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: All cells are cultured in the cell incubator under conditions of 37 &#176;C and 5% CO2.
1. Preparation of Mouse Endothelial Culture in Permeable Support Inserts
NOTE: BEND3 cells are maintained according to manufacturer&#39;s instructions.
<ol>
	<li>Prepare BEND3 cell medium (BM) by adding 50 mL of FBS and 5 mL of penicillin-streptomycin into 500 mL of DMEM and mix well.</li>
	<li>Aspirate the medium from the dish and wash the BEND3 cells culture with 1 mL of PBS once. Add 1 mL of 0.25% Trypsin-EDTA into the cells and incubate the cells for 4 min at 37 &#176;C.</li>
	<li>Add 1 mL of BM into the cells to neutralize Trypsin-EDTA and pipette up and down gently to completely dissociate the cells. Transfer the cell suspension into a new 15 mL conical tube and pellet by centrifugation at room temperature (RT) for 5 min at 400 x g.</li>
	<li>Aspirate the supernatant from the tube and resuspend the cells with 9 mL of fresh BM, then add 1 mL of cell suspension into one permeable support insert. Add another 2 mL of fresh BM per well at the bottom chamber of the matrix. Continue to culture the cells for one day.</li>
</ol>
2. Preparation of Mouse Primary Cortical NSPCs Culture
<ol>
	<li>Preparation of culture plate, papain digestion medium, and cortical adherent culture medium (AM)
	<ol>
		<li>Coat 6-well plates with poly-L-lysine (PLL) by adding 1 mL of PLL solution per well into 6-well plates. Then, incubate the plates at RT for 30 min.</li>
		<li>Transfer the PLL solution into a 15 mL conical tube. Wash the plates 3 times with double distilled water. Airdry the plates and put them aside until use.</li>
		<li>Prepare the papain digestion medium by adding 50 U of papain, 50 &#181;L of L-glutamine, and 50 &#181;L of 100 mg/mL acetyl-L-cysteine into 5 mL of DMEM. Mix the medium briefly and warm it to 37 &#176;C for 30 min for enzyme activation.</li>
		<li>Prepare the cortical cell adherent culture medium (AM): add 500 &#181;L of L-glutamine, 500 &#181;L of sodium pyruvate, 500 &#181;L of 100 mg/mL N-acetyl-L-Cysteine, 500 &#181;L of N2, 1 mL of B27, and 5 &#181;L of 100 &#181;g/mL bFGF into 50 mL of DMEM. Mix the medium well and warm it to 37 &#176;C before use.</li>
	</ol>
	</li>
	<li>Preparation of primary cerebral cortical cells and subsequent plating
	<ol>
		<li>Sacrifice an E10.5 timed pregnant mouse by cervical dislocation. 
		NOTE: At E10.5, a majority of cells are proliferating NSPCs in the cerebral cortex, giving rise to large clones of progeny in vitro.</li>
		<li>Sterilize the abdomen by 75% ethanol. Use fine scissors and micro-serrated forceps to open the abdomen by cutting the skin and underlying muscle along the right side of the middle line. Remove the uterus from the abdominal cavity gently with serrated forceps and cut it out from the abdominal cavity with fine scissors.</li>
		<li>Wash the uterus with 40 mL of pre-chilled HBSS in 10 cm Petri dish. Then, transfer the uterus into a new 10 cm Petri dish and wash it again with 40 mL of pre-chilled HBSS.</li>
		<li>Transfer the uterus into a new 10 cm Petri dish with 40 mL of pre-chilled HBSS. Remove the embryos from the uterus and amniotic membrane, then cut the heads of the embryos off from the trunks with Jewelers microforceps.</li>
		<li>Wash the heads with 40 mL of pre-chilled HBSS and transfer the heads to a new 10 cm Petri dish with 40 mL of pre-chilled HBSS. Use Jewelers microforceps to peel away skin and cartilage covering the brains, then cut the cerebral cortices off and collect them in a 15 mL conical tube with pre-chilled HBSS.</li>
		<li>Pellet the cortices by centrifugation for 3 min at 4 &#176;C and 300 x g. Aspirate the supernatant from the tube, then add activated papain digestion&#160;medium and 15 &#181;L of 4 mg/mL DNase I into the tissue pellet.</li>
		<li>Resuspend the tissue pellet briefly by gentle vortexing. Incubate the tissue at 37 &#176;C for 30 min. During this time, loosen the tissue by brief vortexing every 10 min. 
		NOTE: At the end of the digestion, there should be no visible tissue pieces in the tube.</li>
		<li>Pellet the cortical cells by centrifugation for 10 min at 4 &#176;C and 450 x g. Aspirate the supernatant from the tube and wash the cell pellet with pre-chilled DMEM. Repeat this step once. 
		NOTE: During the digestion and washing, take caution not to pipette the tissues and cell pellet roughly to avoid damaging the cells with a strong shearing force.</li>
		<li>Aspirate the supernatant from the tube then add 1.5 mL of pre-chilled HBSS into the tube. Dissociate the cortical cell pellet into single cells with gentle pipetting. Count the cell number with a hemocytometer.</li>
		<li>Add 2 mL of AM and 2 x 104 cortical cells per well into 6-well plates. Incubate the plate at 37 &#176;C and 5% CO2 for 3 h to let the cells&#160;attach to the plate.</li>
	</ol>
	</li>
</ol>
3. Set-up of Neural-endothelial Co-culture and Ac4ManNAz Labeling System
<ol>
	<li>One day after plating BEND3 cells in the inserts, gently aspirate the medium in the bottom chamber first, then the inserts. Wash the enface of the inserts 3 times with pre-warmed DMEM. Wash the outer surface of the inserts by rinsing with pre-warmed DMEM.</li>
	<li>Add 1 mL of pre-warmed AM into one insert, then transfer the inserts into the wells with primary cortical cells. Incubate the co-culture at 37 &#176;C and 5% CO2 for 12 h.</li>
	<li>Dissolve Ac4ManNAz in DMSO to achieve a stock concentration of 200 mM. 12 h after setting up the neural-endothelial co-culture, add 1 &#181;L of Ac4ManNAz stock per bottom chamber and 0.5 &#181;L of stock per insert into the co-culture. Shake the plates immediately and gently to mix the medium well. For the control cells, add equal volume of DMSO.</li>
	<li>Culture the cells for another 5 days at 37 &#176;C and 5% CO2. Prepare the AM with 10x bFGF as refeeding medium (RM). During this time, add 100 &#181;L of RM per insert and 200 &#181;L of RM per bottom chamber to refeed the endothelial and neural cells every other day. During the refeeding, do not supply Ac4ManNAz or DMSO into the culture.</li>
</ol>
4. Immunofluorescent Staining of Sialoglycoproteins in Expanded Primary NSPCs and Differentiated Neurons
<ol>
	<li>Prepare BTTAA-CuSO4 complex 1 30x stock containing 1.5 mM CuSO4 and 9 mM BTTAA in double-distilled water. Prepare freshly biotin-conjugated buffer 1 containing 50 &#181;M biotin-alkyne, 2.5 mM sodium ascorbate, and 1x BTTAA-CuSO4 complex in PBS.</li>
	<li>Remove the inserts from the co-culture plates. Aspirate the culture medium from the bottom wells and wash the neural cells once with pre-warmed PBS.</li>
	<li>Aspirate the PBS from the wells. Add 1 mL of pre-chilled 4% paraformaldehyde PBS solution per well into the cells and fix the cells at RT for 10 min. Then, wash the cells 3 times with pre-chilled PBS.</li>
	<li>Aspirate PBS from the wells and add 1 mL of freshly prepared biotin-conjugated buffer 1 per well into the cells. Incubate the cells at RT for 10 min.</li>
	<li>Aspirate the reaction buffer from the wells. Wash the cells 3 times with PBS. Prepare the staining buffer containing 1% FBS and 1 &#181;g/mL Alexa Fluor 647-streptavidin. Add 1 mL of staining buffer per well into the cells and incubate the cells at RT for 30 min.</li>
	<li>Aspirate the staining buffer from the wells and washed cells 3 times with pre-chilled PBS. Prepare the blocking buffer containing 5% BSA and 0.3% non-ionic detergent-100 in PBS. Add 1 mL of blocking buffer per well into the cells and incubate at RT for 10 min.</li>
	<li>Prepare a primary antibody solution by diluting the anti-nestin and anti-&#946;-tubulin III antibodies together into the blocking buffer at ratios of 1:20 and 1:1,000, respectively. Remove the blocking buffer from the wells and add 1 mL of primary antibody solution per well into the cells. Incubate the cells at 4 &#176;C overnight.</li>
	<li>Remove the primary antibody solution from the wells. Wash the cells 3 times with pre-chilled PBS. Prepare a secondary antibody solution by diluting Alexa Fluor 488 goat anti-mouse IgG1, Alexa Fluor 546 goat anti-mouse IgG2b, and DAPI together into blocking buffer at a dilution of 1:1,000. Aspirate the PBS from the wells and add 1 mL secondary antibody solution per well into cells. Incubate the cells at RT for 2 h.</li>
	<li>Aspirate the antibody solution from the wells and wash the cells 3 times with pre-chilled PBS. Afterwards, the cells are ready for image capture.</li>
</ol>
5. Purification of Sialoglycoproteins from Expanded Primary NSPCs and Differentiated Neurons
<ol>
	<li>Prepare BTTAA-CuSO4 complex 2 15x stock containing 1.5 mM CuSO4 and 3 mM BTTAA in double distilled water. Prepare protein resuspension buffer A containing 4% SDS and 10 mM EDTA in double distilled water; protein resuspension buffer B containing 150 mM NaCl, 50 mM triethanolamine, and 1% polyoxyethylene oleyl ether (e.g., Brij97) in double distilled water with pH 7.4. Before use, mix buffer A:buffer B = 1:8 (vol/vol) to prepare the full protein resuspension buffer. Prepare protein washing buffer 1 containing 2% SDS in PBS; protein washing buffer 2 containing 8 M urea in 250 mM ammonium bicarbonate (ABC); and protein washing buffer 3 containing 2.5 M NaCl in PBS.</li>
	<li>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.</li>
	<li>Aspirate the PBS from the wells and add 200 &#181;L of pre-chilled RIPA buffer per well into the plates. Incubate the plates on ice for 5 min. Collect the protein lysis into 1.5 mL tubes. Pellet the cell debris by centrifugation for 10 min at 4 &#176;C and 12,000 x g.</li>
	<li>Transfer the supernatant into new 1.5 mL tubes. Determine protein concentration with the BCA kit according to the manufacturer&#39;s instructions. Adjust the protein concentration to 1 mg/mL.</li>
	<li>Add 100 &#181;M alkyne-biotin, 2.5 mM sodium ascorbate, and 1x BTTAA-CuSO4 complex 2 to 1 mL of protein lysis and mix the solution well. Incubate the mix at RT for 1 h.</li>
	<li>Transfer the reaction solution into 20 mL of pre-chilled methanol in a 50 mL conical tube. Mix well and incubate at -30 &#176;C overnight to precipitate the proteins.</li>
	<li>Pellet the protein precipitates by centrifugation for 15 min at 4 &#176;C and 4,500 x g. Wash the protein pellet twice with 20 mL of pre-chilled methanol. Aspirate the supernatant from the tube. Resuspend the protein pellet with 4 mL of protein resuspension buffer and transfer the protein resuspension into a new 15 mL conical tube.</li>
	<li>Take 50 &#181;L of streptavidin beads and wash them 3 times with PBS. Add the washed beads into the protein resuspension. Incubate the solution at 4 &#176;C for 3 h on a vertical rotator at a rotation speed of 20 rpm.</li>
	<li>Wash the beads sequentially with 6 types of buffers: protein washing buffer 1, protein washing buffer 2, protein washing buffer 3, 0.5 M ABC, 0.25 M ABC and 0.05 M ABC.</li>
	<li>After washing, resuspend the beads with 20 &#181;L of PBS and transfer the beads into a new 1.5 mL tube. Add 20 &#181;L of 2x protein loading buffer into the beads and treat at 95 &#176;C for 10 min. The protein samples should then be subjected to SDS-PAGE and stained with Coomassie brilliant blue R-250 according to the manufacturer&#39;s instructions. Cut the proteins in gel as indicated by Coomassie brilliant blue R-250 for mass spectrometry analysis.</li>
</ol>

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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><tbody><tr><td>BEND3</td><td>ATCC</td><td>CRL-229</td><td></td></tr><tr><td>DMEM</td><td>Gibco</td><td>11960044</td><td></td></tr><tr><td>L-glutamine</td><td>Gibco</td><td>25030081</td><td>1%</td></tr><tr><td>Sodium pyruvate</td><td>Sigma</td><td>P5280</td><td>1%</td></tr><tr><td>N2 supplement</td><td>Gibco</td><td>17502048</td><td>1 to 100</td></tr><tr><td>N-acetyl-L-cysteine</td><td>Sigma</td><td>A7250</td><td>1 mM</td></tr><tr><td>Papain</td><td>Worthington</td><td>LS003726</td><td>10 U/mL</td></tr><tr><td>B27 supplement</td><td>Gibco</td><td>17504044</td><td>1 to 50</td></tr><tr><td>Poly-L-lysine</td><td>Sigma</td><td>P4707</td><td></td></tr><tr><td>Basic Fibroblast growth factor</td><td>Gibco</td><td>PHG0261</td><td>10 ng/mL</td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>15140122</td><td>1%</td></tr><tr><td>Fetal bovine serum</td><td>Gibco</td><td>10099141</td><td>10%</td></tr><tr><td>HBSS</td><td>Gibco</td><td>14175095</td><td></td></tr><tr><td>Tripsin-EDTA, 0.25%</td><td>Gibco</td><td>25200056</td><td></td></tr><tr><td>DPBS</td><td>Gibco</td><td>14190094</td><td></td></tr><tr><td>Transwell</td><td>Corning</td><td>3450</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>158127</td><td>4%</td></tr><tr><td>Sucrose</td><td>Sangon</td><td>A100335</td><td></td></tr><tr><td>DAPI</td><td>Gibco</td><td>62248</td><td></td></tr><tr><td>RIPA buffer</td><td>Thermo Scientific</td><td>89900</td><td></td></tr><tr><td>SDS-PAGE loading buffer 2x</td><td>Solarbio</td><td>P1018</td><td></td></tr><tr><td>6-well plate</td><td>Corning</td><td>3335</td><td></td></tr><tr><td>Tris-Glycine protein gel</td><td>invitrogen</td><td>xp00100box</td><td></td></tr><tr><td>Mouse monoclonal anti-Nestin</td><td>Developmental Study Hybridoma Bank</td><td>Rat-401</td><td>1 to 20</td></tr><tr><td>Mouse monoclonal anti-beta-tubulin III</td><td>Sigma</td><td>T8860</td><td>1 to 1,000</td></tr><tr><td>Alexa Fluor 488 goat anti-mouse IgG1</td><td>invitrogen</td><td>A-21121</td><td>1 to 1,000</td></tr><tr><td>Alexa Fluor 546 goat anti-mouse IgG2b</td><td>invitrogen</td><td>A-21143</td><td>1 to 1,000</td></tr><tr><td>Albumin Bovine V</td><td>Amresco</td><td>0332</td><td></td></tr><tr><td>Triton X-100</td><td>Amresco</td><td>0694</td><td></td></tr><tr><td>BCA assay kit</td><td>Thermo Scientific</td><td>23225</td><td></td></tr><tr><td>Dimethyl sulfoxide</td><td>Sigma</td><td>D2650</td><td></td></tr><tr><td>Brij97</td><td>Aladdin</td><td>B129088</td><td></td></tr><tr><td>CuSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>209198</td><td></td></tr><tr><td>Alkyne-biotin</td><td>Click Chemistry Tools</td><td>TA105</td><td></td></tr><tr><td>BTTAA</td><td>Click Chemistry Tools</td><td>1236</td><td></td></tr><tr><td>Ac4ManNAz</td><td>Click Chemistry Tools</td><td>1084</td><td>100 &amp;micro;M</td></tr><tr><td>9AzSia</td><td>synthesized in lab</td><td></td><td></td></tr><tr><td>Sodium ascorbate</td><td>Sigma</td><td>A4034</td><td></td></tr><tr><td>Methanol</td><td>Sigma</td><td>34860</td><td></td></tr><tr><td>EDTA</td><td>Sangon</td><td>A100322</td><td></td></tr><tr><td>NaCl</td><td>Sangon</td><td>A100241</td><td></td></tr><tr><td>SDS</td><td>Sangon</td><td>A100227</td><td></td></tr><tr><td>Alexa Flour 647-conjugated streptavidin</td><td>invitrogen</td><td>S21374</td><td>1 to 1,000</td></tr><tr><td>Triethanolamine</td><td>Sigma</td><td>V900257</td><td></td></tr><tr><td>Dynabeads M-280 Streptavidin&amp;nbsp;</td><td>invitrogen</td><td>60210</td><td></td></tr><tr><td>Ammonium bicarbonate</td><td>Sigma</td><td>9830</td><td></td></tr><tr><td>Coomassie Brilliant Blue R-250</td><td>Thermo Scientific</td><td>20278</td><td></td></tr><tr><td>Isoflurane</td><td>RWD Life Science Co.</td><td>970-00026-00</td><td></td></tr><tr><td>DNase I</td><td>Sigma</td><td>DN25</td><td>12 &amp;micro;g/mL</td></tr><tr><td>Urea</td><td>Sigma</td><td>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...

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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 ...

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

Research

JoVE Journal

Neuroscience

5.3K Views.  Tongji University and Frontier Science Research Center for Stem Cells of Ministry of Education. Neurale stam en voorloper cellen kunnen zichzelf vernieuwen en differentiëren in verschillende neurale celtypes, ondersteuning van het zenuwstelsel ontwikkeling en het aanbieden van een bron voor regeneratieve geneeskunde. Deze cellen zijn heterogeen, en we moeten hun specifieke celoppervlakmarkers kennen om een gezuiverde celpopulaties te krijgen en hun functies te begrijpen. Ons protocol biedt een eenvoudige, gevoelige en efficiënte techniek om membraaneiwitten van primaire neurale stam- ...

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

Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell lineage development. In vitro expansion of neural progenitors from fetal CNS tissue has been well characterized. Despite the identification and isolation of glial progenitors from adult human sub-cortical white matter and development of various culture conditions to direct differentiation of fetal neural progenitors into myelin producing oligodendrocytes, acquiring sufficient human oligodendrocytes for in vitro experimentation remains difficult. Differentiation of galactocerebroside+ (GalC) and O4+ oligodendrocyte precursor or progenitor cells (OPC) from neural precursor cells has been reported using second trimester fetal brain. However, these cells do not proliferate in the absence of support cells including astrocytes and neurons, and are lost quickly over time in culture. The need remains for a culture system to produce cells of the oligodendrocyte lineage suitable for in vitro experimentation.
Culture of primary human oligodendrocytes could, for example, be a useful model to study the pathogenesis of neurotropic infectious agents like the human polyomavirus, JCV, that in vivo infects those cells. These cultured cells could also provide models of other demyelinating diseases of the central nervous system (CNS). Primary, human fetal brain-derived, multipotential neural progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons (progenitor-derived neurons, PDN) and astrocytes (progenitor-derived astrocytes, PDA) This study shows that neural progenitors can be induced to differentiate through many of the stages of oligodendrocytic lineage development (progenitor-derived oligodendrocytes, PDO). We culture neural progenitor cells in DMEM-F12 serum-free media supplemented with basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF-AA), Sonic hedgehog (Shh), neurotrophic factor 3 (NT-3), N-2 and triiodothyronine (T3). The cultured cells are passaged at 2.5e6 cells per 75cm flasks approximately every seven days. Using these conditions, the majority of the cells in culture maintain a morphology characterized by few processes and express markers of pre-oligodendrocyte cells, such as A2B5 and O-4. When we remove the four growth factors (GF) (bFGF, PDGF-AA, Shh, NT-3) and add conditioned media from PDN, the cells start to acquire more processes and express markers specific of oligodendrocyte differentiation, such as GalC and myelin basic protein (MBP). We performed phenotypic characterization using multicolor flow cytometry to identify unique markers of oligodendrocyte.

Primary, human fetal brain-derived, multipotential progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons and astrocytes. This work shows that neural progenitors can be induced to differentiate through stages of the oligodendrocytic lineage by conditioning with select growth factors.

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell ...

Heat-Induced Antigen Retrieval: An Effective Method to Detect and Identify Progenitor Cell Types during Adult Hippocampal Neurogenesis

Traditional methods of immunohistochemistry (IHC) following tissue fixation allow visualization of various cell types. These typically proceed with the application of antibodies to bind antigens and identify cells with characteristics that are a function of the inherent biology and development. Adult hippocampal neurogenesis is a sequential process wherein a quiescent neural stem cell can become activated and proceed through stages of proliferation, differentiation, maturation and functional integration. Each phase is distinct with a characteristic morphology and upregulation of genes. Identification of these phases is important to understand the regulatory mechanisms at play and any alterations in this process that underlie the pathophysiology of debilitating disorders. Our heat-induced antigen retrieval approach improves the intensity of the signal that is detected and allows correct identification of the progenitor cell type. As discussed in this paper, it especially allows us to circumvent current problems in detection of certain progenitor cell types.

During adult hippocampal neurogenesis, a distinct set of genetic markers are expressed when a quiescent neural stem cell sequentially progresses and develops into a functionally integrated neuron in the circuit. Using heat-induced antigen retrieval, progenitor cell types that are otherwise difficult to detect are identified with improved effectiveness.

Traditional  methods of immunohistochemistry (IHC) following tissue fixation allow visualization  of various cell types. These typically proceed with the ...

Growing Neural Stem Cells from Conventional and Nonconventional Regions of the Adult Rodent Brain

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the adult brain. However, the mechanisms these normally quiescent cells employ to ensure proper functioning of neural networks, as well as their role in recovery from injury and mitigation of neurodegenerative processes are little understood. These cells reside in regions referred to as &#34;niches&#34; that provide a sustaining environment involving modulatory signals from both the vascular and immune systems. The isolation, maintenance, and differentiation of CNS SCs under defined culture conditions which exclude unknown factors, makes them accessible to treatment by pharmacological or genetic means, thus providing insight into their in vivo behavior. Here we offer detailed information on the methods for generating cultures of CNS SCs from distinct regions of the adult brain and approaches to assess their differentiation potential into neurons, astrocytes, and oligodendrocytes in vitro. This technique yields a homogeneous cell population as a monolayer culture that can be visualized to study individual SCs and their progeny. Furthermore, it can be applied across different animal model systems and clinical samples, being used previously to predict regenerative responses in the damaged adult nervous system.

Neural stem cells harvested from the adult brain are increasing utilized in applications ranging from the basic research of nervous system development to exploring potential clinical applications in regenerative medicine. This makes rigorous control in the isolation and culturing conditions used to grow these cells critical to sound experimental outcomes.

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the ...

Lineage-reprogramming of Pericyte-derived Cells of the Adult Human Brain into Induced Neurons

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis and enable new strategies for in vitro modeling or repairing the diseased brain. Identifying brain-resident non-neuronal cell types amenable to direct conversion into iNs might allow for launching such an approach in situ, i.e. within the damaged brain tissue. Here we describe a protocol developed in the attempt of identifying cells derived from the adult human brain that fulfill this premise. This protocol involves: (1) the culturing of human cells from the cerebral cortex obtained from adult human brain biopsies; (2) the in vitro expansion (approximately requiring 2-4 weeks) and characterization of the culture by immunocytochemistry and flow cytometry; (3) the enrichment by fluorescence-activated cell sorting (FACS) using anti-PDGF receptor-&#946; and anti-CD146 antibodies; (4) the retrovirus-mediated transduction with the neurogenic transcription factors sox2 and ascl1; (5) and finally the characterization of the resultant pericyte-derived induced neurons (PdiNs) by immunocytochemistry (14 days to 8 weeks following retroviral transduction). At this stage, iNs can be probed for their electrical properties by patch-clamp recording. This protocol provides a highly reproducible procedure for the in vitro lineage conversion of brain-resident pericytes into functional human iNs.

Targeting brain-resident cells for direct lineage-reprogramming offers new perspectives for brain repair. Here we describe a protocol of how to prepare cultures enriched for brain-resident pericytes from the adult human cerebral cortex and convert these into induced neurons by retrovirus-mediated expression of the transcription factors Sox2 and Ascl1.

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis ...

Flow Cytometry Protocols for Surface and Intracellular Antigen Analyses of Neural Cell Types

Flow cytometry has been extensively used to define cell populations in immunology, hematology and oncology. Here, we provide a detailed description of protocols for flow cytometric analysis of the cluster of differentiation (CD) surface antigens and intracellular antigens in neural cell types. Our step-by-step description of the methodological procedures include: the harvesting of neural in vitro cultures, an optional carboxyfluorescein succinimidyl ester (CFSE)-labeling step, followed by surface antigen staining with conjugated CD antibodies (e.g., CD24, CD54), and subsequent intracellar antigen detection via primary/secondary antibodies or fluorescently labeled Fab fragments (Zenon labeling). The video demonstrates the most critical steps. Moreover, principles of experimental planning, the inclusion of critical controls, and fundamentals of flow cytometric analysis (identification of target population and exclusion of debris; gating strategy; compensation for spectral overlap) are briefly explained in order to enable neurobiologists with limited prior knowledge or specific training in flow cytometry to assess its utility and to better exploit this powerful methodology.

We provide a detailed description of a protocol for flow cytometric analysis of surface antigens and/or intracellular antigens in neural cell types. Critical aspects of experimental planning, step-by-step methodological procedures, and fundamental principles of flow cytometry are explained in order to enable neurobiologists to exploit this powerful technology.

Flow cytometry has been extensively used to define cell populations in immunology, hematology and oncology. Here, we provide a detailed description of ...

Immunohistochemistry and Multiple Labeling with Antibodies from the Same Host Species to Study Adult Hippocampal Neurogenesis

Adult neurogenesis is a highly regulated, multi-stage process in which new neurons are generated from an activated neural stem cell via increasingly committed intermediate progenitor subtypes. Each of these subtypes expresses a set of specific molecular markers that, together with specific morphological criteria, can be used for their identification. Typically, immunofluorescent techniques are applied involving subtype-specific antibodies in combination with exo- or endogenous proliferation markers. We herein describe immunolabeling methods for the detection and quantification of all stages of adult hippocampal neurogenesis. These comprise the application of thymidine analogs, transcardial perfusion, tissue processing, heat-induced epitope retrieval, ABC immunohistochemistry, multiple indirect immunofluorescence, confocal microscopy and cell quantification. Furthermore we present&#160;a sequential multiple immunofluorescence protocol which circumvents problems usually arising from the need of using primary antibodies raised in the same host species. It allows an accurate identification of all hippocampal progenitor subtypes together with a proliferation marker within a single section. These techniques are a powerful tool to study the regulation of different progenitor subtypes in parallel, their involvement in brain pathologies and their role in specific brain functions.

This video article illustrates a comprehensive protocol to detect and quantify all stages of adult hippocampal neurogenesis within the same tissue section. We elaborated a method to overcome the limitations of indirect multiple immunofluorescence that arise when suitable antibodies from different host species are unavailable.

Adult neurogenesis is a highly regulated, multi-stage process in which new neurons are generated from an activated neural stem cell via increasingly ...

Enumeration of Neural Stem Cells Using Clonal Assays

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. NSCs and neural progenitors (NPs) are commonly cultured in vitro as neurospheres. This protocol describes in detail how to determine the NSC frequency in a given cell population under clonal conditions. The protocol begins with the seeding of the cells at a density that allows for the generation of clonal neurospheres. The neurospheres are then transferred to chambered coverslips and differentiated under clonal conditions in conditioned medium, which maximizes the differentiation potential of the neurospheres. Finally, the NSC frequency is calculated based on neurosphere formation and multipotency capabilities. Utilities of this protocol include the evaluation of candidate NSC markers, purification of NSCs, and the ability to distinguish NSCs from NPs. This method takes 13 days to perform, which is much shorter than current methods to enumerate NSC frequency.

Neural stem cells (NSCs) refer to cells&#160;which can self-renew and differentiate into the three neural lineages. Here, we describe a protocol to determine NSC frequency in a given cell population using neurosphere formation and differentiation under clonal conditions.

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. ...

Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or postmitotic neurons. Later, some progenitors switch to a gliogenic fate, adding to the astrocyte and oligodendrocyte populations. Using time-lapse video-microscopy of primary cerebral cortex cell cultures, it is possible to study the cellular and molecular mechanisms controlling the mode of cell division and cell cycle parameters of progenitor cells. Similarly, the fate of postmitotic cells can be examined using cell-specific fluorescent reporter proteins or post-imaging immunocytochemistry. More importantly, all these features can be analyzed at the single-cell level, allowing the identification of progenitors committed to the generation of specific cell types. Manipulation of gene expression can also be performed using viral-mediated transfection, allowing the study of cell-autonomous and non-cell-autonomous phenomena. Finally, the use of fusion fluorescent proteins allows the study of symmetric and asymmetric distribution of selected proteins during division and the correlation with daughter cells fate. Here, we describe the time-lapse video-microscopy method to image primary cerebral cortex murine cells for up to several days and analyze the mode of cell division, cell cycle length and fate of newly generated cells. We also describe a simple method to transfect progenitor cells, which can be applied to manipulate genes of interest or simply label cells with reporter proteins.

Live imaging is a powerful tool to study cellular behaviors in real time. Here, we describe a protocol for time-lapse video-microscopy of primary cerebral cortex cells that allows a detailed examination of the phases enacted during the lineage progression from primary neural stem cells to differentiated neurons and glia.

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or ...

Expansion Microscopy for Visualizing Intracellular Sialylated Glycans via MOE

Visualizing Intracellular Sialylation with Click Chemistry and Expansion Microscopy

Metabolic labeling techniques allow the incorporation of bioorthogonal reporters into glycans, enabling the targeted bioconjugation of molecular dyes within cells through click and bioorthogonal chemistry. Metabolic oligosaccharide engineering (MOE) has attracted considerable interest due to the essential role of glycosylation in numerous biological processes that involve molecular recognition and its impact on pathologies ranging from cancer to genetic disorders to viral and bacterial infections.
Although MOE is better known for the detection of cell surface glycoconjugates, it is also a very important methodology for the study of intracellular glycans in physiological and pathological contexts. Such studies greatly benefit from high spatial resolution. However, super-resolution microscopy is not readily available in most laboratories and poses challenges for daily implementation. Expansion microscopy is a recent alternative that enhances the resolution of microscopy by physically enlarging biological specimens labeled with fluorescent markers. By embedding the sample in a swellable gel and causing it to expand isotropically through chemical treatment, subcellular structures can be visualized with enhanced precision and resolution without the need for super-resolution techniques.
In this work, we illustrate the capacity of expansion microscopy to visualize intracellular sialylated glycans through the combined use of MOE and click chemistry. Specifically, we propose a procedure for bioorthogonal labeling and expansion microscopy that employs a reporter targeting sialylation, which may be associated with immunofluorescence for co-localization studies. This protocol enables localization studies of sialoconjugate biosynthesis, intracellular trafficking, and recycling.

Here, we propose a simple protocol combining metabolic oligosaccharide engineering, click chemistry, and expansion microscopy that allows bioimaging of intracellular sialylated N-glycoproteins with improved resolution using routine microscopy equipment.

Metabolic labeling techniques allow the incorporation of bioorthogonal reporters into glycans, enabling the targeted bioconjugation of molecular dyes ...

Biology

Immunofluorescent Staining of Sialoglycoproteins in Neural Progenitor Cells and Neurons

Source: Bai, Q., et al., <a href="https://app.jove.com/v/58945">Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan.</a> J. Vis. Exp. (2019)
This video demonstrates the biotinylation and fluorescent labeling of modified sialoglycoproteins in primary neural stem and progenitor cells, as well as neurons, and their visualization using fluorescence microscopy.

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

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