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 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.