with mouse brain endothelial cell lines using a permeable support matrix insert (e.g., transwell) system11. In contrast, NPSCs cultured alone without endothelial cells generate differentiated progeny11,12. Thus, protein&#160;samples from these two culture systems can be comparatively analyzed to identify proteins that are differentially expressed in NSPCs and differentiated neurons. As most cell surface proteins are modified by sialic acid13, unnatural sialic acid precursor analog N-azidoacetylmannosamine-tetraacylated (Ac4ManNAz) was used to hijack the intrinsic metabolic pathway so that endogenous, newly synthesized sialoglycans are labeled with azido groups, generating a chemical handle14. Through azido-alkyne-mediated bioorthogonal reactions, which conjugate biotin to sialoglycans, cell surface proteins can be visualized and enriched for proteomic identification through a streptavidin-coupled fluorophore or matrix14.
Here, we perform staining of SDS-PAGE gel analysis of the surface sialoglycoproteome from NSPCs expanded in an endothelial co-culture and differentiating cells in a&#160;non-co-culture system. We also selectively purify surface sialoglycoproteome in the two culture systems for proteomic comparison. Our protocol, compared with&#160;the traditional centrifugation-based cell surface purification protocols15, increases extraction efficacy by reducing the surface protein extraction procedures through specific tag conjugation and affinity purification. Meanwhile, it increases the extraction purity of cell surface proteins based on the premise that sialylation happens mostly at the cell surface proteins. Although endothelial factors cannot completely block differentiation of expanded NSPCs, the comparative study between a co-culture and differentiated culture provides a convenient method to pinpoint stem cell-enriched surface proteins without the need to analyze proteins from NPCs purified by FACS16. We believe this approach can be applied to studies of surface proteins in other systems with the appropriate modifications.

<table border="0" cellpadding="0" cellspacing="0" style="width:880px;" width="879">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">BEND3</td>
			<td style="width:283px;">ATCC</td>
			<td style="width:123px;">CRL-229</td>
			<td style="width:172px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">DMEM</td>
			<td style="width:283px;">Gibco</td>
			<td style="width:123px;">11960044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">L-glutamine</td>
			<td style="width:283px;">Gibco</td>
			<td align="right" style="width:123px;">25030081</td>
			<td align="right">1%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Sodium pyruvate</td>
			<td style="width:283px;">Sigma</td>
			<td style="width:123px;">P5280</td>
			<td align="right">1%</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:303px;">N2 supplement</td>
			<td style="width:283px;">Gibco</td>
			<td align="right" style="width:123px;">17502048</td>
			<td>1 to 100</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:303px;">N-acetyl-L-cysteine</td>
			<td style="width:283px;">Sigma</td>
			<td style="width:123px;">A7250</td>
			<td>1 mM</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:303px;">Papain</td>
			<td style="width:283px;">Worthington</td>
			<td style="width:123px;">LS003726</td>
			<td>10 U/mL</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:303px;">B27 supplement</td>
			<td style="width:283px;">Gibco</td>
			<td align="right" style="width:123px;">17504044</td>
			<td>1 to 50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Poly-L-lysine</td>
			<td style="width:283px;">Sigma</td>
			<td style="width:123px;">P4707</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:303px;">basic Fibroblast growth factor</td>
			<td style="width:283px;">Gibco</td>
			<td style="width:123px;">PHG0261</td>
			<td>10 ng/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Penicillin-Streptomycin</td>
			<td style="width:283px;">Gibco</td>
			<td align="right" style="width:123px;">15140122</td>
			<td align="right">1%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Fetal bovine serum</td>
			<td style="width:283px;">Gibco</td>
			<td style="width:123px;">10099141</td>
			<td align="right">10%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">HBSS</td>
			<td style="width:283px;">Gibco</td>
			<td align="right" style="width:123px;">14175095</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Tripsin-EDTA, 0.25%</td>
			<td style="width:283px;">Gibco</td>
			<td align="right" style="width:123px;">25200056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">DPBS</td>
			<td style="width:283px;">Gibco</td>
			<td align="right" style="width:123px;">14190094</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Transwell</td>
			<td style="width:283px;">Corning</td>
			<td align="right" style="width:123px;">3450</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Paraformaldehyde</td>
			<td style="width:283px;">Sigma</td>
			<td align="right" style="width:123px;">158127</td>
			<td align="right">4%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Sucrose</td>
			<td style="width:283px;">Sangon</td>
			<td style="width:123px;">A100335</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">DAPI</td>
			<td style="width:283px;">Gibco</td>
			<td align="right" style="width:123px;">62248</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">RIPA buffer</td>
			<td style="width:283px;">Thermo Scientific</td>
			<td align="right" style="width:123px;">89900</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">SDS-PAGE loading buffer 2X</td>
			<td style="width:283px;">Solarbio</td>
			<td style="width:123px;">P1018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">6-well plate</td>
			<td style="width:283px;">Corning</td>
			<td align="right" style="width:123px;">3335</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Tris-Glycine protein gel</td>
			<td style="width:283px;">invitrogen</td>
			<td style="width:123px;">xp00100box</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:303px;">mouse monoclonal anti-Nestin</td>
			<td style="width:283px;">Developmental Study Hybridoma Bank</td>
			<td style="width:123px;">Rat-401</td>
			<td style="width:172px;">1 to 20</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:303px;">mouse monoclonal anti-beta-tubulin III</td>
			<td style="width:283px;">Sigma</td>
			<td style="width:123px;">T8860</td>
			<td>1 to 1000</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:303px;">Alexa Fluor 488 goat anti-mouse IgG1</td>
			<td style="width:283px;">invitrogen</td>
			<td style="width:123px;">A-21121</td>
			<td>1 to 1000</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:303px;">Alexa Fluor 546 goat anti-mouse IgG2b</td>
			<td style="width:283px;">invitrogen</td>
			<td style="width:123px;">A-21143</td>
			<td>1 to 1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Albumin Bovine V</td>
			<td style="width:283px;">Amresco</td>
			<td style="width:123px;">0332</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Triton X-100</td>
			<td style="width:283px;">Amresco</td>
			<td style="width:123px;">0694</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">BCA assay kit</td>
			<td style="width:283px;">Thermo Scientific</td>
			<td align="right" style="width:123px;">23225</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">dimethyl sulfoxide</td>
			<td style="width:283px;">Sigma</td>
			<td style="width:123px;">D2650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Brij97</td>
			<td style="width:283px;">Aladdin</td>
			<td style="width:123px;">B129088</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">CuSO4</td>
			<td style="width:283px;">Sigma</td>
			<td align="right" style="width:123px;">209198</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">alkyne-biotin</td>
			<td style="width:283px;">Click Chemistry Tools</td>
			<td style="width:123px;">TA105</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">BTTAA</td>
			<td style="width:283px;">Click Chemistry Tools</td>
			<td align="right" style="width:123px;">1236</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Ac4ManNAz</td>
			<td style="width:283px;">Click Chemistry Tools</td>
			<td align="right" style="width:123px;">1084</td>
			<td>100 &#181;M</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">9AzSia</td>
			<td style="width:283px;">synthesized in lab</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">sodium ascorbate</td>
			<td style="width:283px;">Sigma</td>
			<td style="width:123px;">A4034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Methanol</td>
			<td style="width:283px;">Sigma</td>
			<td align="right" style="width:123px;">34860</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">EDTA</td>
			<td style="width:283px;">Sangon</td>
			<td style="width:123px;">A100322</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">NaCl</td>
			<td style="width:283px;">Sangon</td>
			<td style="width:123px;">A100241</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">SDS</td>
			<td style="width:283px;">Sangon</td>
			<td style="width:123px;">A100227</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Alexa Flour 647-conjugated streptavidin</td>
			<td style="width:283px;">invitrogen</td>
			<td style="width:123px;">S21374</td>
			<td>1 to 1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Triethanolamine</td>
			<td style="width:283px;">Sigma</td>
			<td style="width:123px;">V900257</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Dynabeads M-280 Streptavidin&#160;</td>
			<td style="width:283px;">invitrogen</td>
			<td align="right" style="width:123px;">60210</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">ammonium bicarbonate</td>
			<td style="width:283px;">Sigma</td>
			<td style="width:123px;">9830</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Coomassie Brilliant Blue R-250</td>
			<td style="width:283px;">Thermo Scientific</td>
			<td align="right" style="width:123px;">20278</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Isoflurane</td>
			<td style="width:283px;">RWD Life Science Co.</td>
			<td style="width:123px;">970-00026-00</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:303px;">DNase I</td>
			<td style="width:283px;">Sigma</td>
			<td style="width:123px;">DN25</td>
			<td>12 &#181;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">urea</td>
			<td style="width:283px;">Sigma</td>
			<td style="width:123px;">U5378</td>
			<td></td>
		</tr>
	</tbody>
</table>

identifying cell surface markers of primary neural stem and progenitor cells by metabolic labeling of sialoglycan

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

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...

Watch this Scientific Journal Video about Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan at JoVE.com

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

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 ...

Neural stem and progenitor cells can self-renew and differentiate into different neural cell types, supporting nervous system development and offering a resource for regenerative medicine. These cells are heterogeneous, and we need to know their specific cell surface markers in order to get a purified cell populations and understand their functions. Our protocol provides a simple, sensitive, and efficient technique to analyze membrane proteins of primary neural stem and progenitor cells, helping identify novel surface markers for these cells.The main advantage of our protocol is its ability to directly analyze the surface proteome of primary neural stem and progenitor cells with the improved sensitivity and specificity. This is achieved by expanding them through co-culture within endothelial cells in vitro and high tractin intrinsic cellulation MAPK-ERK pathway to label surface porins. With pore pro-modifications on expanding stem cells in vitro, our protocol can easily be applied to identify surface markers of other stem cell types.To prepare the endothelial cells, re-suspend a BEN3 cell pellet from a 10 centimeter Petri dish at 90%confluence with nine milliliters of BEN3 cell medium. Add one milliliter of the cell suspension into one permeable support insert. Add another two milliliters of BEN3 medium per well at the bottom chamber of the matrix.Continue to culture the cells for one day at 37 degrees Celsius with 5%carbon dioxide. One day after plating the cells in the inserts, gently aspirate the medium, first from the bottom chamber and then from the inserts. Wash the face of the inserts three times with pre-warmed DMEM.Wash the outer surface of the inserts by rinsing with pre-warmed DMEM. Next, add one milliliter of pre-warmed AM into one insert. Transfer the inserts into the wells with primary cortical cells.Incubate the co-culture at 37 degrees Celsius with 5%carbon dioxide for 12 hours. Dissolve Ac4ManAz in DMSO to achieve a stock concentration of 200 millimolar. Retrieve the neural-endothelial co-culture plate from the incubator.Add one microliter of the Ac4ManAz stock to each bottom chamber and 0.5 microliters per insert into the co-culture. Culture the cells at 37 degrees Celsius with 5%carbon dioxide for five days. While the cells are culturing add 100 microliters of RM per insert and 200 microliters of RM per bottom chamber to re-feed the endothelial and neural cells every other day.First remove the inserts from the co-culture plates and aspirate the culture medium from the bottom of the wells. Next wash the neural cells once with pre-warmed PBS. Aspirate the PBS from the wells and add one milliliter of a pre-chilled 4%paraformaldehyde in PBS to each well.Fix the cells at room temperature for 10 minutes. Then wash the cells three times with pre-chilled PBS. Aspirate the PBS and add one milliliter of freshly prepared biotin conjugated buffer 1 into each well.Incubate at room temperature for 10 minutes. After this, aspirate the reaction buffer from the wells and wash the cells three times with PBS. Add one milliliter of staining buffer to each well of cells and incubate at room temperature for 30 minutes.Next, aspirate the staining buffer from the wells and wash the cells three times with pre-chilled PBS. Add one milliliter of blocking buffer to each well of cells and incubate at room temperature for 10 minutes. Remove the blocking buffer from the wells and add one milliliter of primary antibody solution to each well.Incubate the cells overnight at four degrees Celsius. The next day, remove the primary antibody solution from the wells. Wash the cells three times with pre-chilled PBS.Aspirate the PBS from the wells and add one milliliter of secondary antibody to each well. Incubate the cells at room temperature for two hours. After this, aspirate the solution from the wells and wash the cells three times with pre-chilled PBS.First, prepare BTTAA cupric sulfate complex 2, full protein re-suspension buffer, protein washing buffer 1, protein washing buffer 2 and protein washing buffer 3 as outlined in the text protocol. Next, remove the inserts from the co-culture plates. Aspirate the culture medium from the bottom wells and wash the neural cells once with pre-chilled PBS.Aspirate the PBS and add 200 microliters of pre-chilled RIPA buffer to each well. Incubate the plates on ice for five minutes then collect the protein lysis into 1.5 milliliter tubes. Centrifuge at 12000 times G and at four degrees Celsius for 10 minutes to pellet the cell debris.Transfer the supernatant into new 1.5 milliliter tubes and determine the protein concentration with a BCA kit according to the manufacturers instructions. Then, adjust the protein concentration to one milligram per milliliter. Add 100 micromolar alkyde biotin, 2.5 millimolar sodium ascorbate and 1XBTTAA cupric sulfate complex 2 to one milliliter of protein lysis.Mix the solution well and incubate it at room temperature for one hour. After this, transfer the reaction solution into 20 milliliters of pre-chilled methanol in a 50 milliliter conical tube. Mix well and incubate overnight at minus 30 degrees Celsius to precipitate the proteins.Centrifuge at 4500 times G and at four degrees Celsius for 15 minutes to pellet the protein precipitates. Wash the pellet twice using 20 milliliters of pre-chilled methanol per wash. Next, aspirate the supernatant and re-suspend the protein pellet in four milliliters of re-suspension buffer.Transfer the protein re-suspension into a new 15 milliliter conical tube. Wash 50 microliters of streptavidin beads three times with PBS. Add the washed beads to the protein re-suspension and incubate the solution at four degrees Celsius for three hours on a vertical rotator at a rotation speed of 20 rpm.After this, wash the beads sequentially with each washing buffer shown here. Re-suspend the washed beads with 20 microliters of PBS and transfer them into a new 1.5 milliliter tube. Add 20 microliters of 2X protein loading buffer and treat at 95 degrees Celsius for 10 minutes.Then process the protein samples with SDS-PAGE and stain them with Coomassie Brilliant Blue as outlined in the text protocol. In this study primary embryonic NSPCs are expanded and metabolically labeled in vitro. Successful endothelial co-culture leads NSPCs to form large sheet-like clones.Immunostaining with antibodies reveals that in the clone, most of the cells are Nestin positive NSPCs, while very few are beta tubulin 3 positive neuronal cells. In contrast, the percentage of Nestin positive cells and beta tubulin 3 positive neuronal cells in clones formed in non co-culture system are nearly the same. The chemical reporter Ac4ManAz is a metabolic analog and can be incorporated into the intrinsic protein sialylation pathway.An optimized labeling concentration of 100 micromolar for primary NSPCs is used and combinatory evaluation of cell death indicated by cellular and nuclei morphology suggest this labeling concentration does not cause obvious cytotoxic effects and is able to efficiently label NSPCs. The clonal morphology, self-renewal and differentiation potential of NSPCs in both the endothelial co-culture and non-co-culture system are not affected. Protein samples prepared from the Ac4ManAz labeled culture by biotin conjugation and streptavidin beads purification show strong Coomassie Brilliant Blue staining signal in SDS-PAGE gels.Meanwhile there were only staining background and non-specific binding signals in the lanes loaded with protein samples from the DMSO control group. This also indicates the efficient labeling of NSPCs by Ac4ManAz. When attempting this protocol, all of the solutions needed for biocellular reaction should be prepared freshly and reaction time should be controlled tightly to prevent over of insufficient reaction.Following our protocol the structure of neuro stem cell enriched surface glycan can be analyzed. Our protocol of neural stem and progenitor cell enriched surface proteins not only rise markers but also play important functions on the raised pattern purification strategy and the illustration of rigiditory signal circuitry for this cell population.

identifying-cell-surface-markers-primary-neural-stem-progenitor-cells

Research

JoVE Journal

Neuroscience

5.2K Görüntüleme.  Tongji University and Frontier Science Research Center for Stem Cells of Ministry of Education. Sinirsel kök ve progenitor hücreleri kendini yenilemek ve farklı nöral hücre tipleri içine ayırt, sinir sistemi gelişimini destekleyen ve rejeneratif tıp için bir kaynak sunan. Bu hücreler heterojendir ve arınmış hücre popülasyonları elde etmek ve işlevlerini anlamak için onların özel hücre yüzeyi işaretlerini bilmemiz gerekir. Protokolümüz, primer nöral kök ve progenitor hücrelerin membran proteinlerini analiz etmek için basit, hassas ve etkili bir teknik sağla...