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

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 brains^1,2. As development proceeds, the neural stem cell population becomes heterogenous^3,4, 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 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 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 progeny^11,12. Thus, protein 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 non-co-culture system. We also selectively purify surface sialoglycoproteome in the two culture systems for proteomic comparison. Our protocol, compared with 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.

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).

NOTE: All cells are cultured in the cell incubator under conditions of 37 °C and 5% CO₂.

1. Preparation of Mouse Endothelial Culture in Permeable Support Inserts

NOTE: BEND3 cells are maintained according to manufacturer's instructions.

1. 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.
2. 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 °C.
3. 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.
4. 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.

2. Preparation of Mouse Primary Cortical NSPCs Culture

1. Preparation of culture plate, papain digestion medium, and cortical adherent culture medium (AM)
   1. 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.
   2. 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.
   3. Prepare the papain digestion medium by adding 50 U of papain, 50 µL of L-glutamine, and 50 µL of 100 mg/mL acetyl-L-cysteine into 5 mL of DMEM. Mix the medium briefly and warm it to 37 °C for 30 min for enzyme activation.
   4. Prepare the cortical cell adherent culture medium (AM): add 500 µL of L-glutamine, 500 µL of sodium pyruvate, 500 µL of 100 mg/mL N-acetyl-L-Cysteine, 500 µL of N2, 1 mL of B27, and 5 µL of 100 µg/mL bFGF into 50 mL of DMEM. Mix the medium well and warm it to 37 °C before use.
2. Preparation of primary cerebral cortical cells and subsequent plating
   1. 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.
   2. 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.
   3. 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.
   4. 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.
   5. 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.
   6. Pellet the cortices by centrifugation for 3 min at 4 °C and 300 x g. Aspirate the supernatant from the tube, then add activated papain digestion medium and 15 µL of 4 mg/mL DNase I into the tissue pellet.
   7. Resuspend the tissue pellet briefly by gentle vortexing. Incubate the tissue at 37 °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.
   8. Pellet the cortical cells by centrifugation for 10 min at 4 °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.
   9. 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.
   10. Add 2 mL of AM and 2 x 10⁴ cortical cells per well into 6-well plates. Incubate the plate at 37 °C and 5% CO₂ for 3 h to let the cells attach to the plate.

3. Set-up of Neural-endothelial Co-culture and Ac₄ManNAz Labeling System

1. 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.
2. 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 °C and 5% CO₂ for 12 h.
3. Dissolve Ac₄ManNAz in DMSO to achieve a stock concentration of 200 mM. 12 h after setting up the neural-endothelial co-culture, add 1 µL of Ac₄ManNAz stock per bottom chamber and 0.5 µL 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.
4. Culture the cells for another 5 days at 37 °C and 5% CO₂. Prepare the AM with 10x bFGF as refeeding medium (RM). During this time, add 100 µL of RM per insert and 200 µL 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.

4. Immunofluorescent Staining of Sialoglycoproteins in Expanded Primary NSPCs and Differentiated Neurons

1. 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 µM biotin-alkyne, 2.5 mM sodium ascorbate, and 1x BTTAA-CuSO₄ complex in PBS.
2. 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.
3. 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.
4. 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.
5. Aspirate the reaction buffer from the wells. Wash the cells 3 times with PBS. Prepare the staining buffer containing 1% FBS and 1 µg/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.
6. 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.
7. Prepare a primary antibody solution by diluting the anti-nestin and anti-β-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 °C overnight.
8. 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.
9. 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.

5. Purification of Sialoglycoproteins from Expanded Primary NSPCs and Differentiated Neurons

1. 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.
2. 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.
3. Aspirate the PBS from the wells and add 200 µL 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 °C and 12,000 x g.
4. Transfer the supernatant into new 1.5 mL tubes. Determine protein concentration with the BCA kit according to the manufacturer's instructions. Adjust the protein concentration to 1 mg/mL.
5. Add 100 µM 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.
6. Transfer the reaction solution into 20 mL of pre-chilled methanol in a 50 mL conical tube. Mix well and incubate at -30 °C overnight to precipitate the proteins.
7. Pellet the protein precipitates by centrifugation for 15 min at 4 °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.
8. Take 50 µL of streptavidin beads and wash them 3 times with PBS. Add the washed beads into the protein resuspension. Incubate the solution at 4 °C for 3 h on a vertical rotator at a rotation speed of 20 rpm.
9. 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.
10. After washing, resuspend the beads with 20 µL of PBS and transfer the beads into a new 1.5 mL tube. Add 20 µL of 2x protein loading buffer into the beads and treat at 95 °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's instructions. Cut the proteins in gel as indicated by Coomassie brilliant blue R-250 for mass spectrometry analysis.

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 vivo^17,18. 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...

Materials

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 1,000
Alexa Fluor 488 goat anti-mouse IgG1	invitrogen	A-21121	1 to 1,000
Alexa Fluor 546 goat anti-mouse IgG2b	invitrogen	A-21143	1 to 1,000
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 1,000
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