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. 2022 Aug 17:13:930804.
doi: 10.3389/fphys.2022.930804. eCollection 2022.

PEDOT: PSS promotes neurogenic commitment of neural crest-derived stem cells

Alessandra Pisciotta  1 , Alice Lunghi  2   3 , Giulia Bertani  1 , Rosanna Di Tinco  1 , Laura Bertoni  1 , Giulia Orlandi  1 , Fabio Biscarini  2   4 , Michele Bianchi  2 , Gianluca Carnevale  1
Affiliations

PEDOT: PSS promotes neurogenic commitment of neural crest-derived stem cells

Alessandra Pisciotta et al. Front Physiol. .

Abstract

Poly (3,4-ethylendioxythiophene) polystyrene sulphonate (PEDOT:PSS) is the workhorse of organic bioelectronics and is steadily gaining interest also in tissue engineering due to the opportunity to endow traditional biomaterials for scaffolds with conductive properties. Biomaterials capable of promoting neural stem cell differentiation by application of suitable electrical stimulation protocols are highly desirable in neural tissue engineering. In this study, we evaluated the adhesion, proliferation, maintenance of neural crest stemness markers and neurogenic commitment of neural crest-derived human dental pulp stem cells (hDPSCs) cultured on PEDOT:PSS nanostructured thin films deposited either by spin coating (SC-PEDOT) or by electropolymerization (ED-PEDOT). In addition, we evaluated the immunomodulatory properties of hDPSCs on PEDOT:PSS by investigating the expression and maintenance of the Fas ligand (FasL). We found that both SC-PEDOT and ED-PEDOT thin films supported hDPSCs adhesion and proliferation; however, the number of cells on the ED-PEDOT after 1 week of culture was significantly higher than that on SC-PEDOT. To be noted, both PEDOT:PSS films did not affect the stemness phenotype of hDPSCs, as indicated by the maintenance of the neural crest markers Nestin and SOX10. Interestingly, neurogenic induction was clearly promoted on ED-PEDOT, as indicated by the strong expression of MAP-2 and β -Tubulin-III as well as evident cytoskeletal reorganisation and appreciable morphology shift towards a neuronal-like shape. In addition, strong FasL expression was detected on both undifferentiated or undergoing neurogenic commitment hDPSCs, suggesting that ED-PEDOT supports the expression and maintenance of FasL under both expansion and differentiation conditions.

Keywords: cell differentiation; conductive polymers; dental pulp stem cells; immunomodulatory properties; nanostructured thin films; stemness.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Surface characterization of SC-PEDOT and ED-PEDOT films. 2D (A,C) and 3D (B,D) AFM topography images of SC-PEDOT (A,B) and ED-PEDOT (C,D) films underlying the nanostructured granular surface; (E) RMS roughness of both the films acquired at different length scales; (F) water contact angle values obtained on both the films; (G) dry and wet sheet resistance of SC-PEDOT and ED-PEDOT; (H) Bode |Z| plot of SC-PEDOT and ED-PEDOT.
FIGURE 2
FIGURE 2
Characterization of STRO-1+/c-Kit+ hDPSCs. Representative confocal fluorescence images showing the expression of the stemness markers STRO-1 and c-Kit (A), the neural crest markers Nestin and SOX10 and the immunomodulatory molecule FasL (B) in immune-selected hDPSCs. Multilineage differentiation potential of STRO-1+/c-Kit+ hDPSCs is shown by the expression of osteogenic, myogenic and glial specific markers (C). Nuclei were counterstained with DAPI (blue). Scale bar: 10 μm.
FIGURE 3
FIGURE 3
Evaluation of cell morphology, adhesion and proliferation of neural crest-derived stem cells on PEDOT:PSS films. (A) Immunofluorescence images showing cell morphology and adhesion of phalloidin-stained hDPSCs (red) grown on PEDOT:PSS films at different time points (16, 24 and 48 h). Nuclei were counterstained with DAPI. hDPSCs cultured on plastic culture plates were taken as controls. Scale bar: 20 μm. (B) Histograms represent cell proliferation of hDPSCs cultured on PEDOT:PSS films (16 h, 1, 2 and 5 days). Cell countings are reported as mean ± standard deviation (SD). *** p < 0.001 hDPSCs cultured on ED-PEDOT vs hDPSCs cultured on SC-PEDOT, ***p < 0.001 hDPSCs cultured on ED-PEDOT vs. control hDPSCs; one-way ANOVA followed by Newman-Keuls post hoc test.
FIGURE 4
FIGURE 4
Evaluation of stemness and neural crest markers in hDPSCs cultured on PEDOT: PSS films. (A) Immunofluorescence images show the expression of the stemness markers STRO-1 (green) and c-Kit (red) after 48 h of culture and (B) the neural crest related markers Nestin (green) and SOX10 (red) in hDPSCs after 48 h and 7 days of culture on both types of PEDOT:PSS films. Nuclei were counterstained with DAPI. Scale bar: 10 μm (A), 20 μm (B).
FIGURE 5
FIGURE 5
Evaluation of neurogenic differentiation of hDPSCs on PEDOT: PSS films. The expression of the neuronal markers β —Tubulin-III (green) and MAP-2 (red) in hDPSCs cultured in neurogenic medium for 7 days on both types of PEDOT:PSS films are shown by representative immunofluorescence images. Nuclei were counterstained with DAPI. hDPSCs differentiated on plastic culture plates were used as controls. Scale bar: 20 μm (A). Real-time PCR analysis showing fold increase of mRNA levels of β —Tubulin-III, MAP-2, NeuN and Synaptophysin (SYP) in hDPSCs after 3 weeks of neurogenic induction on ED-PEDOT films. Data represent mean ± SD of fold change obtained from three independent experiments. * p < 0.05, *** p < 0.001 vs. neurodiff hDPSCs (i.e., on plastic culture plates) (B).
FIGURE 6
FIGURE 6
AFM analysis of cell morphology at the micro- and nanoscale. Topography images acquired on hDPSCs cultured on ED-PEDOT (A) before and (B) after 7 days of differentiation. In panels b, v and b, vi, the profile lines corresponding to the lines in b, iii and b, iv, are reported.
FIGURE 7
FIGURE 7
Evaluation of FasL expression in hDPSCs after culture on PEDOT: PSS films. Immunofluorescence (top) and pseudocolour (bottom) analysis of FasL immunolabeling is shown in hDPSCs after 7 days of culture in the neurogenic medium. Control group consisting in hDPSCs cultured for 7 days in standard expansion medium. Nuclei were counterstained with DAPI. Scale bar: 20 μm.
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