Structurally distinct LewisX glycans distinguish subpopulations of neural stem/progenitor cells

Abstract

There is increasing evidence that the stem and progenitor cell population that builds the central nervous system is very heterogeneous. Stem cell markers with the potential to divide this cell pool into subpopulations with distinct characteristics are sparse. We were looking for new cell type-specific antigens to further subdivide the progenitor pool. Here, we introduce the novel monoclonal antibody clone 5750. We show that it specifically labels cell surfaces of neural stem and progenitor cells. When 5750-expressing cells were isolated by fluorescence-activated cell sorting from embryonic mouse brains, the sorted population showed increased neurosphere forming capacity and multipotency. Neurospheres generated from 5750-positive cells could self-renew and remained multipotent even after prolonged passaging. Carbohydrate binding assays revealed that the 5750 antibody specifically binds to LewisX-related carbohydrates. Interestingly, we found that the LewisX epitope recognized by clone 5750 differs from those detected by other anti-LewisX antibody clones like 487(LeX), SSEA-1(LeX), and MMA(LeX). Our data further reveal that individual anti-LewisX clones can be successfully used to label and deplete different subpopulations of neural cells in vivo and in vitro. In conclusion, we present a new tool for the isolation and characterization of neural subpopulations and provide insights into the complexity of cell surface glycosylation.

FIGURE 1.

The mAb 5750 labels neural stem/progenitor cells. A, bright field image of a sagittal section of an E13.5 mouse embryo. A′, immunohistochemical staining using mAb 5750 of the section shown in A. Note the prominent 5750 immunoreactivity in the dorsal cortex and the weak expression in striatal regions. B and C, E13.5 cortex double labeled with mAb 5750 (red) and the indicated cell type-specific markers (green). The nuclei were counterstained with Hoechst (blue). The 5750-positive signal does not overlap with βIII-tubulin (βIII)-positive neurons, whereas there is a prominent but not complete overlap between 5750- and nestin-positive precursors. D, quantification of immunopositive cells from acutely dissociated forebrain at E10.5 and cortex at E14.5 and E18.5. E, acutely dissociated cells from E14.5 and E18.5 cortex (Ctx) or GE were immunocytochemically labeled with mAb 5750 and quantified. The number of 5750-positive cells decreases between E14.5 and E18.5 in the embryonic cortex. There are significantly fewer 5750-positive cells in the GE than in the cortex (n = 4). F–J, the photomicrographs show immunostainings for 5750 (red) and cell type-specific markers (green) on cryosections of primary neurospheres derived from E13.5 cortical tissue. 5750-positive cells strongly co-label with the neural stem/progenitor cell markers BLBP, GLAST, and nestin. Note that the 5750-positive cell population is not completely identical to the BLBP-, GLAST-, or nestin-positive cell population. The astroglial marker GFAP and the neuronal cell marker βIII-tubulin show no strong overlap with the 5750 signal. K, immunohistochemical staining of cells from E13.5-derived cortical neurospheres cultivated under adherent, differentiating conditions. MAb 5750 (red) labels only a subpopulation of nestin-positive cells (green). The nuclei are shown in blue. The data are expressed as the means ± S.D. (**, p ≤ 0.01; ***, p ≤ 0.001). Scale bars, 1 mm (A) or 50 μm (B, C, F-J, and K). FB, forebrain; βIII, βIII-tubulin.

FIGURE 2.

FACS sorting using mAb 5750 enriches for self-renewing, multipotent neural precursor cells. E14.5 cortices were dissociated to single cell suspensions, stained with mAb 5750 and FITC-conjugated secondary antibody, and subjected to FACS analysis. A, the diagram shows the gate settings of one representative experiment of three for the FACS analysis. A 5750-positive cell population expressing high levels of the 5750 epitope (5750) and a 5750-negative cell population (negative) was isolated. B, immunocytochemical analysis of the sorted cells shows that the 5750-positive cell population (5750) contains mostly nestin-positive precursor cells, whereas the majority of βIII-tubulin-positive neurons are found in the 5750-negative cell population (negative). Unsorted cells were used as a control (control). C, the 5750-positive cell population (5750) shows a 3-fold increased neurosphere forming capacity compared with the unsorted control (control) and 32-fold to the immunodepleted cell population (negative). D, neurospheres obtained from 5750-positive cells are multipotent and retain multipotency over several passages. Immunocytochemical analysis of differentiated neurospheres shows that βIII-tubulin-positive neurons (red), GFAP-positive astrocytes (blue), and O4-positive oligodendrocytes (green) are present. The data are expressed as the means ± S.D. (*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001). P, passage; βIII, βIII-tubulin. Scale bar, 50 μm.

FIGURE 3.

The mAb 5750 detects a glycan on Tenascin-C, L1-CAM, and Phosphacan. A and B, immunoaffinity-purified Tenascin-C (A) or L1-CAM (B) were treated either with (+) or without (−) N-glycosidase F (N-Glyc.F) to remove N-linked glycans. The silver stainings confirm the purity and integrity of the respective proteins. Western blot analysis with polyclonal antibodies against the respective proteins (pk Tnc and pk L1) visualizes migration of the core protein in the gel. Note that both in silver stain and after detection with polyclonal antibodies, the detected bands shift down after N-Glyc.F treatment. N-Glyc.F activity was controlled by the removal of the HNK1 epitope. Probing with mAb 5750 shows that the epitope is present on both Tenascin-C and L1-CAM and disappears following N-Glyc.F treatment. C, immunoaffinity-purified Phosphacan was subjected to SDS-PAGE after treatment (+) with N-Glyc.F and/or in combination with ChABC. By Western blotting, 5750 immunoreactivity on Phosphacan is not altered neither after treatment with N-Glyc.F, ChABC, or both enzymes. Note the shift in migration after ChABC treatment. Blotting for HNK1 shows successful N-Glyc.F digestion.

FIGURE 4.

The 5750 antibody binds to carbohydrates containing the LeX motif. A, a total of 511 structurally defined glycans was tested for binding to mAb 5750 using a standardized glycan array. The graph shows the results of one representative experiment of 5. The numbers indicate the glycans on array version 4.2 that showed high affinity for mAb 5750. Their structural compositions are depicted in C and D. Note that all of the ligands contain the basic LeX motif. The error bars are indicated in gray. B, glycan array version 4.2 performed with the LeX-directed mAb 487^LeX. Note that the detected glycans overlap only to some extent with those detected by mAb 5750. C–E, structural composition of the glycans that showed high affinity exclusively for mAb 5750 (C), for both mAbs 5750 and 487^LeX (D), and for 487^LeX, only (E). The analysis suggests that mAb 5750 is directed against an internal tandem repeat of LeX, whereas mAb 487^LeX binds terminal LeX, also on a short spacer. F, COS7 cells were transfected with a GFP control plasmid (GFP) or in combination with a plasmid coding for Fut9 (Fut9 + GFP). The 5750 epitope was only detectable on Fut9-transfected COS7 cells, indicating that α1–3-linked fucose produced by Fut9 is sufficient for binding of mAb 5750. RFU, relative fluorescent units; Fuc, fucose; Gal, galactose; Sp0, -CH₂CH₂NH₂; Sp8, -CH₂CH₂CH₂NH₂. Scale bar, 50 μm.

FIGURE 5.

The 5750 epitope is related but not identical to the epitope recognized by anti-LeX mAb 487^LeX. A, schematic diagram of a sagittal mouse brain section at E14. The dashed lines indicate coronal planes shown in B–D and B′–D′. B–D and B′–D′, immunofluorescent stainings (white) of coronal sections on different anterior to posterior levels at E14.5. Two adjacent sections are stained with mAb 5750 (B–D) or 487^LeX (B′–D′). Overall, both antibodies show a similar expression pattern. E, Western blot of whole brain lysates of different development stages analyzed with anti-LeX mAb 5750 or 487^LeX. LeX glycosylation peaks at P10 using both antibodies. Note that mAb 487^LeX detects additional LeX-glycosylated proteins below 250 kDa. The arrowhead indicates the border between stacking and resolving gel. α-tub., α-tubulin. F, quantification of acutely dissociated cells immunopositive for mAb 5750 (black bar) or 487^LeX (gray bar) and originating from different developmental stages and brain areas as indicated. No significant difference in the number of 5750- or 487^LeX-positive cells is detectable between E10 and E18. The data are expressed as the means ± S.D. of at least three independent experiments. G, dissociated cells from the telencephalon at postnatal day 2 were stained after 24 h with anti-LeX mAbs 5750 and 487^LeX. Immunopositive cells were quantified. The 487^LeX-positive cell population is 5-fold larger than the 5750-positive cell population (n = 3; **, p ≤ 0.01). H and I, immunofluorescent stainings (white) of coronal sections of the mouse telencephalon at P1 stained with mAbs 5750 (H) or 487^LeX (I). FB, forebrain; Ctx, cortex; LGE, lateral GE; MGE, medial GE; CGE, central GE; Di, diencephalon; LV, lateral ventricle. Scale bars, 800 μm (B–D) or 800 μm (H and I).

FIGURE 6.

Depletion of the 5750-positive cell population by complement mediated immunocytolysis reduces neurosphere forming capacities of cortical cells. A–D, E14.5 acutely dissociated cortical cells were incubated with mAb 5750 (A and B) or mAb 487^LeX (C and D) in the presence of complement-active guinea pig serum (B and D) to eliminate the 5750- or 487^LeX-positive cell population, respectively. As a control, the cells were incubated with antibody alone (A and C). Afterward, the cells were stained with mAbs 487^LeX, 5750 (red), or βIII-tubulin (green). The nuclei are stained with Hoechst (blue). After immunocytolysis, LeX-positive cells disappear. E and F, complement-mediated immunocytolysis with mAb 5750 and anti-LeX mAb clone 487^LeX of E14.5 acutely dissociated cells from cortex (E) or GE (F). Shown is the percentage of neurosphere formation normalized to an untreated control (-ab). After incubation with 5750 antibody in the presence of complement (c+), neurosphere formation of cortical cells is reduced by 8-fold in comparison with cells treated with mAb 5750 alone (c−). This correlates with the 8-fold reduction seen when immunodepletion is performed with anti-LeX mAb 487^LeX. With mAb 487^LeX cells from GE show a reduction by 2-fold, only. The reduction seen with mAb 5750 is not significant. The data are expressed as the means ± S.D. of at least three independent experiments (n = 3). *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001. Scale bar, 50 μm.

FIGURE 7.

Different anti-LeX monoclonal antibody clones are unique and enable to distinguish between cell types. A, mixed neural cultures were cultivated as shown, by dissociating postnatal forebrain tissue and cultivation in the presence of horse serum (HS). B–D, double labeling of mixed neural cultures with mAb 5750 (red) and anti-LeX antibody clones 487^LeX (B), MMA^LeX (C), or SSEA-1^LeX (D) in green. The cell populations labeled with the different anti-LeX mAbs overlap to some extent but are not identical. Note that mAb 487^LeX strongly labels process-bearing cells with small somata, whereas mAb clones 5750, MMA^LeX, and SSEA-1^LeX label cells with flat soma, devoid of processes. However, 5750 overlaps only to some extent with MMA^LeX or SSEA-1^LeX. E, protein lysates from mixed neural cultures (MNC), astrocyte-enriched cultures (Astros), or oligodendrocyte precursor cell enriched cultures (OPC) were analyzed by Western blot with different anti-LeX mAbs. The protein pattern detected in the Western blot is unique for each anti-LeX antibody clone. MAb 487^LeX detects additional proteins on OPCs that are only very weakly or not at all detectable with mAbs 5750 and MMA^LeX (asterisks). The arrowhead indicates the top of the resolving gel. F, mixed neural cultures were incubated with complement-active guinea pig serum alone (control) and in the presence of mAbs 487^LeX or 5750 to deplete the respective cell population. The Western blot shows that 487^LeX immunodepletion leads to a complete elimination of the PDGFRα-positive cell population, whereas 5750 immunodepletion had no effect on PDGFRα-positive cells. The efficiency of the immunocytolysis was verified by blotting for 487^LeX and 5750. G–I, immunofluorescent images of immunodepleted mixed neural cultures, as described for F, stained for 5750, 487^LeX (red), and PDGFRα (green), as indicated. The cell nuclei are labeled in blue. Note that 487^LeX immunodepletion targets OPCs, whereas 5750 immunodepletion does not. NPC, neural precursor cell; α-tub., α-tubulin. Scale bar, 50 μm.