Transcriptome-based screening of ion channels and transporters in a migratory chondroprogenitor cell line isolated from late-stage osteoarthritic cartilage

2.1 Cell culture

Experiments were carried out on a human migratory CPC cell line derived from late-stage OA knee articular cartilage, which has been immortalised by viral transfection of the human telomerase reverse transcriptase (hTERT) as previously described (Koelling et al., 2009). CPCs were cultured in monolayers in 75 cm² cell culture flasks (Nunc; Thermo Fisher Scientific) until approximately 80% confluence in GlutaMax DMEM (1.0 g/L glucose; Gibco; Thermo Fisher Scientific) containing 10% foetal calf serum (FCS; Gibco) and 50 μg/ml gentamycin (Sigma-Aldrich). As a reference cell population, human BM-MSCs (Lonza) were used. The cells were received at passage 2 and were expanded until passage 4 in Lonza hMSC medium at 37°C in a humidified atmosphere of 5% CO₂. MSCs were hTERT-immortalised as previously described in (Okamoto et al., 2002) with the modifications detailed in (Saeed et al., 2015). Immortalised MSCs were expanded in monolayers in 75 cm² cell culture flasks (Nunc) until approximately 80% confluence in GlutaMax DMEM (4.5 g/L glucose; Gibco) containing 10% FCS (Gibco) and 1% P/S (Sigma-Aldrich).

2.2 RNA isolation and reverse transcription

Total RNA was isolated from cells grown in monolayers in 75 cm² cell culture flasks using the RNeasy kit (Qiagen) as per the instructions of the manufacturer and stored at –80°C. RNA concentration and purity were determined by a NanoDrop 2000 UV-Vis spectrophotometer (Thermo Fisher Scientific). For gene expression analyses, 2 μg of RNA was reverse-transcribed into complementary DNA (cDNA) using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific), following the protocol supplied by the manufacturer. cDNA was stored at –20°C.

2.3 Affymetrix microarray analysis

RNA integrity was confirmed using an Agilent 2100 Bioanalyzer with the RNA 6000 Nano Kit (Agilent Technologies). The RNA integrity numbers were ≥9.6 for all samples. Whole-genome transcriptome analysis was conducted by hybridising three biological samples of total RNA per cell type to Affymetrix Human Gene 2.1 ST Arrays Strips (Affymetrix). All steps were conducted at the Nottingham Arabidopsis Stock Centre. Gene expression data were analysed using Partek Genomics Suite 6.6 software (Partek Incorporated). The raw CEL files were normalised using the RMA background correction with quantile normalisation, log base 2 transformation and mean probe-set summarisation with adjustment for GC content. Differentially expressed genes (DEG) were identified by a two-way analysis of variance, and p values were adjusted using the false discovery rate (FDR) method to correct for multiple comparisons. DEG was considered significant if p value with FDR was ≤.05. The expression data of genes coding for selected transporter and ion channel subunits are summarised in Tables S1–S4. The data set is published in a MIAME compliant format in the GEO database (http://www.ncbi.nlm.nih.gov/geo/); accession numbers: GSM4885525-GSM4485530 (GSE160886).

2.4 Pathway analysis

CytoScape v3.4 software with ClueGo v2.3.5 application was used for identifying overrepresented gene ontology (GO) terms. A two-sided hypergeometric test with Bonferroni step-down correction was performed using the list of DEG and the GO Biological process database.

2.5 RT-qPCR analyses

Selected ion channel subunit genes were analysed by RT-qPCR using a custom configured TaqMan 96-Well Fast Gene Expression Array plate (see Tables S5 and S6) and then by absolute quantification using the standard curve method. Primer pairs were ordered from Eurogentec. For sequences of primer pairs please see Table S7. First, standard curves had been generated by conventional PCR using the Promega GoTaq Flexi DNA Polymerase kit (Promega) by adding the following components (per 50 μl reaction): 1.25 U GoTaq polymerase, 3 mM MgCl₂, 0.2 mM dNTP, 200 nM primers and 10 ng cDNA. Amplification was performed in a Techne Prime Thermal Cycler (Techne; Bibby Scientific Ltd) using the following thermal profile: initial denaturation at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 15 s, annealing at 58°C for 20 s, extension at 74°C for 20 s, and then final extension at 74°C for 5 min. PCR products were isolated using a Roche High Pure PCR Product Purification Kit (Roche) according to the instructions of the manufacturer. DNA concentration of purified PCR products was determined using a NanoDrop 2000 UV-Vis spectrophotometer (Thermo Fisher Scientific). Standard curves were prepared by a serial (10-fold) dilution starting from 1 ng/μl.

RT-qPCR reactions were set up using the Promega GoTaq qPCR Master Mix and 20 ng input cDNA per each 10-μl reaction. Reactions were run in a Techne Prime Pro 48 Real-time qPCR machine using the following thermal profile: activation and initial denaturation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 10 s, annealing at 58°C for 30 s, extension at 72°C for 20 s, and then final extension at 72°C for 20 s. Due to spatial limitations in the 48-well qPCR plates, quantification of qPCR products was performed by absolute quantification using the standards prepared in the previous step, followed by normalising the expressional data of genes of interest to those of the most stably expressed reference gene (PPIA), and then the expression levels for the genes were normalised to those in MSCs (set at 1.0). The optimal normalisation gene was selected using the NormFinder algorithm (Andersen et al., 2004). RT-qPCR reactions were performed on three biological replicates (N = 3).

2.6 Cell proliferation and mitochondrial activity assays

To assess the effect of BK channel modulators, cells were plated into 96-well plates at a density of 5000 cells/well. Before the assays, cells were incubated for 48 h in the presence of the selective BK channel blocker iberiotoxin (IBTX) (100 nM; Tocris; Bio-Techne); the potent BK channel inhibitor paxilline (1 μM); the BK channel activator NS1619 (10 μM; Sigma-Andrich); or vehicles at equal volumes (water, dimethyl sulfoxide and ethanol, respectively). The rate of cell proliferation was determined by detecting the amount of incorporated radioactivity from ³H-thymidine. One microcurie per millilitre ³H-thymidine (diluted from methyl-³H-thymidine; 185 GBq/mM; Amersham Biosciences) was added to each well (Wallac, PerkinElmer Life and Analytical Sciences) for 24 h. After washing with phosphate-buffered saline (PBS), proteins were precipitated with ice-cold 5% trichloroacetic acid and rinsed with PBS again. Cells were then air-dried and radioactivity was counted by a liquid scintillation counter (Chameleon; Hidex). Measurements were carried out in seven samples of each experimental group (n = 7) in three independent experiments (N = 3). For mitochondrial activity assays, 10 μl 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent (thiazolyl blue tetrazolium bromide; 5 mg MTT/ml PBS; Sigma-Aldrich) was pipetted into each well. Cells were incubated for 2 h at 37°C, and following the addition of 500 μl MTT solubilizing solution, optical density was measured at 570 nm (Chameleon; Hidex).

2.7 Migration assays

2.8 Electrophysiology

2.9 Measurement of intracellular Ca²⁺ oscillations in CPCs

Cells were loaded with 10 μM Fura-2-AM for 50 min at 37°C in a CO₂ incubator. After loading, cells were kept in Tyrode's solution and placed on the stage of a ZEISS Axiovert 200M inverted microscope (Zeiss) equipped with a Plan-Neofluar ×20 objective (NA = 0.5). Fura-2 was excited with a CoolLED pE-340fura light source (CoolLED Ltd.). Illumination was alternating between 340 and 380 nm wavelength and the emitted fluorescent light was measured through a band-pass filter (505–570 nm) and digitised at 16 bit with an AXIOCam MR3 monochrome camera (Zeiss) at a frequency of 12 frames/min. Image acquisition and postprocessing were carried out with the AxioVision (rel. 4.8) software (Zeiss). The cells were examined in resting conditions for 15 min at room temperature. At the end of the experiments, 180 μM ATP was added to the bath to check the viability of the cells. Cells not responding to the ATP challenge were excluded from the analysis. Intracellular Ca²⁺ concentration ([Ca²⁺]_i) was calculated from the ratio of the images taken at 340 and 380 nm after background correction with constants determined during in vitro calibration of the setup. The cells were classified as active if the increase of [Ca²⁺]_i was more than 10% of the resting level. n = 332 cells were analysed.

2.10 Statistical analysis

All data are representative of at least three independent experiments (biological replicates). RT-qPCR, cell proliferation and mitochondrial activity, as well as cell migration data, are expressed as mean ± SEM, DEP data are expressed as mean ± SD. Statistical analysis was performed using Student's unpaired two-tailed t-test (*p < .05; **p < .01; ***p < .001).

3.1 Affymetrix analysis reveals differentially expressed ion channel genes in migratory CPCs

We performed a principal component analysis of the Affymetrix data on normalised samples to explore their interrelationships (Figure 1). Biological replicates within the CPC/MSC groups were separated into two distinct clusters, showing that the pattern of gene expression was different in CPCs and MSCs. In total, 3214 genes showed significantly different expression levels between CPC and MSC cells, with 2379 genes being downregulated and 835 genes upregulated. Using CytoScape software with the ClueGo application we identified those GO Biological process terms which were overrepresented in our gene list. When we analysed the full gene list (up- and downregulated genes together), and the list of downregulated genes, we found that mainly cell cycle-related categories such as cell cycle, regulation of cell cycle, cell cycle checkpoint and mitotic/meiotic cell cycle processes were enriched. Furthermore, DNA/nucleic acid metabolism-related pathways were also overrepresented. Separate analyses of upregulated genes indicated the enrichment of different types of tissue development and cell proliferation processes, such as skin development and mesenchymal cell proliferation (Figure S1 in the File S1; see also File S2).

To characterise the expression profiles of genes encoding ion channel subunits in CPCs and to identify differences in expression levels compared to MSCs, we further explored the microarray analysis data. We assessed genes encoding ion channels and transporters commonly involved in regulating potassium, sodium and chloride transport and calcium homoeostasis in chondrocytes (Figure 2; see also Tables S1–S4). The expression levels for some of the genes studied were different in migratory CPCs versus MSCs. Genes encoding members of the calcium-activated potassium channels (K_Ca) showed the most prominent expression changes; KCNMA1 and KCNN4, the genes coding for the pore-forming alpha subunit of the large-conductance calcium-activated potassium channel (K_Ca1.1), and the intermediate/small conductance calcium-activated potassium channel (K_Ca3.1), respectively. In both cases, there was a more than 1.5-fold upregulation in CPCs. In contrast, the small conductance calcium-activated potassium channel (K_Ca2.3) encoded by the KCNN3 gene was unchanged. The β4 regulatory subunit of the large-conductance calcium-activated potassium channel (KCNMB4) was significantly downregulated in CPCs. KCNB1 (voltage-gated potassium channel subunit Kv2.1); KCND2 (Kv4.2) and KCNH6 (Kv11.2) were also downregulated in CPCs (Table S1).

Whilst most of the genes coding for the alpha subunits of voltage-gated sodium channels were upregulated in CPCs, with SCN2A encoding the Na_V1.2 sodium channel showing a 1.3-fold upregulation, all epithelial sodium channel subunit genes (SCNN1A, SCNN1B, SCNN1D, SCNN1G) were found to show a trend towards downregulation (Table S2), suggesting an altered K⁺/Na⁺ ion handling in CPCs. The only gene showing a significantly lower expression in CPCs was SCNN1D. We also checked the expression patterns of genes encoding the alpha and beta subunits of the Na⁺/K⁺-ATPase and found that the ATP1A1 gene was significantly downregulated in CPCs. Genes coding for the rest of the subunits were unchanged.

Interesting differences were found also in the expression patterns of genes coding for proteins involved in global calcium handling. The genes coding for molecules that regulate store-operated Ca²⁺ entry (SOCE); inositol 1,4,5-trisphosphate receptors (IP₃Rs) that mediate calcium release from the intracellular calcium stores; as well as the sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase (SERCA) that are responsible for calcium sequestration, were showing a trend for downregulation in CPCs. Transcript levels of both ORAI2 and ATP2A3 (SERCA3) displayed a significant reduction in CPCs. In contrast, genes coding for voltage-gated calcium channel subunits were unchanged. In a similar way, the ATPases and exchangers that mediate calcium extrusion (PMCA2-4, as well as NCX1-2); as well as the metabotropic purinergic receptors were also unchanged with a trend towards upregulation, with the exception of P2RY2, which was significantly downregulated in CPCs. The genes coding for nonselective cation channels, including members of the ionotropic P2X purinergic receptors, as well as the TRP cation channel families TRPC and TRPV, were showing a general trend towards downregulation, in particular, TRPC4 and TRPV2 (Table S3).

We have also looked at other ion channel expression patterns and found that the only gene which had a significantly different expression (downregulation in CPCs) between the two cell types was ASIC1 coding for the acid-sensitive cation channel subunit 1. None of the other transcripts showed significantly different expression levels between CPCs and MSCs (Table S4).

3.2 Validating microarray data by RT-qPCR confirmed the differential expression of ion channel genes in CPCs

The messenger RNA (mRNA) expression profiles of selected candidate genes encoding various ion channel subunits were first validated using custom-configured TaqMan 96-Well Fast Gene Expression Array plates (see Tables S5 and S6). Given their role in chondroprogenitor and chondrocyte physiology, we were particularly interested in studying the roles of the calcium-activated potassium channels, and therefore we looked at the expression patterns of the genes encoding the various subunits of these channels in details. To confirm the expression patterns of these genes, we employed custom-designed primers and performed individual qPCR reactions. The genes coding for the α and β subunits of the large-conductance calcium-activated potassium channel (BK) were all detected; KCNMA1 and KCNMB4 were upregulated; KCNMB1 was downregulated in CPCs; KCNMB2 and KCNMB3 were unchanged. Of the genes encoding the small conductance calcium-activated potassium channel proteins 1–3 (KCNN1–3), KCNN1 was unchanged, whereas KCNN3 was massively downregulated in CPCs. The gene for the intermediate conductance calcium-activated potassium channel protein 4 (KCNN4) was showing a significant downregulation in CPCs (Figure 3). Differences were detected between our qRT-PCR and microarray data. This has been observed before and is largely attributed to the differences in probe sequences (Canales et al., 2006); however, the observed differences could also be attributable to different methodological aspects of the two approaches, such as the fundamentally different data normalisation strategies (Morey et al., 2006).

3.3 Modulation of the BK potassium channel alters cell proliferation, marker gene expression and migration of CPCs

To assess the role of large-conductance calcium-activated potassium channels in CPCs and MSCs on cell proliferation and viability (mitochondrial activity), cells were incubated in the presence of the selective BK channel inhibitors IBTX (100 nM) and paxilline (1 μM); or the BK channel activator NS1619 (10 μM) before assays. Whilst none of the tested compounds interfered with the mitochondrial activity of the cells (i.e., did not have adverse effects on general cell physiology; Figure 4a), both IBTX and paxilline significantly reduced the proliferation rate in CPCs, but had no significant effect in MSCs (Figures 4b and S2). When CPCs were cultured in the presence of 1 μM paxilline, the osteogenic transcription factor RUNX2 was significantly upregulated compared to the control, whereas the expression profile of SOX9, the chondrogenic master regulator, remained unchanged. COL1A1, on the other hand, was upregulated following paxilline treatment (Figure 4c).

Given that the CPCs employed in this study have previously been demonstrated in vitro and in vivo migratory features (Koelling et al., 2009), we performed two different migration assays with or without the BK channel inhibitor paxilline. Fibronectin-guided migration of CPCs in a Boyden chemotaxis chamber was significantly increased in the presence of 1 μM paxilline (Figure 5a). The effect of paxilline was also examined in random cell migration assays. Paxilline at 1 μM enhanced the migratory parameters of chondroprogenitor cells, significantly increased the total path of migration, the maximum distance from the origin; furthermore, the average cell speed was also significantly greater compared to the control (Figure 5b). Next, we shifted the migratory tracks (total paths) of the individual cells to a common origin to generate static wind rose plots (Figure 5c). The bigger diameter of the wind rose plot in paxilline-treated cells indicates the increased motility of these cells.

3.4 Electrophysiological properties and intracellular calcium oscillations of chondroprogenitor cells

CPC and MSC cell lines with a characteristic mesenchymal morphology were grown under standard culturing conditions before preparation for DEP analysis (Figure 6a). The measured DEP spectra of the cell lines were used to determine specific membrane capacitance (C_Spec) and conductance (G_Spec) values; as these calculations take cell size into account (the values of capacitance and conductance are cell size-independent), the cell radii were determined after trypsinisation, which was statistically different. The electrophysiological properties of CPC and MSC cells are shown in Figure 6b, and summarised in Table 1. Effective membrane capacitance (C_Eff) is a measure of the ability of the membrane to store charge and generate a dipole in a frequency-dependent manner in DEP. The C_Eff values of CPC and MSC cells were not significantly different from each other. The membrane capacitance per cell area values were also very similar between CPC and MSC. The intracellular conductivities of CPC and MSC were also almost identical between the two cell types. The greatest difference between the two cell types was seen with regard to the specific membrane conductance (G_Spec) parameter: CPCs were characterised by a significantly higher G_Spec value than MSCs, which equals to a 32.8% difference. Representative DEP spectra for CPC and MSC cells are shown in Figure 6c.

As RT-qPCR analysis revealed changes in the expression of several ion channels at the mRNA level, whole-cell currents were examined. Both CPC and MSC cells were heterogeneous in respect to the ionic current expression. The significant current was recorded in 6 of 27 CPC cells. The voltage-dependent features of these outward currents were reminiscent of that of BK channels. In addition, the time-dependent component was inhibited by paxilline, indicating that the current was partially carried by BK channels. The linear component of the current was not specified further. In a similar way, the paxilline-sensitive composite current was observed in four of seven MSC cells (Figure 7a,b). The RMP of CPCs did not differ significantly from that of MSCs was determined by whole-cell patch-clamp measurements (–24.1 vs. –21.3 mV) (Figure 7c).

Since CPCs have been previously described to display oscillations in cytosolic calcium levels, we also looked at whether paxilline interfered with these spontaneous calcium events. In control cultures, 16% of CPCs exhibited periodic changes in cytosolic Ca²⁺ concentration (n = 112), but 1 μM paxilline completely abolished such events (n = 83) (Figure 7d).

4.1 Differential K⁺ transporter gene expression profiles in CPCs

We chose to study the expression profiles of those ion channels and transporter families that may have relevance in chondroprogenitor cell physiology (Barrett-Jolley et al., 2010; Matta & Zakany, 2013; Mobasheri et al., 2019). The most widely reported ion channels in chondrocytes and MSCs are potassium channels (Mobasheri et al., 2012; Pchelintseva & Djamgoz, 2018). The human genome contains around 70 different potassium channel genes, which makes them the largest family of membrane ion channels (Mobasheri et al., 2012). The α-subunit (KCNMA1) of the large-conductance Ca²⁺-activated potassium channel (BK, BK_Ca, MaxiK), as well as the intermediate Ca²⁺-activated potassium channel (IK, KCNN4, SK4, K_Ca3.1) transcripts, displayed the largest fold changes, with a 50% upregulation in CPCs. BK channels have been detected in undifferentiated MSCs both at the mRNA level and by single-channel recordings (Kawano et al., 2003), and also in mature chondrocytes (Mobasheri et al., 2010). BK channels may play various roles in chondrocyte physiology including volume regulation, oxygen sensing and mechanotransduction (Mobasheri et al., 2012). Since BK channels have been implicated in driving MSC differentiation (Pchelintseva & Djamgoz, 2018), perhaps the fact that CPCs are more committed to the chondrogenic lineage than undifferentiated MSCs may explain the higher levels of KCNMA1 both at the transcript and at the protein level (Matta et al., 2019).

BK channels may also be potential drug targets to protect against joint degeneration in OA (Haidar et al., 2020). In an in vitro model of synovial inflammation, KCNMA1 was found to be upregulated following cytokine treatment in primary synovial fibroblasts (Haidar et al., 2020). BK channel expression was also found to be upregulated in human OA cartilage (Lewis et al., 2013). Given that the migratory CPCs used in this study had been isolated from late-stage OA, the increased KCNMA1 expression may also be a result of their original inflammatory niche.

To further characterise the contribution of BK channels to the chondroprogenitor phenotype, we first looked at whether BK channel function was required for cell division. The role of BK channels in mediating proliferation has been controversial. BK channels are known to stimulate proliferation in BM-MSCs (Zhang et al., 2014); however, their activation may also lead to antiproliferative effects in human-induced pluripotent stem cell-derived MSCs (Zhao et al., 2013). The BK channel inhibitors paxilline and IBTX have significantly lowered the proliferation rate of CPCs, but not in MSCs. This may be attributed to the different sensitivity of the two cell types to BK channel inhibition, and the dose-dependent effect of paxilline on proliferation. Whilst 1 μM paxilline caused approximately 10% reduction in DNA synthesis in BM-MSCs, at 3 μM it resulted in approximately 80% inhibition (Zhang et al., 2014). On the other hand, in line with our data, IBTX did not alter [³H]-thymidine incorporation levels in rat BM-MSCs (Deng et al., 2007).

The increased abundance of KCNN4 transcripts in CPCs may reflect their inherently enhanced cell motility as IK channels have a confirmed role in migration (Pchelintseva & Djamgoz, 2018). In contrast, BK channels have a rather controversial role in cell motility (Catacuzzeno et al., 2015). In migratory CPCs, BK channels may play an inhibitory role in the motility of the cells as paxilline treatment increased every parameter of migration. These findings are rather unexpected as the BK channel inhibitor IBTX was reported to block the platelet lysate-induced migration of MSCs (Echeverry et al., 2020). In glioma cells, however, BK channel openers inhibited migration (Kraft et al., 2003). Given that CPCs express both SOX9 and RUNX2 and that there is a degree of crosstalk between these transcription factors (Koelling et al., 2009), perhaps BK blockade influenced CPC behaviour in such a way that it attenuated their chondrogenic phenotype, which is also reflected in the upregulation of the osteogenic transcription factor RUNX2.

4.2 Ca2⁺ homoeostasis in chondroprogenitor cells

Given that BK channel function depends on both RMP and [Ca²⁺]_i, we undertook to look at the transporters mediating the Ca²⁺ homoeostasis of the chondroprogenitor cells. Calcium plays central roles in cell physiology in nonexcitable cells such as chondrocytes (Suzuki et al., 2020). Dynamic changes in calcium signalling have been shown to be paramount to chondrogenesis (Matta & Zakany, 2013), and we have described earlier the calcium handling in CPCs (Matta et al., 2015). SOCE, one of the main sources of Ca²⁺ influx, is mediated by Ca²⁺ release-activated Ca²⁺ channels formed of ORAI1, ORAI2 and ORAI3 proteins. We found that ORAI2 was present in lower abundance in CPCs; ORAI2 has been reported to modulate the magnitude of SOCE (Inayama et al., 2015; Vaeth et al., 2017), which further pinpoints the important role of SOCE in CPC homoeostasis. Given that purinergic signalling regulates intracellular Ca²⁺ oscillations in CPCs and MSCs (Jiang et al., 2017; Matta et al., 2015), we also looked at differences in purinergic receptor transcript levels. The only gene with a significantly altered expression was P2RY2, which codes for a metabotropic receptor involved in the negative regulation of the osteogenic differentiation of BM-MSCs (Li et al., 2016).

BK channel function was reported to modulate [Ca²⁺]_i oscillations in various cell types (Mizutani et al., 2016; Wakle-Prabagaran et al., 2016). Both CPCs (Matta et al., 2015) and MSCs (Kawano et al., 2003) are known to exhibit periodic fluctuations in resting cytosolic Ca²⁺ levels. We confirmed that BK channels play a central role in mediating the Ca²⁺ homoeostasis in CPCs since the BK channel inhibitor paxilline completely abolished these [Ca²⁺]_i oscillations. This is the first study to report a mechanistic link between the big conductance calcium-sensitive potassium channels and periodic [Ca²⁺]_i oscillations in chondroprogenitor cells.

4.3 Electrophysiological profiling of CPCs

Having established the channelome of CPCs at the transcript level, we then looked at whether the global electrophysiological profile (electrome) of the progenitor cells was different from that of BM-MSCs. In addition to conventional patch clamping, we also employed DEP, which has been shown to be an efficient quantitative method of differentiating between closely related cell types, for example, in the bone marrow (Ismail et al., 2015). DEP can be used for both assessing the passive electrical properties of cellular components and as the basis for a separation method (Mahabadi et al., 2018). This could be especially relevant for CPCs present at very low abundance in arthritic cartilage, given that a truly reliable cell surface marker has still not been identified. Inherent cell properties that do not require the use of specific labelling for detection would provide a unique means to identify progenitors committed to particular cell fates. Whilst the effective membrane capacitance and the intracellular conductivity values did not differ, we report membrane conductance as a specific electrophysiological property that reflects the differentiation stage of human CPCs and MSCs. Membrane conductivity is a parameter that describes the potential of the membrane to transmit charge; and is indicative of ionic flux (Henslee et al., 2017).

We employed patch clamping to establish the RMP of CPCs, which was not statistically different from that of MSCs. The V_m value of MSCs detected in our study (approximately –20 mV) was similar to what has been observed earlier (–10 mV) (Kawano et al., 2003). The RMP of mature chondrocytes is dependent on the coordinated function of different types of ion channels including nonselective cation channels (Lewis et al., 2011) and K⁺ channels (Maleckar et al., 2018; Wilson et al., 2004). The majority of these channels did not show statistically different expression between MSCs and CPCs, which probably explains why there is no difference in RMP as observed in this study. As described above, various K⁺ channels were detected at the transcript level in both cell types; therefore, we studied the outward whole-cell currents in CPC and MSC cells. Both cell populations were heterogeneous with respect to ion current expression, and we found evidence of paxilline-sensitive conductances.