Purification of interstitial cells of Cajal by fluorescence-activated cell sorting

Tamás Ördög,1 Doug Redelman,2,4 Lisa J. Miller,1 Viktor J. Horváth,1 Qiao Zhong,2 Graça Almeida-Porada,3 Esmail D. Zanjani,3 Burton Horowitz,1 and Kenton M. Sanders1

1Department of Physiology and Cell Biology, 2Cytometry Center, and 3Department of Animal Biotechnology, University of Nevada, Reno 89557, and 4Sierra Cytometry, Reno, Nevada 89509

Submitted 30 June 2003 ; accepted in final form 1 October 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Interstitial cells of Cajal (ICC) in the gastrointestinal tract generate and propagate slow waves and mediate neuromuscular neurotransmission. Although damages to ICC have been described in several gastrointestinal motor disorders, analysis of their gene expression in health and disease has been problematic because of the difficulties in isolating these cells. Our goal was to develop techniques for large-scale purification of ICC. Murine ICC were identified in live gastrointestinal muscles with fluorescent Kit antibodies. Because this technique also labels resident macrophages nonspecifically, we attempted to separate ICC from these cells by fluorescence-activated cell sorting with or without immunomagnetic presorting. Efficacy and specificity of ICC purification were tested by quantitative RT-PCR of cell-specific markers. Fluorescence-based separation of small intestinal ICC from unlabeled cells and macrophages tagged with F4/80 antibodies yielded 30,000–40,000 cells and ~60-fold enrichment of c-kit mRNA. However, the macrophage marker CD68 was also enriched ~6-fold. Magnetic presorting of ICC did not significantly improve selectivity. After labeling contaminating cells with additional paramagnetic (anti-CD11b, -CD11c) and fluorescent antibodies (anti-CD11b) and depleting them by magnetic presorting, we harvested ~2,000–4,000 cells from single gastric corpus-antrum muscles and detected an ~30-fold increase in c-kit mRNA, no enrichment of mast cells, and an ~4-fold reduction of CD68 expression. Adding labeled anti-CD45 antibody to our cocktail further increased c-kit enrichment and eliminated mast cells and macrophages. Smooth muscle cells and myenteric neurons were also depleted. We conclude that immunofluorescence-based sorting can yield ICC in sufficiently high numbers and purity to permit detailed molecular analyses.

mouse; c-kit; macrophage; dendritic cell; mast cell


NORMAL GASTROINTESTINAL (GI) motor functions require the coordinated actions of the smooth musculature, which is responsible for contractile activity, the enteric and autonomic nervous systems, which generate motor patterns, and interstitial cells of Cajal (ICC), which drive rhythmic contractions by generating electrical slow waves and mediate neural inputs to smooth muscle cells (12, 31). Electrical pacemaking and mediation of neurotransmission are performed by distinct classes of ICC: 1) Slow waves are generated and propagated by multipolar ICC that form two-dimensional networks in the myenteric region, on the submucosal border of the circular smooth muscle layer, and within intermuscular septa of phasic GI smooth muscles (5, 11, 13, 22, 24, 43). Pacemaking by ICC depends on cycling of Ca2+ between intracellular compartments (endoplasmic reticulum and mitochondria) mediated by specialized channels and pumps (16, 32, 36, 44), and rhythmic electrical activity per se is the consequence of periodic openings of voltage-insensitive, nonselective cation channels driven by the intracellular Ca2+ oscillations (15, 44). Other investigators have suggested a role for Ca2+-regulated Cl- channels in pacemaker activity (14, 40). 2) Mediation of neuromuscular neurotransmission and, possibly, mechanoreception, are functions of elongated or strictly bipolar ICC that occur throughout the GI tract (7, 45). These cells represent the only ICC class found in tonic muscles (45). They form close contacts with varicose processes of enteric motor neurons and mediate excitatory and inhibitory neural inputs to the smooth musculature via electrical coupling (45) and, in the case of nitrergic inhibitory inputs, by releasing additional nitric oxide (27). Despite these functional differences, ICC classes share many features (e.g., immunoreactivity for Kit/CD117, a receptor tyrosine kinase; structural and functional dependence on Kit signaling, ultrastructure) (31), and gene expression studies have so far failed to identify significant differences between them (6, 9).

The recognition that Kit expression can be used as a histological marker for ICC (19) has led to important observations about the role of ICC in human motor pathologies. Depletion of ICC populations have been described in tissues of patients with a variety of GI motor disorders, including congenital (anorectal malformations, Hirschsprung's disease, infantile pyloric stenosis) and acquired diseases (inflammatory bowel disease, diabetic gastroenteropathy, stromal tumors, paraneoplastic, idiopathic or "functional" disorders) (10, 18, 21, 25, 26, 31, 41). While these studies do not go beyond descriptions (and in some cases quantification) of ICC loss, animal models of GI disorders offer exciting opportunities to unravel the changes in gene expression that underlie lesions in ICC networks (3, 4, 17, 22, 23, 33).

Understanding what makes ICC and ICC classes unique, and why these cells undergo damage or phenotypic changes in various disorders, may be aided by large-scale analysis of gene expression. Molecular analyses of ICC have been difficult because of the scarcity and widespread distribution of these cells in the tunica muscularis. ICC are a minor component of GI muscles, and, therefore, molecular analyses of GI muscles are heavily contaminated by the expression patterns of other cell types. In previous studies investigators have harvested small numbers of ICC from primary cultures and suspensions of freshly dispersed cells by sucking visually identified native or immunostained Kit+ cells into micropipettes for RT-PCR analysis (6, 9, 30, 39). Major drawbacks of these approaches might include erroneous identification of nonspecifically stained cells as ICC, experimenter bias, inability to obtain sufficient mRNA for quantitative RT-PCR or large-scale genomic analysis or for reliable detection of genes expressed at low levels, and reliance on data collected from a very small number of cells (which may not be representative of the tens of thousands of ICC populating GI tissues). Therefore, we have sought alternative approaches to isolating ICC based on immunolabeling and large-scale purification by fluorescence-activated cell sorting (FACS).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals

BALB/c mice (9–15 days old) were obtained from breeder pairs purchased from either Simonsen Laboratories (Gilroy, CA) or Charles River Laboratories (Wilmington, MA). BALB/c mice 5–6 wk old were purchased from Charles River Laboratories. The animals were anesthetized by isoflurane inhalation (AErrane, Baxter Healthcare, Deerfield, IL) and killed by decapitation. Mice were maintained and the experiments performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the American Physiological Society's "Guiding Principles in the Care and Use of Animals." All protocols were approved by the Institutional Animal Use and Care Committee at the University of Nevada, Reno.

Tissue Preparation

The entire small intestines from juvenile mice and stomachs from adult animals were excised and opened along the insertion of the mesentery and the lesser curvature, respectively. Contents were washed away with Krebs-Ringer bicarbonate solution (see composition in Solutions). The mucosa and submucosa were removed by peeling, and only the tunica muscularis of the entire jejunum and ileum or gastric corpus and antrum were used.

Preparation of Labeled Cell Suspensions for FACS

FACS of ICC was based on the detection of the ICC marker Kit with extracellularly reacting rat monoclonal (IgG2b) antibodies (ACK2). Because the epitope recognized by ACK2 is destroyed or masked by collagenase treatment, ICC labeling had to be performed in the tissues before cell dispersion (6, 9). We tested four approaches to purify ICC from small intestinal and gastric muscles. After the description of approach 1 below, only the differences are noted.

Approach 1. Small intestinal muscles from 9- to 13-day-old mice were pinned loosely, with the submucosal side up, to pieces of halved PE-50 polyethylene tubing (Fisher Scientific, Pittsburgh, PA) secured to the surface of a 35-mm culture dish coated with Sylgard 184 silicone elastomer (Dow Corning, Midland, MI). This arrangement was meant to ensure that the tissues did not adhere to each other and would be exposed to the antibody solutions on both sides. Because of the unavoidable uptake of the ICC labels by resident macrophages (6, 9), these cells were also labeled with a rat monoclonal (IgG2b) anti-mouse F4/80 (a macrophage marker; clone CI:A3–1) antibody (20) conjugated with the Tri-Color fluorochrome (TC; a tandem conjugate of R-phycoerythrin and Cy5; CALTAG, Burlingame, CA). The macrophage label was diluted to 4 µg/ml with Ca2+-containing, HEPES-buffered physiological salt solution (CaPSS; see Solutions) and applied at room temperature for 2 h. After the tissues were washed with cold CaPSS, ICC were labeled with ACK2 that was conjugated with the fluorescent dye Alexa Fluor 488 (AF 488) using the Alexa Fluor 488 Protein Labeling Kit from Molecular Probes (Eugene, OR). AF 488-ACK2 conjugates were diluted to 10 µg/ml in CaPSS and applied for 3 h at 4°C. After labeling, the tissues were equilibrated with nominally Ca2+-free Hanks' salt solution (CFH; see Solutions) and incubated overnight at 4°C with an enzyme solution containing 1.3 mg/ml collagenase (type II; Worthington Biochemical, Freehold, NJ), 2 mg/ml BSA, 2 mg/ml trypsin inhibitor, and 0.27 mg/ml ATP (all from Sigma, St. Louis, MO) in CFH, and then for an additional 7 min at 37°C. After three washes with CFH, the tissues were triturated through a series of three blunt pipettes of decreasing tip diameter. The resultant cell suspensions were sedimented by centrifugation (300 g, 5 min), resuspended in CFH containing 2% BSA and 2 mM EDTA (sorting buffer), and filtered through a polyester filter with 30-µm mesh size (Miltenyi Biotec, Auburn, CA) to obtain single-cell suspensions. Aliquots (≥25,000 cells) from each suspension were saved in sterile, siliconized microcentrifuge tubes (Fisher Scientific), sedimented by centrifuging at 3,000 g for 30 min, and immediately processed for quantitative RT-PCR as the "unsorted group." The remainder of the suspensions was used for FACS.

Approach 2. We attempted to increase the selectivity of the ICC purification by including positive immunomagnetic selection (MACS) in the protocol. The small intestinal tissues were prepared and labeled as for approach 1. After trituration, the cell suspensions were sedimented by centrifugation (300 g, 5 min, 4°C) and washed with 4 ml of cold, carefully degassed sorting buffer. The labeled ACK2 was then reacted with goat F(ab')2 anti-rat IgG (H+L) conjugated with paramagnetic beads (Miltenyi; 1:5 in sorting buffer, 4°C for 15 min). After washing, the labeled cells were resuspended in 1 ml of sorting buffer and passed through prewetted polyester filters (Miltenyi, 30-µm mesh size), which were then washed three times with 1 ml of sorting buffer. An aliquot of the flow-through (≥25,000 cells) was saved as the unsorted group. The remaining suspension was passed through prewetted MS magnetic columns placed in a strong magnet (Mini-MACS; all from Miltenyi). Cells not retained on the columns by the magnet were washed three times with 0.5 ml of sorting buffer. The columns were then removed from the magnet, and the retained cells were flushed out with sorting buffer (MACS+ cells; 1 ml) and used for FACS.

Approach 3. Gastric corpus and antrum muscles from adult mice were used in these experiments. The tissues were loosely pinned to miniature triangular frames constructed from halved PE-50 tubing, and labeling was carried out in microcentrifuge tubes. Labeling with the TC-anti-F4/80 antibody was performed at 37°C for 1 h. We also employed fluorescent antibodies to the integrin CD11b (Mac-1{alpha}), another macrophage marker (35), and attempted to reduce the number of CD11b+ resident macrophages and CD11c (integrin {alpha}x chain)-positive dendritic cells (28) by immunomagnetic depletion (negative MACS). After trituration, the cells were sedimented by centrifugation (300 g, 5 min, 4°C), resuspended in cold, degassed sorting buffer, and filtered as described for approach 1. Macrophages and dendritic cells were labeled with rat monoclonal (IgG2b) anti-mouse/human CD11b (clone M1/70.15.11.5) conjugated with paramagnetic beads (10 µl/106 cells) and paramagnetic hamster monoclonal (IgG) anti-mouse CD11c (clone N418; 20 µl/106 cells; both from Miltenyi Biotec), respectively, by incubating the cells in a 100-µl total volume at 4°C for 15 min. Fluorescent labeling of macrophages was also reinforced by adding 0.4 µg of TC-conjugated rat monoclonal (IgG2b) anti-mouse CD11b antibody (clone M1/70.15; CALTAG) per 106 cells to the above mixture and incubating the cells for an additional 10 min at 4°C. The cells were then washed and resuspended in 1 ml of sorting buffer. An aliquot (≥25,000 cells) was saved at this step as the unsorted group. The remaining suspension was passed through prewetted MS magnetic columns equipped with 26-gauge needles as flow resistors and placed in a MiniMACS magnet (all from Miltenyi). Cells not retained on the columns by the magnet were washed three times with 0.5 ml of sorting buffer, and the entire flow-through (MACS-cells) was used for FACS.

Approach 4. These experiments were carried out in a manner identical to approach 3, except that cells from six adult stomachs were pooled to obtain a higher number of sorted cells for quantitative RT-PCR analysis. We also attempted to tag mast cells (and further reinforce the labeling of macrophages and other hematopoietic cells) by including TC-conjugated rat monoclonal (IgG2b; k) anti-mouse CD45 (Ly-5 or leukocyte common antigen, LCA) antibodies (0.1 µg/106 cells; clone 30-F11; eBioscience, San Diego, CA) with the anti-CD11b and -CD11c antibodies in the labeling mixture before magnetic depletion.

Flow Cytometry Analysis

For flow cytometry (FCM) analysis, labeled cell suspensions were analyzed with a Beckman-Coulter (Fullerton, CA) XL/MCL flow cytometer equipped with an argon ion laser (excitation wavelength: 488 nm), a photodiode to measure light scattered at low forward angles (forward scatter, FSC), and photomultiplier tubes to measure orthogonally scattered light (side scatter, SSC), plus four wavelengths of fluorescence at 525 nm [used for the detection of AF 488-ACK2 (emission maximum, Em: 519 nm) and FITC-anti-CD11c (Em: 518 nm)]; 575 and 610 nm (unused); and 675 nm [used for TC-anti-F4/80, TC-anti-CD11b, and TC-anti-CD45 (Em: 667 nm)]. Cells were detected by triggering on FSC signals, and data files of ≥20,000 events were collected by using the Coulter System II acquisition software. The green and red emissions from the AF 488 and TC dyes were spectrally distinct and required no fluorescence compensation. List-mode data files were analyzed with SuperCyt Analyst (Sierra Cytometry) or FlowJo (Treestar, San Carlos, CA) software. Regions were created to define clusters with green (presumed ICC), red and red + green fluorescence (single- or double-labeled macrophages and other hematopoietic cells) and with unlabeled cells presumably including enriched populations of smooth muscle cells and myenteric neurons.

Purification of ICC by FACS

Purification of ICC was performed on either a Beckman-Coulter Elite sorter (setting: "purify first") or a Becton Dickinson Immunocytometry Systems (San Jose, CA) FACSVantage instrument (setting: "normal C," which produces high purity, lower recovery, and higher count accuracy). The sorters were set to match the measurements determined on the FCM analytical instrument. Two cell populations (ICC and macrophages or ICC and unlabeled cells, which include smooth muscle cells and myenteric neurons) were sorted using the light scatter and fluorescence parameters determined by FCM in the previous step and collected in sterile, siliconized microcentrifuge tubes (Fisher Scientific), sedimented by centrifuging at 3,000 g for 30 min, and immediately processed for quantitative RT-PCR. Proper instrument operation was verified by analyzing standard reference beads.

Quantitative RT-PCR

Quantitative RT-PCR was employed to assess the efficacy and specificity of the ICC sorting. Total RNA was isolated using the Trizol reagent (Invitrogen, Carlsbad, CA) following the manufacturer's instructions with minor modifications. Genomic DNA was removed by incubating total RNA either with RNase-free DNase (5 U per 1 µl per tube; PanVera, Madison, WI) for 20 min at 25°C, followed by heat inactivation for 5 min at 75°C, or with the Stratagene Absolutely RNA Nanoprep Kit (La Jolla, CA). Total RNA was reverse transcribed with 200 units of SuperScript II RNase H- Reverse Transcriptase (Invitrogen) in a reaction containing 500 ng of oligo d(pT)18 primer (New England Biolabs, Beverly, MA), 10 mM each dNTP, 5x First-Strand Buffer, and 100 mM dithiothreitol (DTT), followed by heat inactivation. The cDNA reverse transcription product was amplified with specific primers (see below) by PCR, using the following amplification profile: 95°C for 10 min to activate the AmpliTaq polymerase (Applied Biosystems, Foster City, CA), and then 40 or 50 cycles of 95°C for 15 s and 60°C for 1 min. Real-time quantitative PCR was performed by using SYBR Green chemistry on a GeneAmp 5700 sequence detector (Applied Biosystems). Standard curves were generated for each primer set by regression analysis of RT-PCRs performed on log10-diluted cDNA. Unknown quantities relative to the standard curve for the housekeeping gene {beta}-actin (GenBank accession no. X03672 [GenBank] ; primers: sense bordering nucleotide positions nt 210–231, antisense bordering nucleotide positions nt 337–360) were calculated to obtain transcriptional quantification of gene products. These values were then normalized to {beta}-actin expression to control for differences in the number of cells used in the assay. The following PCR primers were used to detect cell type-specific mRNA species: c-kit (Y00864 [GenBank] , ICC): sense, nt 2706–2726; antisense, nt 2847–2867; CD68 [NM_009853; macrosialin, a pan-macrophage marker also expressed by some myeloid-derived dendritic cells (1)]: sense, nt 41–65; antisense, nt 201–225; mast cell tryptase (MCT; M57626 [GenBank] ; mast cells): sense, nt 264–283; antisense, nt 433–454; smooth muscle myosin heavy chain (MyHC; NM_013607 [GenBank] ; smooth muscle): sense, nt 5721–5745; antisense, nt 5930–5954; protein gene product 9.5 (PGP 9.5, a pan-neuronal marker; AF172334 [GenBank] ): sense, nt 22–44; antisense, nt 171–190; and prolyl-4-hydroxylase [BC018411; fibroblasts (6, 42)]: sense, nt 1012–1031; antisense, nt 1144–1162. All primers were obtained from Keystone Labs (Camarillo, CA). The amplified products (10 µl) were separated by electrophoresis on a 2% agarose/1x TAE (Tris, acetic acid, EDTA) gel, and the DNA bands were visualized by ethidium bromide staining for qualitative assessment. To confirm the specificity of the primers, PCR products generated from each pair of primers were extracted and sequenced. We tested for genomic DNA contamination in the source RNA by PCR assay with cytoglobin (AJ315163 [GenBank] ) primers that span an intron (sense, nt 268–289; antisense, nt 497–519) (2). Nonspecific amplification and spurious primer-dimer fragments were controlled for by omitting the template from the PCR amplification.

Confocal Microscopy

Live-stained tissues were fixed with 4% paraformaldehyde-saline (pH 7.4; 10 min at room temperature) for verification of labeling by confocal imaging. These specimens were examined with a Bio-Rad MRC 600 confocal microscope (Hercules, CA) equipped with an argon-krypton laser and coupled to a Nikon Diaphot inverted microscope. Images were acquired with Nikon Fluor x40/1.30 NA oil-immersion objective, and Z-series scans were constructed with CoMOS software (version 7.0a; Bio-Rad). Additional images were acquired with an LSM 510 META confocal microscope (Carl Zeiss Microimaging, Thornwood, NY) equipped with argon and heliumneon lasers and coupled to an Axioplan 2 microscope and a x40/1.3 NA Plan-Neofluar lens (Zeiss). The histological images shown are representatives of at least three experiments.

Solutions

Krebs-Ringer bicarbonate solution contained (in mmol/l) 120.35 NaCl, 5.9 KCl, 2.5 CaCl2, 1.2 MgCl2, 15.5 NaHCO3, 1.2 NaH2PO4, and 11.5 glucose, pH 7.3–7.4 when bubbled with 97% O2 and 3% CO2. CaPSS contained (in mmol/l) 135 NaCl, 5 KCl, 2 CaCl2, 1.2 MgCl2, 10 glucose, and 10 HEPES, adjusted to pH 7.4 with Tris. CFH contained (in mmol/l) 125 NaCl, 5.36 KCl, 15.5 NaHCO3, 0.336 Na2HPO4, 0.44 KH2PO4, 10 glucose, 2.9 sucrose, and 11 HEPES, adjusted to pH 7.2 with NaOH. Sorting buffer consisted of CFH containing 2% BSA and 2 mM EDTA.

Statistical Analyses

SigmaStat statistical software for Windows (version 2.03; SPSS Science, Chicago, IL) was used for all statistical analyses. Data are expressed as means ± SE; n signifies the number of tissues in the experiment. In approach 4, we used cells pooled from six stomachs; therefore, the error bars in Fig. 3I reflect the variability among the triplicates in the assay, and no tests of significance were performed. In the other studies, before tests of significance were performed, data were examined for normality and equal variance to determine whether parametric or nonparametric tests should be employed. Unpaired Student's t-test and the Mann-Whitney rank sum test were performed on the raw data. A probability value of P < 0.05 was used as a cut-off for statistical significance in all procedures.



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Fig. 3. Immunofluorescent labeling and sorting of ICC (approach 3 and approach 4). A–C: confocal images of murine gastric distal corpus muscles labeled live with the ICC label AF 488-ACK2 (A) and the macrophage label TC-anti-F4/80 (B). C: composite image. Note single-labeled ICC and double-labeled macrophages (yellow) in C. D: Kit+ ICC and mast cell (MC) in a primary culture prepared from gastric antrum muscles. E–G: effect of immunomagnetic depletion (MACS) of CD11b+ and CD11c+ cells on the identification of ICC for FACS (approach 3). Cells with red (TC) or red and green (TC and AF 488) fluorescence include F4/80+ and/or CD11b+ cells (probably mainly resident macrophages). Cells that only displayed green (AF 488) fluorescence (i.e., Kit+F4/80 —CD11b-cells) were considered ICC. Before MACS, macrophages and dendritic cells were also labeled with paramagnetic anti-CD11b and anti-CD11c antibodies, respectively. Note depletion of cells with bright TC fluorescence in the MACS-fraction (F) and their enrichment in the MACS+ fraction (G). Also note the improved definition of ICC in the MACS-fraction (F). H: quantitative RT-PCR analysis of c-kit and CD68 expression in the ICC fraction sorted from the MACS-suspensions (approach 3). Note that the ~30-fold increase in c-kit expression was accompanied by an ~4-fold decrease in CD68 mRNA. Mast cells were neither enriched nor depleted as indicated by mast cell tryptase (MCT) expression. I: quantitative RT-PCR analysis of gene expression in the ICC fraction sorted from MACS-cell suspensions after including TC-anti-CD45 antibodies with the anti-CD11b and -CD11c antibodies in the labeling mixture (approach 4). Note the improved ICC enrichment, the complete lack of CD68- and MCT-expressing cells, and the depletion of smooth muscle cells (MyHC) and myenteric neurons (PGP 9.5). *P < 0.03 relative to the unsorted group. See MATERIALS AND METHODS for details on experiments shown in I. Scale bar in C applies to AC.

 


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Approach 1

Live immunostaining of small intestinal muscles from juvenile BALB/c mice with the AF 488-ACK2 conjugates resulted in a uniform, quantitative labeling of ICC networks in these tissues (Fig. 1A). Mast cells are also Kit positive, but as in fixed small intestinal specimens (19, 31), we did not observe mast cells in the tunica muscularis devoid of the submucosal layer. However, as described previously (6), this technique also labeled resident macrophages, which appeared to contain the fluorescent antibody in intracellular compartments, although binding to Fc receptors could not be excluded. In double-labeled tissues we selectively identified macrophages with fluorescent (TC) antibodies against F4/80 (20) (Fig. 1, B and C). Morphologically, most of these cells were irregularly shaped with four to six short processes (e.g., in the myenteric and subserosal regions); others were elongated (e.g., within the circular muscle layer) or, rarely, round (on the submucosal surface). Some of the elongated cells may have been fibroblasts, rather than macrophages, because they did not take up fluorescent dextran (not shown).



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Fig. 1. Immunofluorescent labeling and sorting of interstitial cells of Cajal (ICC) (approach 1 and approach 2). A–C: confocal images of murine small intestinal muscles labeled live with the ICC label AF 488-ACK2 (A) and the macrophage label TC-anti-F4/80 (B). C: composite image. Note single-labeled ICC and double-labeled macrophages (yellow) in C. D: flow cytometry (FCM) analysis of cells dispersed from the labeled tissues (approach 1). Each cell in the 2-dimensional logarithmic plot of fluorescence intensities is represented by a single dot. Pseudocolor signifies frequencies of cells with similar fluorescence parameters. Cells with red (TC) or red and green (TC and AF 488) fluorescence are presumed macrophages. Cells that only displayed green (AF 488) fluorescence (i.e., Kit+F4/80-cells) were harvested as ICC. E: quantitative RT-PCR analysis of c-kit and CD68 expression in the sorted ICC fraction (approach 1). Gene expression was normalized to {beta}-actin mRNA levels and expressed relative to the expression levels detected in the unsorted cells. Note the >60-fold increase in c-kit expression and the small enrichment of CD68 mRNA in the ICC fraction. F: effects of magnetic presorting (positive selection) of Kit+ cells (approach 2) on their detection by FCM. Note the massive enrichment of presumed ICC and a smaller increase in the frequency of macrophages in the FCM plot. Also note the leftward shift of mean fluorescence levels in the ICC fraction. G: quantitative RT-PCR analysis of c-kit and CD68 expression in the ICC fraction sorted from MACS+ cell suspensions (approach 2). Magnetic presorting somewhat reduced the enrichment of both c-kit and CD68. **P = 0.002; *P = 0.029 relative to the unsorted group. Scale bar in C applies to A–C.

 

FCM analysis of the cells dispersed from the double-labeled tissues revealed a well-defined cluster of cells with only green (AF 488) fluorescence (i.e., Kit+F4/80-cells; Fig. 1D) representing 1.1–4.3% of the total cell count reported by using this technique. These cells were clearly distinguishable from the unlabeled cells and from the cells with red (TC) or red and green (TC and AF 488) fluorescence (presumed macrophages) and were considered ICC. From each tissue representing the entire jejunal and ileal musculature of 9- to 13-day-old mice (n = 6), we harvested 30,000–40,000 of the presumed ICC. Quantitative RT-PCR analysis in this cell population indicated a 61.7 ± 16.2-fold (P = 0.002) increase of c-kit expression relative to unsorted cells (Fig. 1E). A 54.0 ± 23.8-fold enrichment (P = 0.004) of CD68 expression was detected in the simultaneously sorted "macrophage fraction" (F4/80+ cells), confirming that this group consisted mainly of macrophages (not shown). However, CD68 expression also increased 5.6 ± 3.0-fold (not significant, NS) in the "ICC fraction," indicating that some of the cells that took up ACK2 were not detected by the macrophage label (Fig. 1E).

Approach 2

In an attempt to reduce the enrichment of macrophages, we tried to improve the detection of ICC for FACS by immunomagnetic presorting (positive MACS). As shown in Fig. 1F, MACS increased the proportion of cells in the ICC fraction, although some increase in the macrophage fraction was also noted. Quantitative RT-PCR (n = 4) indicated somewhat reduced enrichment of CD68 mRNA in the ICC fraction (i.e., 3.1 ± 0.5-fold; NS), although this occurred at the expense of lower and more variable ICC recovery (7,000–34,000 cells) and a lower and more variable enrichment of c-kit expression (53.4 ± 20.8-fold; P = 0.029) (Fig. 1G). The latter may have been due to increased contamination from unlabeled cells resulting from an increase in the proportion of ICC with lower fluorescence and a consequent leftward shift in the center of the ICC fraction in the FCM analysis (Fig. 1F). Even though the c-kit/CD68 enrichment ratio in the sorted cells improved somewhat after MACS, this was more than offset by the reduced cell recovery and the added cost.

Identification of F4/80 —CD68+ Cells for FACS

Probably because of the largely intracellular localization of CD68 (macrosialin) (35), our attempts to identify CD68+ cells in murine GI muscles by vital immunostaining invariably failed, forcing us to look for surrogate markers. CD68 is also expressed by some dendritic cells of myeloid origin (1), and some macrophage-like cells in GI muscles express the dendritic cell marker CD11c (20). Therefore, we examined whether F4/80 —CD11c+ cells could take up the ICC label and contribute to the detected contamination. Because of our ongoing interest in gastric ICC, we performed these and the rest of the experiments in gastric corpus and antrum tissues obtained from adult BALB/c mice. FCM of cells dispersed from gastric muscles indeed revealed a small number of F4/80 —CD11c+ cells (Fig. 2, A and B). Vital labeling of CD11c+ cells in gastric tissues with a FITC-conjugated antibody confirmed that these cells were very rare. We identified two cell types that could be colabeled with simultaneously applied AF 594-ACK2 conjugates (Fig. 2, C–H): short, bipolar cells with an asymmetrically positioned nucleus running parallel to the axes of intramuscular ICC in the circular muscle (Fig. 2, C–E), and round, lymphocyte-like cells on the submucosal surface of the circular muscle (Fig. 2, F–H). The mechanism of the labeling of these two cell types with fluorescent ACK2 could not be identified in these specimens.



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Fig. 2. F4/80 —CD11c+ cells take up ACK2 and may contaminate sorted ICC populations. A and B: identification by FCM of F4/80 — CD11c+ cells in dissociated murine gastric tunica muscularis. Note the lack of cells with green (FITC) fluorescence in suspensions labeled with TC-anti-F4/80 only (A). Double labeling with TC-anti-F4/80 and FITC-anti-CD11c antibodies revealed the existence of a small number of F4/80 —CD11c+ cells (i.e., cells with greenonly fluorescence). C–E: a rare CD11c+ cell (C) in the gastric circular muscle layer that took up the ICC label (AF 594-ACK2; D) after vital immunostaining. E: composite image. Note perinuclear staining with the FITC-anti-CD11c antibody in the cell marked with an asterisk. The axis of this short, bipolar cell with an asymmetrically positioned nucleus was parallel to the axes of intramuscular ICC labeled with AF 594-ACK2 only (arrows). F–H: colocalization (H) of FITC-anti-CD11c (F) and AF 594-ACK2 (G) in a rare, round, lymphocyte-like cell on the submucosal surface of the circular muscle of the gastric antrum. Scale bars in E and H apply to C–E and F–H, respectively.

 

Approach 3

We next attempted to improve the selectivity of the ICC sorting by reducing the number of contaminating macrophages and dendritic cells with the aid of immunomagnetic depletion (negative MACS) and by reinforcing macrophage labeling. To this end, we added paramagnetic CD11c and CD11b (another macrophage marker; see Refs. 20, 35) antibodies and TC-anti-CD11b to dispersed cells. In preliminary tests the latter antibody appeared to stain the same cells as the TC-anti-F4/80 antibody in intact muscles (not shown). Labeling with AF 488-ACK2 and TC-anti-F4/80 of the live tissues was carried out as before (Fig. 3, A–C). The morphology and distribution of F4/80+ cells in the stomach were similar to those of cells found in the small intestines (Fig. 3, B and C). However, in addition to ICC (Fig. 3A), we also detected a small number of Kit+ mast cells in the antral musculature and in primary cultures prepared from these tissues (Fig. 3D).

As shown in Fig. 3, E–G, depletion of CD11b+ and CD11c+ cells by MACS reduced the number of cells with bright TC fluorescence and improved the definition of the ICC fraction in the MACS-cells (Fig. 3F). From this fraction we harvested 2,000–4,000 cells per adult murine gastric corpus and antrum by FACS (n = 4). Quantitative RT-PCR in these cells (Fig. 3H) indicated a 30.2 ± 10.0-fold (P = 0.027) enrichment of c-kit expression relative to the unsorted group and a significant reduction of CD68-expressing cells (to 23.3 ± 21.5% of unsorted; P = 0.018). Mast cells were neither enriched nor depleted.

Approach 4

To further reduce the number of contaminating hematopoietic cells, we modified approach 3 by including a TC-labeled antibody against the common leukocyte marker CD45 in the labeling step immediately before magnetic presorting. In a preliminary histological test we found that the TC-anti-CD45 antibody labeled cells in intact gastric muscles that were morphologically similar to the cells detected with the TC-anti-F4/80 or TC-anti-CD11b antibodies (not shown). We tested this approach on presumed ICC sorted and pooled from six adult stomachs (18,600 cells) because the improved techniques reduced the number of cells harvested from single stomachs and thus reduced the abundance of some mRNA species to levels that were difficult to reliably detect by quantitative RT-PCR. As shown in Fig. 3I, we detected a 110.0-fold increase in c-kit expression in the ICC fraction (note that because of the pooling of the tissues, no tests of significance were performed; see MATERIALS AND METHODS for details), whereas CD68 mRNA was completely eliminated. We also did not recover any mast cells, although the level of tryptase expression was also below the level of detectability in the unsorted group. Of the monitored markers for unlabeled cell types, MyHC was only detected in one of the triplicate samples, indicating that contamination from smooth muscle cells was minimal; and the expression of the neuronal marker PGP 9.5 was reduced to 8.1% of the unsorted cells (Fig. 3I). mRNA for the fibroblast marker prolyl-4-hydroxylase was also undetectable in the ICC fraction (not shown).


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In this study we developed and tested techniques for largescale purification of ICC from murine GI muscles. We conclude that immunofluorescent labeling of ICC (and potential contaminating cells) and selection by FACS can yield ICC in sufficiently high numbers and purity to permit detailed molecular analysis. Use of the purification approaches presented in this paper may facilitate studies that could provide important insights into the expression patterns of healthy and diseased ICC. Such studies may help to determine the causes and mechanisms of ICC loss, altered morphology, or dysfunction that have been observed in a variety of human GI disorders (10, 18, 21, 25, 26, 31, 41). Some of these changes in ICC networks have also been demonstrated in animal models (3, 4, 17, 22, 23, 33), and molecular expression studies may help determine the fidelity of animal models in relation to human disorders and may help to establish cause-and-effect between ICC loss and dysfunction and motility disorders in human patients. Largescale analyses may also help to further determine the genes that distinguish ICC from other cell types or to distinguish pacemaker ICC from ICC that mediate neuromuscular neurotransmission (6, 9). [The latter can, for example, be accomplished by purifying ICC from organs or dissected muscle layers that only contain one ICC class (6, 14), since pacemaker and other ICC cannot be distinguished by existing ICC markers.] Studies to date have also created controversy about the expression of specific genes in ICC (e.g., CD34, SK3) (6, 8, 29, 30, 42), and further molecular studies will help settle these debates. Previous investigators have approached some of these problems by harvesting small numbers of visually identified ICC from cultures or suspensions (6, 9, 30, 39). However, significant problems limit the utility of this approach (see Introduction). Others have attempted to identify genes specifically expressed by ICC by comparing whole tissues with or without these cells (e.g., tissues from wild-type and W/WV mice) using cDNA microarrays or more conventional differential display techniques (34, 37, 38). However, in this indirect approach, the detected differences may reflect secondary changes in the W/WV tissues that lack ICC (e.g., changes indicating a super-sensitive phenotype secondary to "denervation" of the muscle), rather than the missing gene expression of the missing ICC (34). Clearly, direct analysis of large numbers of pure ICC could circumvent these technical problems and provide indepth information about the molecular constitution of these cells. In addition, our approach could also provide a roadmap for the purification of ICC from various organs of other species and for the purification and/or quantification of other cell types within the GI tunica muscularis, e.g., immune-related cells, which we have begun characterizing in the present work.

The evolution of our purification techniques largely represents progress in identifying and removing contaminating cells. These include not only Kit+ cells (mast cells in the antrum) but also cells that nonspecifically take up Kit antibodies. In addition to resident macrophages (6), we also identified resident CD11c+ cells as members of the latter group. Approach 4 successfully eliminated contaminating cells by combining magnetic and fluorescent sorting and the application of a cocktail of antibodies that recognize various macrophage-specific antigens (F4/80, CD11b) (20, 35), antigens that are typically expressed by dendritic cells (CD11c) (28), and the general leukocyte marker CD45. Increased selectivity did, however, come at the expense of lower yields. Of all the techniques discussed in this article, we consider approach 1 and approach 4 as potential candidates for use in various studies, because with respect to selectivity and yield, they represent the opposite ends of the spectrum. Approach 1 can yield tens of thousands of ICC and be used for gene expression studies 1) that require greater cell numbers and 2) in which potential contamination from macrophages and dendritic cells does not jeopardize the outcome. The latter can, for example, be verified by simultaneously sorting these contaminating cells (see Fig. 1D) and demonstrating that they do not express mRNAs of interest. This approach would also be useful for comparative expression studies of ICC under different conditions and may permit the analysis of functional properties (e.g., electrical slow wave activity, membrane currents, intracellular Ca2+ dynamics) of the sorted ICC in culture. For studies that require higher purity, cells could be sorted from multiple tissues, using approach 4, and pooled for molecular analysis. It is also possible to make approach 1 more selective without significantly reducing its yield by including the fluorescent CD11b and CD45 antibodies and not immunomagnetic presorting. Thus the protocol used in different experiments can, to some extent, be adapted to the requirements of the particular study.

Finally, it is very important to mention that the purity of the sorted ICC must be tested with RT-PCR of markers for potential contaminating cells and cannot be inferred from the enrichment of c-kit expression, even though the detected enrichment values (~30- to 110-fold) were not inconsistent with concentrating ICC to near purity from the frequencies observed in the unsorted populations (1.1–4.3%). This is because the actual enrichment values can be dramatically influenced by small changes in c-kit mRNA in the unsorted cells (i.e., the denominator in the calculation of the enrichment), and the relatively low ICC frequency and c-kit mRNA levels in this fraction may lead to less reliable measurements. The enrichment values are, however, useful for comparing the effect of the sorting technique on various cell types within the same experiment, as demonstrated in RESULTS. They should also be used as benchmark in the identification of genes expressed exclusively by ICC, because their enrichment in any given experiment should be very similar to that of c-kit.


    ACKNOWLEDGMENTS
 
We thank Nancy Horowitz, Donna M. Colón, and Ann Marie Butler for aid in the conduct of this study.

GRANTS

This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-58185 (to T. Ördög) and DK-40569 (to K. M. Sanders), a Research Grant Award from the American Motility Society and Janssen Pharmaceutica and Research Foundation (to T. Ördög), and a Seed Grant from the University of Nevada (UNR), Reno Sanford Center for Aging (to T. Ördög). The UNR Cytometry Center was supported in part by the Nevada Biomedical Research Infrastructure Network Grant P20 RR-16464 from the National Institutes of Health.


    FOOTNOTES
 

Address for reprint requests and other correspondence: T. Ördög, Dept. of Physiology and Cell Biology, Univ. of Nevada, Reno School of Medicine, Anderson Bldg., Mail Stop 352, Reno, NV 89557 (E-mail: tamas{at}physiology.unr.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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