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First published online February 28, 2008
Stem Cells Vol. 26 No. 5 May 2008, pp. 1356 -1365
doi:10.1634/stemcells.2007-0333; www.StemCells.com
© 2008 AlphaMed Press

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TRANSLATIONAL AND CLINICAL RESEARCH

Transplantation of Embryonic Stem Cells Improves Nerve Repair and Functional Recovery After Severe Sciatic Nerve Axotomy in Rats

Lin Cuia,c, Jun Jianga,c, Ling Weia,d, Xin Zhoub, Jamie L. Frasera, B. Joy Sniderd, Shan Ping Yua,b

aDepartment of Pathology and
bDepartment of Pharmaceutical and Biomedical Sciences, Medical University of South Carolina, Charleston, South Carolina, USA,
cDepartment of Cardiology, Second Affiliated Hospital, Zhejiang University College of Medicine, Hangzhou, 310016, China,
dDepartment of Neurology and Hope Center for Neurological Disorders, Washington University School of Medicine, St. Louis, Missouri, USA

Key Words. Sciatic nerve axotomy • Embryonic stem cell • Axonal regeneration • Myelination • Growth factors

Correspondence: Correspondence: Shan Ping Yu, M.D., Ph.D., Department of Pharmaceutical Sciences, Medical University of South Carolina, Charleston, South Carolina 29425, USA; Telephone: 843-792-2992; Fax: 843-792-1712; e-mail: yusp{at}musc.edu

Received on May 3, 2007; accepted for publication on January 28, 2008.

First published online in STEM CELLS EXPRESS  February 28, 2008.

   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Extensive research has focused on transplantation of pluripotent stem cells for the treatment of central nervous system disorders, the therapeutic potential of stem cell therapy for injured peripheral nerves is largely unknown. We used a rat sciatic nerve transection model to test the ability of implanted embryonic stem (ES) cell-derived neural progenitor cells (ES-NPCs) in promoting repair of a severely injured peripheral nerve. Mouse ES cells were neurally induced in vitro; enhanced expression and/or secretion of growth factors were detected in differentiating ES cells. One hour after removal of a 1-cm segment of the left sciatic nerve, ES-NPCs were implanted into the gap between the nerve stumps with the surrounding epineurium as a natural conduit. The transplantation resulted in substantial axonal regrowth and nerve repair, which were not seen in culture medium controls. One to 3 months after axotomy, co-immunostaining with the mouse neural cell membrane specific antibody M2/M6 and the Schwann cell marker S100 suggested that transplanted ES-NPCs had survived and differentiated into myelinating cells. Regenerated axons were myelinated and showed a uniform connection between proximal and distal stumps. Nerve stumps had near normal diameter with longitudinally oriented, densely packed Schwann cell-like phenotype. Fluoro-Gold retrogradely labeled neurons were found in the spinal cord (T12–13) and DRG (L4-L6), suggesting reconnection of axons across the transection. Electrophysiological recordings showed functional activity recovered across the injury gap. These data suggest that transplanted neurally induced ES cells differentiate into myelin-forming cells and provide a potential therapy for severely injured peripheral nerves.

Disclosure of potential conflicts of interest is found at the end of this article.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Transplantation of progenitor and stem cells into the central nervous system (CNS) after ischemic stroke and in a variety of neurodegenerative diseases has been under extensive investigation in recent years, based on the hypothesis that the implanted cells might ultimately replace lost neurons and non-neuronal cells and/or provide trophic support for the repair process [1–3]. On the other hand, few investigations have been done to explore the possibility of applying stem cell transplantation therapy for the treatment of peripheral nerve injury.

Peripheral nerve injuries are common and often result in incomplete or no functional recovery, particularly after a complete transection [4]. Functional recovery usually requires guided axonal regeneration into the growth environment of the distal nerve stump [5]. In many cases, however, the distance between the proximal and distal stumps is too large to allow direct approximation of transected nerves. For such severe injuries, the nerve autograft remains the gold standard for treatment, but use of autografts is accompanied by donor-site morbidity such as denervation distal to the donor site, scarring, and neuroma formation [6]. Furthermore, there are a limited number of suitable sites available for nerve harvesting [7]. Allogenic nerve graft with immunosuppression is another choice; however, outcomes with the use of allografts are not as good as those obtained with autografts. As an alternative to autologous grafting, efforts have been made to develop bioartificial nerve grafts [8]. A conduit, support cells, neurotrophic factors, and extracellular matrices are four important elements of a bioengineered nerve. Although both natural and synthetic materials have been used to make conduits, it appears clear that acellular conduits failed to facilitate the nerve regeneration across long gaps [7].

Even though the exact underlying mechanisms regulating axon-Schwann cell interactions are not well understood, current data support the concept that Schwann cells offer a highly preferred substrate for axon migration and release neurotrophic factors that further enhance nerve regeneration. Schwann cells transplanted into transected nerves can survive and promote axonal regeneration [9]. The limited supply of Schwann cells, however, hinders clinical utilization of these cells. Embryonic stem (ES) cells can be maintained in vitro without any apparent loss of differentiation potential [10, 11]. These pluripotent cells can differentiate into virtually any cell lineage, including germ line, neurons, and non-neuronal nervous system cells [12–14]. Compared with Schwann cells, ES cells proliferate actively; the doubling times of human and mouse ES cells are only 30–35 hours and 12–15 hours, respectively [15]. We have demonstrated that neurally induced ES cell transplantation promotes remyelination, tissue repair, and functional recovery in a rodent model of cerebral ischemia [3]. In the present investigation, we explored the possibility that transplantation of ES cell-derived neural progenitor cells (ES-NPCs) into the severely injured peripheral nerve could promote axonal regeneration, remyelination, and functional recovery. The regenerating effects of transplanted ES-NPCs in the sciatic nerve model are comparable with previous studies of transplantation of bone marrow stromal cells into the injured sciatic nerve [16].


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Animal Model of Severe Sciatic Nerve Injury
Adult Long Evans rats (300–350 g) were anesthetized with intraperitoneal injection of 4% chloral hydrate. The left sciatic nerve was exposed via a 3-cm skin incision, and two tiny cuts with 1 cm distance were made in epineurium at the mid-thigh level. The sciatic nerve was then truncated underneath the cuts and the isolated 1-cm nerve segment was removed through one of the cut windows. After the nerve axotomy, the two cuts on epineurium were closed with 10–0 microsurgical sutures. The surgery wound was sutured after ES cell transplantation (see below and the Supplemental Figure). Animals had free access to food and water after recovery from anesthesia. All rats were kept in air-ventilated cages with room temperature maintained at 21 ± 0.5°C and a lighting condition of 12 hour light/dark cycle. Surgery and animal care were performed in compliance with institutional guidelines.

Embryonic Stem Cell Cultures and Neural Induction
Mouse ES cells were prepared from stocks of the wild-type D3 ES cell line. Cells were grown and differentiated as previously described [17, 18]. Briefly, undifferentiated cells were maintained in T25 flasks in ES cell growth media (ESGM) consisting of Dulbecco's modified eagle media (with L-glutamine and without pyruvate), supplemented with 10% fetal bovine serum, 10% newborn calf serum, 8 µg/ml adenosine, 8.5 µg/ml guanosine, 7.3 µg/ml cytidine, 7.3 µg/ml uridine, 2.4 µg/ml thymidine, leukemia inhibitory factor (LIF) at 1,000 units/ml, and 10–4 M β-mercaptoethanol. For induction of neural differentiation, cells were harvested from the growth flasks by trypsinization with 0.25% trypsin and EDTA in Hank's salts solution (Gibco, Carlsbad, CA, http://www.invitrogen.com) for 10 minutes. One quarter of the cells from a T25 flask were seeded into a standard 100 mm bacterial Petri dish in ESGM lacking LIF and β-mercaptoethanol (ESIM). After 2 days, the media and cell aggregates were removed from the dish and the aggregated cells were allowed to settle for 10 minute in a 15-ml centrifuge tube; the media were aspirated and replaced with fresh media. Cells were then returned to the culture dish for an additional 2 days. The culture media were then replaced with ESIM containing 5 x 10–7 M retinoic acid (RA) (all-trans retinoic acid, Sigma, St. Louis, http://www.sigmaaldrich.com) and the cells were cultured for an additional 4 days. Cells treated by this method, designated the "4–/4+" procedure, were ready for transplantation into axotomized sciatic nerve. After the 4–/4+ protocol, selected ES-NPC cultures were checked for viability with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay [19] and by trypan blue exclusion.

ES-NPC Transplantation
ES-NPC transplantation and medium injection control were performed 1 hour after the onset of axotomy. Cell density in transplantation solutions was adjusted to ~25,000 viable cells per µl. The relatively low cell density was selected based on our previous investigations on ES-NPC transplantation after ischemic stroke, which was sufficient for tissue repair in the CNS [3]. Neurally induced ES cells were injected into the 1-cm gap between the sciatic nerve stumps, the surrounding epineurium functioned as a natural conduit for transplants (see supplemental online Fig. 1). Control rats received injections of the culture medium. Each animal received a total injection of 3 µl transplantation solution. The solution was injected at a pace of 1 µl/minutes using a glass pipette with a tip diameter of ~80 µm mated to a 5-µl Hamilton syringe (Hamilton Company, Reno, NV, http://www.hamiltoncompany.com). A 2-minute waiting period allowed the ES cells to settle before needle removal. Vehicle was injected in a similar manner as ES cells. To suppress rejection of the mouse ES cells, rats in both groups received cyclosporine A (15 mg/kg/day, s.c.; Sandoz Pharmaceutical Corp., Princeton, NJ, http://www.sandoz.com) diluted in extra virgin oil, starting with a double-dose 1-day before surgery. Ten days after transplantation, the dosage was reduced to 10 mg/kg/day until the day before sacrifice.

Electrophysiological Recording
Electrophysiological evaluation (evoked action potential of the sciatic nerve) was performed before, immediately after, and 90 days after the sciatic axotomy and ES cell transplantation. Under anesthesia, the rat's left sciatic nerve and fourth digital nerve were exposed. Bipolar hooked platinum recording and stimulating electrodes were used to induce and record electrical activity. The stimulating electrode was placed under the proximal sciatic nerve and the recording electrode was placed under the fourth digital nerve. The evoked action potential in responding to the stimuli (one ms, 500 mV) in the ipsilateral sciatic nerve was recorded using Powerlab-800 system (AD Instruments, Colorado Springs, CO, http://www.adinstrumentsinc.com).

Retrograde Tracing After Sciatic Axotomy
Eighty-three days after the sciatic nerve axotomy, animals were anesthetized and 1 µl of 4% Fluoro-Gold (Molecular Probes, Carlsbad, CA) in deionized-distilled water was infused slowly using a Hamilton syringe over 10 minute into the ipsilateral sciatic nerve 1 cm away from the distal suture [20]. Seven days after Fluoro-Gold injection, rats were perfused and fixed with 10% formalin for immunohistochemical examination.

Histological Evaluation
Morphological assessments were performed different days after sciatic nerve transection. The rats were perfused and fixed with 10% formalin, and segments of the sciatic nerve were cut into 10-µm sections and examined by hematoxylin and eosin (H&E) stain and immunohistochemistry using antibodies against the myelin protein and neurofilament (Chemicon International, Inc., Temecula, CA, http://www.millipore.com). The dorsal root ganglia (DRG) and spinal cord (T10–13) were transected and examined by immunohistochemistry and retrograde labeling under light microscopy. Fresh frozen sections of 10-µm thickness were cut on a cryostat microtome (Vibratome 5,040, MetriCore, Inc., St. Louis, http://www.vibratome.com) at –18°C. Primary antibodies were applied overnight at room temperature. Sections were washed 3–5 minute in phosphate buffered solution (PBS) and then incubated with a cross-adsorbed secondary antibody for 45 minute at room temperature. Sections were washed 3–5 minute in PBS before coverslips were applied with Vectashield for immunofluorescence under a fluorescence microscope (Eclipse, TE2000-S, Nikon, Tokyo, http://www.nikon.com). Fluorescein-5-isothiocyanate (FITC)-conjugated anti-mouse IgG (green color in RGB channels) or Cy3-conjugated anti-mouse IgG antibodies (red color in RGB channels; Molecular Probes, Eugene, OR, http://www.probes.invitrogen.com) were used as secondary antibodies. For immunofluorescence double-labeled coronal sections, FITC and Cy3 fluorochromes on the sections were excited at 488 and 647 nm. Emissions were acquired sequentially with two separate photomultiplier tubes at 522 and 680 nm, respectively. Confocal microscopy was performed using a Zeiss LSM5 PASCAL laser scanning confocal microscope equipped with a plan-Apochromat 63 X/1.4 oil DIC objective (Carl Zeiss, Gottingen, Germany http://www.zeiss.com). Images were scanned at scales of 0.02 µm (X), and 0.4 µm (Z), with the pinhole size of 106 µm (channel one) and 158 µm (channel 2). Fluorescence data were collected using LP560 (channel one) and BP 505–550 (channel two) filters and the PASCAL software (Carl Zeiss). Off-line analysis was performed with Adobe Photoshop CS (Adobe Systems, Mountain View, CA, http://www.adobe.com).

Enzyme-Linked Immunosorbent Assay
Enzyme-Linked Immunosorbent Assay (ELISA) was performed on the medium collected from ES cell cultures after RA-induction, using a commercial brain-derived neurotrophic factor (BDNF) Sandwich ELISA kit (Chemicon). According to manufacturer's protocols, cell culture medium was collected 48 hours after media exchange. The culture medium was frozen at –80°C until the time of assessment. Medium was thawed on ice, diluted by 1:2, and then serial 1:2 dilutions were made. Dilutions of media were added to the ELISA plate in duplicate, along with a BDNF standard. The medium was incubated on the plate at 4°C overnight on a shaker. The following day, the plate was washed with the wash buffer provided, the detection antibody, biotinylated monoclonal mouse anti-BDNF, was applied for 3 hours at room temperature on a shaker. The plate was then washed and streptavidin-HRP conjugate was applied to each well. The plate was incubated at room temperature on a shaker for 1 hour and then washed again. Tetramethylbenzidine was applied to each well and incubated for 15 minutes. A hydrochloric acid-based stop solution was added, the plate developed, and the plate was read on a plate reader at 450 nm. Using the BDNF standard, regression analysis was used to calculate the equation for the regression line: y = 0.0026x + 0.0635; R2 = 0.9829. The sample dilutions were subjected to regression analysis to determine the mathematically derived optical density of BNDF in undiluted medium. The ODs were then applied to the regression equation for the BDNF standard and the actual concentration of BDNF for each condition was determined.

Statistics Analysis
Student's two-tailed t-test was used for comparison of two experimental groups. Multiple comparisons were done using one-way ANOVA followed by Tukey's test for multiple pairwise examinations. Changes were identified as significant if p was less than 0.05. Mean values were reported together with the standard error of mean (SEM).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
ES Cell Neural Induction and Trophic Factor Expression
ES cells were differentiated into ES-NPCs, using the 4–/4+ protocol (4 days of LIF withdrawal followed by 4 days in RA). This protocol has been used to prepare ES cells for transplantation into the CNS [3, 13, 21, 22]. One day after 4–/4+ differentiation, differentiating cells were positive to the DNA-binding transcription factor AP2, which was consistent with previous reports that AP2 expressed in RA-induced neurons and glial cells [23]. As shown before [21], many of these cells were also labeled by oligodendrocyte marker O4 and glial cell marker GFAP (Fig. 1A). By 3 days after RA induction, 35–38% of total cells express O1 and O4, suggestive of progression toward oligodendrocyte/Schwann cell lineage. Interestingly, some differentiating ES cells co-stained with both the neuronal marker NeuN and the glia/Schwann cell marker S100, implying their potential flexibility in differentiating into neuronal or non-neuronal cells (Fig. 1B).


Figure 1
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  		Figure 1. Phenotype characterization of neurally differentiated ES cells. (A): Fluorescent images of neurally induced ES cells one day after the 4–/4+ RA protocol. (Aa–Ac) show Hoechst (blue), AP-2 (green) and the merge image of these two immunostaining, respectively. (Ad–Ag) show Hoechst alone, double staining of O4 (green)/Hoechst, GFAP (red)/Hoechst, and triple staining of these three immunohistochemical labels, respectively. (B): Confocal imaging shows the cell phenotype 3 days after the RA induction. Images demonstrate double staining of NeuN (red) and S-100 (green) in ES-NPCs. NeuN positive cells (red) and S-100 positive cells (green) were revealed by immunostaining with specific antibodies (Ba, Bb). Overlay images (Bc, Bd) illustrate co-labeling of both NeuN and S-100 in the same cells, implying that the RA-induced ES cells may have the progenitor cell property to further differentiate into either neuronal or glia/Schwann cells. The co-staining of NeuN/S-100 was not observed among transplanted cells 1-month after transplantation. (Bd) is an enlargement of the dotted frame in (Bc). Bar in (A) represents 50 µm; bars in (B) equal to 40 µm (Ba, Bb) and 20 µm (Bc, Bd), respectively. Abbreviation: NeuN, neuronal nuclei.

 
Western blot showed that ES-NPCs expressed several trophic factors including GDNF, NGF, FGF, BDNF and angiopoietin-1 (Ang1) (Fig. 2), that have been reported to support cell survival, axonal regeneration and remyelination [24]. Using ELISA assay, we identified the BDNF concentration secreted from ES-NPCs into the culture medium at different days after RA induction (Fig. 2).


Figure 2
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  		Figure 2. Expression of growth factors in ES-NPCs. (A): Increased expression of FGF-2 and GDNF in differentiating ES cells. Western blot analysis was performed 1–5 days after the end of RA induction. (B): Expression of several trophic and angiogenic factors in ES-NPCs. Western blot showed expression of tested genes in ES cells at the last day (day 8) of 4–/4+ inducing protocol. n = 3 assays for (A) and (B). (C): Different days after RA induction, significant amount of BDNF released from differentiating ES cells was detected by ELISA in the culture media. n = 4 assays. Abbreviations: BDNF, brain-derived neurotrophic factor; FGF-2, fibroblast growth factor-2; NGF, nerve growth factor.

 
Severe Sciatic Nerve Transection Injury and ES Cell Transplantation
Under anesthesia, the left sciatic nerve was exposed at the mid-thigh level and a 1 cm segment of the nerve was completely removed (see supplemental online Fig. 1). The surrounding epineurium was then re-anastomosed, which provided a natural conduit for transplants. Neurally induced mouse ES-NPCs were implanted into the epineurium conduit 1 hour after the nerve axotomy. Culture medium injected into the conduit served as a control. A sham control group was performed with opening and closing the epineurium but without the actual axotomy. In our experiments up to 3 months, no tumor growth or unusual tissue formation was detected in ES-NPC transplanted rats.

Axonal Regeneration and Remyelination After Sciatic Nerve Axotomy
Three months after nerve axotomy, gross examination showed striking differences between rats that received vehicle injection (cultured medium) and those that received ES cell transplantation. Shrunken nerve diameter of the damaged segment implied lack of nerve regeneration (Fig. 3A), which was verified by loss or deteriorated nerve fibers in immunohistochemical examination with neurofilaments and H&E (Fig. 3F and 3I). Axonal degeneration and demyelination in vehicle control rats were obvious (Fig. 3L). In ES-NPC transplantation group, the damaged sciatic nerve regenerated and the nerve diameter recovered to near normal level (Fig. 3B, 3D and 3G). Histological examination with H&E staining showed little repair in control rats that received culture medium, whereas significant nerve regeneration and apparent reconnection of the transected nerve was seen in rats that received ES-NPC implants (Fig. 3H–3J). Immunostaining with SMI-31, a marker for phosphorylated neurofilaments, and myelin showed that ES-NPC transplantation promoted extensive axonal regeneration and nerve repair (Fig. 3K–3M). The presence of cells that stain for both the mouse cell specific marker M2/M6 and the glia/Schwann cell marker S100 support the idea that transplanted mouse ES-NPCs may differentiate into Schwann-like cells and myelinated the regenerated axons (Fig. 4). These cells survived in vivo for at least 3 months (Fig. 4E–4G) and the double staining of S100/NeuN was no longer observed at this time. The number of cells co-labeled with M2/M6 and S100 increased 1–3 months after ES-NPC transplantation (Fig. 4H). Confocal imaging of the sciatic nerve 3 months after injury confirmed that rats that received ES-NPC transplantation showed increased number of myelinated axons (Fig. 5). The nerve regeneration was also examined in a separate experiment as the percentage of myelinated axons (number of meylinated axons at the midconduit of the ipsilateral nerve/number of meylinated axons at a corresponding location of the contralateral nerve). The percentage was 64 ± 15% in rats received ES-NPC transplantation whereas the rats received medium control only had a percentage of 7 ± 3% (p < .05, n = 3 per group).


Figure 3
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  		Figure 3. Sciatic nerve damage, regeneration, remyelination and effects of ES-NPC transplantation. (A–C): Gross anatomy of the sciatic nerve after axotomy and removal of 1 cm nerve segment. (A): shows that, 3 months after axotomy, a significant decrease in the diameter of the sciatic nerve in rats received culture medium. (B): demonstrates the repaired, normal looking sciatic nerve in the rat 3 months after axotomy and ES-NPC transplantation. Photos were taken from anesthetized rats; they are representative for at least five rats per group. (C): shows normal (non-transected) sciatic nerve (left) in comparison with the sciatic nerve 3 months after axotomy plus ES-NPC transplantation (right). Photos were taken after the nerves were dissected from the animals. (D): Quantification of sciatic nerve diameter at the proximal stump site 1 and 3 months after axotomy and ES-NPC transplantation. In rats received ES-NPCs, nerve diameter recovered to the normal levels whereasmedium injection alone showed no such effect. n = 3 rats in sham control group, six in medium control group, and six in ES-NPC transplantation groups, respectively. p < .05 compared with sham controls. (E–G): Sciatic nerve regeneration and remyelination. Three months after sciatic nerve injury, nerve regeneration and remyelination were revealed by immunohistochemical double-staining with the neurofilament marker SMI-31 (green) and myelin (red, appeared as orange). (E): shows normal nerve after sham operation. (F): demonstrates the lack of regeneration of the transected nerve, showing scatted SMI-31 and myelin staining within deficient nerve gap. (G): shows a repaired sciatic nerve after nerve axotomy and ES-NPC transplantation. Continuous SMI-31 and myelin staining demonstrates vigorous axonal regeneration and remyelination across the transection gap. (H–J): H&E histology of sciatic nerve damage and the effect of ES-NPC transplantation. Three months after sciatic transection, H&E staining showed visible differences in the diameter and axonal density of the sciatic nerve in rats. Bar = 150 µm. (H): is a sciatic nerve from sham-operated animal. (I): shows a sciatic nerve from animal that underwent transection and injection with culture medium. (J): is an example of the sciatic nerve after axotomy plus ES-NPC transplantation, showing regenerating nerve fibers extending from the proximal stump (arrow). (K–M): Immunostaining with SMI-31 (green) and myelin (red, appeared as orange) at the midpoint of the sciatic nerve of sham control (K), medium control (L) and ES-NPC transplanted (M) rats, imaged at a high magnification. Bar = 100 µm. (K): shows parallel nerve fiber regrowth and remyelination three mounts after ES-NPC transplantation. (L): is an image showing no organized regeneration in the transection segment from the culture medium vehicle control rat. (M): was taken from the same transection segment of a rat received axotomy and ES-NPC transplantation 3 months ago. Abbreviation: ES-NPC, embryonic stem cell-derived neural progenitor cells.

 


Figure 4
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  		Figure 4. Transplanted ES-NPCs and their differentiation into Schwann-like cells Immunostaining with mouse cell specific antibody (M2/M6) showed mouse ES-NPCs 1-month after their transplantation into rats of sciatic nerve axotomy. (A): M2/M6 positive cells demonstrate transplanted ES-NPCs; these cells are seen within segment of sciatic nerve defect. (B): Staining of the glia/Schwann cell marker S-100 in the same section of the sciatic nerve. (C): Merged image of (A) and (B). (D): Similar to C but in higher magnification; from a different section of the sciatic nerve. (E–G): M2/M6 and S-100 staining in transplant location of the sciatic nerve 3 months after transplantation. The majority of M2/M6 positive cells were also S-100 positive (see overlapping image in G), indicating that transplanted ES-NPCs survived and differentiated into Schwann like cells. (H): Quantification of S100 and M2/M6 double stained cells. ES-NPC transplanted rats showed increasing number of double staining during the three months recovery. The bar graph shows the number of positive cells per microscopic field (~0.037 mm2). n = 6. Values shown are mean ± SEM. M2/M6 are the membrane surface monoclonal antibody specific for mouse CNS cells [39, 40]. Of note, M2 antigen expression on the cell membrane is age dependent and it may not express in oligodendrocytes express O4 antigen but not O1 antigen [40]. Thus the M2/M6 staining might overlook some cells that were lack of O1 antigen. Abbreviation: ES-NPC, embryonic stem cell-derived neural progenitor cells.

 


Figure 5
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  		Figure 5. Remyelination of sciatic nerve. Confocal imaging shows double labeling of the neurofilament marker SMI-31 (green) and myelin (red) in transverse section of sciatic nerves. (A): Immunohistochemical staining shows normally organized axons circled by myelin/Schwann cells in an uninjured nerve. Bar = 8 µm. (B): Three months after sciatic nerve transection, partial regeneration occurred from proximal stump in animals treated with cultured medium only. The image was taken ~0.2 cm beyond the transection site toward the distal direction. Many nerve fibers lacked the surrounding myelin. (C): In a similar location as in B, axonal regeneration and myelination was observed 3 months after injury and ES-NPC transplantation. Axonal diameter was close to the normal size, and nerve fibers were myelinated. (D): Quantification of myelinated axons of the sciatic nerve. One and 3 months after axotomy, myelinated axons remained scarce in rats received medium injection. ES-NPC transplantation significantly increased axonal myelination 3 months after axotomy. Shown are numbers of myelinated axons per field (~0.037 mm2). n = 3 for sham control, six for medium control, and six for ES cell transplantation groups, respectively. Values shown are mean ± SEM; p < .05 compared with medium control. Abbreviation: ES-NPC, embryonic stem cell-derived neural progenitor cells.

 
Re-Connection of the Transected Sciatic Nerve and Functional Restoration
Three months after nerve transection, we injected Fluoro-Gold (FG, 1 µl, 4%) into the sciatic nerve 1 cm distal to the transection site. Animals were sacrificed 7 days later and examined for retrograde staining of spinal cord (T12–13) and DRG (L4–L6) neurons to determine whether the observed axonal regeneration resulted in anatomical reconnection of the transected nerve. Whereas no staining was detected in animals treated with culture medium (Fig. 6A), in ES-NPC transplanted animals FG labeling was detected in spinal cord and DRG neurons (Fig. 6B–6D). Double staining with FG and neurofilament was seen in the middle point of the transected region of rats received ES-NPCs but not in rats received medium injection (Fig. 6E–6H). These findings suggest that in ES-NPC-transplanted animals transected axons extended from the proximal stump into the distal stump that provided the pathway for retrograde passage of FG.


Figure 6
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  		Figure 6. Retrograde labeling of spinal cord neurons Fluoro-Gold (FG) positive labeling in the spinal cord and ipsilateral side of L4 and L5 DRG neurons 3 months after axotomy and ES-NPC transplantation and 7 days after FG injection into the sciatic nerve. The injection location was 1 cm further away from the distal suture. From this location the retrograde tracer should selectively label motor neuron axons and motor neurons in the spinal cord. (A): Medium control. (B): Motor neurons labeled with FG (light blue) after ES cell transplantation. (C): Enlarged image of FG labeling (light blue). The arrow points to a FG-positive neuron. (D): FG positive labeling in the ipsilateral side of L4 and L5 DRG neurons at a lower magnification. (E–H): At midpoint of transected sciatic nerve, FG passed through regenerated axons shown as overlapping images of FG and NF (G, arrows). In culture medium control experiments shown in (H), the lack of NF and FG staining indicated the absence of axonal regrowth and FG passage in this region. Bar = 500 µm in (A) and (B), 50 µm in (C), 40 µm in (E–H). Representative of four experiments.

 
The evoked action potential across the transection gap was examined using in vivo electrophysiological recordings. The action potential propagation along the sciatic nerve virtually disappeared upon the nerve axotomy. Three months after injury, the amplitude of the evoked action potentials showed little recovery in culture medium treated rats, but was near pre-injury levels in rats that received ES-NPC transplantation (Fig. 7).


Figure 7
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  		Figure 7. Electrophysiological evaluation. Evoked action potentials were recorded before axotomy, immediately after axotomy and 90 days after injury in medium treated rats (A) and in rats treated with ES-NPC transplants (B). The initial spike was recorded before axotomy from the left fourth digital nerve upon stimulation of the ipsilateral sciatic nerve (one ms and 500 mV). The evoked action potential disappeared soon after the injury. (C): Amplitude of action potentials recorded at the fourth digital nerve before and immediately after the sciatic nerve axotomy. (D): Amplitude of action potentials recorded 3 months after axotomy in animals that underwent sham axotomy (sham control), or axotomy + medium only (medium control) or axotomy + ES-NPC transplantation. Values shown are mean ± SEM, n = 6. p < .05 compared with sham controls. p < .05 compared with medium controls. Abbreviation: ES-NPC, embryonic stem cell-derived neural progenitor cells.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 Acknowledgments
 References
 
Peripheral nerves have some capability for repair and regeneration after injury; extensive research has been done to understand and develop strategies to promote this regeneration process. However, the recovery from a severe nerve transection is usually far more difficult and the results are far from satisfactory [25]. The experiments presented here suggest that ES-NPC transplantation might be beneficial in promoting nerve repair after severe injuries. The sciatic nerve transection-excision animal model provides a feasible system for studying the nerve regeneration, remyelination, and functional alterations associated with severe peripheral never injury.

Schwann cells provide a structural and adhesive extracellular matrix as well as neurotrophic factors [4]. Both syngeneic and allogeneic Schwann cell transplantation have been shown to enhance peripheral nerve regeneration [9, 26]. However, it is difficult and time-consuming to obtain sufficient quantities of primary human Schwann cells for clinical utilization. Neuronal progenitor cells grafted in a peripheral nerve gap could differentiate into Schwann cell-like supportive cells [27]. ES cells are totipotent and proliferate quite actively. Their plasticity and differentiation are regulated by the microenvironment in which they reside. We recently demonstrated that neurally-differentiated mouse ES cells transplanted into the post-ischemic rat brain survived and differentiated into several cell types present in normal brain: neurons, astrocytes, oligodendrocytes, and endothelial cells [3]. The present study suggests that neurally induced ES cells can also differentiate into Schwann-like cells after implanted into damaged sciatic nerve. However, we noticed that regeneration of myelinated axons stimulated by transplanted ES-NPCs was not sufficient enough to restore the number of myelinated axons to the normal control levels. Further investigations on identifying more specific Schwann cell differentiation protocol, the optimal density of transplanted cells, and supplemental supports such as trophic factors are expected to improve the therapeutic effects of ES-NPC transplantation.

Axotomy of a peripheral nerve leads to degeneration of the distal nerve stump, a process referred to as Wallerian degeneration (WD). During WD Schwann cells respond to loss of axons by extrusion of their myelin sheaths, downregulation of myelin genes, dedifferentiation and proliferation [28, 29]. They finally express surface molecules that guide regenerating fibers. Hematogenous macrophages are rapidly recruited to the distal stump and remove the vast majority of myelin debris. Molecular changes in the distal stump include upregulation of neurotrophins, cell adhesion molecules, cytokines and their corresponding receptors [28]. In rat models of sciatic nerve axotomy, defects of 10 mm and smaller can be bridged and repaired by a non-biological material such as a silicone tube. However, it is quite common that functional recovery does not ensue unless transected nerves are surgically repaired to guide regenerating axons into the growth environment of the distal nerve stump [5]. Even then, surgical repair often fails to achieve full functional recovery, particularly if there is a long gap between the two stumps, that is, size of the defects >1 cm in rats or >3 cm in nonhuman primates [30]. This is likely because of the progressive decline in the ability of neurons to sustain axon growth, and chronic Schwann cell atrophy with subsequent failure to provide a supportive growth environment [31]. To promote axonal regeneration and remyelination, cell transplantation therapy using Schwann cells and neural progenitor cells has been tested with some success. For example, neuronal progenitor cells derived from the fetal rat hippocampus were embedded in collagen gel and this transplantation strategy largely repaired the sciatic nerve defect of a 15 mm gap, partly due to the implanted cell-derived Schwann cell-like supportive cells [27]. Our study using ES-NPCs now provides another source of neural progenitor cells that are abundantly available and show similar nerve regenerative capability.

Nerve autografts have been used as a positive control for comparison with experimental groups. We did not compare the effect of ES-NPC transplantation with that of autologous nerve grafting due to the concern that a severe transection model (removal of 1 cm nerve segment) was tested in the investigation and autografts were basically used for axotomy of much less severe (e.g., <10 mm deficit in rats). In this case, autograts might not serve as a suitable positive control. In stead, we used the culture medium as a negative control to verify the nerve repair ability of ES-NPC transplantation. We agree that a detailed comparison between autologous nerve grafting and ES-NPC transplantation will help to appreciate advantages and disadvantages of these two transplantation therapies.

In an investigation on the treatment of spinal cord injury, Liu and colleagues showed that using the 4–/4+ RA protocol, mouse ES cells could differentiate into neurons, astrocytes, and oligodendrocytes, and suggested that some ES cells might become myelinating cells [21]. The ES cell-derived myelin-forming cells provide the key rationale for using them in repairing damaged and demyelinated nerves. Interestingly and somehow surprisingly, some ES-NPCs were co-stained with both the neuronal marker NeuN and the glia/Schwann cell marker S100. It is likely that these cells would decide their phenotype destiny later according to the cues in microenvironment. Our data support this hypothesis. In a recent investigation, human fetal neural stem cell (hNSC) was transplanted to replace lost motoneurons in an animal model of chronic motoneuron deficiency [29]. In this study of relatively mild sciatic nerve axotomy, axons of hNSC-derived motoneurons in spinal ventral horns pass through ventral root and sciatic nerve to form neuromuscular junctions with their peripheral muscle targets. This new cholinergic innervation correlates with partial improvement of motor function [29]. The stunning capability of axonal growth of stem cell-derived neurons and establishment of neuronal transmission provide another model for repairing damaged spinal cord and sciatic nerve. Together, available data strongly endorse the therapeutic potential of stem cell transplantation in CNS and peripheral nerve injury.

In addition to cells supporting myelination, studies have shown that neurotrophins are absolutely required for peripheral axon outgrowth during development [32]. It is well known that expression of several neurotrophin, GDNF, and IGF family members is upregulated in distal peripheral nerve after injury [33]. A study showed that neurotrophin-4-impregnated fibronectin conduits significantly improved slow motor unit recovery [34]. Cultured embryonic cortical progenitor cells have also been shown to express BDNF and NT-3, which in turn regulate their own survival and differentiation [35]. In several investigations, it was noticed that factors in addition to differentiated Schwann cells must contribute significantly to the repair of sciatic nerve injury because only a small portion (~5%) of transplanted stem cells became Schwann cell-like cells [16, 27]. Delineation of the molecular and cellular mechanisms for effective nerve regeneration will be necessary and important for tissue repair in CNS and PNS systems.

A growing body of evidence supports the concept that inflammatory responses within the CNS promote the survival, proliferation, migration, and differentiation of transplanted oligodendrocyte progenitor cells [36, 37]. In the present investigation, the immunosuppression reagent cyclosporine A was administrated at relatively high doses before and after ES-NPC transplantation (15 mg/kg/day for 11 days followed by 10 mg/kg/day until sacrifice). Under this cyclosporine A treatment, inflammatory responses and its contribution to the sciatic nerve repair in our experiments are expected to be minimum. This proposition is supported by our observation that although both medium control and ES-NPC groups received the cyclosporine A treatment, only the rats received ES-NPC transplantation showed marked nerve regeneration.

The remarkable repair capability of transplanted ES cells signifies an exciting possibility for clinical application. A number of issues, however, remain to be resolved before the repair model can be transferred to clinical therapy. For instance, the sciatic nerve transection-excision model in our investigation uses the remaining epineurium as a natural scaffold. This model of epineural conduit presumably provides a better nourishing and guiding environment; however, nerve damage in clinical settings may not allow the utilization of epineurium as a guiding conduit. A previous study showed that an epineural tube placed into the 1 cm gaps of the transected sciatic nerve resulted in comparable functional recovery and nerve regeneration with autologous nerve transplantation [38]. Thus, the epineural conduit and a number of options of biodegradable and non-biodegradable materials may be used for supporting transplanted cells, accumulating neurotrophic factors, and providing extracellular matrix after peripheral nerve injury. Demonstration of the effectiveness of these materials in ES-NPC transplantation will be necessary and significantly increase clinical applications of the stem cell therapy for peripheral nerve injury. The exact fate of transplanted ES-NPCs and molecular/environmental mechanisms that control ES-NPC differentiation in vivo, specifically differentiating towards Schwann cell lineage, remain poorly defined. The long-term survival and intergradations of transplanted ES-NPCs with host neuronal and vascular networks should be fully elucidated at cellular and ultrastructural levels. Improvements of motor and sensory functions of sciatic nerve transected rats after ES-NPC transplantation need to be evaluated in long-term investigations. Functional side effects such as altered pain sensation should be tested, which can be an important issue in cell therapy of peripheral nerve injury. Human stem cells remain to be examined for their Schwann cell differentiation capacity in vitro and in vivo studies. Finally, it is necessary to exclude even remote possibility of graft associated side effects, such as, teratoma formation or formation of other inappropriate neural or non-neural tissues. To this end, no tumorigenesis occurred in the present investigation of up to 3 months after transplantation. We thus suggest that ES cell differentiation and ES-NPC transplantation should be further explored as a potential therapy and novel mechanism for the treatment of peripheral nerve injury.


   DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
References
 
This work was supported by grants from NIH (NS NS42236, SPY; NS37372, LW; NS 39,577, BJS) and AHA-Bugher foundation (0170063, LW, 017,0064, SPY). The work was also supported by the NIH grant C06 RR015455 from the Extramural Research Facilities Program of the National Center for Research Resources. The authors thank Adrian D. Sproul for his assistance in editing the manuscript. L.C. and J.J. contributed equally to this work.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 

  1. Dezawa M, Takahashi I, Esaki M et al. Sciatic nerve regeneration in rats induced by transplantation of in vitro differentiated bone-marrow stromal cells. Eur J Neurosci 2001;14:1771–1776.[CrossRef][Medline]

  2. Heine W, Conant K, Griffin JW et al. Transplanted neural stem cells promote axonal regeneration through chronically denervated peripheral nerves. Exp Neurol 2004;189:231–240.[CrossRef][Medline]

  3. Wei L, Cui L, Snider BJ et al. Transplantation of embryonic stem cells overexpressing Bcl-2 promotes functional recovery after transient cerebral ischemia. Neurobiol Dis 2005;19:183–193.[CrossRef][Medline]

  4. Evans GR. Peripheral nerve injury: a review and approach to tissue engineered constructs. Anat Rec 2001;263:396–404.[CrossRef][Medline]

  5. Kim DH, Connolly SE, Kline DG et al. Labeled Schwann cell transplants versus sural nerve grafts in nerve repair. J Neurosurg 1994;80:254–260.[Medline]

  6. Vanderhooft E. Functional outcomes of nerve grafts for the upper and lower extremities. Hand Clin 2000;16:93–104, ix.[Medline]

  7. Evans GR. Challenges to nerve regeneration. Semin Surg Oncol 2000;19:312–318.[CrossRef][Medline]

  8. Francel PC, Smith KS, Stevens FA et al. Regeneration of rat sciatic nerve across a LactoSorb bioresorbable conduit with interposed short-segment nerve grafts. J Neurosurg 2003;99:549–554.[Medline]

  9. Mosahebi A, Fuller P, Wiberg M et al. Effect of allogeneic Schwann cell transplantation on peripheral nerve regeneration. Exp Neurol 2002;173:213–223.[CrossRef][Medline]

  10. Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos. Nature 1981;292:154–156.[CrossRef][Medline]

  11. Martin GR. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci U S A 1981;78:7634–7638.[Abstract/Free Full Text]

  12. Bradley A, Evans M, Kaufman MH et al. Formation of germ-line chimaeras from embryo-derived teratocarcinoma cell lines. Nature 1984;309:255–256.[CrossRef][Medline]

  13. McDonald JW, Liu XZ, Qu Y et al. Transplanted embryonic stem cells survive, differentiate and promote recovery in injured rat spinal cord. Nat Med 1999;5:1410–1412.[CrossRef][Medline]

  14. Wobus AM, Boheler KR. Embryonic stem cells: prospects for developmental biology and cell therapy. Physiol Rev 2005;85:635–678.[Abstract/Free Full Text]

  15. Itskovitz-Eldor J, Schuldiner M, Karsenti D et al. Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers. Mol Med 2000;6:88–95.[Medline]

  16. Cuevas P, Carceller F, Dujovny M et al. Peripheral nerve regeneration by bone marrow stromal cells. Neurol Res 2002;24:634–638.[CrossRef][Medline]

  17. Bain G, Gottlieb DI. Neural cells derived by in vitro differentiation of P19 and embryonic stem cells. Perspect Dev Neurobiol 1998;5:175–178.[Medline]

  18. Bain G, Ray WJ, Yao M et al. Retinoic acid promotes neural and represses mesodermal gene expression in mouse embryonic stem cells in culture. Biochem Biophys Res Commun 1996;223:691–694.[CrossRef][Medline]

  19. Xu J, He L, Ahmed SH et al. Oxygen-glucose deprivation induces inducible nitric oxide synthase and nitrotyrosine expression in cerebral endothelial cells. Stroke 2000;31:1744–1751.[Abstract/Free Full Text]

  20. Puigdellivol-Sanchez A, Valero-Cabre A, Prats-Galino A et al. On the use of fast blue, fluoro-gold and diamidino yellow for retrograde tracing after peripheral nerve injury: uptake, fading, dye interactions, and toxicity. J Neurosci Methods 2002;115:115–127.[CrossRef][Medline]

  21. Liu S, Qu Y, Stewart TJ et al. Embryonic stem cells differentiate into oligodendrocytes and myelinate in culture and after spinal cord transplantation. Proc Natl Acad Sci U S A 2000;97:6126–6131.[Abstract/Free Full Text]

  22. Bain G, Kitchens D, Yao M et al. Embryonic stem cells express neuronal properties in vitro. Dev Biol 1995;168:342–357.[CrossRef][Medline]

  23. Philipp J, Mitchell PJ, Malipiero U et al. Cell type-specific regulation of expression of transcription factor AP-2 in neuroectodermal cells. Dev Biol 1994;165:602–614.[CrossRef][Medline]

  24. Frostick SP, Yin Q, Kemp GJ. Schwann cells, neurotrophic factors, and peripheral nerve regeneration. Microsurgery 1998;18:397–405.[CrossRef][Medline]

  25. Millest H. In: Gilbert A, Tubiana R, eds. Tendon, Nerve and Other Disorders. Peripheral nerve repair: techniques and results. Lxsondon, New York: Taylor & Francis, 2005:105-124.

  26. Guenard V, Kleitman N, Morrissey TK et al. Syngeneic Schwann cells derived from adult nerves seeded in semipermeable guidance channels enhance peripheral nerve regeneration. J Neurosci 1992;12:3310–3320.[Abstract]

  27. Murakami T, Fujimoto Y, Yasunaga Y et al. Transplanted neuronal progenitor cells in a peripheral nerve gap promote nerve repair. Brain Res 2003;974:17–24.[CrossRef][Medline]

  28. Stoll G, Muller HW. Nerve injury, axonal degeneration and neural regeneration: basic insights. Brain Pathol 1999;9:313–325.[Medline]

  29. Gao J, Coggeshall RE, Tarasenko YI et al. Human neural stem cell-derived cholinergic neurons innervate muscle in motoneuron deficient adult rats. J Neurosci 2005;131:257–262.[CrossRef]

  30. Heath CA, Rutkowski GE. The development of bioartificial nerve grafts for peripheral-nerve regeneration. Trends Biotechnol 1998;16:163–168.[CrossRef][Medline]

  31. Bush MS, Tonge DA, Woolf C et al. Expression of a developmentally regulated, phosphorylated isoform of microtubule-associated protein 1B in regenerating axons of the sciatic nerve. Neuroscience 1996;73:553–563.[CrossRef][Medline]

  32. Markus A, Zhong J, Snider WD. Raf and akt mediate distinct aspects of sensory axon growth. Neuron 2002;35:65–76.[CrossRef][Medline]

  33. Snider WD, Zhou FQ, Zhong J et al. Signaling the pathway to regeneration. Neuron 2002;35:13–16.[CrossRef][Medline]

  34. Simon M, Porter R, Brown R et al. Effect of NT-4 and BDNF delivery to damaged sciatic nerves on phenotypic recovery of fast and slow muscles fibres. Eur J Neurosci 2003;18:2460–2466.[CrossRef][Medline]

  35. Barnabe-Heider F, Miller FD. Endogenously produced neurotrophins regulate survival and differentiation of cortical progenitors via distinct signaling pathways. J Neurosci 2003;23:5149–5160.[Abstract/Free Full Text]

  36. Tourbah A, Linnington C, Bachelin C et al. Inflammation promotes survival and migration of the CG4 oligodendrocyte progenitors transplanted in the spinal cord of both inflammatory and demyelinated EAE rats. J Neurosci Res 1997;50:853–861.[CrossRef][Medline]

  37. Foote AK, Blakemore WF. Inflammation stimulates remyelination in areas of chronic demyelination. Brain 2005;128:528–539.[Abstract/Free Full Text]

  38. Karacaoglu E, Yuksel F, Peker F et al. Nerve regeneration through an epineurial sheath: its functional aspect compared with nerve and vein grafts. Microsurgery 2001;21:196–201.[Medline]

  39. Lund RD, Chang FL, Hankin MH et al. Use of a species-specific antibody for demonstrating mouse neurons transplanted to rat brains. Neurosci Lett 1985;61:221–226.[CrossRef][Medline]

  40. Lagenaur C, Schachner M. Monoclonal antibody (M2) to glial and neuronal cell surfaces. J Supramol Struct Cell Biochem 1981;15:335–346.[CrossRef][Medline]





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