Analysis of Primary Cilium‐Bearing Human Neuroprogenitors Using Flow Cytometry

TL;DR

This paper introduces a new flow cytometry method to detect primary cilia on human neuroprogenitor cells, offering a faster and more objective alternative to traditional microscopy.

Contribution

The study demonstrates the first use of flow cytometry to detect cilia on human neuroprogenitors using colocalized axoneme and basal body markers.

Findings

Flow cytometry successfully detected ciliated cells using ARL13B and PERICENTRIN markers.

The method showed changes in cilium frequency between ciliopathy patients and healthy controls.

The approach is scalable, objective, and reduces operator dependency compared to microscopy.

Abstract

The primary cilium is a protruding organelle present on many cell types with important roles for cell signaling. Defects in primary cilium formation and function are linked to numerous pathological conditions including neurodevelopmental defects, aging and cancer. The evaluation of ciliated cells within a cell sample traditionally relies on the visual assessment of cilia in fluorescence/confocal microscopy, after immunolabeling for ciliary markers highlighting the organelle for cilium counting. This can be influenced by operator‐dependent factors, notwithstanding advanced image analysis tools developed to facilitate this labor‐intensive evaluation. To address these limitations, a flow cytometry approach was trialed for neuroprogenitor cells (NPCs) differentiated from human iPSCs and stained for the ciliary markers ARL13B and PERICENTRIN measured on a flow cytometer, which detected positively‐labeled ciliated cells. Specific staining was confirmed by microscopy and imaging flow cytometry, demonstrating for the first time the feasibility of cilium detection with axoneme and basal body markers colocalized on a single spot on human neuroprogenitor cell surface using a scalable, objective, and quantitative modality. Flow cytometry was able to measure changes in cilium frequency in a comparative analysis of neuroprogenitors derived from ciliopathy patients and healthy controls, underlining the discriminating capacity of this streamlined approach for the study of ciliary defects in a scalable and operator‐independent manner.

Figures

Cilium‐specific staining of neuroprogenitors using anti‐ARL13B immunostaining performed with conventional staining on cell‐seeded coverslips (A), on cells stained in suspension for flow cytometry analysis (B, C) before cytospin deposition for microscopy confirmation (D). (C) Shows imaging flow cytometry analysis of the sample confirming ciliary labeling on the membrane. Images (A, D) acquired with a Leica DM6B microscope. ARL13B is shown in green, and DAPI nuclear counterstain is shown in blue. Scale bar: 5 μm.

Flow cytometry analysis of ARL13B and PCNT‐stained neuroprogenitor cells. (A) Fluorescence microscopy observation of NPCs in culture stained for ARL13B (red) and PCNT (green), with DAPI nuclear counterstaining. Scale bar:5 μm. (B) Overlay dot plot of the dual (blue), single (green and red), and unstained (purple) samples, and (C) corresponding quantification, acquired with a BD FACS ARIA III. (D) Visualization of the dual‐stained FACS samples by imaging flow cytometry (Amnis ImageStream) confirming colocalized membrane signal of both cilium markers. Scale bar: 10 μm.

Comparative cytometry analysis of ciliated populations from a CPLANE1‐mutated and a healthy control neuroprogenitor line. (A) Fluorescence microscopy observation of cultured neuroprogenitors stained for ARL13B (green) and PCNT (red), with DAPI (blue) nuclear counterstaining in control (top) and mutated (bottom) neuroprogenitor lines. Scale bar: 25 μm. (B) Samples analysed by flow cytometry (BD FACS Lyric) showing the control (blue) and CPLANE1‐mutated (red) samples. (C) Corresponding quantitation of the ciliated populations measured by flow cytometry (grey) and microscopy (orange) methods (data normalised to healthy controls,  ∗∗∗∗ p < 0.00001).

Full text

1. Introduction

The primary cilium is a protruding organelle present on a wide array of cell lineages including adipocytes, chondrocytes, and neuroprogenitors [1–3]. Impaired ciliation is associated with numerous pathological conditions including neurodevelopmental diseases [4] but also aging [5] and cancer [6]. Experimentally, ciliation is typically assessed by fluorescence microscopy imaging, in widefield or confocal mode, following immunostaining for ciliary markers to label the axoneme and basal body parts of the cilium [7], allowing morphological analyses of cilium characteristics. Recently, additional approaches have been developed for the qualitative analysis of isolated and purified cilia including by flow cytometry [8]. For quantitative evaluations, the presence of ciliated cells is assessed on fluorescent microscopy images, analyzing multiple images per sample, typically in triplicates, amounting to up to hundreds of cells counted per sample to calculate the percentage of ciliated cells [9]. This widely used methodology has practical limitations, in primis, the number of cells that can be counted per sample. Its outcome can also be influenced by operator‐dependent variability in the way cilia are considered and counted.

Although some image analysis models have recently emerged to mitigate these issues [9–12], their rollout has been somewhat limited in some cases by segmentation challenges [13] and variable cilium positioning [14] particularly relevant for neuroprogenitors forming high‐density clusters in vitro [15], or by their computational requirements. Here, an alternative modality was explored for cilium, the analysis of cilium‐bearing human neuroprogenitors through a flow cytometry and imaging flow cytometry approach.

2. Materials and Methods

All reagents were purchased from Thermo Fisher Scientific unless otherwise stated.

2.1. Cell Culture

Neuroprogenitor cells (NPCs) were produced from a wild‐type human iPSC line [16] and from an iPSC line carrying a CPLANE1 mutation [17] using an established protocol [18]. NPCs were maintained on vitronectin‐coated plates in medium composed of DMEM/F12 and Neurobasal medium (1:1), supplemented with Glutamax (1% v/v), N2 (0.5% v/v), B27 (1% v/v), penicillin/streptomycin (1% v/v, Euroclone), CHIR (3 mM, MedChemExpress), puromorphamine (0.5 mM, Merck), and ascorbic acid (150 mM, Merck). To reduce the number of ciliated cells, cells were incubated for 20 min with 10 mM CaCl_2_ as described elsewhere [8, 19] before fixation in 4% ice‐cold PFA for 30 min and storage in PBS at 4 °C until analysis.

2.2. Immunofluorescence

Cells were seeded at a density of 5.2 × 10^5^ cells/mL on vitronectin‐coated coverslips and incubated overnight before fixation with 10% formalin for 15 min and storage in PBS at 4 °C until analysis. Immunostaining was performed after 45 min in blocking solution (0.1% of Triton X and 2% BSA in PBS). Primary antibodies (mouse anti‐ARL13B, Antibodies Inc, 1:2000; rabbit anti‐PCNT, Abcam 1:2000; rabbit anti‐INPP5E, Proteintech, 1:500) were diluted in blocking solution and left for overnight incubation on the samples, at 4 °C. Coverslips were then washed 3 times in PBS for 5 min each and incubated for 1 h at room temperature, in the dark, in dilutions (1:500) of secondary antibodies as indicated (goat anti‐rabbit Alexa Fluor 488 (1:500), goat anti‐mouse DyLight 550 (1:500), and goat anti‐rabbit DyLight 650 (1:250), Thermo Fisher Scientific) prepared in blocking solution. After a further 3 PBS washes, coverslips were counterstained with DAPI (Merck) and mounted using Mowiol. Fluorescent signal was observed with a Leica SP8 or a Leica DM6B microscope (Centro Grandi Strumenti, University of Pavia) as stated and analyzed using Image J. For cilium quantification, a minimum of 10 random images were acquired per sample, with at least 100 cells per image. For each, the number of cells positive for ARL13B and PCNT was normalized to the number of nuclei (marked by DAPI) to calculate the percentage of ciliated cells. Nuclei and signal colocalization were counted manually using the “multi‐point” ImageJ tool.

2.3. Flow Cytometry

Cells were detached using Accutase (Stem Cell Technologies), washed in PBS, and fixed in suspension using 4% ice‐cold PFA for 15 min before 4 °C storage in PBS until analysis. The staining was conducted in suspension phase (2 × 10^6^ cells per sample, suspended in 500 μL) using the solutions and dilutions described for the immunostaining protocol, with centrifugation (300 g for 10 min) to pellet cells in between each incubation or washing step. After 45 min in blocking solution at RT, mild agitation was applied for first 15 min of the primary antibody incubation phase before storing the samples at 4 °C overnight. The next day, cells were washed in PBS, pelleted, and resuspended in 250 μL of secondary antibody solution for a 1 h incubation under mild agitation at RT in the dark. After 2 rounds of PBS washes, samples were resuspended in 200 μL PBS and run on a cytometer (BD FACS Lyric or BD FACS Aria III, Becton Dickinson) or an imaging cytometer (ImageStreamX Mark II, Amnis/Luminex) as stated. Data analysis was performed with Floreada (https://floreada.io/) using the control samples stained with secondary antibodies to gate positive cells. Samples were also used to prepare cytospin slides by centrifugation at 500 g for 5 min using a Hettich Cyto‐System (Seneco, Milano) used for microscopy analysis as previously described.

3. Results

In order to test the principle of flow cytometry detection of ciliated cells, human NPCs were immunostained for the axoneme marker ARL13B using a green fluorescent secondary antibody and analyzed both by microscopy and on a flow cytometer (Figure 1A, B). Signal was clearly detected for a significant population of cells, and comparison with the secondary only control showed a clear shift in signal (Figure 1B). Ciliary specificity of the signal was confirmed by parallel microscopy observation of the same sample after cytospin deposition on a slide and analysis performed on an imaging flow cytometer (Figure 1C, D). In both cases, the signal was observed to be specific to the cilium present on the membrane, mirroring the pattern obtained with conventional microscopy on adherent cultures (Figure 1A).

Cilium‐specific staining of neuroprogenitors using anti‐ARL13B immunostaining performed with conventional staining on cell‐seeded coverslips (A), on cells stained in suspension for flow cytometry analysis (B, C) before cytospin deposition for microscopy confirmation (D). (C) Shows imaging flow cytometry analysis of the sample confirming ciliary labeling on the membrane. Images (A, D) acquired with a Leica DM6B microscope. ARL13B is shown in green, and DAPI nuclear counterstain is shown in blue. Scale bar: 5 μm.

Since conventional cilium analysis methods rely on the microscopy‐based counting of primary cilia labeled with an axoneme and a basal body marker, flow cytometry was also attempted combining ARL13B (axoneme marker [20]) and PERICENTRIN (basal body marker [21]) staining, assessed in parallel by microscopy and flow cytometry. Double‐labeling with these markers provided a highly specific ciliary staining visible in conventional immunofluorescence (Figure 2A) and resulted in clearly visible positive signal in flow cytometry (Figure 2B). Both markers were detected individually, and when used together (Figure 2C), a result was unequivocally confirmed by the imaging cytometer, which demonstrated specific detection of the two labels colocalized on a single spot on the cell membrane (Figure 2D). All cells in the ARL13B^+^ fraction were PCNT^+^, while some PCNT^+^ cells were ARL13B^−^, a result mirroring microscopy observations (Figure S1) and confirming ARL13B as a more discriminating markers for cilium detection [22–24]. In addition, cilium detection was confirmed by combined staining with antibodies against ARL13B and INPP5E, another known cilium‐associated marker [25] which showed signal overlap both in flow cytometry and microscopy analyses (Figure S2).

Flow cytometry analysis of ARL13B and PCNT‐stained neuroprogenitor cells. (A) Fluorescence microscopy observation of NPCs in culture stained for ARL13B (red) and PCNT (green), with DAPI nuclear counterstaining. Scale bar:5 μm. (B) Overlay dot plot of the dual (blue), single (green and red), and unstained (purple) samples, and (C) corresponding quantification, acquired with a BD FACS ARIA III. (D) Visualization of the dual‐stained FACS samples by imaging flow cytometry (Amnis ImageStream) confirming colocalized membrane signal of both cilium markers. Scale bar: 10 μm.

This method was used to compare samples from untreated controls to cells that had been subjected to a short treatment with CaCl_2_, known to reduce the percentage of cilium‐bearing cells. The ciliated cell fraction in the treated sample analyzed by flow cytometry was indeed found to be lower than the control (Figure 3), aligning with the microscopy analysis performed on the same samples (15% and 22% reduction upon treatment, respectively).

Figure 3. Comparative cytometry analysis of ciliated populations from untreated and CaCl_2_‐treated neuroprogenitors. (A) Fluorescence microscopy observation of neuroprogenitors stained for ARL13B (green) with DAPI (blue) nuclear counterstaining in untreated and CaCl_2_‐treated neuroprogenitor samples. Scale bar: 10 μm. (B) Corresponding analysis of ciliated population percentage by flow cytometry (BD FACS Lyric, gray) and microscopy‐based (blue) methods (data normalized to untreated controls,  ^∗∗^ p < 0.001).(A)(B)

This approach was also tested to analyze the ciliated population in neuroprogenitors produced from iPSCs presenting a genetic mutation in CPLANE1 [17] and compare it to that seen in neuroprogenitors produced from control iPSCs. CPLANE1‐mutated cells, which were reprogrammed from a Joubert Syndrome patient, were previously observed to yield a significantly lower ciliated cell population than healthy controls after early cerebellar differentiation, based on immunostaining using AcTUBULIN and PCNT [26]. Here, neuroprogenitor cultures produced from the same CPLANE1‐mutated iPSC line and analyzed using the newly established flow cytometry‐based protocol showed a notable reduction in the percentage of the ciliated fraction compared to the control cells (Figure 4).

Comparative cytometry analysis of ciliated populations from a CPLANE1‐mutated and a healthy control neuroprogenitor line. (A) Fluorescence microscopy observation of cultured neuroprogenitors stained for ARL13B (green) and PCNT (red), with DAPI (blue) nuclear counterstaining in control (top) and mutated (bottom) neuroprogenitor lines. Scale bar: 25 μm. (B) Samples analysed by flow cytometry (BD FACS Lyric) showing the control (blue) and CPLANE1‐mutated (red) samples. (C) Corresponding quantitation of the ciliated populations measured by flow cytometry (grey) and microscopy (orange) methods (data normalised to healthy controls,  ∗∗∗∗ p < 0.00001).

4. Discussion

Traditional measurements of ciliated cell frequency rely on visual evaluation and counting [27–30] and are therefore time‐consuming and potentially prone to operator‐dependent variability. Over the past few years, examples of semiautomated approaches have been reported for 2D and 3D cell samples [31] using computational pipelines and plugins for cilium counting on different image analysis platforms [10, 12, 32]. However, these still require the acquisition of large numbers of images to be sufficiently representative and often require access to specialized computational resources and expertise which may not be widely accessible. In addition, for neuroprogenitor cultures forming high‐density cell clusters [15], precise cilium imaging and segmentation represent particular challenges. Here, populations of ciliated human neuroprogenitors were successfully detected and analyzed using flow cytometry, with a protocol for cilium labeling using anti‐ARL13B and anti‐PCNT antibodies to label the axoneme and basal body, respectively. Signal specificity was confirmed by imaging flow cytometry, which provides both sensitivity and spatial resolution, establishing the first proof of concept that this cell‐by‐cell modality is suitable for the assessment of ciliated cell populations. The use of ARL13B labeling to mark ciliated cells was more discriminant compared to the centrosomal PCNT and specific for the axoneme, in line with previous reports [22−24,33, 34]. Results from flow cytometry were found to be in agreement with the classical microscopy‐based cilium counting method when trialed on two experimental cases affecting cilia, one arising from chemical deciliation [8] and one from a ciliopathy model [26], demonstrating the technique’s ability to identify and quantify changes in cilium frequency.

To our knowledge, this is the first report for the application of flow cytometry to analyze whole, intact ciliated neuroprogenitor cells. While a previous attempt has been reported for shedding primary cilia after their isolation from cells through a deciliation step [8, 19], the present study demonstrates detection and measurement of whole, cilium‐bearing human neuroprogenitors by flow cytometry. Experimental advantages are substantial in terms of both sample throughput and accuracy of ciliation measurements. For each sample, the number of cells that can be assessed by cytometry is orders of magnitude higher than with existing microscopy‐based approaches, providing more representative data outputs. Objective measurements of fluorescent signal on large cell samples on a cytometer also provide a way to overcome possible operator‐dependent biases that are inherently associated with the visual numeration of cilia in selected fields of views. In that respect, flow cytometry’s speed and high throughput are particularly fitting for the quantification of ciliated cell frequency in large cell samples with the acquisition of statistically reliable datasets. Furthermore, the availability of multiple fluorescence channels simultaneously measurable on a cell‐by‐cell basis opens the possibility of combining two or more immunolabels to detect phenotypic, proliferation, or cell cycle markers, such as cyclins, Ki67, and PCNA, as well as other cell status markers (e.g., senescence or apoptosis assays) compatible with fluorescence readouts [35] to further characterize ciliated subpopulations.

Samples analyzed in flow cytometry can be processed in parallel for cytospin slides to provide a complementary qualitative control for cilium staining specificity and phenotype. Alternatively, the use of imaging flow cytometry can add precise spatial resolution to provide an immediate qualitative validation of the signal. This advanced modality could be of particular benefit for pharmacological screening seeking to find new ciliogenesis modulators [36, 37] and could also provide increased throughput for preclinical studies using ciliated cell measurements to assess neurodevelopmental disease phenotype. In particular, translational approaches based on in vitro models of ciliopathy patients’ samples [26, 29, 38, 39] could take advantage of this cell‐by‐cell flow modality, achievable with a standard cytometry setup. The ability to identify and possibly purify ciliated cells for further characterization by flow cytometry will likely facilitate new experimental strategies and modeling for neurodevelopmental disease [40, 41]. In conclusion, these results provide the first evidence that flow cytometry can be used to detect primary cilium on neuroprogenitors in a quantitative, high‐throughput, and operator‐independent modality, opening new possibilities to streamline and refine research on ciliated cell populations.

Funding

This research was funded by the European Union – Next Generation EU (Missione 4 Componente 1 CUP F53D23003800006 [Grant 2022JT5PWC_002]), with support from the Italian Ministry for University and Research (MUR) through a doctoral studentship (PhD in Translational and Precision Medicine), the National Recovery and Resilience Plan (PNRR), project MNESYS (PE0000006—A Multiscale integrated approach to the study of the nervous system in health and disease), and a grant under the initiative “Dipartimenti di Eccellenza (2023–2027)” to the Department of Molecular Medicine (University of Pavia). Open access publishing facilitated by Universita di Pavia, as part of the Wiley ‐ CRUI‐CARE agreement.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting Information

Additional supporting information can be found online in the Supporting Information section.

Supporting information

Supporting Information Figure S1: Fluorescence microscopy analysis of ARL13B (red) and PCNT (green) stained neuroprogenitors with (A) or without (B) DAPI nuclear counterstain, showing double‐positive (white arrow) and single PCNT‐positive (yellow arrow) cells. Boxed area in (B) is enlarged in (C). Scale bar: 25 μm. Figure S2: Validation of cilium detection in human neuroprogenitors using ARL13B and INPP5E staining. (A, B) Flow cytometry detection of ciliated NPCs marked with ARL13B (FITC) and INPP5E (APC) used singularly and together (A) with corresponding quantitation of costaining (B). (C) Fluorescence microscopy confirmation of cilium specificity (arrows) and overlap for the signal obtained using ARL13B (green) and INPP5E (red) immunodetection. Scale bar: 20 μm.