The beneficial effects of chick embryo extract preconditioning on hair follicle stem cells: A promising strategy to generate Schwann cells

Abstract The beneficial effects of hair follicle stem cells in different animal models of nervous system conditions have been extensively studied. While chick embryo extract (CEE) has been used as a growth medium supplement for these stem cells, this is the first study to show the effect of CEE on them. The rat hair follicle stem cells were isolated and supplemented with 10% fetal bovine serum plus 10% CEE. The migration rate, proliferative capacity and multipotency were evaluated along with morphometric alteration and differentiation direction. The proteome analysis of CEE content identified effective factors of CEE that probably regulate fate and function of stem cells. The CEE enhances the migration rate of stem cells from explanted bulges as well as their proliferation, likely due to activation of AP‐1 and translationally controlled tumour protein (TCTP) by thioredoxin found in CEE. The increased length of outgrowth may be the result of cyclic AMP response element binding protein (CREB) phosphorylation triggered by active CamKII contained in CEE. Further, CEE supplementation upregulates the expression of vascular endothelial growth factor (VEGF), brain‐derived neurotrophic factor and glial cell line‐derived neurotrophic factor. The elevated expression of target genes and proteins may be due to CREB, AP‐1 and c‐Myc activation in these stem cells. Given the increased transcript levels of neurotrophins, VEGF, and the expression of PDGFR‐α, S100B, MBP and SOX‐10 protein, it is possible that CEE promotes the fate of these stem cells towards Schwann cells.


| BACKGROUND
Cell-based therapy has evolved in several aspects over the last decades and hundreds of clinical trials have been initiated to treat a large panel of pathological indications. Besides the therapeutic effects elicited by the direct presence of stem cells, cell-derivatives such as extracellular vesicles and other secretome components can also benefit the damaged tissue or organ as critical mediators of trophic factors. 1 Up until now, various cell sources have been investigated for cell-based therapies and regenerative medicine. 2 One of the adult multipotent stem cells that has been considered in the treatment of various neurological conditions is a specific type of hair follicle stem cells called epidermal neural crest stem cells (EPI-NCSCs). These cells are remnants of the embryonic neural crest, 3 residing in the bulge of adult hair follicle 4 that are ontologically related to the central nervous system (CNS) and display a high level of physiological plasticity. 5,6 These stem cells can differentiate into various cell types, such as neurons [7][8][9][10][11] and glial cells, [12][13][14] and express a variety of neurotrophic factors. 15 Accordingly, they can significantly contribute to the recovery of sensory and motor functions in a mouse and rat model of spinal cord injury. [16][17][18] In addition, transplantation of hair follicle stem cells supports the repair of peripheral nerve defects [19][20][21] and has benefits in a rat model of ischemic stroke. 22,23 Nowadays, several strategies are employed to comply with large-scale production of stem cells and their derivatives in the most efficient fashion possible to maximize their therapeutic effects.
Among introduced approaches, preconditioning has attracted much interest as it increases cell survival rate, differentiation potential, homing and proliferative and secretory capacities of the stem cells. 24 Preconditioning methods include hypoxia, incubation with trophic factors/cytokines, conditioned medium from functional cells or serum-free medium, and pretreatment with pharmacological/ chemical agents, physical factor assistance and gene manipulation. 25,26 The pre-exposure of stem cells to hypoxic conditions triggers various protective signalling pathways, and increases cell survival and cytokine secretion that can greatly improve the benefit of in vivo stem cells therapy. 27 Using pharmacological/chemical agents, such as various off-label drugs, is another promising approach that can optimize the restorative potential of transplanted stem cells. 28 The modulation of the biochemical and biophysical microenvironment, mediated by either naturally derived or synthetic biomaterials, is another way to influence stem cell fate and to enhance their therapeutic potential. 29 An additional strategy to achieve successful therapeutic outcomes is the genetic modification that induces overexpression or knock out/down of a certain gene to improve the stem cells' inherent restorative properties. 30 Although genetically engineered stem cells are widely used in pre-clinical investigations, there are lots of limitations and unsolved issues hampering their clinical application. 31 In addition, priming of stem cells with different cytokines and growth factors is commonly used to stimulate the secretion of anti-inflammatory and immunomodulatory factors and to improve the immunosuppressive functions of stem cells to withstand the harsh microenvironment of target tissues.
However, a major challenge of using this preconditioning tool is the high costs of priming with recombinant cytokines and trophic factors. 32 Thus far, several preconditioning strategies including pharmacological/chemical agents 13,15 as well as substrate stiffness priming 33 have optimized the functionality of EPI-NCSCs in vitro. This study was designed to assess the chick embryo extract (CEE) preconditioning effect on the fate and function of EPI-NCSCs in culture.
CEE is a complex cocktail of growth factors that has been widely used to supplement the growth media of various cell types, such as neural crest stem cells (NCSCs). [34][35][36] In the present study, CEE was collected and used to determine whether its presence can stimulate migration of stem cells from the hair bulge, proliferation rate of migrated stem cells, and alter their morphology. Also, the influence of CEE treatment on expression of key cell surface markers, differentiation into osteoblast and adipocyte as well as colony-forming efficiency were assessed. The potential of stem cells to form the three-dimensional (3D) structure of spheroids was another subject that was evaluated following CEE preconditioning. In addition, analysis of gene expression involved in the fate and secretion of trophic factors was performed using reverse transcription (RT)-polymerase chain reaction (PCR). Following gene expression analysis, immunostaining against specific lineage markers was carried out to define stem cells commitment. To identify CEE relevant proteins that possibly affect migration, proliferation, gene and protein expression, and morphological alteration of hair follicle stem cells, the proteome content of CEE was analysed by tandem mass spectrometry.
Indeed, the beneficial role of hair follicle stem cells to reduce pathological indications and improving tissue repair has been proven in animal models of various neurological conditions. We, therefore, sought to investigate if preconditioning of these stem cells with CEE could help to acquire some desirable traits that can enhance their therapeutic benefit in preclinical studies of central and peripheral nervous system damage. We, thus provide first evidence that CEE preconditioning can promote lineage commitment of hair follicle stem cells and owing to specific proteome content might improve transplantation success.

| Preparation of CEE
To prepare CEE, 11-day-old fertilized chick eggs were disinfected using 70% ethanol. The embryos were removed from the shell and washed with ice cold Hanks balanced salt solution, their heads were cut off and the rest of the embryo was chopped and centrifuged as previously described. 35,37 Finally, the extract was filtered through 0.45 and 0.22-μm filters and samples were stored at À80 C until use. The sterility test for bacterial, fungi and yeast contamination revealed the samples are free from contamination.
To compare the number of migrated stem cells between groups, cells were counted following first subculture, using trypan blue staining.

| Cell viability assay
To assess cellular viability, the 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay was performed after third subculture. Cells from the different experimental groups were seeded in a 96-well plate at an equal density of 10 4 cells per well and cultivated in their respective complete growth medium at 37 C in the CO 2 incubator. On the third day, the medium was discarded and 0.5 mg/mL MTT (Sigma-Aldrich, Cat No: M5655) prepared in α-MEM was added to each well. After 3 h incubation, the MTT solution was gently aspirated and acidic isopropanol (0.01 N HCl in absolute isopropanol, 100 μl/ well) added to dissolve the blue formazan crystals. The developed colour was measured at 570 nm using a microplate reader (BioTek).

| Colony-forming assay
To compare colony-forming efficiency between experimental groups, an in vitro colony formation assay was performed. First, 300 cells were seeded into 6-cm plates and cultured with their corresponding growth medium for 10 days. The colonies were fixed with 4% paraformaldehyde (PFA) and stained with crystal violet. The number of colonies and their sizes were defined for each individual plate and analysed with Prism software. This experiment was performed in triplicate and repeated trice.

| Immunofluorescent staining
To verify the identity of migrated stem cells and their fate following

| Spheroid formation assay
To assess the ability of these stem cells to form spheres in different experimental groups, following second subculture, the trypsinized stem cells were seeded on 1% agarose coated 96-well plates at a density of 5 Â 10 3 cells in a volume of 200 μl per well, as previously described. 39 2.11 | RNA isolation and quantitative RT-PCR

| Proteome analysis of CEE content
Freshly prepared CEE was flash-frozen and subjected to proteome analysis as previously described. 40 Briefly, proteins were extracted and trypsinated using gel-assisted sample preparation. Resulting peptides were separated by liquid chromatography prior to peptide sequence analysis by tandem mass spectrometry in data-dependent acquisition mode on an AB Sciex TripleTOF 5600+. For protein identification, sequence data were blasted against both, the non-curated

UniProtKB/TrEMBLE and the manually annotated and non-redundant
UniProtKB/Swiss-Prot database.

| Statistical analysis
Statistical analysis was performed using GraphPad Prism (Version 7.03, GraphPad Software Inc.). One-and two-way analysis of variance with Tukey post hoc correction were performed, and, where appropriate, t-test to detect statistical differences among the experimental groups. p < 0.05 was considered to be statistically significant. The data are presented as means ± SEM.

| Characterization of EPI-NCSCs
A few days after bulge isolation from whisker pads ( Figure 1A,B) and explantation on collagen coated wells, migrating stem cells with stellate morphology were observed around the bulges in all primary experimental groups (i, ii, iii) ( Figure 1C). Phalloidin conjugated to Alexa Fluor 488 was used for staining filamentous actin (F-actin) ( Figure 1D), while immunostaining of β-tubulin revealed the general morphology of migrating stem cells ( Figure 1E). Also, indirect immunofluorescent staining against Nestin (marker of NCSCs) ( Figure 1F) and other NCSC markers, such as SOX-10 ( Figure 1G), β-III Tubulin ( Figure 1H) and GFAP (data not shown) verified the identity of migrating cells as EPI-NCSCs. 14,33 It is worth noting, that representative immunostaining micrographs were provided from CEE supplemented experimental group after the first subculture.

| EPI-NCSC migration and expansion enhanced in presence of CEE
Following the treatment of explanted bulges with different growth media, hair bulges were evaluated at DIV 3, 5, 7 and 11 ( Figure 2A).
Captured images from the same bulges at different days of culture revealed the presence of migrating stem cells at DIV 3 in all three experimental groups with increasing cell numbers over culture time ( Figure 2B). However, the percentage of bulges with migrating stem cells was higher in experimental group 3, which was supplemented with 10% FBS + 10% CEE and it significantly increased at DIV 7 and 11, compared to experimental group 2 that was supplemented with 20% FBS ( Figure 2C). In addition, the number of stem cells after the first subculture was significantly higher than in experimental group 3 than in the other groups ( Figure 2D). Further, an MTT assay per-

| CEE supplement preservers the multipotency of hair follicle stem cells
Regarding the cranial origin of isolated NCSCs, these hair follicle-  Consistent with these histological and gene expression findings, an enhanced activity of ALP enzyme was detected in CEE group

| CEE induces spheroid formation in EPI-NCSCs
Growth medium composition also affected the ability of hair follicle stem cells to form spheroids in vitro. To define the capacity of stem cells to create spheroids on agarose-coated 96-well plates ( Figure 6A), initial seeding cell numbers of 750, 2000, 3000 and 5000 were selected.
According to the captured images, 5000 cells were needed to form tight spheroids ( Figure 6B). Next, the same initial cell number of 5000 was

| CEE affects the cell fate of EPI-NCSCs
Immunostaining was performed to determine the cell fate of CEE treated stem cells. We found that in concordance with gene expression analysis, MAP2 and GFAP proteins were also expressed at very low levels. In contrast, the majority of cells expressed PDGFR-α, S100 B and MBP (myelin basic protein) at the protein level ( Figure 8A). S100 B is a calcium binding protein that is prevalently expressed in glial cells. 44,45 Also, MBP is one of the major proteins of the CNS that is abundantly expressed in the myelin sheath and is essential for the formation of the dense line. Considering the expression of Nestin ( Figure 1F), enhanced levels of PDGFR-α transcript and protein and expression of S100 B and MBP in this experimental group, it can be hypothesized that CEE content may govern the differentiation of these stem cells towards the glial lineage and more specifically into Schwann cells.
Besides evaluation of the aforementioned genes and proteins, the expression of SOX10 as a key transcriptional regulator of neural crest development was assessed. SOX10 is preferentially and abundantly expressed in glial cells and it is involved in lineage specification. 46 To define the influence of CEE on SOX10 activity, we evaluated the expression of SOX10 in our three experimental groups. SOX10 expression was significantly decreased in the CEE supplemented group at passage 3 ( Figure 8B). The comparison of SOX10 transcript levels between passages 1 (after their first subculture) and 3 by RT-PCR, revealed that SOX10 transcript levels were approximately 9-fold higher in passage 1 than passage 3 ( Figure 8C) nucleus and cytoplasm or exclusively found in the cytoplasm of CEE treated stem cells in passage 1 ( Figure 8D).  Tables 2, 3, and 4). In order to identify relevant transcription factors in Table 2, we performed an in silico analysis of the top transcription factor binding sites of our genes of interest that we identified to be significantly regulated by CEE treatment (Table 5) and

| Proteome content of the CEE
referenced those with factors in CEE that are known to biochemically Intriguingly, TCTP can also be released into the extracellular space via exosomes 59 and re-enter our target cells in a similar way like thioredoxin. A third candidate for an extracellular mitogenic and promigratory factor is Y-box-binding protein 1 (YB-1). YB-1 inhibits apoptosis but promotes proliferation, migration, invasion and angiogenesis. YB-1 is a transcription co-factor, 60 but can also be secreted and acts as an extracellular mitogen. 61 Secretion is not mediated by classical exosomic pathways, but rather in a non-classical way via microvesicles and ATP-binding cassette transporters. 61  SMADs also form physical complexes with AP-1 to drive gene transcription, which would also affect the regulation of the above-mentioned AP-1-dependent genes. 62 Moreover, exosome recognition via the TGFß pathway has been shown to activate c-Myc, 63 providing a secondary mechanism how thioredoxin-, or fortilin-containing exosomes can activate mitogenic, migratory, angiogenic, or morphological factors. However, we like to emphasize that gene expression is a multi-factorial process, with dozens of different transcription factors tightly regulating the expression of one single gene. Therefore, our findings on CEE-borne factors that are able to bind the promoters of our target genes do not claim to be comprehensive but provide a first insight into a set of essential factors that are present in the CEE.

| DISCUSSION
The role of EPI-NCSCs, isolated from different animal species, has been extensively studied over the last two decades. 64  In addition to the successful preclinical application of hair follicle stem cells, isolated from different animal species, in various neurological conditions, human hair follicle stem cells are attractive candidates for disease modelling, drug discovery and cell-based therapies. 80,81 Two essential parameters that need to be considered before human  16 with VEGF and BDNF being among them. VEGF signalling guides neuronal migration and axon pathfinding independently from its vascular effects. 83 Besides the neuroprotective effects of VEGF in neurodegenerative disease, it has several contextual roles in various neurological diseases, including trauma, stroke and multiple sclerosis. 84 BDNF expression was also increased in these stem cells by CEE treatment. BDNF is the most common neurotrophin in the brain and its expression is affected in several neurological conditions. 85,86 To date, various strategies based on BDNF administration are designed to restore BDNF function in neurodegenerative diseases. 87 One of the possible therapeutic strategies to increase BDNF levels in the brain is to graft cells that are engineered to stably express this trophic factor. 88 Expression of BDNF has been confirmed in naïve hair follicle stem cells. 16 Thus, CEE treatment might serve as a preconditioning strategy to enhance the baseline expression of BDNF, which makes them a proper cell type for transplantation under conditions like ischemic stroke. Another trophic factor whose expression was elevated by CEE treatment was GDNF. The therapeutic potential of GDNF has been extensively studied in different disorders with disturbed dopamine homeostasis 89 Note: AP-1: AP-1 activity regulates Sox-10, S100B and MBP expression and is induced by Thioredoxin (see Table 2). BDNF, GDNF, as well as VEGF expression is regulated by the transcription factor CREB (Jeon et al., 2007), which in turn is activated by γCamKII (see Table 2). Nestin, PDGFR and VEGF expression are regulated by the transcription factor c-Myc, which is under the control of the signalling factor SMAD3 (see Table 2).
of differentiation and myelination. 90  TCTP is another key element that can be regulated by AP-1. The capacity of this protein to modulate cellular stress, such as oxidative stress, imbalance of ion metabolism that are main hallmarks of injury site and its antiapoptotic effect, prompt speculation that CEE preconditioning might improve transplantation success of hair follicle stem cells in injury sites.

| CONCLUSION
Collectively, hair follicle stem cells are multipotent adult stem cells that present a valuable resource for nervous system cell-based therapies.
Our findings demonstrate that CEE contains a complex cocktail of trophic factors and cytokines that help hair follicle stem cells to acquire some desirable traits that can enhance their therapeutic benefit without the need for genetic manipulation. Given the increased level of neurotrophins, the angiogenic factor VEGF, and the expression of PDGFR-α, S100B, MBP and SOX-10 protein, it is tempting to speculate that CEE treatment directs the fate of these stem cells towards the glial lineage, and more specifically towards Schwann cells. In addition to our findings on CEE-directed cell fate, we hope to prompt further research to narrow down the effective factors in the CEE cocktail to allow the recombinant synthesis of an animal-free extract with similar capacities.