# Immunological isolation and characterization of neuronal progenitors from human dental pulp: A laboratory-based investigation

Hayley P Mcmillan, Fionnuala T Lundy, Orla M Dunne, Kiran John

Mcloughlin, Imad About, T M Curtis, Ikhlas El Karim

► To cite this version:

Hayley P Mcmillan, Fionnuala T Lundy, Orla M Dunne, Kiran John Mcloughlin, Imad About, et al. Immunological isolation and characterization of neuronal progenitors from human dental pulp: A laboratory-based investigation. International Endodontic Journal, 2024, 57 (8), pp.1136-1146. 10.1111/iej.14077. hal-04967251

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Distributed under a Creative Commons Attribution 4.0 International License
# Immunological isolation and characterization of neuronal progenitors from human dental pulp: A laboratory-based investigation

Hayley P. McMillan<fnref n="1" />

Fionnuala T. Lundy<fnref n="1" /> ID

Orla M. Dunne<fnref n="1" /> ID

Kiran John McLoughlin<fnref n="1" /> ID

Imad About<fnref n="2" /> ID

T. M. Curtis<fnref n="1" />

Ikhlas El Karim<fnref n="1" /> ID

## Correspondence

Ikhlas El Karim, Centre for Experimental Medicine, School of Medicine, Dentistry and Biomedical Sciences, Queen's University Belfast, The Wellcome-Wolfson Institute of Experimental Medicine, 97 Lisburn Road, Belfast, BT9 7AE, Northern Ireland, UK.

Email: i.elkarim@qub.ac.uk

## Funding information

Animal Free Research UK, Grant/Award Number: NA

## Abstract

**Aims:** Dental pulp stem cells (DPSCs) contain a population of stem cells with a broad range of differentiation potentials, as well as more lineage-committed progenitors. Such heterogeneity is a significant obstacle to experimental and clinical applications. The aim of this study is to isolate and characterize a homogenous neuronal progenitor cell population from human DPSCs.

**Methodology:** Polysialylated-neural cell adhesion molecule (PSA-NCAM+) neural progenitors were isolated from the dental pulp of three independent donors using magnetic-activated cell sorting (MACS) technology. Immunofluorescent staining with a panel of neural and non-neural markers was used to characterize the magnetically isolated PSA-NCAM+ fraction. PSA-NCAM+ cells were then cultured in Neurobasal A supplemented with neurotrophic factors: dibutyryl cyclic-AMP, neurotrophin-3, B27 and N2 supplements to induce neuronal differentiation. Both PSA-NCAM+ and differentiated PSA-NCAM+ cells were used in Ca<fnref n="2" />⁺ imaging studies to assess the functionality of P2X3 receptors as well as membrane depolarization.

**Results:** PSA-NCAM+ neural progenitors were isolated from a heterogeneous population of hDPSCs using magnetic-activated cell sorting and anti-PSA-NCAM MicroBeads. Flow cytometry analysis demonstrated that immunomagnetic sorting significantly increased the purity of PSA-NCAM+ cells. Immunofluorescent staining revealed expression of pan-neuronal and mature neuronal markers, PGP9.5 and MAP2, respectively, as well as weak expression of the mature sensory markers, peripherin and islet1. ATP-induced response was mediated predominately by P2X3 receptors in both undifferentiated and differentiated cells, with a greater magnitude observed in the latter. In addition, membrane depolarizations were also detected in cells before and after differentiation when loaded with fast-voltage-responding fluorescent molecule, FluoVolt™ in response to potassium chloride. Interestingly, only differentiated PSA-NCAM+ cells were capable of spontaneous membrane oscillations.

<figure><img src="image_2.png" /></figure>

<footnote n="1">¹School of Medicine, Dentistry and Biomedical Sciences, Queens University Belfast, Northern Ireland, UK</footnote>

<footnote n="2">²Aix Marseille University, CNRS, Institute of Movement Sciences, Marseille, France</footnote>
**Conclusions:** In summary, DPSCs contain a population of neuronal progenitors with enhanced neural differentiation and functional neural-like properties that can be effectively isolated with magnetic-activated cell sorting (MACS).

KEYWORDS

dental pulp, neurons, progenitors, regenerative, stem cells

INTRODUCTION

The dental pulp is a source for heterogeneous MSC pop-
ulations of neural crest origin with a remarkable poten-
tial for differentiation into multiple lineages (Gronthos
et al., 2000). Because of their neural crest origin, DPSCs
possess many of the features of neural stem cells and ex-
press key neurotropic factors including neurotrophin 3
(NT-3), brain-derived neurotrophic factor (BDNF) and
neurotrophin 4 (NT-4) as well as some neuronal lineage
markers such as Nestin, Sox2 and Glial Fibrillary Acidic
Protein (Karaöz et al., 2011). These unique properties
together with their ready accessibility make DPSCs an
attractive cell source for nerve regeneration and nerve
tissue engineering. Indeed, transplantation of DPSCs has
demonstrated promise at improving functional outcome
in experimental models of nervous system diseases (Király
et al., 2009; Sakai et al., 2012). However, these effects are
mostly protective, and it is, yet, unclear whether DPSCs
can differentiate into, and functionally compensate for,
neuronal and glial cell types after transplantation.

The dental pulp itself is a highly vascularized and in-
nervated connective tissue that hosts stem cells within
its neurovascular niches. It has been reported that neu-
rovascular niches can activate MSC proliferation and
differentiation and promote these cells to participate in
pulp tissue repair and regeneration (Huang et al., 2020).
Nerves produce signals, such as vascular endothelial
growth factor (VEGF) that could guide vessel formation,
maturation and remodelling (Wang et al., 2016); therefore,
integrating neurogenesis would benefit dental pulp regen-
eration strategies. Although many studies reported pulp
regeneration using heterogeneous cell populations, fabri-
cating prevascularized and preinnervated pulp tissue con-
structs for pulp regeneration has recently been reported
(Basabrain et al., 2024; Ruan et al., 2023), suggesting that
differentiation of DPSCs to specific lineage may be useful
for future development of such tissue constructs.

In vitro studies have indicated the potential of DPSCs
to differentiate into neuronal-like cells; however, only
a small fraction of cells within such cultures develop
a functional phenotype (Aanismaa et al., 2012; Arthur
et al., 2008; Gervois et al., 2015; Li et al., 2019). Such short-
comings are largely due to cellular heterogeneity, which
is a significant obstacle to the clinical implementation of

cell-based therapies. Mixed cultures may contain a combi-
nation of stem cells with a broad range of differentiation
potentials and long-term proliferative abilities, as well as
more lineage-committed progenitor cells. Such variability
among cells in the same transplantable population can
lead to the adoption of adverse phenotypes, potentially
limiting improvements in outcome.

Attempts at optimizing neuronal differentiation have employed selective sorting of neural stem cells to produce a more homogenous population of neurons from embryonic stem cells. Both flow cytometer and magnetic bead-based protocols have been applied to enrich embryonic stem cell-derived neural precursors (Chung et al., 2011; Pruszak et al., 2007; Yuan et al., 2011). However, in comparison to flow cytometry, magnetic sorting has been reported to allow faster separation and gentler handling of cells, and can be used under sterile conditions (Bosio et al., 2009; Pruszak et al., 2007). Specific cell surface markers and their respective antibodies have also been used to sort undifferentiated pluripotent cells and to isolate differentiated cells (Pruszak et al., 2007).

An example of such specific cell markers is the neu-
ral cell adhesion molecule (NCAM). NCAM is one of the
major adhesion molecules in the nervous system where
it establishes cell–cell adhesion through homophilic in-
teractions of its extracellular domains. The functional
properties of NCAM are strongly influenced by poly-
sialylation. Polysialylic acid (PSA), which is a polymer
of derivatives of neuraminic acids, polysialylates NCAM
to produce the polysialylated form (PSA-NCAM).
Polysialylation of NCAM plays an important role in
neurogenesis, neuronal cell migration and neural plas-
ticity (Cremer et al., 1998; Cremer & Durbec, 2001; El
Maarouf & Rutishauser, 2003; Kiss & Rougon, 1997;
Muller et al., 1996; Rousselot et al., 1995). PSA-NCAM
is therefore considered a marker for immature neural
precursors (Nguyen et al., 2003) and has been shown to
successfully isolate neural progenitors that could be dif-
ferentiated into functional neurons (Barral et al., 2013;
Guo et al., 2012; Welzel et al., 2015). PSA-NCAM is
reportedly expressed in DPSCs (Arthur et al., 2008;
Nourbakhsh et al., 2011) indicating the existence of
neuronal progenitors as a subpopulation within DPSCs.
To date, immunomagnetic sorting with PSA-NCAM
has not been used to isolate neuronal progenitors from
hDPSCs. In the present study, we hypothesized that DPSCs contain a subset of neuronal progenitors that can be isolated magnetically based on surface expression of PSA-NCAM. Our aim was therefore to investigate the utility of the PSA-NCAM sorting method for isolation, characterization and neuronal differentiation of the neural progenitor subpopulation within human dental pulp.

## MATERIALS AND METHODS

The manuscript of this laboratory study has been written according to Preferred Reporting Items for Laboratory studies in Endodontology (PRILE) 2021 guidelines (Figure S1).

### Cell culture

Human dental pulp cells were isolated by explant culture from immature permanent third molar teeth as previously described (About et al., 2000), in accordance with French ethics legislation and covered by the Office for Research Ethics Committees (Northern Ireland) ethical approval number 08/NIR03/15. Donors were aged between 18 and 35 years old. Cells were grown in minimal essential medium with L-glutamine supplemented with 10% foetal bovine serum (FBS), 100UI/ml penicillin and 100µg/mL streptomycin. The cells were used between the second and fifth passages. All experiments were repeated a minimum of three times, with three or more technical replicates per batch of cells.

### Flow cytometry

Flow cytometric analysis was used to determine the percentage of neural progenitors within populations of hDPCs before and after magnetic-activated cell sorting (MACS). Approximately, 10⁶ cells were resuspended in 100 µL FACs buffer (phosphate-buffered saline containing 1% foetal bovine serum and 0.09% sodium azide; Invitrogen) and incubated with 10 µL anti-PSA-NCAM-APC (Miltenyi Biotec) for 10 min in the dark at 4°C. To identify dead cells, 7-Aminoactinomyosin (7-AAD) (FisherScientific) was added to each tube 5 min before running on the cytometer. Cells were run on an Attune NxT cytometer (Life Technologies). APC-labelled cells were excited by the 637 nm laser and detected in the RL1 channel (670/14 bandpass filter). 7-AAD was excited by the 488 nm laser, and the emitted fluorescence was detected in the BL2 channel (590/40 bandpass filter). Dead

cells identified by 7-AAD were excluded from the analysis, and APC+ events were determined by control gating according to the unstained and isotype IgM (Miltenyi Biotec) controls Flow cytometry standard (FCS) files were exported and analysed using FlowJo software (BD Biosciences).

## Magnetic isolation of PSA-NCAM-positive neural progenitors from hDPCs

PSA-NCAM+ neural progenitors were isolated from a heterogeneous population of hDPCs using MACS and anti-PSA-NCAM MicroBeads (Miltenyi Biotec) according to the manufacturer's protocol. Cell suspensions containing approximately 10⁶ cells were passed through a sterile filter and resuspended in 60 µL of cold selection buffer (PBS containing 0.5% bovine serum albumin (BSA) and 2 mM EDTA), incubated for 15 min at 4°C with 20 µL of anti-PSA-NCAM MicroBeads per 60 µL of cell suspension. Cells were then washed by adding 2 mL of selection buffer and mixed by gentle pipetting and centrifuged at 300×g for 10 min. Cells were resuspended in 500 µL selection buffer and loaded onto a MACS Column (LS type: Miltenyi Biotec), which was placed in the magnetic field of a MACS Separator (Miltenyi Biotec). The effluent was collected as the unlabelled negative fraction. The column was then washed three times with 3 mL selection buffer. The magnetically labelled cells were retained in the column and eluted out with 1 mL selection buffer as the positive fraction. The positive fraction was either used immediately to determine the efficiency of MACS using flow cytometric analysis or seeded onto poly-L-ornithine (PLO)/laminin-coated culture vessels to drive proliferation followed by differentiation.

## Neuronal differentiation of positively selected PSA-NCAM neural progenitors

The neuronal differentiation protocol used was based on reports by (Clarke et al., 2017; Gervois et al., 2015), with some minor modifications. The present study omitted neurosphere culture and instead opted for MACS to enrich neural progenitors. The isolated PSA-NCAM+ cells were seeded onto PLO/laminin-coated glass coverslips (VWR International) or glass bottom dishes (WillCo) for immunofluorescent and confocal Ca²⁺ imaging studies, respectively, initially in proliferation media (DMEM/F12 supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, 2% B27 supplement (all purchased from Gibco, Life Technologies), 20 ng/mL EGF and 20 ng/mL
FGF (Peprotech Ltd.)). Cells remained in proliferation medium for 1–2 days (referred to as PSA-NCAM+ cells), before being changed to neuronal differentiation medium (Neurobasal A medium supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, 2 mM L-glutamine, 2% B27 supplement, 1% N2 supplement (all purchased from Gibco, Life Technologies), 1 mM dibutyryl-cAMP and 30 ng/mL Neurotrophin-3) (StemCell Technologies) to induce neuronal maturation. This differentiation medium was changed every 2–3 days, and cells were kept under these conditions for 7 days. Cells that were cultured under differentiation conditions for 7 days will be referred to as d-PSA-NCAM herein.

## Intracellular Ca²⁺ measurements

PSA-NCAM+ and d-PSA-NCAM+ cells were incubated for 1 h at 37°C in 2 µM Fluo-4 AM. Glass bottom dishes were placed in a recording chamber mounted on the stage of a Leica SP5 inverted confocal microscope and continuously perfused with normal Hanks' solution (see Drugs and Solutions). Fluo-4 was excited at 488 nm, and the emitted fluorescence was collected at 526 nm. XY images over time were acquired using ×20 objective (HC PL Fluotar 20.0×0.50 DRY) at a frame rate of 15 frames per second using LASX software. A final concentration of 100 µM ATP was delivered manually into the recording bath using a pipette. For ATP antagonist experiments, PSA-NCAM+ and d-PSA-NCAM cells were pre-treated with 10 µM AF-353 for 7 min before starting the experiment, followed by restimulation with 100 µM ATP. Drugs were added 20 s after the start of the experiment and the recording continued for a further 270 s. Changes in intracellular Ca²⁺ were expressed as F/F₀, where F represents fluorescence intensity and F₀ is the average baseline fluorescence measured before addition of ATP. Data were analysed by measuring area under the curve (AUC) for each F/F₀ plot using Prism V8.4.2 (GraphPad Software, CA, USA).

## Measurement of membrane potential

Membrane potential was measured as previously described (McMillan et al., 2021). Briefly, PSA-NCAM+ and d-PSA-NCAM+ cells were loaded with FluoVolt™ loading solution (100X PowerLoad™ concentrate and FluoVolt™ dye, 1000X) (Invitrogen), in normal Hanks' solution for 20 min at room temperature, as recommended by the manufacturer. Membrane potential changes were measured with confocal microscopy, as described previously by McMillan et al. (2021). Initial recordings were taken to

detect any spontaneous activity. For potassium chloride (KCl) experiments, normal Hanks' was added to show baseline fluorescence before the addition of 100 mM KCl, which was used as a positive control. Membrane fluorescence was expressed as F/F₀ ratios, where F represents fluorescence intensity and F₀ is the baseline fluorescence prior to the addition of KCl.

## Immunocytochemistry

Cells were fixed in 4% paraformaldehyde (PFA) for 15–20 min at room temperature, and residual PFA was quenched with 0.1 M glycine in PBS. Non-specific binding sites were blocked with phosphate-buffered saline supplemented with 0.01% Triton 100-X (PBS-TX) containing 10% goat serum for 1 h at room temperature. Cells were then incubated with primary antibody overnight at 4°C. They were then incubated for 1 h with an appropriate secondary antibody. The primary and secondary antibodies used in this study are listed in Table S1. Cells were mounted using Prolong Gold with DAPI (4',6 diamidino-2-phenylindole) (Invitrogen, UK) to counterstain the nuclei. Images were captured using Leica SP8 confocal laser scanning microscope (Leica Microsystems). Negative controls were included in each staining, whereby the staining procedure was performed in parallel but with the omission of the primary antibody.

## Drugs and solutions

Normal Hanks' solution contained 140 mmol/L NaCl, 5 mmol/L KCl, 2 mmol/L CaCl₂, 1 mmol/L MgCl₂, 5 mmol/L D-glucose and 10 mmol/L 4-(2-hydroxyethyl)-1-piperazin-2-ethylhexanoic acid [HEPES] (pH 7.4 with NaOH). Reagents in this study were purchased from the following sources: Fluo-4 AM (Life Technologies, California, USA), Adenosine 5'-triphosphate disodium salt (ATP) (Sigma Aldrich, St Louis, Missouri, USA) and AF-353 was a gift from Dr Anthony Ford of Afferent Pharmaceuticals.

## Statistical analysis

Summary data are presented as mean ± SD. Statistical comparisons were performed on raw data using Prism V10.1.0 (GraphPad Software, San Diego, CA, USA). All data were parametric, unless otherwise stated and were analysed using Student's t-tests or one-way ANOVA. Results were obtained from at least three independent experiments. In all experiments, *p* < .05 was considered statistically significant.
# RESULTS

## Enrichment and characterization of PSA-NCAM+ subpopulation of hDPSCs

Several studies have reported PSA-NCAM expression in human DPSCs, but to our knowledge, there are no reports which have isolated the PSA-NCAM+ subpopulation from hDPSCs using MACS technology to refine existing neural differentiation protocols. We therefore performed flow cytometry analysis of DPSCs collected following explant outgrowth. An unstained control sample was used to set a baseline for the detection of PSA-NCAM+ events. Representative dot plots show the general workflow, whereby debris is first excluded with forward (FSC) versus side scatter (SSC) and doublets were then excluded by FSC-Area versus FSC-Height. Non-viable cells were then excluded by selecting cells which do not retain 7-AAD, which represented almost ~92% of cells. Of the viable cells, a control gate could then be set for the detection of cells expressing PSA-NCAM (Figure 1a). With the gating strategy optimized, we then evaluated the percentage of hDPCs expressing PSA-NCAM in the original fraction (ori frac), that is, before MACS to be 6%, which

could be enriched to 58.3%, shown in the positive fraction (pos frac) after MACS (Figure 1b). Summary results show enrichment of PSA-NCAM+ cells from three independent donors. Next, we used immunofluorescent staining to confirm that the PSA-NCAM+ fraction was neuronal in origin. As expected, the cells expressed PSA-NCAM as well as nestin, another neural progenitor marker. The pan-neuronal marker PGP9.5, immature neuronal marker and βIII-tubulin were also present. Interestingly, the mature neuronal marker, MAP2 and mature sensory markers islet1 and peripherin were also detected. As fibroblasts make up a large proportion of the hDPC population, FSP was also included. The lack of FSP suggests that there was no fibroblast contamination within our positive fraction.

## Neuron-specific P2X3 channels are functional in PSA-NCAM+ and d-PSA-NCAM+ cells

PSA-NCAM status of DPSCs drastically altered cell morphology from fibroblastic to more progenitor-like, with a swollen cell body and processes extending from opposite poles. The switch to neurobasal medium as well as

<figure><img src="image_3.png" /><figcaption>**FIGURE 1** Enrichment and characterization of PSA-NCAM+ subpopulation of hDPSCs: (a) Representative gating strategy for the detection of PSA-NCAM+ cells following MACS. Forward scattering area (FSC-A) and side scatter area (SSC-A) were used to initially gate cells and exclude cell debris (Gate 1). Singlets were then selected based on forward scatter height (FSC-H) and FSC-A (Gate 2). Dead cells were excluded with viable dye 7-AAD (Gate 3). Baseline for PSA-NCAM detection (Gate 4). Histogram showing percentage of PSA-NCAM+ cells before (Ori frac) and after (Pos frac) compared to isotype IgM control from representative donor sample. (b) Summary graph of PSA-NCAM+ cells from three independent donors. Data presented as mean (±SD), **$p$≤.001 for indicated comparison (paired t-test). (c) Cells collected as the positive fraction were cultured on PLO/Laminin. After 2–3 days in culture, cells expressed neural progenitor markers, PSA-NCAM and Nestin; immature neuronal marker, βIII-Tubulin; pan-neuronal marker, protein gene product 9.5 (PGP9.5) and mature neuronal marker, microtubule-associated protein (MAP2). Expressions of mature sensory markers, peripherin and islet1 were also present, and fibroblast surface protein (FSP) was absent. Nuclei were counterstained with DAPI (blue). White scale bars: 50, 75, 100 and 250 µm.</figcaption></figure>
activation of dibutyryl cyclic-AMP and neurotrophin-3-
mediated signalling pathways induced further maturation
of these cells in terms of morphology, made evident from
axon-like and dendrite-like extensions making connec-
tions with neighbouring cells, which is typical of mature
neurons (Figure 2a). However, a favourable morphology
does not necessarily guarantee functionality. We there-
fore moved on to Ca²⁺ imaging studies to investigate this.
P2X3 channels are found predominately in nociceptive

neurons (Ford, 2012). We therefore used drugs that tar-
get P2X3 in our Ca²⁺ imaging experiments to build on
our immunofluorescent data and confirm the neuronal
identity of the cells. To test whether these channels were
functionally expressed, confocal Ca²⁺ imaging was per-
formed on PSA-NCAM+ cells and their differentiated
progeny. The P2X3 agonist, ATP, elicited single Ca²⁺ tran-
sients in all PSA-NCAM+ (Figure 2a) and d-PSA-NCAM+
cells (Figure 2b) within the field of view, although Ca²⁺

<figure><img src="image_4.png" /><figcaption>**FIGURE 2** PSA-NCAM+ and d-PSA-NCAM+ cells express functional P2X3 receptors. (a) Explant outgrowth derived hDPSCs cultured as a monolayer (left), PSA-NCAM+ fraction cultured in neural induction media at day 1 (middle) and differentiation media (right) d-PSA-NCAM+ cells were characterized by a large cell body (encircled) and multiple cytoplasmic extensions (arrowheads) that formed connections with neighbouring cells. Scale bars: 100 µm. (b) Confocal Ca²⁺ imaging in Fluo-4 AM-loaded PSA-NCAM+ cells and (c) d-PSA-NCAM+ cells. Representative F/F₀ plots of four individual cells from a single field of view (left). Summary box and violin plots showing AUC of ATP-induced Ca²⁺ signals in the absence and presence of P2X3 antagonist, AF-353. Data presented as mean (±SD), N=9–13 cells per treatment, ****p≤.0001 for indicated comparisons (One-way ANOVA).</figcaption></figure>
transients observed in PSA-NCAM+ cells were statisti-
cally smaller in amplitude (Figure S2), consistent with
their identity as developing neurons. Nonetheless, these
responses were absent when cells were pre-treated with
the P2X3 antagonist, AF-353, and stimulated a second
time with ATP Desensitization of P2X3 receptor, and re-
covery within 4 min is reported in the literature (Richards
et al., 2019) in line with our data. Summary AUC data
showing ATP-induced Ca²⁺ responses in the presence and
absence of AF-353 in PSA-NCAM+ and d-PSA-NCAM+
cells are presented in Figure 2b,c. Taken together, these
data suggest that Ca²⁺ responses induced by ATP were
primarily a result of P2X3 channel opening.

PSA-NCAM+ neural progenitors show
spontaneous fluctuations in membrane
potential only when differentiated

To further confirm that both PSA-NCAM+ and d-
PSA-NCAM+ cells display functional characteristics of
neuronal cells in vitro, we loaded the cells with fast-voltage-
responding fluorescent molecule, FluoVolt™, to study

membrane potential changes within our cells. Interestingly,
on recording we noted that in d-PSA-NCAM+ cells, there
were transient fluctuations in F/F₀ which persisted over
100s of recording, indicating spontaneous depolarization
and repolarization of the membrane potential (Figure 3b).
This response, however, was absent in PSA-NCAM+ cells
(Figure 3a). For PSA-NCAM+ cells, 100 mM KCL induced
an rapid increase in F/F₀ which plateaued at around 50s and
remained until 250s when it began to decline (Figure 3a).
These results indicated that the lack of spontaneous mem-
brane potential activity in the PSA-NCAM+ cells could not
be attributed to insufficient dye loading or the health of the
cells. As expected, 100 mM KCL also elicited a rapid rise in
F/F₀ in the d-PSA-NCAM cells, although the magnitude of
the response appeared smaller than that observed in the un-
differentiated cells (Figure 3b).

DISCUSSION

Cellular heterogeneity is a major problem with all stem
cell types, and the existence of committed progenitors spe-
cifically contributes to the heterogeneity and variability

<figure><img src="image_5.png" /><figcaption>FIGURE 3 PSA-NCAM+ neural progenitors fire spontaneously only when differentiated. (a) Baseline recordings of PSA-NCAM+ cells (top left) with representative montage of pseudocolour images taken at 15 s intervals. Four individual PSA-NCAM+ cells in a single field of view showing changes in membrane potential following the addition of KCl (bottom left). (b) Baseline recordings of d-PSA-NCAM+ cells (top right) with representative montage pseudocolour images taken at 1 s intervals. Membrane potential changes in four d-PSA-NCAM+ cells following the addition of KCl (bottom right).</figcaption></figure>
within stem cell populations. Therefore, strategies and approaches to isolate tissue-specific progenitors may help reduce variability in cell-based therapies. In this regard, isolation of neural progenitors has been shown to produce desirable outcomes for neuronal differentiation and also has potential for transplantation (Barral et al., 2013). The neuro-regenerative potential of hDPSCs has been highlighted for dental pulp regeneration (Lambrichts et al., 2017), and recent studies showed the potential use of prevascularized and preinnervated pulp tissue constructs for pulp regeneration (Basabrain et al., 2024; Ruan et al., 2023); however, methods for isolation of homogenous neuronal cell population from the DPSCs are still lacking.

The present study aimed to address heterogeneity within hDPSCs to help improve their utility for tissue regeneration applications (Alraies et al., 2017; Hwang et al., 2015). The results confirmed the feasibility of isolating neuronal progenitors outside the brain using neural progenitor marker PSA-NCAM. DPSCs have neural crest origin and were also considered to be of glial origin (Kaukua et al., 2014; Peters & Balling, 1999; Thesleff & Åberg, 1999). The fact that approximately 6% of cells derived from dental pulp expressed PSA-NCAM further supports the neural stem properties of these cells and highlights the necessity of their characterization for future applicability in regenerative therapies. The 6% expression shown in this study is considerably lower than that of Arthur et al. (>40%) (Arthur et al., 2008). Nourbakhsh et al. also reported a higher percentage of PSA-NCAM-positive cells (11.6%), which is more in line with that of the present study but lower than Arthur et al. who used stem cells derived from the pulp human deciduous (baby) teeth (Nourbakhsh et al., 2011). Although MACS significantly increased the purity of PSA-NCAM+ cells, it was still less than expected. Despite passing the cells through a cell strainer and using EDTA to ensure single-cell suspension, this may be due to the adhesive nature of DPSCs as others using the same technology have yielded ≥70% purity with embryonic stem cells from mice and zebrafish (Barral et al., 2013; Welzel et al., 2015).

It was expected that PSA-NCAM+ cells would express immature markers, PSA-NCAM and nestin. However, interestingly immunofluorescence results revealed weak expression of accepted markers of sensory neurons, namely, peripherin and islet1 (Bae et al., 2015; Sun et al., 2008). These results suggest early sensory neurons lineage commitment of the hDPSC-positive fraction in line with their neural crest origin (Le Douarin & Kalcheim, 1999; Pisciotta et al., 2020). Because of their neural crest and glial origin (Kaukua et al., 2014), it is also likely that our isolated progenitors contain glial cells as previously reported (Al-Zer & Kalbouneh, 2015).

Next, we observed the functional activity to further characterize the neuronal lineage of these cells. In the view that PSA-NCAM+ cells differentiate towards sensory neurons, we studied the functional expression of the P2X3 receptor which is known to be strongly expressed in sensory neuronal cells (Woolf & Ma, 2007). We assessed Ca²⁺ responses in PSA-NCAM+ cells and d-PSA-NCAM+ cells differentiated using media sourced from a report by Gervois and colleagues (Gervois et al., 2015), to determine whether differentiation induced larger Ca²⁺ responses. In PSA-NCAM+ cells, the response to ATP was modest in comparison to d-PSA-NCAM+ cells. P2X3 receptor expression has also been shown in progenitors derived from embryonic stem cells (Ulrich & Illes, 2014). This class of receptor is among the first cell surface receptors expressed during embryonic development (Burnstock & Ulrich, 2011). In addition, both P2X and P2Y receptors induce Ca²⁺ transients that activate transcription factors responsible for neuronal maturation (Glaser et al., 2013). Based on these findings, the response to ATP in PSA-NCAM+ cells observed in this study is not unexpected and highlights their role in developmental and neuro-regenerative processes. The larger Ca²⁺ response shown in d-PSA-NCAM+ cells is consistent with reports that P2X3 expression increases with neuronal differentiation (Glaser et al., 2013). For both PSA-NCAM+ and d-PSA-NCAM+ cells, ATP-induced Ca²⁺ responses were inhibited by a specific P2X3 blocker, AF353. The lack of ATP-induced Ca²⁺ response shown when cells were pre-treated with AF-353 suggests that Ca²⁺ elevations in response to ATP were largely a result of P2X3 activation. Interestingly, d-PSA-NCAM+ cells, but not PSA-NCAM+ cells when loaded with FluoVolt™ membrane potential dye, displayed spontaneous membrane potential oscillations in two cells. We are confident that this is genuine as oscillations (demonstrated by increase in warmer colours) in the two cells adjacent to each other occurred asynchronously (Video S1). Although the sample size is small, to our knowledge, spontaneous (i.e. without external stimulus) membrane potential oscillations have yet to be shown from neuronal cultures derived from dental tissues. In fact, (Gervois et al., 2015; Li et al., 2019) have come the closest to achieving an action potential from neural-like cells derived from dental stem cell cultures. Both managed to show the depolarization phase of the action potential followed by incomplete repolarization. Unlike the present study, both groups opted for the neurosphere method to enrich progenitors, suggesting that MACS may be superior when achieving functional neural-like cells. However, future work is needed to explore the full functional capabilities of these cells.

In summary, the present study provided an innovative efficient method that relied on the endogenous cell
surface expression of PSA-NCAM to selectively enrich a neural progenitor subpopulation within DPSCs cultures. The dental pulp-derived PSA-NCAM+ cells, due to their homogeneity and potential to enhance neurological differentiation, may be an ideal cell source for pulp regeneration, and importantly, provide an excellent in vitro model to study dental pain and neuroimmune and neurovascular interactions in dental pulp health and disease.

## AUTHOR CONTRIBUTIONS

HMcM, IEK, FTL, I A and TC, conceptualization, HMcM, OD, KMCL, generated and analysed data. HMcM and IEK drafted the manuscript. All authors contributed to reviewing and editing the manuscript. All authors have read and approved the final manuscript.

## FUNDING INFORMATION

This study was funded by Animal Free Research UK.

## CONFLICT OF INTEREST STATEMENT

All authors declare no conflict in relation to this study.

## ETHICS STATEMENT

The dental pulp cells used in this study were collected in accordance with French Ethics legislations and covered by the Office for Research Ethics Committees (Northern Ireland) ethical approval number 08/NIR03/15.

## DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.

## ORCID

Fionnuala T. Lundy https://orcid.

org/0000-0003-3150-1150

Orla M. Dunne https://orcid.org/0000-0003-0769-4247

Kiran John McLoughlin https://orcid.

org/0000-0003-3815-2600

Imad About https://orcid.org/0000-0002-7453-3921

Ikhlas El Karim https://orcid.org/0000-0002-5314-7378

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## SUPPORTING INFORMATION

Additional supporting information can be found online in the Supporting Information section at the end of this article.

**How to cite this article:** McMillan, H.P., Lundy, F.T., Dunne, O.M., McLoughlin, K.J., About, I., Curtis, T.M. et al. (2024) Immunological isolation and characterization of neuronal progenitors from human dental pulp: A laboratory-based investigation. *International Endodontic Journal*, 57, 1136–1146. Available from: https://doi.org/10.1111/iej.14077