Expression of a Distinct Set of Chemokine Receptors in Adipose Tissue-Derived Stem Cells is Responsible for In Vitro Migration Toward Chemokines Appearing in the Major Pelvic Ganglion Following Cavernous Nerve Injury

Abstract

Introduction: Adipose tissue-derived stem cells (ADSCs) herald tremendous promise for clinical application in a wide range of injuries and diseases. Several preclinical reports demonstrate their efficacy in the treatment of cavernous nerve (CN) injury-induced erectile dysfunction in rats. It was recently illustrated that these effects were established as a result of ADSC migration to the major pelvic ganglion (MPG) where these cells induced neuroregeneration in loco.

Aims: The study aims to identify chemotactic factors in the MPG following injury and to match upregulated chemokines to their respective receptors in human ADSC on the genomic, structural, and functional levels.

Methods: Quantitative real-time polymerase chain reaction, fluorescence-activated cell sorting (FACS), intracellular FACS, immunofluorescence microscopy, migration assays, and calcium imaging were used in this study.

Main outcome measures: The main outcomes are chemokine expression in the MPG following CN injury, and the functional and structural presence of chemokine receptors in ADSC.

Results: CCR4, CX3CR1, and XCR1 are functionally and structurally present in human ADSC, and are activated by the chemokines CCL2, CX3CL1, and XCL1 respectively, which are upregulated in the MPG following CN injury. CXCR4 and its ligand CXCL12 (SDF1) are likely no major homing factors for ADSC. Expression of chemokine receptor mRNA in ADSC did not necessarily translate into receptor presence at the cell surface and/or functional activation of these receptors. Most of the expressed chemokine receptors were detected in the intracellular compartment of these cells.

Conclusions: We identified the ligand/chemokine receptor pairs CCL2/CCR4, CX3CL1/CX3CR1, and XCL1/XCR1 as potentially responsible for ADSC homing toward the MPG following CN injury. The intracellular localization of various chemokine receptors likely indicates redirecting of chemokine receptors to the cell surface under specific cellular conditions. Furthermore, modification of expression of these receptors at the genomic level may potentially lead to improved migration toward injury sites and thus enhancement of treatment efficacy. Albersen M, Berkers J, Dekoninck P, Deprest J, Lue TF, Hedlund P, Lin C-S, Bivalacqua TJ, Van Poppel H, De Ridder D, and Van der Aa F. Expression of a distinct set of chemokine receptors in adipose tissue-derived stem cells is responsible for in vitro migration toward chemokines appearing in the major pelvic ganglion following cavernous nerve injury. Sex Med 2013;1:3-15.

Keywords: Adipose Tissue-Derived Stem Cells; Cavernous Nerve Injury; Cell Surface; Chemokine Receptors; Chemokines; Erectile Dysfunction; Intracellular; Migration.

Figure 1

(A) Flow cytometry analysis on ADSCs isolated from female human donors shows the vast majority of cells express an CD14−, CD34−, CD45−, HLA-DR− and CD29+, CD90+, CD166+, CD73+, CD105+, CD44+ HLA-ABC+ phenotype, consistent with the typical mesenchymal multipotent stromal cell surface marker profile (light gray area: isotype control, black line: sample). (B) Cultured human ADSCs display a typical fibroblast-like morphology and are able to differentiate toward adipogenic and osteogenic lineages in vitro. Figure 1 is adapted from reference [14] with permission. ADSC = adipose tissue-derived stem cell.

Figure 2

Representative immunofluorescence image of the rat MPG 24 hours after cavernous nerve injury. Original magnification ×400. 4E2 (red signal) is a rat-specific peripheral glia marker, and MCP1 (monocyte chemoattractant protein) is CCL2, a chemokine. Nuclear staining was performed with DAPI (blue). Note the colocalization of MCP1 and 4E2, indicating this chemokine is mainly expressed in glia rather than neurons. MPG = major pelvic ganglion.

Figure 3

(A) FACS of nonpermeabilized cells shows low expression of chemokine receptors at the cell surface. (B) After cell permeabilization, it becomes evident that cells express most of the receptors that were detected by qPCR. CCR4, CCR10, CX3CR1, and XCR1 are strongly expressed, while expression of the remaining chemokine receptors is rather low. (Light gray: isotype control, black line: sample.) FACS = fluorescence-activated cell sorting; qPCR = quantitative real-time polymerase chain reaction.

Figure 4

Immunofluorescence staining of human attached and suspended ADSC. (A, B) Cells were cultured on coverslips, permeabilized using saponin, and stained against f-actin (red), CCR4 (green), and DAPI (blue). Note that the staining pattern of CCR4 is perinuclear and morphologically consistent with expression in the endoplasmic reticulum. (C) Cells in suspension were permeabilized using saponin and stained CCR4 (green) and DAPI (blue). Note the intracellular staining of CCR4. ADSC = adipose tissue-derived stem cell.

Figure 5

Functional activation of chemokine receptors in human ADSC. (A, B) Chemotaxis assay: ADSCs were incubated with increasing concentrations of chemokines in standard medium. Serum-free medium was used as a negative control. FBS-supplemented medium was used as a positive control (green bar). Migration was assessed by counting the number of cells migrated toward the middle area of a well after removal of a central stopper. Values are expressed as ratio of migrated cells over migrated cells in negative control medium. anova: +P < 0.05 compared with other concentration of same chemokine, *P < 0.05 compared with negative control, §P < 0.05 compared with all other samples. (C) Live cell calcium imaging: ADSCs were plated on a coverslip and loaded with FURA2. Coverslips were mounted in a perfusion chamber and cells were exposed to DMEM medium supplemented with chemokines at a concentration of 50 nM for 2 minutes each. The ratio of light emission during 340/380 nM excitation was recorded as a measure of intracellular calcium concentrations. At conclusion of the experiment, cell viability and calcium influx were assessed using ionomycin as a positive control. n = 20–30. ADSC = adipose tissue-derived stem cell; DMEM = Dulbecco's modified Eagle medium; FBS = fetal bovine serum.