1 Introduction Neurological diseases (ND) of degeneration and injury such as Alzheimer’s disease (AD),Parkinson’s disease (PD),amyotrophic lateral sclerosis (ALS),and spinal cord injury (SCI) severely affect human health. In spite of the tremendous efforts in improving medication of such devastating ND,it is still challenging to develop effective treatment for successful cure of such disorders.

Stem cells can self‐renew,proliferate,and differentiate into specialized cells for cell replacement therapy of a wide variety of diseases,including neurological disorders. In a mouse model of PD,grafts of neural stem cells (NSC) survive in the midbrains and significantly improve behavior performance^[1]. The human counterparts of such stem cells are also functional in pre‐clinical investigations^[2]. Because of a lack of quality control for such type of cell products,in August of 2010,the Food and Drug Administration (FDA) approved the first proposed clinical trials of human embryonic stem cells (hESC)‐derived oligodendrocyte progenitor cells. As the first product of hESC derivatives approved by the FDA for clinical trials, GRNOPC1 were primarily examined for the treatment of SCI^[3]. Other clinical trials were approved shortly after to evaluate the use of hESC‐derived retinal pigment epithelium in age‐related macular degeneration by ACT,Inc.,and NSC in treating Pelizaeus‐ Merzbacher disease by SCI,Inc.^[4] The past 10 years has witnessed an increase in the number of clinical trials involving stem cell products in United States (US),as shown in Table 1. Although not all trials will generate positive results for a specific entity of product, these Phase Ⅰ or Ⅱ trials will accumulate valuable data that will be informative for future stem cell‐based therapy^[5]. Here,we summarize recent advances in cell transplantation for ND and discuss the policies and regulations of cell therapy in the United States (US).

2 Cell therapies of ND The human nervous system (NS) is composed of highly specialized structures that are difficult to regenerate once damaged. ND such as acute injuries (e.g.,stroke and traumatic brain injury) and chronic neurodegeneration (e.g.,AD,PD,and Huntington’s disease), which all cause loss or malfunction of neural cells,are the leading cause of human death and disability^{[6, 7]}. The limited ability to regenerate requires alternative avenues such as cell transplantation for repair. Given their capacity of self‐renewal and multi‐lineage differentiation, stem cells are thought to be promising sources of cells that will achieve the goal of cell therapy for ND.

2.1 Types of stem cells for cell therapies of ND Over the past few decades,a growing number of stem cells have been tested or applied for treating severe ND. On top of the list are mesenchymal stem cells (MSC),hematopoietic stem cells (HSC),umbilical cord blood‐derived stem cells (UCBSC),neuroprogenitorcell (NPC),olfactory ensheathing cells (OEC),and hESC‐ or induced pluripotent stem cells (iPSC)‐derived nerve cells.

2.2 MSC for cell therapies of ND MSC exist in somatic tissues and can be isolated from various tissues of the adult or fetus^{[8, 9]},including fat, bone marrow,umbilical cord blood,liver,dental pulp^{[10, 11]},placenta,and muscle^[12]. These cells exhibit advantages such as their abundance and ease of harvesting; additionally,few ethical considerations limit their use. MSC can differentiate into a variety of mesenchymal tissues,such as bone,adipose,and cartilage,under appropriate culture conditions. Additionally,MSC exhibit several immunomodulatory characteristics and the plasticity to differentiate in petri‐dish into other lineages such as neurons or glia^{[13, 14, 15]}. In previous clinical trials,respective transplantation of MSC in patients with MS,ALS^[16],and stroke^[17] all are clinically feasible and safe. Recently,a study confirmed that umbilical cord MSC improved neurological function and self‐care in patients with traumatic brain injury^[18]. Based on the capacity for direct fate conversion of somatic cells,it is not surprising that MSC are considered one of the most promising sources of cells for treating human neurodegenerative diseases^[19].

2.4 HSC for cell therapies of ND HSC therapy,which is a conventional cell‐based therapy,has shown substantial clinical validation in patients with ND. In clinical cases,HSC,particularly autogenous HSC,have been shown to have an positive effect following transplantation into SCI patients^[20]. A Phase Ⅱ trial demonstrated that intense immunosuppression followed by autologous HSC transplantation was significantly superior to mitoxantrone in reducing magnetic resonance imaging activity in severe cases of MS^[21]. However,the limited functional integration of transplanted cells into the target tissue is a limitation for HSC therapies because the number of cells that become integrated is rather low.

2.5 UCBSC for cell therapies of ND UCBSC as an alternative source of such cells have recently been recognized for their ease of collection and relatively low immunogenicity. Such properties allow for direct intravenous delivery for therapy without the need considering possible immune rejection^[22]. UCBSC exhibit significant trophic effects and have demonstrated neural protective effects in rodent models of neonatal hypoxia‐ischemia^{[23, 24]}. UCBSC may also be capable of direct fate conversion to other lineages such as CD34+ hematopoietic and endothelial progenitor cells (~1%)^[25] which can secrete angiogenic factors to promote neurogenesis^[26],neural and glia‐like cells that may be directly used for cell replacement therapy in ND^[27].

2.6 NPC for cell therapies of ND In specific specialized areas of mammalian brains, such as the hippocampus,epidermal region or the ventricular zone,and sub‐ventricular zone,produce proliferating NPC^{[28, 29]}. Neurogenesis in the adult brain has been identified in various species,including rodents,primates^[30],and humans^[31]. NPC have the capacity to proliferate and can differentiate into specific types of neurons and glia,and thus are suitable for application in regenerative medicine^[32]. NPC primarily reside in the sub‐ventricular zone of the adult human brain,indicating that the mammalian brain can repair itself by recruiting endogenous NPC following injury or degeneration. Within the NS,astrocytes exhibit substantial heterogeneity,and a certain class of astrocytes has been demonstrated to be another source of NPC^[33]. In addition to the significant variation among NPC lines,because of a lack of standardization and species into which NPC are transplanted, different lines of NPC function distinctively,which may explain why migration does not always occur^[34]. However,how to obtain a sufficient number of NPC used for patients,since primary cultured NPC can only be used in preclinical animal studies because of ethical considerations. Human pluripotent stem cells, including hESC and iPSC,can differentiate into all three germ layers,making hESC and iPSC the best potential source of NPC.

2.7 OEC for cells therapies of ND OEC are specialized glial cells within the olfactory system that ensheathe bundles of nonmyelinated olfactory nerve axons. These cells support axonal regeneration and remyelination with the appropriate formation of axonal nodes of Ranvier and improve nerve conduction velocity. Numerous clinical studies have used OEC for transplantation into SCI patients[35‐38]. These studies have generally indicated the feasibility and safety of using these cells,but several of factors must be addressed,including cell derivation sources, methods for evaluating efficacy,and long‐term large‐scale evaluation. In addition,OEC have been transplanted into other ND patients,including cerebral palsy^[39] and ALS^[40] patients. The transplantation of OEC resulted in functional improvement in children and adolescent with cerebral palsy,but further evaluation in large‐scale clinical trials is necessary^[39]. However,there are no indications after OEC treatment in ALS patients^[40].

2.8 hESC for cell therapies of ND hESC have the capacity to differentiate into any cell type in the body (pluripotency). Since the first hESC line was established in 1998^[41],hESC have been widely used as an unlimited source of cells for developmental biology,drug discovery,transplantation,and regenerative medicine studies. Before this cell replacement therapy can be implemented,the cells must be differentiated into more specialized progenies and require substantial evaluation to determine their safety and efficacy to overcome their inherent hurdles such as genetic stability,risk of teratoma formation,and functional integration.

In August 2010,as the first product of hESC derivatives,hESC‐derived oligodendrocyte progenitor cells were approved by the FDA for clinical trial, and GRNOPC1 was to be used for the treatment of SCI^[3]. However,in the preclinical study,cystogenesis occurred when cells were transplanted into animals, and the clinical trial was postponed for two years until this issue was resolved. Subsequently,other clinical trials ofhESC‐NSC in treating Pelizaeus‐Merzbacher disease were conducted by SCI^[4],and the specific treatment effect is being examined.

3 Preclinical evaluations of neural cells differentiated from human stem cells In order to cure ND using hESC‐derived neural cells, a growing number of preclinical studies have been carried out in animal models varying from rodents to primates. The types of cells used in these studies include neural progenitors and terminal differentiated neural cells. As described above,MSC can be differentiated into neural‐like cells^{[42, 43]} and have shown positive effects when transplanted into animal models of ND^[44].

Neural cells derived from other types of pluripotent stem cells as well were proved functional in preclinical studies. For instance,glial‐rich neural progenitors derived from human iPSC were transplanted into rodent models of mutant superoxide dismutase 1 (SOD1)‐mediated ALS^[45]. These transplanted cells then converted into astrocytes and expanded the lifespan of recipient mice,indicating the efficacy of human iPSC‐derived glial progenitors for cell therapy of ALS. In animal models of SCI,human glial restricted progenitors (hGRP) and hGRP‐derived astrocytes survived following engraftment into the injured spinal cord,migrated extensively in the rostral and caudal directions into the adjacent white matter,and expressed a variety of phenotypic markers including GFAP^[46]. This research also showed that embryonic astrocytes alone were not sufficient to promote functional recovery through long‐distance axonal regeneration,suggesting a necessity of combining cell transplantation and other therapeutic interventions such as neurotrophins^[47],cAMP^[48] and chondroitinase^[49]. Nevertheless,because of the high heterogeneity of glia,it should be noted that not all astroglia have the same function in curing ND. Both hESC‐derived Olig2+ and Olig2‐ progenitors could be differentiated into astroglia,respectively. However, upon transplanted into rat model of ischemic stroke, Olig2+/‐ astroglia exhibit distinctions from the Olig2‐ and the mixed population of astroglia and significantly improved learning and memory of the ischemic stroke rats^[50].

4 Regulatory agencies of stem cells and translational medicine in America In the 2000s,the United States (US) informed the world that work with stem cells is immoral and should be stopped. The stage of stem cell research is rudderless, and this greatly slowed the research on stem cells. Finally,the ban on using federal funding for human embryonic stem cell research was lifted on March 9, 2009,when Barack Hussein Obama Jr.,the new President,issued executive order 13505. A door was re‐opened for human embryonic stem cell research and regenerative medicine in the US triggering the advance of reliable scientific research involving human stem cells and the approval of the first clinical study of hESC by the FDA^[51]. In the same year,an extensive and detailed guideline: “National Institutes of Health Guidelines for Human Stem Cell Research” (Guidelines) was finalized by the National Institutes of Health (NIH)^[52]. Thereafter,with the advance of stem cell research and clinical translation,a series of customized guidelines,regulations,and laws were mandated in the following years. For example,two were published for cellular therapy for cardiac disease and for allogeneic pancreatic islet cell products (Cellular Therapy for Cardiac Disease,Considerations for Allogeneic Pancreatic Islet Cell Products). These documents emphasize that any processing and manufacture of cellular products should be conducted with stringent,independent review and supervision to ensure the safety,integrity, and efficacy for use in patients.

4.1 Regulations on sourcing material hESC lines are derived from pre‐implanted embryos; this is fraught with disputes regarding the onset of human personhood and reproduction. hESC researches are surrounded by ethical and political controversy (Table 2). According to the guidelines,the current regulatory structure of the Institutional Review Board (IRB) review under the common rule (45 CFR Part 46, Subpart A) addresses the core ethical principles needed for appropriate oversight of hESC derivation. Several other methods of deriving stem cells have fewer ethical concerns,for example,the reprogramming of somatic cells to produce iPSC avoids the ethical problems specific to hESC. In addition,iPSC used for the original donor can go through a minimum screening and has less immunogenicity compared with hESC. Nevertheless,iPSC have not been used in clinical trial in the US iPSC and their derivatives should be subjected to additional preclinical tests because of the potential risks of genomic instability caused by reprogramming or the possible functional defects owing to the nature of unnatural entities.

The only way to establish eligible hESC lines can be registered on NIH (http://grants.nih.gov/stem_cells/ registry/current.htm) to avoid burdensome and repetitive assurances from multiple funding applicants, or to demonstrate compliance with the specific pro‐ cedural requirements of guidelines by submitting an assurance with supporting information for administrative review by the NIH. In President Bush’s era, only certain existing embryonic stem cell lines were recognized by the NIH. Currently,increasing number of new hESC lines are eligible for NIH funding,and federal funding is also available to carry out research with hESC lines not on the NIH list and to derive new hESC lines from frozen embryos donated for research if a woman who received in vitro fertilization (IVF) has determined they are no longer needed for reproductive purposes. However,derivation of stem cells using somatic cell nuclear transfer is still not permitted under federal funding^{[53, 54]}.

Currently,cells used in studies from a wide variety of sources including autologous or allogeneic,undifferentiated stem/progenitor cells,or terminally differentiated cells have been submitted to the FDA for review. To ensure safety of the manufacturing process of cells,several aspects need to be considered, including the eligibility of donors selected to provide the source material through screening and testing. Moreover,the information of the donor should be a specific conservation (Informed Consent/Institutional Review Boards: 21 CFR Parts 50/56). In detail,(1) the donor will be screened for infectious and possibly genetic diseases. Pre‐implantation genetic diagnosis (PGD) of the donation of embryos is allowed by NIH, although this may result in the identification of chromosomal abnormalities that would make the embryos medically unsuitable for clinical use; (2) the donated cells may be subject to genetic modification by the investigator; (3) disclosure of medical and other relevant information that will be retained,and the specific steps that will be taken to protect donor privacy and confidentiality of retained information, including the date at which donor information will be destroyed,if applicable; (4) the donor should be informed that in the case of pluripotent stem cells,the ability to destroy all samples may be limited and that with newer genetic techniques,complete anonymity may not be feasible; (5) disclosure that any resulting cells,lines,or other stem cell‐derived products may have commercial potential,and whether any commercial and intellectual property rights will reside with the institution conducting the research. The FDA may be agnostic about the source of stem cell,apart from assurance of product characteristics. The investigator and research institution do not have access to identifiable private information related to the cell line, and a written agreement is obtained from the holder of the identifiable private information related to the cell line provided that such information will not be released to the investigator under any circumstances (45 CFR Part 46). In this case,the research may be considered to not involve human subjects.

4.2 Regulations for stem cell manufacturing and characterization As for the production process,a detailed description of all programs of standard operating procedures (SOPs) must be given related to product quality as a traditional medicinal product. The procurement of tissue from a human donor may not fall under the jurisdiction of current Good Manufacturing Practice (cGMP,21 CFR 211),but should be conducted using Good Laboratory practices (GLP,21 CFR Part 58). Various types of reagents may be used to manufacture the cellular component of a product. These include reagents that promote cellular replication and induce differentiation,used to select targeted cell populations, specifically,serum,peptides,cytokines,and monoclonal antibodies,and culture medium. The reagents must be duly qualified. Demonstration of manufacturing controls was evidenced by strict adherence to SOPs and quality control assessment of manufacturing intermediates and testing of the final cellular preparation.

Criteria for release of cells for transfer to research subjects in trials must minimize risk from cultureacquired abnormalities. It should also follow regulatory guidelines including universal precautions to minimize the risks of contamination,infection,and pathogen transmission. One of the crucial requirements to enable clinical use of these cells and embryo is to eliminate the risk of xeno‐transmitted infections and immunoreactions caused by animal products. This would enable better expansion of cells in xeno culture system,including medium,digestive tool, and cryoprotectant,prior to transplant. The FDA explains a testing approach for characterization of cells. This involves development of quantitative analytical techniques for conducting a panel of tests including morphology evaluation,detection of phenotype‐specific cell surface antigens,genomic/proteomic analysis, and assessment of unique biochemical markers to determine the characterization of stem cells.

4.3 Regulations for preclinical cell therapy products (CTPs) For the overall consideration of the design of preclinical studies to support early‐phase clinical trials, the FDA has published guidance entitled “Guidance for Industry: Preclinical Assessment of Investigational Cellular and Gene Therapy Products”. All preclinical studies testing the safety and efficacy should be designed in a way that supports a precise,accurate, and unbiased measure of clinical promise. CTPs vary with respect to characteristics such as the formulation including combination with a scaffold or other non‐cellular component,genetic relationship of the cells to the patient (autologous,allogeneic,xenogeneic), and cell source. CTPs can be generally classified as stem cell‐derived CTPs and mature/functionally differentiated cell‐derived CTPs. CTPs must be in compliance with cGMP,including quality control and quality assurance programs during translation from “bench to bedside”,to minimize the risks of contamination, infection,and pathogen transmission.

To determine whether it is reasonable to provide granting permissions for clinical trials,the FDA evaluates the potential risk based on results derived from the analytical assessment of CTP characteristics and safety testing. Preclinical in vitro and in vivo proof‐of‐concept,pharmacology,and toxicology studies are conducted to establish feasibility and rationale for clinical use. These studies also provide the scientific basis to support the conclusion that it is reasonably safe to conduct the proposed clinical investigations (21 CFR 312.23(a) (8)). Preclinical in vitro animal model studies are the principal means of testing. Various models that have been considered include immunosuppressive agents in immune‐competent animals; genetically immunodeficient animals; humanized animals; administration into an immuneprivileged site; or a combination of these scenarios (PHS Guideline on Infectious Disease Issues in Xenotransplantation; January 2001). Important design elements for preclinical animal studies,include (1) selection of relevant disease/injury models; (2) testing of product intended for clinical administration; (3) using route and method of delivery comparable to clinical plan; (4) optimal timing of intervention relative to disease/ injury onset; (5) duration necessary to assess potential adverse safety events and durable biological activity; (6) need for immunocompetence modification of animal model^[55]. In addition to the plasticity and unlimited capacity of stem cells,ES cells can themselves form teratomas. Overall,CTPs to be used in clinical trials must first be rigorously characterized to assess potential toxicities through in vitro studies and in animal studies. In some cases,the donor may receive a treatment prior to the harvest of the source material. If the donor is also the trial subject,such pretreatment may add to the overall risk to the subject. Similarly, some CTPs require pretreatment of the recipient, such as with immune modification or myeloablative conditioning to facilitate cell survival. In such cases, the risks associated with the pre‐treatment should be considered in the overall benefit‐risk assessment.

4.4 Regulations for clinical trial In the United States,all clinical research involving cells or test articles regulated as drugs,devices,and biological products are subject to the FDA regulations governing investigational new drugs (INDs) or devices (IDEs) (21 CFR Parts 312 or 812),biologics regulations (21 CFR 600),and cGMP. In particular,the US federal regulations divide cellular therapy into two sections of the Public Health Service Act (PHSA),referred as “361 produces” and “351 produces”. Traditional blood, bone marrow progenitor cells,and other tissues for transplantation are classified into section 361 produces. The FDA regulates that cells or tissues used for therapeutic purposes and the regulation that pertains to process of 361 produces are codified under the Good Tissue Practice (GTP)^[56]. Part of CTPs that is only used for safety and effectiveness assessment, can be pertained to process of 351 produces,the requirements of which are not as strict as those for 361 produces.

For early phase clinical trials,especially first‐inhuman trials,the regulations in 21 CFR Part 312 emphasize the importance of the assessment of trial risks and the safeguards for trial subjects. Regulations on cells,tissue,and cellular and tissue‐based products (HCT/Ps) are ruled in CFR 21,part 1271. Moreover, guidance: “Considerations for the Design of Early‐Phase Clinical Trials of Cellular and Gene Therapy Products” published by the FDA is the explicit guide for clinical trial design of CTPs. The objectives of early‐phase clinical include (1) dose exploration: initial tests of a novel product should be tested under low risk condition of low dose before escalating to a higher dose to determine the relationship with potential adverse reactions,and the best route of administration; (2) feasibility assessments: CTPs sometimes require specialized equipment or novel procedures for customized preparation of products,special handling of products administration,or adjunctive therapy. In these cases,sponsors should consider designing early phase trials to identify and characterize any technical or logistic issues with manufacturing and administering the product; (3) activity assessments: a common secondary objective of early phase trials is to obtain preliminary assessments of product activity,and such trials can use both short‐term responses and longterm outcome. It could suggest the potential efficacy of the products. Such proof‐of‐concept data can support subsequent clinical development. For CTPs,activity assessments might include specialized measures such as gene expression,cell engrafting,or morphologic alterations,and more common measures such as changes in immune function,tumor shrinkage,or physiologic responses of various types. In addition, choice of study population is stringent. The potential for benefit might depend on the choice of study population,including healthy volunteers,disease stage,or severity of patients or other special group such as pediatrics. For CTPs,the potential instability of cells deemed the trials unacceptable for healthy volunteers. Selection of sick volunteers involves consideration of the potential benefits and the ability of the study population to provide interpretable data. For the principle of scientific necessity,children should not be enrolled in a clinical investigation unless necessary to answer an important scientific question about the health and welfare of children (21 CFR 56.111 (a)(1) and (b)).

Late phase trials are aimed at providing decisive evidence of clinical utility. Currently,most of CTPs are tested in the early phase,and very few in phase Ⅲ. Late phase trials benefit by the use of clinical measures and response by monitoring over a longer,more clinically relevant period. To preserve the ability to draw valid conclusions about the clinical benefit of late phase clinical trials,use of generally randomized and comparator arms to avoid artificial factor makes the trials more reasonable,objective,and true. Moreover, careful consideration of appropriate product features, proof‐of‐concept testing,and the design of clinical trials will enable balanced case‐by‐case consideration of proposed therapeutic trials^[57].

5 Conclusion and outlook Thus far,the policy of the US seems obscure and unclear. The regulatory permitting activities of the US in the sphere of stem cells,technologies of regenerative medicine,and substitutive cell therapy are selective,theoretical,and do not fit the existing norm and rules,for example,set for the pharmaceuticals. More clear and detailed policy should be formulated. Currently,detailed policies have been formulated for cardiac disease (Guidance for Industry: Cellular therapy for Cardiac Disease) and diabetes (Considerations for Allogeneic Pancreatic Islet Cell Products). However,there is no tailor‐made guidance for cell therapies of ND thus far.

As a whole,the field of cell transplantation is advancing swiftly with the prosperous banking and manufacturing of stem cells,which will lead to effective therapies for patients. In a framework that facilitates the healthy translation of stem cell research for ND,clinical trials with stem cell products should consider scientific evidence and strictly follow the FDA regulatory guidelines to ensure safety and efficacy. More reliable approaches for precise evaluation of safety and efficacy should be developed so that the most valuable information could be collected for prompt assessment of innovative cell therapy for disorders such as ND.

The authors have no financial interest to disclose regarding the article.