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- Yi-Chao Hsu1, Su-Liang Chen2, Tai-Yu Hsu2,3 and Ing-Ming Chiu2,3,*
Running title: NSCs and iNs for Neural Repair
*Corresponding Author: Ing-Ming Chiu
Keywords: cell therapy, neural stem cells, FGF1, induced neurons, peripheral nerve injury
Cell transplantation can relieve symptoms or even reverse the neurodegenerative diseases and repair nerve injuries. Fibroblast growth factor 1 (FGF1) has been shown to promote neuronal survival and stimulate axonal growth. Combination of FGF1 and cell-based therapy could be a very promising application for nerve repair. For future cell-based treatment, several key issues should be concerned: (i) the source of the cells should be autologous, (ii) consistent method and protocols for cell isolation, and (iii) test in suitable animal models; (iv) the microenvironment of cells implanted should be better characterized. In addition, developing high temporal and spatial resolution images for cell tracking is very important for evaluating the efficacy of cell transplantation. In this review, we also summarize recent progress in cellular reprogramming, such as induced neural stem cells and induced neurons, and future development of cell-based therapy that includes conduits and growth factors in peripheral nerve injury and spinal cord injury (SCI).
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Fibroblast growth factor 1 for nerve injury repair
There are 22 mammalian fibroblast growth factors (FGFs) which are grouped into seven subfamilies based on differences in sequence homology and phylogeny. Notably, FGF1 and FGF2 share sequence and structure similarities and belong to the FGF1 sub-family . The FGF ligands carry out their diverse functions by binding and activating the FGFR family of tyrosine kinase receptors in a heparan sulphate glycosaminoglycan (HSGAG)-dependent manner. There are four FGFR genes (FGFR1-FGFR4) that encode receptors consisting of three extracellular immunoglobulin domains (D1-D3), a single-pass transmembrane domain and a cytoplasmic tyrosine kinase domain . Several FGFR isoforms exist, as exon skipping removes the D1 domain and/or acid box in FGFR1-FGFR . Alternative splicing in the second half of the D3 domain of FGFR1-3 yields b (FGFR1b-3b) and c (FGFR1c-3c) isoforms that have distinct FGF binding specificities  and are predominantly in epithelial and mesenchymal cells, respectively. Each FGF binds to either epithelial or mesenchymal FGFRs, with the exception of FGF1, which activates both splicing isoforms .
The involvement of FGF signaling in human diseases is well documented. Deregulated FGF signaling can contribute to pathological conditions either through gain- or loss-of-function mutations in the FGFRs themselves. As both Fgf1–/– and Fgf1–/–Fgf2–/– mice are fertile and apparently completely normal , the physiological roles of FGF1 and FGF2 remain to be explored. However, it is likely that FGF1 and FGF2 play some physiological part in the maintenance of vascular tone, as administration of FGF1 and FGF2 lowers blood pressure in rats  and can restore nitric oxide synthase activity in spontaneously hypertensive rats . In addition, isolated vessels from Fgf2–/– mice have a reduced response to vasoconstrictors. Although Fgf2–/– mice showed some hypotension owing to decreased smooth muscle contractility , they are still able to regulate their blood pressure . Interestingly, it has been shown that FGF1 plays an very important role in the remodeling process of adipose tissues . FGF1 is significantly induced in gonadal white adipose tissue in the animal model of high-fat diet (HFD) feeding . Furthermore, Fgf1–/– knockout mice developed a phenotype of profound diabetes, when fed with an HFD , suggesting that the importance of FGF1 especially when challenged in different nutritional situations. Similar situation is also observed in collagen VI null mice that only have different phenotype when fed with an HFD [11, 12].
FGF1 has been shown its therapeutic potential for cardiovascular disorders. Phase I trials have shown that intramyocardial injection of FGF1 during coronary artery bypass graft surgery improves collateral artery growth and capillary proliferation  . Beneficial effects of FGF1 on the peripheral circulation have also been shown. Injection of a plasmid that encodes FGF1 into the leg improved perfusion of end-stage lower-extremity ischemia in a Phase I trial  and led to a twofold reduction in the need for amputation in patients with critical limb ischemia in a recent Phase II study . Interestingly, distal blood and oxygen pressure are similar after injection of either FGF1 plasmid or placebo , and the mode of action of FGF1 might not have been primarily angiogenic.
FGF1 can repair nerve injuries. It enabled functional regeneration of transected spinal cords in rats  and restored some motor function to paralyzed limbs in a 6-month-old boy with brachial plexus avulsion  has benefited patients with chronic transverse myelitis . FGF1 administration and the combination of sural nerve grafts with FGF1 treatment partly restored ambulation to a paraplegic . When tested the combination of peripheral nerve grafts and FGF1 in spinal cord-transected rats, it retores the function of hindlimb locomotor [17, 21, 22]. The expression of arginase I, the macrophage M2 marker and the recruitment of M2 marcophages were both observed in the repaired site . Furthermore, FGF1 and the nerve grafts induce IL-4 expression and NGF/BDNF expression in the repaired site, respectively. Therefore, an ideal repair strategy should consider the both beneficial effects of FGF1 and nerve grafts . We have previously shown that the combination of FGF1 and neural stem cells (NSCs) and micropatterned poly(D,L-lactide) (PLA) conduits could facilitate nerve repair and functional recovery in rats [24, 25].We have ongoing studies to combine different growth factors and adult NSCs and test their efficacies in peripheral-nerve regeneration .
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Neural stem cells for Peripheral Nerve Injury Repair
Sources of NSCs for Clinical Application
Cell transplantation has been expected to relieve symptoms or even reverse the progression different kinds of neural diseases. Mesenchymal stem cells (MSCs), embryonic stem cells or brain stem cells have been tested their efficacies in different animal models, such as Parkinson’s disease, Huntington’s disease and Alzheimer’s disease along with multiple sclerosis and cerebral ischemia . In addition to direct cell transplantation, the ideal therapeutic approaches should also consider to stimulate endogenous stem cells and induced the expression of the active molecules in situ simutaneously. several key issues should be concerned: (i) the source of the cells should be autologous, (ii) consistent method and protocols for cell isolation, and (iii) test in suitable animal models; (iv) the microenvironment of cells implanted should be better characterized. In addition, developing high temporal and spatial resolution images for cell tracking is very important for evaluating the efficacy of cell transplantation .
The ideal NSC sources for cell transplantation should better be patient-derived cells for autologous transplantation that could avoid immune rejection. The potential cell sources for the repair of neural diseases include brain tissues-derived NSCs [28, 29], blood or bone marrow-derived MSCs [30-32], skin or blood-derived induced pluripotent stem cells (iPSCs) [33-36], skin or urine-derived induced neural stem cells (iNSCs) [37-43] and skin-derived induced neurons (iNs) [44-53].
Isolation and characterization of NSCs
Flow cytometry and fluorescence-activated cell sorting has been applied extensively in stem cell biology, including the isolation of different precursor and progenitor populations from the hematopoietic and nervous system. NSCs that had been isolated from brain tissues by NSC-specific cell surface markers CD133 and NSC-specifc genes, such as Sox1, Sox2, Nestin and FGF1 [54, 55]. NSCs can be isolated by such approaches and then cultured to form neurospheres, which was defined as an indication of self-renewal. For determining the multipotency, the EGF and FGF2 in the culture of are withdrawal or other inducing factors are added .
The human FGF1 gene was first cloned in our laboratory . FGF1 is expressed in neurons in different regions, including ventral cochlear, olfactory bulbs and hippocampus . The FGF–1B promoter is brain-specific and only active in the brain [59, 60]. Interestingly, FGF-1B mRNA is elevated for supporting the NSCs in hippocampus neurogenic region in exercise-induced neurogenesis . Furthermore, FGF-1B promoter (-540 to +31)-driven GFP reporter (F1B-GFP) could be used to isolate self-renewal and multipotent NSCs from human , and mouse brain sources [54, 55]. A series of patented technology have been developed in which NSCs could be isolated as GFP positive cells when adult mouse brain cells were transfected with F1B-GFP plasmid (USA patent No. 6,984,518; 7,045,678; and 7,745,214). This F1B-GFP plasmid comprises the GFP coding sequences driven by the human FGF-1B promoter [54, 55]. When applying F1B-GFP-selected NSCs in the damaged sciatic nerve of paraplegic rats, it showed significant repairing efficacy. Combination using nerve conduits, together with NSCs and FGF1, could further repair peripheral nerve injury in animals [25, 62]. A novel material named ultra-nanocrystalline diamond (UNCD) has been also demonstrated by our laboratory to apply as the biomaterial for NSC transplantation in peripheral nerve injuries [63, 64]. Recently, we demonstrated that F1B-GFP-selected NSCs with nerve conduits could significantly improve functional recovery of sciatic nerve injury in mice (Figure 1) through the secretion of a cytokine (unpublished data). As a proof of concept, direct combination of this cytokine along with F1B-GFP NSCs in nerve conduits could improve motor function recovery, promoted nerve regeneration, and increased the diameter of newly regenerated nerve up to 4.5 fold. Our data suggested that the likelihood for administration of an immune factor in clinical settings of sciatic nerve injury repair.
Cellular Reprogramming for iNs and iNSCs
The promising cellular sources for regenerative medicine should be personalized. The ideal sources are from patients’ somatic cells, such as skin fibroblasts or peripheral blood. These personalized sources have been demonstrated to be reprogrammed in to iPSCs . Although the generation of iPSCs from patients with amyotrophic lateral sclerosis demonstrate the accessibility of patient-derived iPSCs . iPSCs have also been shown sizeable genetic and epigenetic abnormalities . Recently, direct reprogramming approach provides a straightforward, fast and reliable platform to produce different types of functional cells. In Table 1, we summarized reports of cellular reprogramming of myocytes , macrophages , cardiomyocytes  and hepatocytes . For example, combination of miRNA124, Brn2 and Myt1l could reprogramm human fibroblasts into functional neurons (iN) [49, 72]. Notably, these functional iN cells expressed a variety of mature neuronal markers and were shown there capability to fire action potentials. In Table 2, we summarized recent reports of cellular reprogramming of neurons. Recently, a signaling adaptor protein SH2B1 has been shown by our group to enhance neurite outgrowth of iNs. These enhanced iNs were demonstrated to express mature neuronal markers, such as NeuN, synapsin, GABA, vGluT2, and tyrosine hydroxylase. Importantly, these SH2B1-enhanced iNs showed accelerated reprogramming of iNs (Figure 2) . Our findings will facilitate the application of iNs for the disease modelling and treatment of neural diseases. Most recently, it has been reported that NSC-specific transcription factors, especially SOX2, can reprogram mouse and human fibroblasts into iNSCs with self-renewing ability and multipotency [37-41]. Future studies remain to be done to adapt this iNSC protocol to eventually substitute viral NSC factors with nonintegrating delivery modes such as Sendai viruses [73, 74], or small molecules. We anticipate that iNSCs could provide a safe and robust cellular platform for the generation of patient-specific neural cells for nerve injury repair. Here we summarized recent reports of iNSCs (Table 3).
Combination of biomaterials, growth factors, iNSCs/iNs for peripheral nerve injury repair
For the purpose of nerve regeneration, it has been shown that nature polymers including gelatin, collagen, chitosan, chitin, as well as synthetic biodegradable polymers such as PLA or poly (D,L-lactic-co-glycolic acid) (PLGA) are commonly used as their neuro-compatible properties . However, when using non-degradable materials, it should be avoided in long-term neural repair to prevent the nerve damage and chronic inflammation. Therefore, biodegradable materials are more acceptable in neural repair . In addition, poly(glycerol sebacate) has surface erodible and elastomeric properties when using as the material of nerve conduit . Notably, using new techniques such as electrospinning or polymer blending could attach neurotrophic factors in the nerve conduit , thus increasing the neural-compatible properties in the surface of conduit .
When nerve injuries occur, growth factors in situ plays an important and multiple roles in regulating local neural and non-neural cells. Although the endogenous growth factors secreted by neural cells in the distal nerve stump can support axon regeneration, the supportive action may not be sustained indefinitely due to an obvious decline with time in cellular production of growth factors, and hence the continuous supply of growth factors is critically required, which is mainly dependent on the addition of exogenous growth factors. During tissue remodeling, growth factors can initiate signaling pathways for repair. In addition to stimulating endogenous neural stem/progenitor cells, some growth factors are also critical for their differentiation into different neural cells . Therefore, using the nerve conduit for nerve repair also provides a channel for growth factors diffusion . Another approach using the combination of liposomes and neurotrophic factor genes also showed their efficacy in nerve injury [82, 83]. Furthermore, when using PLGA as microspheres to carry growth factors, the release of acidic products may result in protein inactivation . In order to control the delivery of growth factor, electrospun nanofibrous scaffold is the ideal delivery vehicle since it can serve as a scaffold and has better contact guidance .
Possible application in SCI and traumatic brain injury (TBI)
To enhance the outcome of peripheral nerve regeneration through the use of scaffolds alone, efforts have concentrated on the optimal incorporation within biomaterials of biochemical cues, including supporting cells, growth factors, and/or cytokines. Combination of FGF1, biomaterials and iNSCs/iN cells is promising for clinical application. Of note, using peripheral nerve grafts and FGF1 not only repair the hind limb of adult paraplegic rats [17, 21], but also exert therapeutic effect on the patients with SCI and common peroneal nerve lesions [86, 87]. Thus, direct FGF1 treatment of FGF1 may also benefit the patients. Notably, Chen et al. developed a FGF1-based SCI repair strategy and designed a clinical trial to test the efficacy and safety of using FGF1 in combination with surgical intervention in human SCI. They demonstrated that the use of FGF1 for SCI is safe and feasible in the clinical trial . There are significant improvements in American Spinal Injury Association motor and sensory scale scores, ASIA impairment scales, neurological levels, and functional independence measure at 24 months after treatment . Given the significance of FGF1 in the treatment of SCI and peripheral nerve injury, future efforts generating FGF1-expressing iNSCs or iNs will have promising potential in the treatments of peripheral nerve injury and central nervous system diseases, such as SCI and TBI.
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