Epidermal neural crest stem cell transplantation as a promising therapeutic strategy for ischemic stroke

Abstract Introduction Cell‐based therapy is considered as promising strategy to cure stroke. However, employing appropriate type of stem cell to fulfill many therapeutic needs of cerebral ischemia is still challenging. In this regard, the current study was designed to elucidate therapeutic potential of epidermal neural crest stem cells (EPI‐NCSCs) compared to bone marrow mesenchymal stem cells (BM‐MSCs) in rat model of ischemic stroke. Methods Ischemic stroke was induced by middle cerebral artery occlusion (MCAO) for 45 minutes. Immediately after reperfusion, EPI‐NCSCs or BM‐MSCs were transplanted via intra‐arterial or intravenous route. A test for neurological function was performed before ischemia and 1, 3, and 7 days after MCAO. Also, infarct volume ratio and relative expression of 15 selected target genes were evaluated 7 days after transplantation. Results EPI‐NCSCs transplantation (both intra‐arterial and intravenous) and BM‐MSCs transplantation (only intra‐arterial) tended to result in a better functional outcome, compared to the MCAO group; however, this difference was not statistically significant. The infarct volume ratio significantly decreased in NCSC‐intra‐arterial, NCSC‐intravenous and MSC‐intra‐arterial groups compared to the control. EPI‐NCSCs interventions led to higher expression levels of Bdnf, nestin, Sox10, doublecortin, β‐III tubulin, Gfap, and interleukin‐6, whereas neurotrophin‐3 and interleukin‐10 were decreased. On the other hand, BM‐MSCs therapy resulted in upregulation of Gdnf, β‐III tubulin, and Gfap and down‐regulation of neurotrophin‐3, interleukin‐1, and interleukin‐10. Conclusion These findings highlight the therapeutic effects of EPI‐NCSCs transplantation, probably through simultaneous induction of neuronal and glial formation, as well as Bdnf over‐expression in a rat model of ischemic stroke.


| INTRODUC TI ON
Globally, stroke is among the main causes of death and disability.
Although thrombolysis and mechanical thrombectomy have revolutionized the treatment of ischemic stroke, the issues of availability, narrow time windows, risk of hemorrhage, and treatment failure are serious drawbacks. [1][2][3][4] Hence, seeking new alternatives to treat ischemic stroke in order to ameliorate neurological function and reduce mortality is of paramount necessity. Stem cell-based therapies have the potential to induce angiogenesis, neurogenesis, and synaptic plasticity, and represent a novel and promising regenerative strategy. In this regard, various types of stem cells, including embryonic, neural, induced pluripotent, and mesenchymal stem cells (MSCs), as well as endothelial progenitor and vascular progenitor cells, have been employed and their curative potentials have been evaluated in the treatment of ischemic stroke. 5 Among them, bone marrow-derived MSCs (BM-MSCs) are the most commonly used MSCs, due to their safety, weak immunogenicity, and easyto-culture capabilities. 6 Several lines of evidence indicate that BM-MSCs affect the pathological processes underlying ischemic stroke through multiple mechanisms, including inhibition of apoptosis, secreting neurotrophic factors, inducing angiogenesis, and modulating the immune system. 7 However, bone marrow aspiration is a highly invasive procedure, causing severe pain at the harvesting site. This procedure-associated pain is considered as one of the major limitations of intraoperative stem cell therapy approaches 8 ; hence, alternative sources from which to isolate autologous stem cells should be considered.
Epidermal neural crest stem cells (EPI-NCSCs) are remnants of the embryonic neural crest, residing in the bulge of adult hair follicle. These cells, similar to their neural crest origin, can be differentiated into various cell types, such as neurons, 9 glial cells, 10 osteocytes, and melanocytes. 11 EPI-NCSCs were initially introduced in 2004, 12 with advantages such as high plasticity, abundancy, and accessibility through a minimal invasive procedure, as well as not having ethical issues and graft rejection. 13 Furthermore, EPI-NCSCs express a variety of neurotrophic factors, such as brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), glial cell-derived neurotrophic factor (GDNF), neurotrophin-3 (NT-3), as well as angiogenic factors such as vascular endothelial growth factor (VEGF) and extracellular proteases that have the capability of supporting cell survival and neo-vascularization. [14][15][16][17] In this regard, EPI-NCSCs might be promising donor cells in the treatment of ischemic stroke, as its beneficial effects have been reported in animals 18 and ex vivo 19 models of spinal cord injury, peripheral nerve injury, 13 as well as Alzheimer's disease. 20 Intracranial, intra-arterial, and intravenous application are three effective routes of postcerebral ischemia stem cell administration.
Intracranial transplantation is of more invasive nature and can do damage to healthy tissue, as a result of the direct application of cells.
Therefore, in the present study, the therapeutic effect of BM-MSCs and EPI-NCSCs following intra-arterial and intravenous administration was compared in a rat model of transient middle cerebral artery occlusion (MCAO). In this regard, neurological function was evaluated before ischemia and 1, 3, and 7 days after transplantation. In addition, infarct volume ratio and relative expression of 15 selected target genes in three categories of trophic factors, cellular markers, and inflammatory cytokines were evaluated 7 days after cell therapy.

| Animals and ethics statement
In the present study, 82 Sprague Dawley male rats weighing 240-260 g at the beginning of the experiment were used. All rats were housed under controlled conditions and allowed ad libitum access to standard food and water. This experiment was approved by the Animal Care Committee of Shiraz University of Medical Sciences, Shiraz, Iran, and was carried out in compliance with the recommendations of the Care and Use of Laboratory Animals (National Academy Press, 1996, Washington, USA).

| Experimental groups
Experimental animals were randomly divided into 6 groups: (a) sham

| Stem cell preparation
To isolate EPI-NCSCs, rat whiskers pad (n = 10) were micro-dissected to obtain individual hair follicles. After several washes, the capsule of follicles was cut longitudinally and the bulge region within the follicles rolled out. Bulges of hair follicles were explanted on collagencoated 12-well cell culture plates and fed with minimum essential medium-α (α-MEM, Sigma-Aldrich) contained 5% day-11 chick embryo extract, 10% fetal bovine serum (FBS, Gibco), and 1% penicillin/streptomycin (P/S, Gibco) and were incubated in a humidified atmosphere at 37°C with 5% CO 2 . Half of the culture medium was renewed every day, and 7 days after stem cell migration, cells were detached using 0.25% trypsin/EDTA (Gibco) and passaged. This procedure was described in detail in previous publications. 21,22 Verified rat BM-MSCs were purchased from the Iranian Biological Resource Center (Tehran, Iran) and expanded in high glucose Dulbecco's modified Eagle's medium (DMEM-HG) supplemented with 10% FBS and 1% P/S. Both BM-MSCs and EPI-NCSCs were isolated from 10-to 14-week old male Sprague Dawley rats and harvested at passage number 4-6 for grafting.

| MCAO procedure
Experimental rats were subjected to transient MCAO as it was described earlier. 23 In brief, animals were anesthetized with chloral hydrate (320 mg/kg, intraperitoneally) and following midline incision of the neck, right common carotid artery, right external carotid artery, and right pterygopalatine artery were ligated. A silicone rubber-coated monofilament (#403556, Doccol Corporation) was inserted into the right common carotid artery and advanced cranially to the internal carotid artery until a mild resistance felt in order to occlude the blood flow of the right middle cerebral artery. For reperfusion, the filament was carefully removed after 45 minutes. Laser Doppler was used to monitor microvascular blood flow reduction during the surgery. Also, during the surgical procedure, rectal temperature was monitored and maintained at 37°C using a heating lamp and heating pad.

| Transplantation approaches
In the intra-arterial groups, immediately after removing the suture, the common carotid artery ipsilateral to the MCAO was cannulated using PE 20 (Clay Adams lnc.), and 2 × 10 6 cells (BM-MSCs or EPI-NCSCs) in 0.5 mL PBS were injected directly into the artery over the course of 1 minute. For intravenous delivery, 2 × 10 6 cells were suspended in 0.5 mL PBS and injected into the tail vein immediately after suture withdrawal.

| Behavioral test
In all experimental groups, the behavioral test was performed before ischemia (day 0) and 1, 3 and 7 days after the surgery/stem cell transplantation. In doing so, neurological function was graded on a scale of 0-4 as follows, 0: no neurological deficit; 1: unable to fully extend left forepaw (mild); 2: leftward circling (moderate); 3: falling to the left (severe); and 4: minimal level of consciousness without spontaneous walking. 24 In all experimental groups, MCAO rats with scores 3 or 4 died within 2-7 days after the ischemia and were excluded from the experiment. Furthermore, MCAO rats with score 0 at day 1 were also excluded from the experiment.

| Measurement of infarct volume ratio by TTC (2,3,5-triphenyltetrazolium chloride) staining
Seven days after the surgery/stem cell transplantation, half of the animals in each experimental group were subjected to quantification of infarct volume ratio. Under deep anesthesia, rats were killed, brains were removed quickly and coronal sections with 2 mm thickness prepared. Then, brain sections were incubated for 30 minutes at 37°C in 1% TTC (Sigma) and infarct volume ratio was evaluated using ImageJ software. In the present study, relative expression of 15 genes in three categories was evaluated as follows: 1-trophic factors including BDNF, NGF, GDNF, NT-3, and VEGF; 2-cellular markers including nestin, SOX10, doublecortin (DCX), β-III tubulin, GFAP, β-actin, and 3-inflammatory cytokines including tumor necrosis factor-α (TNFα), interleukin (IL)-1β, IL-6, and IL-10. To evaluate target genes, qRT-PCR was performed using first-strand cDNA template, specific primer sets (presented in Table 1), and SYBR green Master Mix (RealQ Plus 2X, Ampliqon). All samples were run in triplicate.

| Evaluation of the target genes using qRT-PCR
Amplification conditions included 95°C for 15 minutes, and then, 40 cycles of 95°C for 20 seconds and 60°C for 1 minutes were performed on the Applied Biosystems StepOnePlus (ABI, USA).
Melting curve analysis revealed just one amplification peak for each reaction, while nontemplate as well as minus reverse transcriptase controls confirmed the absence of genomic contamination. The Ct value for each target gene was normalized to the Ct of hypoxanthine phosphoribosyltransferase-1 (HPRT1) transcript as a suitable housekeeping gene, according to the previous reports in the in vivo model of MCAO. 25,26 In addition, 5 μL of amplified products was subjected to electrophoresis on a 1% agarose gel to observe a single band of the expected size. The arithmetic formula 2 −ΔΔCT was used to calculate fold changes. 27

| Verification of EPI-NCSCs
In the current investigation, 2-3 days after explantation, the migrated cells were observed around the bulges, which increased over time.
Immunostaining against nestin, SOX10, DCX, β-III tubulin, and GFAP revealed the expression of these markers that verified the type of migrated cells as EPI-NCSCs ( Figure 1).

| Functional deficits
In the present study, neurological function was assessed before surgeries (day 0) and 1, 3, and 7 days postischemia/cell therapy. Before surgery, no deficits were observed in the experimental groups. At 1 and 3 days postsurgery, the MCAO group as well as all the stem cell transplantation groups exhibited significant functional deficits in comparison to the sham group. Seven days after transplantation, the intra-arterial administrations of EPI-NCSCs and BM-MSCs as well as intravenous administration of EPI-NCSCs led to a better functional outcome compared to the MCAO group; however, the differences were not statistically significant (Figure 2A).

| Infarct volume ratio
Seven days after surgery/stem cell transplantation, the infarct volume ratio was assessed by TTC staining ( Figure 2B). Here, the ipsilateral hemisphere was severely damaged in the MCAO group and MSC-IA (2.9 ± 0.22%) groups, but not in the MSC-IV (17 ± 2.3%) group, compared to the control ( Figure 2C).

| Relative expression of target genes
Seven days after surgery/stem cell transplantation, relative expres-  Figure 6.
F I G U R E 2 A, Neurological deficit before surgeries (day 0) and 1, 3, and 7 d postischemia/cell therapy. ** P < .01 (n = 12 in each experimental group); B, Representative photographs of coronal brain sections 7 days postischemia/cell therapy in six experimental groups stained with 2,3,5-triphenyltetrazolium chloride and C, Bar graph showing %infarct volumes in each group. ### P < .001 (only significant differences compared to MCAO group are pointed; n = 6 in each experimental group) In the present investigation, neurological deficits were assessed without functional outcomes has also been reported in drug-based therapy of cerebral ischemia. 35 Striatum and neocortex are two main brain regions that always affected by mild (30 minutes) MCAO. 36 Hence, we evaluated the F I G U R E 3 Relative expression of nerve growth factor (NGF), neurotrophin-3 (NT-3), brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), and vascular endothelial growth factor (VEGF) 7 d postischemia/ cell therapy in the striatum as well as cortex of six experimental groups. * P < .05, ** P < .01, *** P < .001 significant differences compared to sham group; ## P < .01, ### P < .001 significant differences Nestin is known as a neuronal progenitor cell marker in the adult brain, and it is well established that nestin-positive cells can ultimately differentiate into a variety of CNS cell types, including oligodendrocytes, astrocytes, and neurons. 41,42 Increased nestin expression following ischemic stroke was reported in several investigations, and it was suggested that nestin-positive cells induced by MCAO eventually shifted toward reactive astrocytes. 43,44 In line with these reports, our F I G U R E 4 Relative expression of nestin, SOX10, doublecortin (DCX), β-III tubulin, glial fibrillary acidic protein (GFAP), and β-actin 7 d postischemia/ cell therapy in the striatum as well as cortex of six experimental groups. ** P < .01, *** P < .001 significant differences compared to sham group; # P < .05, ## P < .01, ### P < .001 significant differences compared to MCAO group (n = 6 in each experimental group) results revealed the upregulation of Nestin and Gfap (as glial marker) after ischemia. This finding was highlighted in the striatum region after EPI-NCSCs transplantation, which might be one of the approaches that these stem cells employed to protect injured sites. In this regard, we showed previously that EPI-NCSCs grafting led to over-expression of GFAP in an ex vivo model of spinal cord injury, which ultimately ameliorated the devastating condition of damaged tissue. 19 Furthermore, it was suggested that SOX10 plays a crucial role to direct the fate of neural precursor cells toward the oligodendrocyte lineage. 45 Here, although cerebral ischemia did not affect Sox10 expression, EPI-NCSCs transplantation caused elevated expression of this transcript suggesting formation of different glial cells types.

| D ISCUSS I ON
DCX is widely considered as a marker of neurogenesis as well as neuronal precursor cells. A positive correlation between DCX F I G U R E 5 Relative expression of tumor necrosis factor-α (TNFα), interleukin (IL)-1β, IL-6, and IL-10 7 d postischemia/cell therapy in the striatum as well as cortex of six experimental groups. * P < .05, ** P < .01, *** P < .001 significant differences compared to sham group; # P < .05, ## P < .01, ### P < .001 significant differences compared to MCAO group (n = 6 in each experimental group) expression and the extent of adult neurogenesis has been demonstrated previously. 46,47 Moreover, it was reported that DCX expression in the lesioned brain area following stroke correlates with the recovery of functional deficits. 48 Transgenic ablation of DCX resulted in exacerbation of stroke outcome and attenuation motor function recovery. 49,50 Interestingly, our findings of upregulated Dcx expression in the brain following EPI-NCSCs transplantation can be due to the expression of this marker by EPI-NCSCs and/or enhanced levels of endogenous DCX expression as a result of stem cell grafting. Here, both scenarios might eventually improve the function of the injured area. Remarkably, in the current investigation, we have found that β-III tubulin (as an immature neuronal marker), Dcx, Nestin, Gfap, and Sox10 are upregulated in the EPI-NCSCs transplanted groups, suggesting the simultaneous induction of neuronal and glial formation. Lastly, although increased expression of inflammatory cytokines after ischemia was expected, 51 the pathways through which stem cells modulate inflammatory processes following stroke require further investigation. 52 One of the main issues in cell transplantation for cerebral ischemia is the selection of suitable types of stem cells. It has been proposed that the ideal cell should have the ability to proliferate and expand ex vivo from the minimal numbers of donor cells. Also, cell transplants should be phenotypically plastic, bear minimal risk of rejection, be free of ethical controversies, and possess the ability to differentiate into appropriate neural and glial cells. 53 In this regard, EPI-NCSCs offer several advantages: They possess a high degree of plasticity, generate all major neural crest derivatives, can be isolated as a highly pure population, are abundant and easily accessible, can be expanded in vitro into millions of cells, do not raise ethical concerns, and last but not least show absence of tumorigenicity. 14,21,54 It is important to note that stroke mostly occurs in elderly people, and it is known that BM-MSC populations, as well as their differentiation and/or proliferation capacity, dramatically decline with age. 6,55 To the contrary, recent reports revealed that NCSCs from human epidermis of aged donors maintain their multipotency in vitro and in vivo. 56 Therefor, EPI-NCSCs, due to abundance and easy accessibility in the hairy skin, might be a good candidate for elderly patients.
One critical limitation of EPI-NCSC therapy for cerebral ischemia is lack of sufficient information regarding their mechanism of action. However, potential mechanisms might be growth factor secretion, synaptogenesis, angiogenesis, neurogenesis, normalizing metabolic/microenvironmental profiles, enhanced autophagy, reduced scar thickness, immunomodulation, neural circuit reconstruction, apoptosis inhibition, and possibly replacing damaged cells. 7,57,58 F I G U R E 6 Heat map representation of all evaluated target genes expression in the striatum as well as cortex Nevertheless, further investigations are required to clarify the exact mechanism involved. Additionally, therapeutic effects of EPI-NCSCs should be assessed in aging rats to mimic the conditions of elderly stroke patients.

| CON CLUS ION
In summary, the present findings suggest the therapeutic potential of EPI-NCSCs in a rat model of ischemic stroke induced by middle cerebral artery occlusion. We also found that administration of EPI-NCSCs via IA or IV routes immediately after reperfusion had created a comparable outcome to MSC-IA, 7 days after transplantation.

ACK N OWLED G M ENT
Authors wish to thank Mr H. Argasi at the Research Consultation Center (RCC) of Shiraz University of Medical Sciences for his invaluable assistance in editing this manuscript.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.