The influence of cerebrospinal fluid on epidermal neural crest stem cells may pave the path for cell-based therapy
- Sareh Pandamooz†1, 2,
- Mohammad Naji†3,
- Farid Alinezhad1, 4,
- Amin Zarghami1, 4 and
- Mohsen Pourghasem1Email author
© Pandamooz et al.; licensee BioMed Central Ltd. 2013
Received: 18 April 2013
Accepted: 10 July 2013
Published: 18 July 2013
Epidermal neural crest stem cells (EPI-NCSCs) in the bulge of hair follicles are a promising source for cell-replacement therapies in neurodegenerative diseases. A prominent factor in cell-based therapy is the practicalities of different routes of administration. Cerebrospinal fluid (CSF), owing to its adaptive library of secreted growth factors, can provide a trophic environment for transplanted cells. Thus, the effect of CSF on the behavior of EPI-NCSC was studied here.
In this study, the highly pure population of EPI-NCSCs was obtained from the bulge of mouse hair follicle. Migrated cells were characterized with real-time polymerase chain reaction (RT-PCR) and immunocytochemistry. Subsequently isolated stem cells were cultured in CSF, which was collected from the cisterna magna of the adult rat. The expression of pertinent markers was assessed at the gene and protein levels with RT-PCR and immunocytochemistry, respectively. Colorimetric immunoassay was used to quantify the rate of proliferation of EPI-NCSCs after cultivation in CSF.
Isolated EPI-NCSCs could survive in the CSF, and they maintained the expression of nestin, β–tubulin ІІІ (early neuronal marker), and glial fibrillary acidic protein (GFAP, glia marker) in this environment. In addition, CSF decreased the proliferation rate of EPI-NCSCs significantly in comparison to primary and expansion culture medium.
Our findings demonstrate that CSF as a cocktail of growth factors helps EPI-NCSCs to acquire some desirable traits, and because of its circulatory system that is in close contact with different parts of the central nervous system (CNS), can be a practical route of administration for delivery of injected stem cells.
KeywordsEpidermal neural crest stem cell Cerebrospinal fluid Hair follicle Bulge
Epidermal neural crest stem cells (EPI-NCSCs) are multipotent stem cells that persist in the bulge of hair follicles through adulthood, and they have the capacity to generate various types of differentiated cells under appropriate culture conditions . EPI-NCSCs are one of the main derivatives of transient embryonic neural crest that retain the neurologic differentiation potential of their neural crest origin. The neural crest forms during gastrulation and locates in the boundary between somatic ectoderm and neuroectoderm. It leaves the closing neural tube during late neurulation, invades the embryo, and give rise to distinct cell types and tissues, such as craniofacial bone/cartilage, meninges, tooth papillae, the autonomic and enteric nervous systems, sensory ganglia, endocrine cells of the adrenal medulla, smooth musculature of the cardiac outflow tract, and great vessels and pigment cells (melanocytes) of the skin and internal organs. The bulge region within the outer root sheath of the hair follicle is one of the prime targets of the neural crest during development that serves as a specialized niche for epidermal stem cells [2–6]. EPI-NCSCs exhibit several characteristics of embryonic and adult stem cells. Similar to embryonic stem cells, these cells show a high level of physiological plasticity, and they can be easily expanded under in vitro condition. Similar to other kinds of adult stem cells, they are a promising group of stem cells that do not elevate ethical concern. Despite all these similarities, this unique type of stem cells can circumvent several setbacks associated with embryonic stem cells, such as immunologic incompatibility. Moreover, they are relatively abundant and accessible in the bulge area of hairy skin and can be isolated by a minimally invasive procedure. However, most of other types of adult stem cells are fairly sparse and approachable with difficulty [7–9].
Previous studies have established that local signaling and regional identity during migration of neural crest cells play a crucial role in cell-type specification, and several investigations have emphasized on the importance of the concerted action of a combination of growth factors on survival, proliferation, and differentiation of neural crest cells at multiple levels [10, 11]. Therefore, it is quite conceivable that the CSF, as a cocktail of secreted growth factors, can provide a trophic environment for survival and proliferation of these multipotent stem cells. This issue has received support from numerous studies that examined the critical influence of CSF-borne signals not only on neuroectodermal cells during brain development but also on survival, proliferation, and fate specification of neural stem cells in adult brain throughout life [12–18]. Furthermore, the close ontologic relation between EPI-NCSCs and stem cells of the central nervous system (CNS) has fueled this hypothesis that the CSF can be an instructive milieu for these cells because the fate of neural progenitor cells at the brain-CSF interface is governed by CSF [19, 20].
Based on these facts, in this experiment, the influence of CSF on the EPI-NCSCs was studied to demonstrate whether it can help these cells to acquire some desirable traits that establish them as an appealing candidate for cell-replacement therapy in different CNS injuries and neurodegenerative diseases.
Materials and methods
All experimental protocols of this study were approved by local ethics committee at Babol University of medical sciences.
Cerebrospinal fluid collection
CSF was collected from the cisterna magna (CM) of Wistar rats with 200 to 300 g of body weight by using a fire-polished 1-ml syringe connected to a 27G dental needle. Here the animal was anesthetized with xylazine 2% and ketamine 50 mg/kg per body weight intraperitoneally and placed on the stereotaxic instrument (Stoelting, Wood Dale, IL, USA). Specially constructed ear bars were placed in the external auditory meatus, and the head was flexed downward at approximately 90 degrees so that the occipital bone was almost horizontal. A median incision was made, and the cervicospinal muscle was reflected and the posterior atlanto-occipital membrane exposed.The needle was inserted vertically and centrally to the depressible surface with a rhomboid appearance between the occipital protuberance and the spine of the atlas. A gentle aspiration flow the CSF through the syringe. Collected CSF was transferred to a sterile microtube on ice and centrifuged (Sigma, Osterode am Harz, Germany) at 10,000 rpm for 10 minutes to remove cells or debris, and ultimately all supernatants were stored at −80°C until use. Because the volume of collected CSF from each rat was approximately 100 μl, to provide adequate volume of CSF for the experiment, it all was pooled.
Chick embryo extract preparation
The head of the day-11 chick embryo was cut off. Then the embryo was chopped, homogenized with an equal volume of HBSS (PAA, Austria), and the mixture was incubated for 30 minutes on ice. Subsequently, it was centrifuged at 12,000 rpm for 30 minutes at 4°C, and the supernatant was removed and passed through 0.45-μm and 0.22-μm filters sequentially.
Dissection of the bulge from adult whisker follicle
Isolation and in - vitro expansion of EPI-NCSCs
Explanted bulges were cultured in Alpha-modified MEM (PAA, Pasching, Austria) supplemented with 10% fetal bovine serum (FBS) (PAA), 5% day-11 chick embryo extract (CEE), and 1% penicillin/streptomycin (PAA). Fifty percent of the culture medium was exchanged every other day. After observation of migrated cells, the bulge explants were carefully removed with a 27G needle to minimize the rate of contamination with other undesirable later-migrating cell types, such as keratinocytes. Adhering EPI-NCSCs were resuspended by trypsinization, placed in fresh collagen-coated plates at 7 × 104 cells per each well of four-well plate (SPL Life Sciences, Pochun, South Korea), and cultured for another 24 hours. Thereafter, isolated EPI-NCSCs were cultured in two different mediums. The first group of cells was expanded in the culture medium that consisted of 90% Alpha-modified MEM plus 10% day-11 chick embryo extract supplemented with fibroblast growth factor-2 (FGF-2, 20 ng/ml; Sigma-Aldrich, St. Louis, MO, USA). Likewise, a second group of cells were cultivated in the collected CSF for 72 hours.
Indirect immunofluorescent staining was performed to demonstrate the presence of Nestin, β-tubulin ІІІ and GFAP-positive cells in the population of migrated cells or cells after their cultivation in different media. Cultures were washed twice with phosphate-buffered saline (PBS; PAA) for 2 minutes and fixed with 4% paraformaldehyde (Merck, Germany) in PBS at room temperature (RT) for 12 minutes, followed by three 5-minute TPBS washes (0.05% Tween-20 in PBS, Sigma-Aldrich). They were permeabilized with 0.2% Triton X-100 (Merck) in PBS at RT for 10 minutes and then were washed again in TPBS for three 5–minute intervals, and blocked with 1% BSA in TPBS at RT for 1 hour. Primary antibody was added, diluted appropriately in blocking buffer, and incubated overnight at 4°C. Cultures were then rinsed 3 times with TPBS for 5 minutes each, followed by addition of secondary conjugated antibody diluted (1:250) in blocking buffer and incubated in the dark for 1 hour at RT. Secondary antibody was removed, and three, 5-minute TPBS washes were performed, followed by counterstaining with propidium iodide (PI) (Sigma-Aldrich). Primary antibodies used included rabbit anti-Nestin (1:200) (Abcam, Cambridge, UK.), rabbit anti-β-tubulin ІІІ (1:50) (Sigma-Aldrich), and rabbit anti-GFAP (1:1,000) (Abcam). Goat anti-rabbit IgG was used as secondary antibody (Sigma-Aldrich).
RNA extraction, cDNA synthesis and polymerase chain reaction
Real-time PCR primers
Forward primer (5′-3′)
Reverse primer (5′-3′)
β – Tubulin ІІІ
For quantitative comparison, compatibility of each primer set efficiency with Hprt primers was validated by qRT-PCR of dilution series of cDNA templates. Each reaction (20 μl) consisted of 10 μl 2× SYBR Premix Ex TaqII (Takara), 0.8 μl forward primer, 0.8 μl reverse primer, 2 μl first-strand cDNA template (1:3 in distilled water), and 6.4 μl distilled water. Thermocycling was conducted as follows: 95°C for 30 seconds to activate HotStart enzyme, 40 cycles of 95°C for 5 seconds followed by 60°C for 35 seconds by using a Rotor-Gene Q instrument (Qiagen). At the end of each run, melting-curve analysis was performed, and a single amplification peak was considered specific amplification. Ct values of target genes were normalized against Hprt Ct (∆Ct), and the relative expression of each target was determined by using the ∆∆Ct method.
Colorimetric immunoassay of cell proliferation
To quantify the proliferation of EPI-NCSCs in different medium, after trypsinization and counting, they were seeded at a density of 2.5 × 104 cells/μl in a 96-well plate (Orange Scientific, Belgium) and after 24 hours, they were cultivated in four different mediums, (a) α-MEM, (b) α-MEM with 10% FBS and 5% CEE, (c) α-MEM supplemented with 5% CEE and FGF, and (d) 100% CSF. Cultures were maintained at 37°C in a 5% CO2 atmosphere for a further 72 hours. Cell proliferation was determined by using the colorimetric BrdU enzyme-linked immunosorbent assay (ELISA) kit (Roche, Mannheim, Germany), based on incorporation of BrdU during DNA synthesis in proliferating cells. Measurement was made on an ELISA reader (Rayto, China) at 450 nm.
Imaging and statistical analysis
Images were obtained with the Olympus Stereomicroscope (SZX16) and invert florescence microscope (CKX41). Statistical analyses were performed on version 18 of SPSS statistical software (SPSS Inc. Chicago, IL, USA) and GraphPad Prism (Version 6.02, 1992–2013 GraphPad Software, Inc.) by using one-way ANOVA and the Tukey post hoc test. A value of P < 0.05 was considered significant.
Expression of nestin, SOX10, β-tubulin ІІІ, and GFAP in culture of primary explants
Expression of EPI-NCSC markers proceeds in CSF environment
CSF decreases proliferation of isolated EPI-NCSCs
A wide range of CNS injuries and neurodegenerative diseases results in various degree of cell death and neuroinflammation. Several therapeutic approaches have been evaluated for treatment of CNS impairment, and stem cell therapy is one of the promising means to achieve this aim. Cell-based therapies have recruited different types of stem cells to replace lost cells or to repair damaged areas. Studying the behavior of these cells after implantation and the feasibility of the mode of administration are two main debatable topics in cell-based therapies .
This investigation revealed that CSF, due to its beneficial environment, can retain viability of epidermal neural crest stem cells, and these cells continue to express a neural crest stem cell molecular signature after 72 hours of cultivation in the CSF milieu. According to our findings in the current study, CSF can be a suitable route of administration for EPI-NCSCs in CNS injuries and neurodegenerative diseases.
Previous studies have shown that EPI-NCSCs, as adult-resident stem cells in the bulge of the hair follicle, presents a number of advantages that make it an appropriate cell type for autologous transplantation. These readily accessible stem cells can generate several types of cells without known tumorigenic effects. Furthermore, their potential for regeneration of peripheral nerves and spinal cord injuries was demonstrated previously . As neural crest stem cells are ontologically related to spinal cord stem cells, EPI-NCSCs are particularly attractive types of stem cell for treatment of spinal cord injury . Several studies in mouse models of spinal cord injury showed that EPI-NCSC grafts resulted in significant improvement in sensory connectivity and touch perception. These cells modulate scar formation by contributing to the vascularization and by producing multiple metalloproteases and other extracellular proteases that degrade different types of extracellular matrix molecules [24–28]. In this study, a highly pure population of EPI-NCSCs were obtained by virtue of their migratory ability through a minimally invasive procedure from the bulge of hair follicles. Isolated cells expressed both neural crest marker SOX10 and stem cell marker Nestin abundantly, which verifies their origin and multipotency.
Besides an appropriate cell type, the practicality of different routes of administration is another prominent factor in cell-based therapy. Cerebrospinal fluid, beyond its important role in the maintenance of extracellular ionic balance and providing a fluid cushion for the CNS, was recently implicated in carrying secreted proteins widely throughout life. Lately several studies have demonstrated the crucial role of CSF in neurogenesis at the brain-cerebrospinal fluid interface, regarding its various signaling factors [17, 20]. Therefore it is not considered just as a watery fluid that bathes the brain and spinal cord. Moreover, CSF, owing to its circulatory system, which is in close contact with different parts of the CNS, provides a practical way for EPI-NCSCs transplantation. Above all, CSF-constituent proteins can play an instructive role in fate determination of EPI-NCSCs, as longSAGE gene-expression profile of these cells earlier revealed, EPI-NCSCs express some relevant growth-factor receptors that can convey CSF signals inside the cells .
Our data from culturing EPI-NCSCs in CSF has indicated that these cells can survive in this environment, and the expression of their pertinent markers proceeds for at least 72 hours, adequate for delivering cells to different parts of brain and spinal cord under in vivo conditions. Interestingly, EPI-NCSCs express early lineage markers like β-tubulin ІІІ and GFAP, which demonstrate that these cells can differentiate into either a neuronal or a glial lineage. Moreover, our investigation disclosed that CSF provides a trophic environment for proliferation of isolated EPI-NCSCs. However, the proliferation rate of these cells in CSF was significantly lower than that of cells in primary explants and expansion medium. This acquired trait of EPI-NCSCs after their cultivation in CSF is an attractive feature in cell-based therapy, because tumorigenicity of stem cells is one of the main setbacks of this approach.
Furthermore, our data show that CSF not only decreases the proliferation of EPI-NCSCs but also does not promote their differentiation toward any specific destiny, because the expression of early lineage genes in this medium diminished comparison with the primary explant. This condition can be appropriate for transplanted cells because it allows cells to differentiate according to instructive signals of their prospective target site.
It is noteworthy that the behavior of EPI-NCSCs in the current investigation was studied after their cultivation in healthy CSF, and this result may vary in different pathologic conditions. However, previously, Bai and his colleagues [29, 30] showed that the injection of neural stem cells through the CSF is a practical method to graft cells into traumatic and diseased lesions of the spinal cord. Consistently, Satake et al. in 2004  reported that transplanted mesenchymal stem cells can survive after a lumbar CSF injection and migrate into a previously created thoracic spinal cord injury.
CSF, as a cocktail of growth factors, helps EPI-NCSCs to acquire some desirable traits. CSF, due to its circulatory system in close contact with different parts of the CNS, can be a practical route of administration for delivery of injected stem cells into various parts of the CNS. Further experiments are required to determine the fate, destiny, and behavior of EPI-NCSCs after their administration through CSF in spinal cord-injured animals or other models of neurodegenerative diseases.
Complementary deoxynucleic acid
Chick embryo extract
Central nervous system
Enzyme-linked immunosorbent assay
Epidermal neural crest stem cell
Fetal bovine serum
Fibroblast growth factor
Hank balanced salt solution
Hypoxanthine guanine phosphoribosyl transferase
Phosphate buffered saline
Quantitative real-time polymerase chain reaction
Reverse transcriptase polymerase chain reaction
α-modified Eagle medium.
The authors thank Mr. Hamid Bagheri for providing chick embryos, and all members of the Cellular and Molecular Biology Research Center of Babol University of Medical Sciences for their practical help.
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