Comparative study of xeno-free induction protocols for neural differentiation of human dental pulp stem cells in vitro

Archives of Oral Biology 109 (2020) 104572

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Comparative study of xeno-free induction protocols for neural differentiation of human dental pulp stem cells in vitro Thulasi Thiruvallur Madanagopala,b, Alfredo Franco-Obregónc,d, Vinicius Rosaa,e,

T ⁎

a

Faculty of Dentistry, National University of Singapore, Singapore Campbell Family Mental Health Research Institute, Centre for Addiction and Mental Health, Toronto, Canada c Department of Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore d Institute for Health Innovation & Technology, iHealthtech, National University of Singapore, Singapore e National University Centre For Oral Health Singapore, National University Hospital System, Singapore b

A R T I C LE I N FO

A B S T R A C T

Keywords: Neuron Tissue engineering Regeneration Neurofilament Neurogenesis

Objective: To compare three different xeno-free protocols for neural differentiation of human dental pulp stem cells (DPSC). Methods: DPSC were treated with three different media to induce neural differentiation namely N1 (DMEM for 5 days), N2 (PSC neural induction media for 7 days) and N3 (neural media with B27 supplement, 40 ng/ml bFGF and 20 ng/ml EGF for 21 days). Cell proliferation (MTS assay), morphology, gene (qPCR for NESTIN, VIMENTIN, TUB-3, ENO2, NF-M and NF-H) and protein expression (flow cytometry) of neurogenic markers were assessed at different time points and compared to untreated cells (DMEM supplemented with 10% FBS). Statistical analysis was performed with global significance level of 5%. Results: N1 and N2 formulations increased the genetic expression of two out of six genes TUB-3, NF-M and TUB3, NF-H, respectively, whereas N3 elevated the expression of all genes by the late stage. N3 also stimulated protein expression for NESTIN, TUB-3 and NF-H. Cells treated with both N2 and N3 presented neuron-like morphology, decreased proliferation and expression of stemness genes at protocol end point. Conclusion: N3 was the most effective formulation in promoting a neurogenic shift in gene and protein expression. Cells provided with the N3 formulation exhibited neuron-like morphology, elaborating axonal-like projections concomitant with cell cycle withdrawal and reduced expression of stemness genes indicating greater commitment to a neurogenic lineage.

1. Introduction According to the World Health Organization, approximately 1 billion people worldwide are affected by neurological disorders including epilepsy (50 million), Alzheimer (24 million) or other forms of dementias of which nearly 7 million succumb each year. Moreover, those suffering spinal cord injuries (500,000 per year) are 2–5% more likely to die prematurely (Singh, Tetreault, Kalsi-Ryan, Nouri, & Fehlings, 2014; WHO-Report, 2007, 2013a, 2013b). In response to the human cost associated with said neurological disorders, various approaches have been forwarded including neurotransmitter modulators (agonists or inhibitors), surgical intervention, and deep brain stimulation (Volkmann et al., 2009; Young, 2009). Unfortunately, adverse side effects often accompany these treatments as a result of the employed pharmacology or are disfiguring, leaving permanent scaring, and pain in the case of surgery (Anderson & Spencer, 2003; Kehlet, Jensen, & ⁎

Woolf, 2006). Stem cell-based therapies have been advanced to circumvent some of the limitations and drawbacks associated with conventional acellular interventions (Giordano et al., 2014; Greene, 2009). The use of embryonic stem cells (ESC), however, faces ethical issues as well as has been implicated in the formation of teratomas (Li, Christophersen, Hall, Soulet, & Brundin, 2008), whereas the use of pluripotent stem cells (iPSC) is burdened by low reprogramming efficiency (Rolletschek & Wobus, 2009). Bone marrow-derived mesenchymal stem cells (MSC) require surgical extraction under general anaesthesia and heterologous neural stem cell pose a risk of immune rejection and inadequate migration (Bonnamain, Neveu, & Naveilhan, 2012; Ramos-Zúñiga, González-Pérez, Macías-Ornelas, Capilla-González, & QuiñonesHinojosa, 2012; Wright, Masri, Osman, Chowdhury, & Johnson, 2011). Alternatively, dental pulp stem cells (DPSC) may represent a viable source for neuronal replenishment (Luo et al., 2018; Rosa, Della Bona,

Corresponding author at: Faculty of Dentistry, National University of Singapore, Singapore. E-mail address: [email protected] (V. Rosa).

https://doi.org/10.1016/j.archoralbio.2019.104572 Received 11 May 2019; Received in revised form 18 September 2019; Accepted 22 September 2019 0003-9969/ © 2019 Elsevier Ltd. All rights reserved.

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chain reaction. RNA was extracted (Purelink RNA Mini Kit, Invitrogen) and cDNA was synthesised (iScript RT Supermix, Bio-Rad, USA) from samples harvested at the indicated time points. The relative gene expression of markers for neurogenic differentiation and stemness genes was obtained from quantification cycle values against the expression of housekeeping gene GAPDH. The oligonucleotide primer sequences are listed in Appendix Table 1 in Supplementary materials. Protein expression was evaluated by flow cytometry analysis during late differentiation (Table 2) using FITC-conjugated mouse monoclonal antibody against human NESTIN (1:300, Abcam, Hong Kong), TUB 3 (1:50, Abcam) and neurofilament heavy (NF-H, 1 μg/1 × 106 cells, Abcam). Briefly, the cells were fixed and permeabilized (Intracellular fixation & permeabilization buffer set, eBioscience, USA) for 20 min each. Cells were next incubated with primary antibody for 30 min followed by incubation in FITC-conjugated mouse monoclonal secondary antibody (1:2000, 30 min, Abcam). Suspensions of cells in PBS were analysed using a FACScan flow cytometer (BD LSRFortessa X-20 cell analyser, BD Biosciences, USA) and the data were analysed with the FlowJo 10.0.8 software (Tree star Inc., USA). The experiments were performed three times in three independent biological triplicates.

Cavalcanti, & Nör, 2012). Due to their ectomesodermic origin, DPSC can differentiate into neuronal cells and glial cells in response to appropriate environmental clues and stimulation (Arthur, Rychkov, Shi, Koblar, & Gronthos, 2008; Ellis, O’Carroll, Lewis, Rychkov, & Koblar, 2014; Sasaki et al., 2008). Several protocols have been developed to assist the neuronal differentiation potential of DPSC. Media deprivation of serum and trophic factors is one method to stimulate the innate potential of DPSC for neuronal differentiation. Mouse DPSC cultured under serum deprivation for 5 days exhibited increased Nestin and β TUB-3 gene expression (Zainal Ariffin et al., 2013). By contrast, others protocols require growth factor supplementation (e.g. retinoic acid, epidermal growth factor and others) to promote neurogenesis (Arthur et al., 2008; Sasaki et al., 2008). One study showed that a serum‐free neural induction medium promoted rapid derivation of primitive neural stem cells from human pluripotent stem cells after 7 days (Yan et al., 2013). Perhaps the most widely used protocol relies on the use of growth factors such as basic fibroblast growth factor (bFGF) and epithelial growth factor (EGF) for neuronal induction (Arthur et al., 2008; Ferro, Spelat, & Baheney, 2014; Hori et al., 2002). This protocol has been shown to induce stem cells from human deciduous teeth (SHED) to exhibit neural morphology and to express neural markers (e.g. NESTIN, TUB-3, GAD, NeuN, GFAP, NFM, and CNPase) after 28 days (Miura et al., 2003). Human DPSC treated with EGF and FGF also exhibited increased genetic expression of NF-M and NF-H and displayed neural morphology after 21 days (Arthur et al., 2008). In summary, diverse protocols have been developed that differ in the use of temporal and trophic cues to promote neuronal differentiation. Nonetheless, a systematic comparison of the neuronal induction of DPSC under the different protocols has not been undertaken. Our objective in this study was to compare three different neuronal induction protocols on human DPSC.

2.3. Cell proliferation and morphology Cells (2 × 103) were seeded onto tissue culture plates and treated the different media formulations described in Table 1. For proliferation under N3, DPSC P7 was thawed, passaged twice and seeded for the test (P9). Cells of P9 were used as the assay was long (21 days) and the doubling time of P6 is short (2.4 days). The results from N3 were compared with untreated DPSC of the same passage (P9). The proliferation was evaluated using MTS assay (CellTiter 96 AQueous One Solution Assay, Promega, USA) at time points mentioned in Table 2. Briefly, MTS reagent (20 μl / 100 μl of media) was added to the cells and incubated at 37 °C for 2 h and the absorbance was obtained using a plate reader (Tecan, Switzerland) at 490 nm. The experiment was performed in three independent samples. For morphology, cells were stained with 2 μM calcein AM (excitation/emission 494/517 nm) and 4 μM ethidium homodimer-1 (excitation/emission 517/617 nm) following manufacturer’s instructions (LIVE/DEAD Viability/Cytotoxicity Kit for mammalian cells, Invitrogen). Nucleus was stained with NucBlue Live ReadyProbes Reagent (excitation/emission 360/460 nm, Invitrogen) for 25 min at room temperature. The morphology was imaged with Olympus FluoView FV1000 (Olympus, Japan) laser scanning confocal microscope with 543 nm HeNe laser as the excitation source.

2. Materials and methods 2.1. Human dental pulp stem cells culture The use of human dental pulp stem cells from permanent teeth (DPSC) was approved by NUS Institutional Review Board (Approval Number: NUS 2094). The DPSC were isolated from the third molar from single donor (DPF003, AllCells, USA). Due to ethical requirements, the age, gender and ethnic group of the donor cannot be disclosed. The protein expression of CD34, CD73, CD90 and CD105 are available elsewhere (Rosa et al., 2016). The cells were thawed at P4 and cultured in basal growth media [Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (all from Invitrogen, USA)] and passaged at 70% confluence. All assays were performed with cells at P6 (experimental and controls) unless stated otherwise.

2.4. Statistical analysis Lavene’s tests was performed for checking normality and homogeneity. Statistical analysis was done using one way ANOVA and posthoc Bonferroni test for proliferation, pair-wise student’s T test for RTPCR (α = 0.05, SPSS v23, IBM corporation, USA).

2.2. Neural differentiation of human DPSC: gene and protein expression

3. Results

The genetic and protein expressions for marker of neurogenic differentiation were assessed at different time points that were defined to depict the evolution of the differentiation process for each neurogenic media tested. For the early stage, cells were treated with the media for 1 day and the biological outcomes assessed. Likewise, for the intermediate stage the markers were assessed after 3 days (N1 and N2) or 14 days (N3) whereas for the late stage after 5, 7 and 21 days for N1, N2 and N3, respectively (Table 2). DPSC (5 × 104) were seeded on 24 well plate and cultured with basal growth media for 24 h. Afterward, cells were washed with phosphate buffered saline (PBS, Invitrogen) and treated with one of the neural induction media described in Table 1 (all consumables from Invitrogen). The media was changed completely every two days. Gene expression was assessed by quantitative real time polymerase

3.1. Expression of neural markers at genetic and protein levels Quantitative RT-PCR results evaluating neural genes are shown in Fig. 1. At the early stage, media formulations N1 and N2 resulted in significantly increased expression levels of NESTIN, VIMENTIN, and NFH. At the late stage, N1 resulted in increased expression of TUB-3 and NF-M, whereas N2 increased the expression of TUB-3 and NF-H. Notably, the N3 formulation promoted significant increases in the expression of all genes at all time points (except NF-H at early stage) compared with control (p < 0.05). On the other hand, the expression of the stemness genes OCT-4 and NANOG were significantly downregulated at the late stage with N3 and NANOG and SOX-2 were 2

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Table 1 Composition of the neurogenic induction media and basal growth media (control). Group

Neurogenic induction medium

Duration (reference)

N1

DMEM and 1% penicillin/streptomycin

N2 N3

Neurobasal media, 1x neural induction supplement and 1% penicillin/streptomycin Neurobasal-A medium, 1xB-27 supplements, 20 ng/ml EGF 40 ng/ml bFGF, 1% penicillin/streptomycin

Control

DMEM supplemented with 10% FBS and 1% penicillin/streptomycin

5 days (Zainal Ariffin et al., 2013) 7 days (Yan et al., 2013) 21 days (Arthur et al., 2008) 5 for N1, 7 for N2 and 21 days for N3

3.2. Cell morphology and proliferation

Table 2 Stages (in days) used to quantify the gene and protein expression and morphology of the cells according to the neurogenic induction media used. The expressions were compared with cells kept under basal growth media (Control) for that same time point. Group

Early

Intermediate

Late

N1 N2 N3 Control

1 1 1 1

3 3 14 3 for N1 and N2, 14 for N3

5 7 21 5 for N1, 7 for N2 and 21 for N3

Cell morphology was assessed by fluorescence microscopy and revealed that cells in N1 maintained a spindle-shaped fibroblastoid morphology. Cells treated with N2 and N3 elaborated elongated and bifurcated morphologies (Fig. 5). The proliferation rates of cells treated with N1 and N2 were significantly lower than controls at the late stage, whereas N3 arrested proliferation at both intermediate and late stages (Appendix Fig. 2 in Supplementary materials).

4. Discussion significantly down-regulated with N2 relative to late stage controls (Appendix Fig. 1 in Supplementary materials). The protein expressions of NESTIN, TUB-3, NF-H were assessed at the late stages using flow cytometry. N3 induced higher expressions of all proteins analysed at late stage (Figs. 2–4).

Dental pulp stem cells (DPSC) can be induced to undergo neurogenic differentiation upon appropriate stimulation (Arthur et al., 2008; Ellis et al., 2014; Sasaki et al., 2008). Different clinical scenarios could benefit from neurons derived from DPSC. For instance, the transplantation of scaffold populated with DPSC have reduced inflammatory

Fig. 1. Effects of different induction protocol in the neurogenic differentiation of dental pulp stem cells (DPSC) in vitro. DPSC were treated with N1, N2 or N3 and the gene expression of neurogenic markers was assessed at different stages of differentiation described in Table 2. The quantitative real-time polymerase chain reaction analysis showed that N3 increased significantly the expression of all genes tested at intermediate and late stage compared with the DPSC under basal growth media (control) cultured for the same period (* denotes statistical difference for that group compared to the controls at p < 0.05). 3

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Fig. 2. The expression of NESTIN was measured by flow cytometry before (Control) and at the late stage of stimulation with different neurogenic medium (Treated). DPSC treated with N3 presented the highest shift in the expression.

tested (Fig. 1). At late stage, the expressions of most of the genes for N1 and N2 were similar to the control levels, whereas N3 presented generally higher expression throughout the study. NESTIN is essential for cell survival and renewal, whereas VIMENTIN is a negative regulator of neuron projection and suppresses the formation of neuronal processes, such as axons and dendrites (UniProtConsortium, 2016a, 2016d). These are progenitor markers that are expressed at stages preceding cell cycle withdrawal and commitment to a specific lineage (Suzuki, Namiki, Shibata, Mastuzaki, & Okano, 2010). Although NESTIN is downregulated when progenitor cells differentiate into neurons or glial cells (Frederiksen & McKay, 1988), genetic expression depends on the region of origin of the central nervous system progenitor cells (Dahlstrand, Lardelli, & Lendahl, 1995). N2 induced elevated expression of ENO-2 and TUB-3 predominantly at the early stage, whereas N3 treated cells exhibited elevated

injury and facilitated axonal regeneration after complete section of spinal cord in rats (Yang, Li, Sun, Guo, & Tian, 2017). Also, DPSC can be used to recover peripheral nerve injuries as previously shown with DPSC-embedded nerve conduits that have promoted regeneration and improved the functional recovery of injured facial nerve (Sasaki et al., 2011). Available strategies to derive neurons differ in the provided cues and times of exposure needed to induce differentiation (Arthur et al., 2008; Ferro et al., 2014; Hori et al., 2002; Sasaki et al., 2008; Yan et al., 2013; Zainal Ariffin et al., 2013). The protocols tested herein induced varying levels of response in DPSC undergoing neurogenic differentiation. We assessed the expression of markers indicative of neurogenic differentiation. The genetic expression of NESTIN and VIMENTIN were significantly upregulated at early time points for all induction media 4

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Fig. 3. The expression of TUB-3 was measured by flow cytometry before (Control) and at the late stage of stimulation with different neurogenic medium (Treated). DPSC treated with N3 presented the highest shift in the expression.

increased both NF-M and NF-H (Fig. 1). Notably, N3 promoted greatest increases in NF-H protein expression (Fig. 4) which is necessary for de novo extension of axons and functionally does not overlap with NF-L or NF-M (UniProtConsortium, 2016b, 2016c). Fig. 5 shows the morphological changes of cells upon the neurogenic induction. It was previously shown that murine DPSC exposed to N1 for 5 days presented with neuron-like structures (e.g., dendrites and axons) (Zainal Ariffin et al., 2013). This aspect of the N1 formulation could not be reproduced here and may relate to inherent differences (genetic and physiological) between DPSC of murine and human origin (Chinwalla et al., 2002). Nevertheless, N2 and N3 were shown to promote changes in the morphology at protocol end points. Previously, ESC and iPSC treated with N2 did not show any neural morphology after 7 days (Yan et al., 2013). The changes observed here with DPSC

expression of said genes at all time points (Fig. 1). ENO-2 is neuroprotective and neuro-tropic, binding in calcium-dependent manner to neocortical neurons to promote cell survival (UniProtConsortium, 2016d). Most importantly, N3 stimulated late expression of TUB-3 which is essential for proper axon extension, guidance and maintenance (UniProtConsortium, 2016a). A similar trend to that reported here was observed for TUB-3 in stem cells from human exfoliated deciduous teeth (SHED) when treated with N3 after four weeks (Miura et al., 2003). Neurofilaments have been correlated with effective differentiation to neuronal phenotypes and are involved in the maintenance of axonal structure (Eyer & Peterson, 1994; Gingras, Champigny, & Berthod, 2007; Podrygajlo et al., 2009). At the late stage, N2 promoted a significant increase in the genetic expression of NF-H, whereas N3

5

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Fig. 4. The expression of NF-H was measured by flow cytometry before (Control) and at the late stage of stimulation with different neurogenic medium (Treated). DPSC treated with N3 presented the highest shift in the expression.

Supplementary materials and Figs. 1–4). This can be explained by the fact that cell proliferation and differentiation, although temporally linked, are mutually exclusive stages of development. In this respect, the serum deprivation associated with the N1 protocol may have inadvertently blunted differentiation prematurely. Despite our reporting differences in the performance of the distinct induction protocols, our work is limited by the fact that the hDPSC were isolated from single donor of unknown differentiation potential relative to other donors. Alternatively, “pooled cell” populations may be used but they also have limitations like the presence of different phenotypes which can give higher variability in numerical data. Finally, the evaluation could be performed with independent cell populations from different donors, but this strategy incurs high cost in terms of consumables and manpower. Despite the limitation of our single donor paradigm, there is a consensus in the literature that DPSC can undergo neurogenic differentiation that is very susceptible to pharmacological

administered N2 may be related to the fact that they are derived from cranial neural crest (Miletich & Sharpe, 2004). Interestingly, DPSC treated with N3 presented neuron-like morphology with projections linking different cell as previously observed (Arthur et al., 2008). To substantiate the neurogenic shift, we evaluated the expression of markers associated with proliferative capacity and undifferentiated MSC status. The N2 and N3 protocols promoted significant decreases in two of the three genes analysed at the late stage (Appendix Table 1 in Supplementary materials). The decreased expression of stemness genes is correlated with cell differentiation and upregulated expression of tissue specific genes (D’Ippolito et al., 2004; Huang, Gronthos, & Shi, 2009; Pierdomenico et al., 2005). Also observed were significant decreases in the proliferative capacity of cells with all the induction media relative to control. Notably, DPSC treated withN3 exhibited greatest suppression of proliferation concomitant with greatest increases in neurogenic gene and protein expression (Appendix Fig. 2 in 6

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Fig. 5. Cell morphology was assessed by fluorescence microscopy and revealed that cells in N1 maintained a spindle-shaped fibroblastoid morphology lacking the branching pattern typical of neurons in the control condition. Cells treated with N2 and N3 elaborated elongated and bifurcated morphologies (scale bar = 25 μm).

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Acknowledgements The authors wish to thank Dr. Paul Edward Hutchinson (Flow Cytometry Laboratory/Life Sciences Institute NUS) for the assistance with the flow cytometry assays. This research was supported by grants from the National Medical Research Council, Singapore (NMRC/CNIG/ 1107/2013) and National University Health System, Singapore (R-221000-074-515).

Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.archoralbio.2019. 104572. 7

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