A combinatorial panel for flow cytometry-based isolation of enteric nervous system cells from human intestine

Abstract

Efficient isolation of neurons and glia from the human enteric nervous system (ENS) is challenging because of their rare and fragile nature. Here, we describe a staining panel to enrich ENS cells from the human intestine by fluorescence-activated cell sorting (FACS). We find that CD56/CD90/CD24 co-expression labels ENS cells with higher specificity and resolution than previous methods. Surprisingly, neuronal (CD24, TUBB3) and glial (SOX10) selective markers appear co-expressed by all ENS cells. We demonstrate that this contradictory staining pattern is mainly driven by neuronal fragments, either free or attached to glial cells, which are the most abundant cell types. Live neurons can be enriched by the highest CD24 and CD90 levels. By applying our protocol to isolate ENS cells for single-cell RNA sequencing, we show that these cells can be obtained with high quality, enabling interrogation of the human ENS transcriptome. Taken together, we present a selective FACS protocol that allows enrichment and discrimination of human ENS cells, opening up new avenues to study this complex system in health and disease.

Keywords: ENS; FACS; enteric glia; enteric neurons; flow cytometry; human intestine.

Figure 1. Identification of the ENS cluster from pediatric intestinal material

1. Maximum intensity projections of the pediatric (0–12 months) small intestine showing the distribution of CD56, CD90, and CD24 within the ENS. The first image of each row shows a low‐magnification overview and an inset depicting the DAPI channel in high magnification. The remaining panels correspond to the high magnification inset. Scale bar in overview images = 200 μm. Scale bar in high magnification panels = 40 μm.

2. Initial identification of the ENS cluster as CD56⁺CD271⁺. ENS cells are in orange and non‐ENS cells in gray. The ENS gate is outlined in orange.

3. Gating on the single markers CD271 and CD56 (upper left and bottom left plot, respectively) versus SSC, followed by the analysis of the match of CD271 and CD56 single positive cells with the previously defined double positive CD271⁺CD56⁺ ENS cluster. CD56 shows a much better match than CD271.

4. The ENS cluster identified as CD56⁺CD90⁺, highlighted in red to differentiate it from the CD56⁺CD271⁺ defined ENS cluster, shows two subpopulations here defined as CD90^R and CD90^L (upper inset) and has a complete match with the CD271⁺CD56⁺ cluster (lower inset). The average percentage (mean ± SD) of ENS cells in representative ileum preparations was 7.1 ± 4.2% (mean ± SD, N = 20 biological replicates).

5. Histogram plot showing the percentage (mean ± SD, N = 5 biological replicates) of positive cells for the single markers CD271 and CD56, and CD56⁺CD90⁺, with the CD56⁺CD271⁺ cluster used in this initial phase, as “high purity standard”.

6. RT–qPCR from CD56⁺CD90⁺ sorted cells versus control non‐ENS cells for representative neuronal and glial markers, confirms high enrichment of both neurons and glial cells (mean ± SD, N = 4 biological replicates, *P < 0.05, ***P < 0.001, multiple Student's t‐test, FDR correction for multiple comparisons).

7. Single‐cell preparations from the human colon show the same staining pattern observed in the small intestine and a comparable amount of ENS cells (8.9 ± 3.4%; mean ± SD, N = 11 biological replicates).

Figure 2. Further validation and subdivision of the ENS cluster

1. ENS cells from the human ileum, selected as CD56⁺CD90⁺ (upper left) are additionally stained for CD24 (bottom left). ENS cells are CD24+ (in optimal conditions CD24⁺ ENS cells were 98 ± 1%, mean ± SD, N = 8 biological replicates) with evident subdivision in two main subpopulations, here defined CD24^LOW and CD24^HI. Right lower inset: magnification of the ENS cells in a CD56/CD24 plot with the subpopulations labeled in cyan (CD24^LOW) and dark yellow (CD24^HI). The average percentage of CD24^LOW and CD24^HI is indicated inside the plot (mean ± SD of N = 6 biological replicates). Right upper inset: a magnification of the ENS cluster in a CD56/CD90 plot shows a good, although not perfect, match of CD24^LOW (cyan) with CD90^L, and CD24^HI (dark yellow) with CD90^R. For additional details and statistics see Appendix Fig S2B–E.

2. Representative plot showing full co‐expression of TUBB3 (neuronal marker) and SOX10 (glial marker), on ENS cells (bottom) and only minimal and low‐intensity background on non‐ENS cells (Top; N = 1 representative sample, for statistics, see Fig 2C). Intracellular staining was performed on formalin‐fixed cells from the ileum, after prestaining for the extracellular markers. Selection of the ENS cluster in fixed cells is shown in Appendix Fig S2F.

3. Percentage of TUBB3⁺ and SOX10⁺ cells in ENS and non‐ENS cells (mean ± SD, TUBB3: N = 4 biological replicates, SOX10: N = 3 biological replicates). The single markers TUBB3 and SOX10 were gated versus SSC‐A as shown in Appendix Fig S2G.

Figure 3. Live nuclear staining of the ENS cluster

1. Live human intestinal cells (ileum) previously bulk sorted as Live/Lin‐ (as shown in Appendix Fig S1A), are further stained with the cell‐permeant and selective DNA‐binding dye Hoechst 33342, and analyzed. Hoechst 33342 staining subdivides the ENS cluster into nucleated cells (30.6 ± 7.8%) and non‐nucleated particles (68.5 ± 8.6%; mean ± SD, N = 6 biological replicates), largely overlapping in size (FSC‐A). Cells can be further refined by means of stringent doublet exclusion and cell cycle analysis, as shown in Appendix Fig S3A.

2. The distribution of ENS cells and non‐nucleated particles are represented in a CD56/CD90 plot. Left: initial selection of ENS cells. Right: zoom on the ENS cluster, where ENS events are shown as non‐nucleated, nucleated, or merge. For additional details and statistics see Appendix Fig S3E.

3. ENS cells and non‐nucleated particles are shown in a CD56/CD24 plot, as in Fig 3B. Nucleated and non‐nucleated events largely overlap and define the same subclusters in CD56/CD24 and CD56/CD90 plots, although the average staining intensity of the three markers is lower for non‐nucleated events. For additional details and statistics see Appendix Fig S3E.

4. ENS cells and non‐nucleated ENS particles are largely overlapping in an FSC‐A versus SSC‐A plot.

Figure 4. Microscopy of sorted ENS cells

Confocal images of ENS events sorted as nucleated and non‐nucleated, as shown in Fig 3.

1. A, B

   (A) Most of the staining belongs to what appears to be non‐nucleated (DAPI⁻) cellular debris (putative neuronal terminations) attached to a nucleated cell (putative glial cell). This is particularly true for TUBB3 and CD24, which show (as expected for neuronal selective markers) a starker distinction than CD56 (also expressed by glial cells). At a very low frequency among sorted ENS cells, we could observe putative neurons as represented in (B), with a more homogenous distribution of the three markers.

2. C

   Examples of sorted Hoechst‐negative ENS debris, which are confirmed to be non‐nucleated (thus validating our gates on Hoechst 33342‐A) but positive for all neuronal markers.

Figure 5. Discrimination of enteric neurons from EGCs

1. Upper left plot: selection of ENS cells as CD56⁺CD90⁺ in a methanol fixed sample from human ileum. The intensity of CD56 and CD90 (both stained before fixation) is much lower than in live cells but sufficient for ENS cell identification. Bottom left: ELAVL4 staining shows the presence of 2.5 ± 1.1% (mean ± SD, N = 3 biological replicates) ELAVL4⁺ ENS cells (purple “large” dots according to FACS DIVA software). Dots were enlarged to improve visibility in small‐size plots. Normal size dots are reported in Appendix Fig S4A for comparison. Right plots: ELAVL4⁺ cells, as defined by SSC‐A versus ELAVL4, correspond to the tip of the CD90^R ENS subcluster in a CD56/CD90 plot. Upper right inset: all ENS cells are shown, and CD24^TIP cells are highlighted in purple; middle‐right inset: only CD24^TIP cells are shown. Bottom right: an FSC‐A versus SSC‐A plot shows higher scattering‐distribution of putative neurons versus glial cells.

2. Selection of ENS cells in a live sample from human ileum, previously sorted as Live/Lin⁻ (upper left plot), is followed by gating single nucleated cells in G0/G1 (bottom left plot). Upper right plot: among ENS cells in G1/G0, a small subpopulation is visible on the right tip of the CD24‐based distribution, here defined as CD24^TIP and highlighted in purple using again “large” dots for better visibility. Bottom right plots: zoom on ENS cells in a CD56/CD90 plot. CD24^TIP cells are localized in the upper part of the CD90^R ENS subcluster. The percentage of each CD24‐based subpopulation is indicated in the CD24 versus CD90 plot as mean ± SD, N = 6 biological replicates. Further details are given in Appendix Fig S4B–D.

3. RT–qPCR for neuronal markers on cells sorted for CD24^TIP versus all the other ENS cells, confirms strong neuronal enrichment (mean ± SD, N = 5 biological replicates, ***P < 0.001, multiple Student's t‐test, FDR correction for multiple comparisons).

4. Comparison of an aganglionic (right) versus ganglionic colon (left) preparation from an HSCR patient (surgery was performed at the age of 4 months), using density plots that show all ENS events (cells + debris). A striking difference can be appreciated not only in the CD24^TIP, which is totally absent as expected, but also in the CD24^LOW cluster. This cluster, which is also absent, is paralleled by a similar unbalance of the corresponding CD90^R and CD90^L subclusters.

5. Graph showing the proportion of the CD24‐based subclusters in the nucleated versus non‐nucleated fraction of ganglionic and aganglionic colon (mean ± SD, N = 4 biological replicates, ***P < 0.001, multiple Student's t‐test, FDR correction for multiple comparisons). Additional details and statistics are shown in Appendix Fig S4E–J.

Figure 6. Characterization of sorted ENS cells by scRNAseq

1. Schematic representation of the sorting procedure for scRNAseq. Standard selection of Live/Lin⁻/CD56⁺CD90⁺ ENS cluster cells was followed by the selection of nucleated events. Negative selection of DAPI‐ (live) and Lin⁻ cells was performed as shown in Appendix Fig S1A. The procedure is as shown in Fig 3, with the difference that here nuclear staining was based on Cycle Green instead of Hoechst3342, and was performed in a single staining step. This is because the blue emitting viability dye DAPI (and also the Lin antibodies) allowed co‐staining with the green emitting cell cycle dye Cycle Green.

2. Uniform manifold approximation and projection (UMAP) of CD56⁺CD90⁺ sorted cells. The majority of captured cells are EGCs with minor contributions from contaminating cell types. Enteric neurons are also captured, albeit at a low frequency (1.6%).

3. UMAP of CD56⁺CD90⁺ sorted cells colored by sample origin. Relatively equal contribution of cells is observed from both colonic and ileal samples to the individual clusters.

4. Dot plot of top markers from each of the captured cell types. Each cell cluster can be defined by its own set of canonical markers.

5. Expression of CD24 across the entire dataset shows the increased expression in neurons and epithelial cells, and limited expression in glia, as expected.

6. UMAP of a subset of enteric neurons. Four subtypes of enteric neurons can be discerned.

7. Stacked violin plots showing enriched genes in specific neuronal subtypes.