A, Dissociated induced pluripotent stem cell (iPSC)–derived motor neurons were transplanted into the tibial branch of the distal sciatic nerve stump approximately 5 mm proximal to the sciatic trifurcation. B and C, One day after transplant, longitudinal and axial sections of tibial nerve demonstrate survival of implanted human iPSC motor nerve cell bodies, as labeled with anti-human nuclear antigen antibody (HuNu) shown in green. D-I, One week after transplant, longitudinal sections of tibial nerve from 2 separate specimens (D-F from one specimen and G-I from another) demonstrate that surviving iPSC motor neurons (labeled with human antimitochondrial antibody [HuMito] in D and with HuNu in G) begin to extend very short neurites (labeled with anti–β-tubulin III/Tuj1 antibody [Tuj1] in E and H). F, The area indicated by the arrowheads emphasizes the presence of HuMito with relative absence of Tuj1 staining. I, Colocalization is noted when using an alternative HuNu.
A-F, Two weeks after transplant, longitudinal sections of tibial nerve demonstrate surviving human iPSC-derived motor neurons labeled in green with anti-human nuclear antigen antibody (HuNu). These surviving neurons show obvious directed neurite outgrowth (labeled with anti–β-tubulin III/Tuj1 antibody [Tuj1] in red) toward muscle, which is not depicted but located further downstream of the neurite in this image. In addition to Tuj1, the surviving neurons were demonstrated to stain positively for anti-HB9 antibody, a transcription factor only expressed in committed motor neurons, confirming the successful motor neuron differentiation of the transplanted cells. G-I, Three weeks after transplant, surviving motor neurons are seen to extend progressively lengthening neurites.
A and B, At 4 weeks after transplant, 1 mouse demonstrated particularly robust neurite outgrowth (indicated in red by anti–β-tubulin III/Tuj1 antibody [Tuj1]) that measured up to 751 µm in length. A magnified view (original magnification ×40) of the white boxed area in part A is depicted in part B. C-H, At 6 weeks after transplant, surviving iPSC motor neuron cell bodies (indicated in red by anti-human nuclear antigen antibody [HuNu] in C) extend long neurites of up to 784 µm in length (indicated in green by Tuj1 in part D) down the tibial nerve. A magnified view (original magnification ×40) of the white boxed area in parts C, D, and E is seen in parts F, G, and H.
A, Six weeks after transplant, iPSC-derived motor neurons clearly demonstrated robust survival and growth at the site of injection and down to the triceps surae muscle. Anti-human nuclear antigen antibody (HuNu)–labeled (red) iPSC motor neuron cell bodies and anti–β-tubulin III/Tuj1 antibody (Tuj1)–labeled (green) neuronal processes are present diffusely within the tibial nerve and reach the tibial-triceps surae junction. B, Enlargement of the blue boxed area in part A. A small number of neurites were also shown to form neuromuscular junctions with the triceps surae muscle. Fluorescent rhodamine-conjugated α-bungarotoxin (α-BTX) was used to demarcate motor end plates to demonstrate synapse formation between the implanted motor neurons and the murine postsynaptic acetylcholine receptors. C-E, Enlargements of the white boxed area in part A.
A, After humane killing of the mice at 10 weeks after the intervention, the mass of the triceps surae muscle is significantly higher in the hind limb of mice that had received a cellular implant vs controls that received growth factor only. Mean values are expressed as a percentage of the contralateral hind limb, in which the sciatic nerve was not injured. Error bars indicate SD. Differences between mean values were determined using a 1-sided Wilcoxon rank sum test, with P < .05 considered as significant. B, Photograph of triceps surae muscle after human induced pluripotent stem cell (iPSC) motor neuron transplant. Normal hind limb muscle is shown on the left. Hind limb muscle dissected 10 weeks after iPSC motor neuron implant is shown on the right. C, Photograph of triceps surae muscle after negative control implant (growth factors and growth media). Normal hind limb muscle is shown on the left. Hind limb muscle dissected 10 weeks after growth factor and media injection is shown on the right.
eFigure. Histogram of the Longest Representative Neurite, Plotted by Time Since Cellular Transplant
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Pepper J, Wang TV, Hennes V, Sun SY, Ichida JK. Human Induced Pluripotent Stem Cell–Derived Motor Neuron Transplant for Neuromuscular Atrophy in a Mouse Model of Sciatic Nerve Injury. JAMA Facial Plast Surg. 2017;19(3):197–205. doi:10.1001/jamafacial.2016.1544
Are human induced pluripotent stem cells a potential source of replacement motor neurons in the setting of peripheral motor nerve injury?
In this experimental study, motor neurons derived from human induced pluripotent stem cells successfully engrafted and extended neurites to target denervated muscle in 13 immunodeficient mice with sciatic nerve injury. These motor neurons reduced denervation-induced muscular atrophy compared with negative controls.
Motor neurons derived from human induced pluripotent stem cells may have future use in the treatment of peripheral motor nerve injury, including facial paralysis.
Human motor neurons may be reliably derived from induced pluripotent stem cells (iPSCs). In vivo transplant studies of human iPSCs and their cellular derivatives are essential to gauging their clinical utility.
To determine whether human iPSC-derived motor neurons can engraft in an immunodeficient mouse model of sciatic nerve injury.
Design, Setting, and Subjects
This nonblinded interventional study with negative controls was performed at a biomedical research institute using an immunodeficient, transgenic mouse model. Induced pluripotent stem cell–derived motor neurons were cultured and differentiated. Cells were transplanted into 32 immunodeficient mice with sciatic nerve injury aged 6 to 15 weeks. Tissue analysis was performed at predetermined points after the mice were killed humanely. Animal experiments were performed from February 24, 2015, to May 2, 2016, and data were analyzed from April 7, 2015, to May 27, 2016.
Human iPSCs were used to derive motor neurons in vitro before transplant.
Main Outcomes and Measures
Evidence of engraftment based on immunohistochemical analysis (primary outcome measure); evidence of neurite outgrowth and neuromuscular junction formation (secondary outcome measure); therapeutic effect based on wet muscle mass preservation and/or electrophysiological evidence of nerve and muscle function (exploratory end point).
In 13 of the 32 mice undergoing the experiment, human iPSC-derived motor neurons successfully engrafted and extended neurites to target denervated muscle. Human iPSC-derived motor neurons reduced denervation-induced muscular atrophy (mean [SD] muscle mass preservation, 54.2% [4.0%]) compared with negative controls (mean [SD] muscle mass preservation, 33.4% [2.3%]) (P = .04). No electrophysiological evidence of muscle recovery was found.
Conclusions and Relevance
Human iPSC-derived motor neurons may have future use in the treatment of peripheral motor nerve injury, including facial paralysis.
Level of Evidence
Peripheral injury to motor nerves is surprisingly common, with worldwide estimates of as many as 1 million cases per annum.1-3 Surgical repair of an injured motor nerve is the preferred method of initial treatment; however, in many clinical scenarios, this is difficult or impossible. In many cases of motor nerve injury, the true extent of injury is unknown at the initial presentation. Clinicians often recommend an observation period of 6 months or more, during which time it is hoped that the motor axons will regenerate from the point of injury to reach denervated muscle.4,5 However, prolonged periods of denervation have a significant functional effect despite motor neuron regrowth.6-8 In the setting of facial paralysis, partial recovery can leave patients with significant functional deficits and psychosocial disability.9,10
In 2006 and 2007, Takashashi et al11,12 described the reprogramming of adult somatic cells (ie, skin fibroblasts) into induced pluripotent stem cells (iPSCs). Induced pluripotent stem cells retain their pluripotency for long periods in standard culture conditions and therefore offer access to an unlimited supply of donor cells that are derived from a nonembryonic tissue source. Induced pluripotent stem cells can be used to differentiate patient-specific cells of any lineage, which can in turn be used for cellular therapy or for in vitro modeling. In particular, notable successes producing motor neurons from many different iPSC lines have been achieved.13-16
The central hypothesis of this research was that human iPSCs may be used as a source of motor neurons that can sustain nerve and muscle after traumatic injury to a peripheral motor nerve. Indeed, recent studies17 have reported mouse iPSC-derived motor neurons surviving transplant and preserving neuromuscular function in mouse models of sciatic nerve injury. Although transplant of mouse iPSC-derived motor neurons into mouse recipients represents a landmark, important differences may exist between human and mouse iPSCs.18-20 Therefore, to evaluate the therapeutic potential of human iPSC-derived motor neurons, we attempted the differentiation and transplant of human motor neurons from a healthy iPSC control line into an immunodeficient mouse model of sciatic nerve injury.
We generated a control iPSC line used for motor neuron differentiation from a lymphoblastoid cell line (National Institute of Neurological and Communicative Disease and Stroke repository ND03231) using episomal vectors as previously described.21 Induced pluripotent stem cell–derived motor neurons were cultured and differentiated as previously described15,22 with small modifications. Overall, this protocol permits the accelerated differentiation of lateral motor column spinal motor neurons from iPSCs using a series of molecular cues that mimic the signaling pathways of normal embryologic development. Animal experiments were performed from February 24, 2015, to May 2, 2015, and were conducted in accordance with protocols and guidelines approved by the University of Southern California Institutional Animal Care and Use Committee.
Induced pluripotent stem cells were passaged in feeder-free conditions using growth factor basement membrane matrix (Geltrex; Thermo Fisher Scientific) with culture medium (mTeSR; Stem Cell Technologies). Cells were dissociated and then maintained as embryoid bodies. These small clumps of cells allow cell-to-cell contact. Medium changes were performed daily, and the colony was assessed via phase contrast microscopy until colony confluency was grossly 80% or greater. Cells were then passaged in cell detachment solution (Accutase; Sigma) with Rho-associated kinase inhibitor (Abcam) to promote cell survival and then resuspended in the stem cell media. Neuralization was begun on the following day (day 3) using dual activin receptor-like kinase inhibitors LDN193189 (Stemgent) in 100nM solution and SB435142 (Sigma) in 10µM solution. Neuralization of the embryoid bodies was continued until day 7. The media was transitioned from stem cell medium to a supplemented medium (KnockOut Serum Replacement; Thermo Fisher Scientific) on days 3 to 5 and then switched to neural induction medium on day 7. Neural induction medium included Dulbecco modified Eagle medium/nutrient mixture F-12 (Thermo Fisher), L-alanyl-L-glutamine (GlutaMax; Thermo Fisher Scientific), nonessential amino acids (MEM Non-Essential Amino Acids solution; Gibco), N2 supplement (Gibco), penicillin and streptomycin (Corning), retinoic acid (Sigma), and smoothened agonist (Cayman Chemical).
At days 21 to 28, cells were prepared for transplant and again assessed for general colony morphology. A sample of cells was passaged using papain enzyme (Worthington Biochemical) or mechanical trituration with a P200 pipette. Neural induction medium was used for transplant in all cases. Neural induction medium was supplemented with ciliary neurotrophic factor (20 µg/mL), brain-derived neurotrophic factor (20 µg/mL), and glial cell line–derived neurotrophic factor (10 µg/mL; R&D Systems) for transplant or replating of cells for in vitro study. For the parallel in vitro study, cells were cultured on a monolayer culture (Geltrex; Thermo Fisher Scientific) to assess in vitro growth after implantation and to ensure that cells remained viable during the course of in vivo implantation.
We used the NSG immunodeficient mouse strain NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (nonobese diabetic.severe combined immunodeficiency mutation of the PRKDC gene [NCBI Entrez Gene 19090] [protein kinase, DNA activated, catalytic peptide]/targeted mutation 1 [Warren J. Leonard] of the interleukin 2 receptor γ chain/SzJ), which is bred specifically to tolerate human stem cell xenograft transplant (Jackson Laboratories). Mice aged 6 to 15 weeks were deeply sedated using inhaled isoflurane anesthesia. Under sterile surgical conditions, the sciatic nerve was identified. Two 7-0 polypropylene sutures (Prolene; Ethicon) were placed approximately 5 mm from the sciatic notch, with subsequent sharp transection between the 2 ligatures. To minimize native axonal regeneration, the proximal nerve stump underwent high-temperature cautery and was reflected medially, then sutured to the paraspinal muscles. Wounds were closed with interrupted 6-0 polyglactin 910 (Vicryl; Ethicon) sutures. Buprenorphine hydrochloride was injected during recovery to provide analgesia.
Approximately 50 000 to 500 000 dissociated iPSC-derived motor neurons within 5 μL of medium were injected into the tibial branch of the sciatic nerve using an aseptic, custom 31-gauge syringe (Hamilton; Sigma-Aldrich). The injection site was approximately 5 mm proximal to the sciatic trifurcation. Surgical control animals underwent the same surgical procedure but received injection of 5 µL of neural induction media with identical concentrations of the growth factors listed above.
Data were analyzed from April 7, 2015, to May 27, 2016. At predetermined time points after implantation, animals were killed humanely. The triceps surae muscles and the attached tibial nerve were dissected free from soft-tissue attachments using a stereomicroscope. The triceps surae and tibial nerve were weighed and then immersion fixed in 4% paraformaldehyde for 2 hours, with cryoprotection using 30% sucrose for 24 to 72 hours. Longitudinal 20-µm sections were taken through the entire nerve-muscle preparation using a cryostat (Leica).
Primary antibodies consisted of anti-human nuclear antigen antibody (1:250; EMD Millipore) and human antimitochondrial antibody (1:100; EMD Millipore) to confirm graft survival, anti–β-tubulin III antibody (1:1000; Sigma) to visualize neurite extension from the transplanted neurons, anti-HB9 antibody (1:5; DSHB) to confirm successful differentiation of transplanted neurons into committed motor neurons, anti-NeuN antibody (1:100; EMD Millipore) to confirm successful differentiation of transplanted neurons into postmitotic neurons, and rhodamine-conjugated α-bungarotoxin antibody (1:300; Thermo Fisher Scientific) to visualize postsynaptic acetylcholine receptors. Slides were incubated overnight at 4°C with primary antibodies in a solution of 0.3% surfactant (Triton X-100; Thermo Fisher Scientific) and phosphate-buffered saline solution with 10% fetal bovine serum. Slides were incubated for 1.0 to 1.5 hours at room temperature with the following secondary antibodies in 0.3% surfactant and phosphate-buffered saline with 10% fetal bovine serum solution at a dilution of 1:500: goat anti–mouse IgG2b (Alexa Fluor 350; Invitrogen), donkey anti–mouse IgG (Alexa Fluor 488; Invitrogen), donkey anti–mouse IgG (Alexa Fluor 594; Invitrogen), donkey anti–mouse IgG (Alexa Fluor 647; Invitrogen), goat anti–rabbit IgG (Alexa Fluor 450; Invitrogen), donkey anti–rabbit IgG (Alexa Fluor 488; Invitrogen), donkey anti–rabbit IgG (Alexa Fluor 594; Invitrogen), donkey anti–rabbit IgG (Alexa Fluor 647; Invitrogen), and donkey anti–goat IgG (Alexa Fluor 647; Invitrogen). All digital images were captured on a fluorescent microscope (BZ-X710; Keyence) with a digital camera and digital imaging acquisition software (BZ-X Viewer; Keyence).
Analyses of neurite projections from implanted neurons were performed for each sample with analysis of representative slides for the presence of neurites (as stained with anti–β-tubulin III antibody). For a representative image at each time point, the longest neurite was measured from soma to distal end. Once identified, the neurite length was quantified using Image J open-source software (https://imagej.nih.gov/ij/download.html).
Bipolar stimulation of the sciatic nerve was performed using insulated titanium electrodes, applied under deep sedation as described above, using identical surgical methods for exposure of the sciatic nerve. We delivered stimulation (S88 model stimulator; Gras) with single pulses, an amplitude of 10 V, and a pulse duration of 80 to 100 microseconds. A force transducer was used to detect and measure the force of evoked twitch, if present.
Statistical analysis was performed using a 1-sided Wilcoxon rank sum test. P < .05 indicated significance.
To assess whether human iPSC-derived motor neurons can survive in vivo transplant, we used a mouse model of sciatic nerve injury. Thirty-two mice underwent the experimental procedure. After ligation and division of the sciatic nerve, dissociated iPSC-derived motor neurons were transplanted into the tibial branch of the distal sciatic nerve stump approximately 5 mm proximal to the sciatic trifurcation (Figure 1A).
After surgery, mice were killed humanely at predetermined intervals to assess the viability and in vivo behavior of the transplanted cells via immunohistochemistry. Analysis of tissue at 24 hours after initial transplant showed clear evidence of surviving human cells within the recipient nerve (Figure 1B and C). At 1 week after cell transplant, evidence of viable human cells persisted (Figure 1D and G), now colocalized with short neurites that originated from the bolus of human cells (Figure 1E, F, H, and I).
At 2 and 3 weeks after transplant, longer neurite processes were visible. Neurites showed preferential growth down the distal portion of the sciatic nerve sheath or toward the triceps surae muscle (Figure 2). Tissue sampled at 4 weeks (Figure 3A and B) and 6 weeks (Figure 3C-H) after transplant showed continued growth of neurites toward target muscle. The longest visible neurite was isolated in representative high-powered fields at each time point and quantified. The growth rate was statistically significant between time points (Pearson correlation coefficient, r = 0.8191; P < .05), and a line of best fit appeared to demonstrate a linear growth rate (eFigure in the Supplement). Most neurites do not course completely within the plane of tissue section for their entire distance; therefore, this measure is only a relative estimate of neurite outgrowth.
Tissue analyzed at 6 weeks after transplant exhibited robust growth of immature neurites within the tibial nerve and into the hilum of the triceps surae muscle (Figure 4A-D). Higher-magnification views from within the muscle demonstrated colocalization of acetylcholine receptors with terminal neurites, indicative of early neuromuscular junction formation (Figure 4E). Based on this finding, we extended the period of observation to 10 weeks and assessed whether iPSC-derived motor neurons are capable of reducing muscular atrophy after denervation injury. We compared the wet muscle weights of the triceps surae muscle between the experimental group (cell implantation) and surgical control group (neural induction media and growth factor without cells) 10 weeks after cell transplant. The wet muscle weight of the injured hind limb was expressed as a percentage of the contralateral, noninjured hind limb muscle mass. Compared with the surgical control group, the mean (SD) muscle weight ratio of the experimental group (n = 3) was about 20% higher (54.2% [4.0%]) than that of the control group (n = 3) (33.4% [2.3%]) (P = .04, Wilcoxon rank sum test). Thus, we found significant muscle mass preservation in the hind limbs of mice that had received iPSC motor neuron transplants at 10 weeks after sciatic nerve injury (Figure 5A). Gross inspection of the implanted hind limb muscle (Figure 5B) showed obvious preservation of muscle bulk and mass compared with hind limbs that received growth factor and medium alone (Figure 5C). Electromyography was attempted in these animals at 10 weeks after cellular transplant; however, no detectable electrical activity or muscle twitch was observed in these muscles in response to bipolar stimulation of the sciatic nerve near the site of iPSC motor neuron transplant.
The results of this study highlight the therapeutic potential of iPSC-derived motor neuron transplant in the setting of peripheral nerve injury. We found clear histologic evidence of cellular engraftment in this xenograft model of sciatic nerve injury. Furthermore, the transplanted cells appeared to respond to endogenous cues and extend neurites toward denervated muscle targets. We also found an apparent therapeutic effect, with preserved muscle mass of the triceps surae muscle compared with negative controls. However, the transplanted motor neurons were not electrically excitable because we found no evidence of restored muscle twitch in response to bipolar stimulation near the site of cellular implant.
Given the clear evidence of cell survival, the lack of electrical response after transplant of human iPSC-derived motor neurons is surprising. Our in vitro differentiation protocol likely yields a heterogeneous cell population that is composed of neuronal precursors and mature motor neurons. At least a portion of the motor neurons produced by this method will depolarize appropriately when exposed to glutamate agonists and will fire action potentials when depolarized via patch clamp techniques.15 Therefore, the cells surviving transplant in our model of murine sciatic nerve injury may have been neuronal precursors that did not reach complete functional maturity, such that electrical current applied to the nerve could not induce detectable depolarization. Bipolar electrical stimulation of the sciatic nerve likely requires relatively mature and functional motor neurons to induce an evoked twitch of triceps surae muscle. At the very least, motor neurons within the nerve sheath must have elongated sufficiently to create an anatomic connection at the neuromuscular junction. Despite histologic evidence of motor end plate formation, the degree of connectivity may be insufficient to induce contraction or a detectable motor potential within the muscle. Given the slow neurite growth rate observed in this study, the neurites detected on immunohistochemistry may have lacked the structural maturity and/or connectivity to effectively conduct an electrical signal to target motor end plates.
These results contrast with recent findings that used cellular isografts of mouse iPSC-derived motor neurons in similar models of nerve injury.17,22 These studies found that inducible muscle twitch was reestablished after mouse iPSC-motor neuron transplant into mouse recipients. This finding may reflect important qualitative differences between murine and human iPSC lines and their cellular derivatives. Alternatively, the xenograft transplant model used in this study may have led to more unpredictable cell survival and function and thereby reduced the in vivo functionality of these transplanted cells.
The lack of electrical response of the triceps muscle is a significant barrier to translational application of human stem cell–derived motor neurons. Simply preventing skeletal muscle fiber atrophy and preserving muscle mass is insufficient. The true therapeutic end point is restoration of inducible movement. In addition, this transplant model offers no synaptic connection to the central nervous system. Nerve depolarization would have to be driven from an external source, which limits the translational potential of these findings. Last, although the human iPSC-derived motor neurons engrafted and extended neurites into target muscle, in a scenario of incomplete nerve injury, such a transplant could exert inhibitory effects on native neurons as they regenerate. New motor end plates, once formed, are unlikely to be displaced even if native nerve fibers are able to regenerate the length of the nerve. This scenario is particularly important for facial musculature, which is known to tolerate relatively lengthy periods of denervation and still retain some ability to respond to reinnervation surgery.
Despite these significant limitations, the overall clinical potential of this form of cellular therapy remains enticing, given the right clinical scenario. A potential application for this research includes the transplant of patient-derived motor neurons into selected distal nerve branches in the setting of proximal motor nerve injury. Based on these results, use of patient-derived motor neurons as a cellular crutch may be possible when transplanted into a motor nerve that has a poor prognosis for spontaneous recovery. The finding of preserved muscle mass attests to a potential therapeutic window, whereby cellular implants may preserve nerve and muscle during a critical period after nerve injury.
Such a treatment strategy could be applied to clinical scenarios of facial nerve injury. For patients with high-grade injury to the proximal facial nerve, such a cellular transplant could maintain facial tone and possibly improve the results of subsequent nerve transfer surgery by minimizing the extent of nerve and muscle atrophy after injury. Future studies will be required to characterize the interaction between transplanted iPSC-derived motor neurons and native motor neurons that attempt to regenerate toward target muscles.
Alternatively, this treatment modality may be useful in the treatment of very distal motor nerve injury. This application may obviate concerns about the interaction between regenerating native neurons and transplanted motor neurons. In facial paralysis, blink rehabilitation has long been treated with simple upper eyelid loading. Although loading enables improved eye closure, it fails to restore normal blink. This rationale is cited in efforts to develop an implantable electrode array that may be used to induce blink.23-25 Patient-specific motor neurons may offer an alternative strategy because they could be implanted to select branches of the distal facial nerve. This strategy is technically feasible based on the results presented above, with the relative diameter of the mouse sciatic nerve being comparable to that of a distal facial nerve branch. Furthermore, the use of iPSCs as the source of cellular material allows for genetic modification of the cell line in ways that may improve functionality. For instance, the introduction of channel rhodopsin photoreceptors may offer a means of producing graded depolarization of motor neurons in response to light stimulus.17 This strategy could offer not only on-demand contraction but graded contraction of target muscle, which is a clinically important end point for restoring the function of facial musculature. This strategy would arguably be superior to a permanent implantable electrode.
However, for iPSC-derived motor neurons to move closer toward clinical use, larger mammal models of peripheral nerve injury will likely be required to determine the performance of transplanted cells in a larger anatomic system. The use of species-matched or donor-matched cells will help to resolve questions about xenograft studies such as this. Recent progress in the generation of primate iPSC lines will permit study of these cells in primate models of peripheral nerve injury.26 The results of this study, although an intriguing proof of concept, do not represent validation of a viable model for preparing cells for transplant. Other potentially superior in vitro methods of generating motor neurons from iPSC precursors exist, and targeted modifications of the iPSC lines and/or the factors used to differentiate motor neurons will likely improve engraftment and in vivo functionality.
Human iPSC-derived motor neurons successfully engrafted and extended neurites to target denervated muscle and reduced denervation-induced muscle atrophy. Human iPSC-derived motor neurons may have future use in the treatment of peripheral motor nerve injury, including facial paralysis.
Corresponding Author: Jon-Paul Pepper, MD, USC Caruso Department of Otolaryngology–Head and and Neck Surgery, Keck School of Medicine, University of Southern California, 1540 Alcazar St, Ste 204, Los Angeles, CA 90033 (firstname.lastname@example.org).
Accepted for Publication: August 11, 2016.
Published Online: December 15, 2016. doi:10.1001/jamafacial.2016.1544
Author Contributions: Dr Pepper and Ms Wang contributed equally to this work. Dr Pepper had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study concept and design: Pepper, Ichida.
Acquisition, analysis, or interpretation of data: Pepper, Wang, Hennes, Sun.
Drafting of the manuscript: Pepper, Wang, Ichida.
Critical revision of the manuscript for important intellectual content: Pepper, Hennes, Sun, Ichida.
Statistical analysis: Wang.
Obtained funding: Pepper, Ichida.
Administrative, technical, or material support: Pepper, Hennes, Sun, Ichida.
Study supervision: Pepper, Ichida.
Conflict of Interest Disclosures: None reported.
Funding/Support: This study was supported by a research scholar award from the American Association of Facial Plastic Surgery (Dr Pepper); a fellowship award from the Donald E. and Delia B. Baxter Foundation (Dr Pepper); an individual grant from the Zumberge Fund (Dr Pepper); grant R00NS077435 from the National Institutes of Health; grant W81XWH-15-1-0187 from the US Department of Defense; grants from the Donald E. and Delia B. Baxter Foundation, Tau Consortium, Frick Foundation for ALS Research, Muscular Dystrophy Association, New York Stem Cell Foundation, University of Southern California (USC) Keck School of Medicine Regenerative Medicine Initiative, USC Broad Innovation Award, and Southern California Clinical and Translational Science Institute (Dr Ichida); and a Robertson investigator award from the New York Stem Cell Foundation (Dr Ichida).
Role of the Funder/Sponsor: The funding sources had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.
Additional Contributions: Nina Bradley, PhD, and Jerry Loeb, MD, contributed to the planning and design of the experimental protocol. Neither received compensation for these contributions.
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