Concise Review: Laying the Groundwork for a First-In-Human Study of an Induced Pluripotent Stem Cell-Based Intervention for Spinal Cord Injury (original) (raw)

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Department of Orthopaedic Surgery, Keio University School of Medicine

, Tokyo,

Japan

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Department of Orthopaedic Surgery, Keio University School of Medicine

, Tokyo,

Japan

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Department of Orthopaedic Surgery, Keio University School of Medicine

, Tokyo,

Japan

Department of Physiology, Keio University School of Medicine

, Tokyo,

Japan

Regenerative & Cellular Medicine Office, Sumitomo Dainippon Pharma Co., Ltd.

, Kobe,

Japan

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Department of Rehabilitation Medicine, Keio University School of Medicine

, Tokyo,

Japan

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Department of Orthopaedic Surgery, Keio University School of Medicine

, Tokyo,

Japan

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Department of Physiology, Keio University School of Medicine

, Tokyo,

Japan

Search for other works by this author on:

Department of Orthopaedic Surgery, Keio University School of Medicine

, Tokyo,

Japan

Department of Physiology, Keio University School of Medicine

, Tokyo,

Japan

Search for other works by this author on:

Department of Orthopaedic Surgery, Keio University School of Medicine

, Tokyo,

Japan

Department of Physiology, Keio University School of Medicine

, Tokyo,

Japan

Search for other works by this author on:

Department of Orthopaedic Surgery, Keio University School of Medicine

, Tokyo,

Japan

Department of Physiology, Keio University School of Medicine

, Tokyo,

Japan

Search for other works by this author on:

Department of Physiology, Keio University School of Medicine

, Tokyo,

Japan

Regenerative & Cellular Medicine Office, Sumitomo Dainippon Pharma Co., Ltd.

, Kobe,

Japan

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Revision received:

16 August 2018

Accepted:

22 September 2018

Published:

12 November 2018

Cite

Osahiko Tsuji, Keiko Sugai, Ryo Yamaguchi, Syoichi Tashiro, Narihito Nagoshi, Jun Kohyama, Tsuyoshi Iida, Toshiki Ohkubo, Go Itakura, Miho Isoda, Munehisa Shinozaki, Kanehiro Fujiyoshi, Yonehiro Kanemura, Shinya Yamanaka, Masaya Nakamura, Hideyuki Okano, Concise Review: Laying the Groundwork for a First-In-Human Study of an Induced Pluripotent Stem Cell-Based Intervention for Spinal Cord Injury, Stem Cells, Volume 37, Issue 1, January 2019, Pages 6–13, https://doi.org/10.1002/stem.2926
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Abstract

There have been numerous attempts to develop stem cell transplantation approaches to promote the regeneration of spinal cord injury (SCI). Our multicenter team is currently planning to launch a first-in-human clinical study of an induced pluripotent stem cell (iPSC)-based cell transplant intervention for subacute SCI. This trial was conducted as class I regenerative medicine protocol as provided for under Japan's Act on the Safety of Regenerative Medicine, using neural stem/progenitor cells derived from a clinical-grade, integration-free human “iPSC stock” generated by the Kyoto University Center for iPS Cell Research and Application. In the present article, we describe how we are preparing to initiate this clinical study, including addressing the issues of safety and tumorigenesis as well as practical problems that must be overcome to enable the development of therapeutic interventions for patients with chronic SCI. Stem Cells 2019;37:6–13

Significance Statement

There is an increasing interest in the regenerative medicine using induced pluripotent stem cells (iPSCs). This article reviews the process in preparation for the first human trial of iPSC-based cell therapy for spinal cord injury, including addressing the issues of safety and tumorigenesis. Practical problems, which must be overcome to enable the development of therapeutic interventions for patients with chronic spinal cord injury, are described.

Ash1l can epigenetically promote the expression of essential osteogenic and chondrogenic transcription factors in C3H10T1/2 cells. It exerts this impact via modifications in the enrichment of H3K4me3 on their promoter regions. Ash1l hampered adipogenesis by enhancing H3K4me3 enrichment in promoter of Creb gene, which was reported to be a repressive gene of PPARγ.

Introduction

Spinal cord injury (SCI) results in severe neurological dysfunction, including motor, sensory, and autonomic paralysis. The annual incidence of SCI in Japan is approximately 5,000, and the prevalence of chronic SCI is approximately 150,000 [1]. This is consistent with a report that the annual incidence of SCI worldwide varies from 8.0 to 246.0 cases per million [2]. In current practice, treatments for acute SCI are limited to stabilization of the injured spine and decompression of the injured spinal cord in the acute stage, followed by rehabilitation to restore physical function and activity to the extent possible. No therapeutic approach has been established for regenerating injured spinal cord. However, the most recent guidelines suggested that blood pressure augmentation and high-dose steroids within 8 hours of injury would be treatment options for SCI [3].

In recent years, there have been many attempts to develop cell transplantation or drug therapies to promote regeneration of the damaged spinal cord. There are two major principal currents of research in regenerative medicine for SCI. The first involves stem cell-based interventions using neural stem/progenitor cells (NS/PCs) derived from fetal tissues [4], embryonic stem cells (ESCs) [5], Schwann cells [6], mesenchymal stem cells [7], olfactory ensheathing cells [8], or induced pluripotent stem cells (iPSCs) [9-11]. Recently, direct reprogramming techniques have also attracted significant attention as a source of NS/PCs [12-14]. The second focus is on the use of neurotrophic factors, such as hepatocyte growth factor [15], IL-10 [16], granulocyte colony stimulating factor [17], and reagents against axon growth inhibitors typified by Semaphorin 3A [18], Nogo [19], Rho [20], and chondroitin sulfate proteoglycans [21]. More effective treatments for SCI may be achievable through a combinatorial application involving both cells and neurotrophic factors.

Although the majority of SCI patients are in the chronic phase, most therapeutically focused studies to date have focused on the acute or subacute phase. Several recent reports showed that stem cell transplantation alone does not yield positive effects in chronic SCI models [22, 23], an issue that must be addressed in future studies. The establishment of effective treatments for chronic SCI patients with decreased therapeutic reactivity thus remains an urgent issue [24].

We have been conducting preclinical research to establish an iPSC-based approach to spinal regenerative medicine. The reason we chose to use iPSC is largely influenced by the current Japanese regulatory framework as well as a number of ethical considerations. Clinical trials using cells from other sources for treatment of SCI have already been started in the U.S., E.U., Pakistan, People's Republic of China, Korea, and elsewhere [25-30]. In these clinical trials, the safety and effectiveness of cells such as fetal-derived NS/PCs, ESC-derived NS/PCs, autologous mesenchymal stromal cells, umbilical cord blood mononuclear cells are being examined. However, clinical trials using iPSCs targeting SCI have not yet been conducted at this point.

In this review, we outline the preparations for first-in-human trials of iPSCs-based cell transplant interventions for subacute SCI under the new Japanese legal system for regenerative medicine and prospects for the development of new interventions for chronic SCI.

iPSCs-Based Cell Therapy and Japan's New Law Systems on Regenerative Medicine

In 2006, it was discovered that mature somatic cells can be reprogrammed to a pluripotent state by gene transfer, generating iPSCs [31]. These cells exhibit pluripotent stemness similar to that of ESCs, and were first established by introducing four reprogramming factors (Sox2, Oct3/4, c-Myc, and Klf4) into somatic cells. Another attractive feature of iPSCs is that they can be derived from a patient's own somatic cells; a clinical research study of autologous iPSC-derived retinal pigment epithelium (RPE) sheet transplantation for exudative age-related macular degeneration patient was initiated in September 2014 by Masayo Takahashi's group in Kobe, Japan, representing the first clinical study involving an iPSC-derived product. The engrafted iPSC-RPE sheet showed no sign of immune rejection for the 1-year monitoring period following transplantation [32].

However, the costs of quality testing and safety concerns for autologous cell transplants have led to increased interest in allogeneic strategies. In an effort to mitigate these practical problems, Japan has launched a program to develop an “iPSC stock” derived from “human leukocyte antigen (HLA) super-donors,” who are homozygous at the three major HLA gene loci, to maintain a pool of safe iPSC clones corresponding to various HLA types. This program is performed mainly by Center for iPS Cell Research and Application (CiRA) at Kyoto University, which contributed greatly to the development of iPSC technology. For example, frequencies of HLA-A*24:02; HLA-B*52:01; HLA-DRB1*15:02 haplotypes in the Japanese population are reported to be the most frequent (8.5%), and iPSCs established from the individuals who are homozygous for this haplotype are expected to be immunologically matched to approximately 17% of the Japanese population. It has been estimated that an iPSC bank comprised of ~140 unique HLA-homozygous donors would cover ~90% of the Japanese population [33]. Globally, an iPSC bank comprising 100 iPSC lines representing the most frequent HLA types from each major ethnic background would cover 78% of U.S. residents of European heritage, 63% of Asian, 52% of Hispanic, and 45% of African American [34]. Masayo Taka shashi's group recently transplanted an allogeneic cell suspension of iPSC-RPE derived from a super-donor carrying the most common HLA haplotype in Japan, which matched the recipient's HLA type, following in vivo animal studies in which macaque monkey iPSC-RPE cells derived from Major Histocompatibility Complex (MHC) homozygote iPSC lines were successfully transplanted into the eyes of MHC-matched heterozygote donors without immunosuppression [35].

Other research groups plan to use allogenic and HLA-unmatched iPSCs as donors of transplantation. The Australian company Cynata Therapeutics plans to study potential clinical uses of iPSC-derived mesenchymal stromal cells (MSCs) generated from healthy donors as immune suppressor cells in graft-versus-host disease patients in the UK and other countries (http://cynata.com/). Curtis et al. transplanted unmatched fetus-derived NS/PCs into chronic SCI patients (n = 4) and reported that no rejection reaction was observed, even after the termination of a temporary immunosuppressant [25]. Recently, the Japanese government health ministry approved a plan proposed by Yoshiki Sawa's group at Osaka University group to begin a pilot study of iPSC-derived cardiomyocyte cell sheet transplantation in heart failure patients; this study will use allogenic iPSCs from the CiRA cell bank. Our own group is currently proposing the first human trial of allogenic iPSC-based cell transplantation for subacute SCI, also using CiRA-derived iPSC. If approved, this study was conducted under the terms set forth in the Act on the Safety of Regenerative Medicine (ASRM).

In 2014, Japan introduced two legal reforms; the ASRM and a set of amendments to the Pharmaceuticals, Medical Devices and Other Therapeutic Products Act (PMD Act). The PMD Act governs the review and approval of “regenerative medical products” intended for commercial distribution, although acknowledging the heterogeneity of cells used in medical products [36]. Notably, the PMD Act introduced a new pathway for conditional and time-limited approval of regenerative medical products.

In contrast, the ASRM governs the development and use of regenerative medicine in both noncommercialized academic clinical studies and private medical practices operating outside the national health insurance system. It adopts a risk-based approach to strengthen safety oversight [37]. The Act classifies regenerative medicine in three categories: class I (high risk); class II (medium risk); and class III (low risk) [36, 37]). iPSC-based cell transplantation falls in the high-risk group (class I), along with approaches using ESCs, transgenic or genetically modified cells, xenogeneic cells, and allogeneic cells [36]. Under ASRM, any medical institution that plans to conduct a clinical study of or offer iPSC-based cell therapies must undergo review by a Certified Special Committee for Regenerative Medicine (CSCRM) as class I regenerative medicine techniques [37].

Preparing the First Human Trial of iPSCs-Based Cell Therapy for SCI of Subacute Phase

To date our group has focused on applications of NS/PCs and reported positive therapeutic effects from the use of rodent (rat/mouse) fetus-derived NS/PCs for mouse/rat injured spinal cord (contusion injury model) at the subacute phase [38, 39]. We additionally transplanted human fetal-derived NS/PCs into nonhuman primate (common marmoset) injured spinal cord, and observed positive effects on motor function recovery [4]. Likewise, significant motor function recovery was reported for NS/PCs derived from mouse ESCs in a mouse SCI model at the subacute phase [40]. However, Japanese governmental guidelines over stem cell research and development that were in effect from 2006 to 2014 prevented us from initiating clinical research using fetus- or ESC-derived NS/PCs, as such studies would involve harvesting cells from aborted fetuses or surplus embryos from in vitro fertilization attempts. iPSCs, which were established in 2006 [31], have made it possible to avoid some of the ethical and regulatory issues surrounding the use of ESC- or fetus-derived cells.

Together with the invention of the iPSC-technology, the above-mentioned restricted situation led us to start the research into clinical application of iPSC-derived NS/PCs (iPSC-NS/PCs)-transplantation for SCI in collaboration with Kyoto University since 2006. As a first step, we developed a method for preparing NS/PCs from mouse [41] and human iPSCs [10], and determined that transplantation of mouse iPSC-NS/PCs into a mouse SCI model and of human iPSC-NS/PCs into an immune-deficient mouse SCI model at the subacute phase promotes motor function recovery and improves motor evoked potential [9, 10]. Moreover, we transplanted human iPSC-NS/PCs into a common marmoset SCI model [4] and found that human iPSC-NC/PCs are able to differentiate into neural trilinage cells (neurons: 52%; astrocytes: 31%; and oligodendrocytes: 27%), form synaptic connections with host neurons, reduce post-SCI demyelination (preservation of 1.5–2 times larger myelinated areas), and consequently promote better motor function recovery [11]. Several mechanisms may support this functional recovery after stem cell-derived NS/PCs transplantation, including (a) creating a permissive substrate for axonal growth; (b) providing cells that remyelinate spared but demyelinated axons; (c) supplying trophic support reducing the damage and rescuing neurons and oligodendrocytes; and (d) enhancing axonal plasticity and replacing lost neurons to reconstruct local circuitry [42-44]. These iPSC-NS/PCs transplantation experiments were conducted in models at the subacute phase of SCI, not at the chronic phase (Fig. 1).

Figure 1

Therapeutic strategy for spinal cord injury (SCI) based on changes in the microenvironment. The microenvironment within SCI changes over the time course following the primary mechanical trauma resulting in the SCI (modified from [45]). Analysis of this time course suggests that 14–28 days postinjury, that is, the subacute phase, is the most appropriate for stem cell transplantation in human patients. It has been reported that neural stem/progenitor cell (NS/PC) transplantation alone is not sufficient to induce significant functional recovery in chronic SCI. However, given the synergistic effects of rehabilitation and NS/PC transplantation in chronic SCI model mice [46], a combination intervention involving transplantation of induced pluripotent stem cell-NS/PCs and use of a robotic device are being planned for future clinical studies.

At the same time, we discovered that there are “safe” and “dangerous” human iPSC clones, and that transplantation of “dangerous” human iPSC-clone-derived NS/PCs resulted in neural tumor-like proliferation of transplants by 103 days postinjury (dpi) [47]. The proliferated tissue was substantially different from teratoma (a form of benign tumor that results from contamination of undifferentiated human iPSCs), and consisted of immature neural cells exhibiting differentiation-resistance. The following analysis revealed that such “dangerous human iPSC-NSPCs” can result from genetic instabilities, such as transgene activation [47], epigenetic events [48], or other genetic modifications such as abnormal karyotype or copy number variations (CNVs) [49] that occur or increase over the course of the culture process. Pre-evaluation of iPSCs and iPSC-NSPCs before transplantation is thus essential for the detection of “dangerous” iPSC-NS/PCs, as is the establishment of fail-safe systems after human iPSC-NS/PC transplantation.

There are five key issues in improving the safety of human iPSC-NS/PCs: (a) preventing the use of genetically unstable human iPSCs; (b) preventing contamination by undifferentiated pluripotent cells in iPSC-NS/PCs; (c) preventing the transformation of progenitor cells into “dangerous iPSC-NS/PCs;” (d) minimizing the risk of proliferation of differentiation-resistant “dangerous iPSC-NS/PCs” in vivo; and (5) removal/ablation of cells after the transplant. In (a)–(c), quality monitoring of iPSC clones and derivative iPSC-NS/PCs in vitro is indispensable. in vitro quality checks of iPSCs require evaluation of the completeness of reprograming status (surface markers, capacity of cellular differentiation, and so on) and genetic stability (karyotypes, CNVs, and so on).

Additionally, to prepare clinical grade human iPSC-NS/PCs, it is important to establish a procedure for obtaining NS/PCs from feeder-free human iPSCs using a xeno-free medium [50]. The evaluation of each iPSC clone-derived NS/PCs in vitro must include confirmation of completeness of the cellular induction process, by using the target NS/PC markers (e.g., SOX1 and PSA-NCAM) and pluripotency markers for the original iPSCs (e.g., OCT3/4 and TRA1-60), preferably by more than two methods, such as fluorescence activated cell sorting (using surface markers), sequencing of RNAs, and immunocytochemistry of intracellular phenotypic markers. The function of induced cells (cellular shape, cell cycle status, proliferation speed, survival rate, and so on), genomic information (karyotype, gene sequences, CNVs, and so on), and the cellular terminal differentiation capacity should also be evaluated. Additionally, since DNA methylation status is known to change over the culture period, the number of days required for the culture process and the DNA methylation status of the final iPSC-NS/PCs should also be monitored [48].

To minimize the risk of proliferation of differentiation-resistant “dangerous iPSC-NSPCs” in vivo (the fourth issue mentioned above), we focused on gamma secretase inhibitor (GSI), which inhibits Notch signaling, which is crucial for maintaining the undifferentiated state. We found that pretreatment of iPSC-NS/PCs with GSI before transplantation promoted neuronal differentiation and prevented tumor formation, even for “dangerous” clone-derived NS/PCs [51, 52].

As the last issue to enhance cell safety, the simplest approach for removal of cells is surgical resection of the transplant. However, in cases in which the transplanted cells invade or metastasize to other sites, the effectiveness of surgical approaches may be limited. Thus, other approaches, including the use of immune-rejection [53], or the use of a “suicide transgene” [54] may be needed for human clinical studies in the future. However, the introduction of a suicide gene system in the clinical setting would involve heightened oversight, since this would require viral (AAV or lentivirus) transduction of the gene into the graft.

In vivo analyses are also essential in preclinical safety testing of iPSC-based cell interventions for SCI. For this, we adopted two methods: transplanting the cells into intact striata of immunodeficient mice, or transplanting the cells into the injured spinal cords of immunodeficient mice [49]. By comparing histology 12–26 weeks after the injection of iPSC-NSPCs, we confirmed that both methods produce equivalent results. Use of intact striata is technically easier, and the mice survive longer, which makes it easier to study long-term safety. On the other hand, the use of injured mice has the advantage of being more similar to the state of human patients. However, the injured spinal cord in mouse is quite different in terms of graft volume, that is, the estimated maximum cell number that can be transplanted into the injured cord was less than that of the brain (striata). Moreover, in both model cases, it is extremely difficult to obtain longer survival (over 1 year) for immune-deficient mice. The method will thus need to continue to be optimized in the future.

Although recently the generation time of autologous human iPSC-NS/PCs has been drastically reduced in the research setting [55, 56], the establishment of autologous human iPSC-NS/PCs takes at least 6 months, and several additional months are needed for safety testing. If quality testing were to be performed for all SCI patient-derived autologous iPSCs, the costs would be extremely high. Thus the issues of time and expense represent significant obstacles to autologous transplantation of iPSC-NS/PCs to SCI patients in the subacute phase [57]. Given these issues, in our planned first-in-human clinical study, we opted to use clinical-grade allogenic iPSC clones produced by CiRA, which have already passed through quality and safety tests. Based on our series of investigations on the safety issues, we developed the draft of standard operating procedures to prepare iPSC-NS/PCs for use in the proposed clinical study, in which cells were transplanted to SCI patients (Fig. 2). These plans are currently under evaluation as a class I regenerative medicine protocol by a CSCRM, pursuant to the ASRM.

Figure 2

Overview of induced pluripotent stem cell-neural stem/progenitor cell (iPSCs-NS/PCs) “first-in-man” clinical trial. The iPSCs-NS/PCs “first-in-man” clinical trial for SCI patients at subacute phase are being planned using the clinical grade iPSCs-NS/PCs generated at Keio University School of Medicine and Osaka National Hospital, National Hospital Organization from human “iPSC stock” prepared in CiRA [37, 57]. The clinical grade iPSC-NS/PCs were kept at frozen state until a few days before the surgery. iPSCs-NS/PCs were transplanted into injured spinal cords of the patients of subacute phase (14–28 days postinjury). After the transplantation, patients were followed-up in terms of safety.

We believe that our proposed clinical trial program must meet higher safety standards than interventions using cells from other sources, due to the gene transfer process used in generating iPSCs and the long culture period for differentiation. However, even allowing for these heightened safety considerations, the use of iPSC-derived cells remains attractive for several reasons, including their pluripotency, lower ethical tensions, clearer regulatory provisions, and in the case of autologous iPSCs, the reduced risk of immune rejection [58]. Indeed, iPSCs are used in spinal cord regeneration studies worldwide. Lu et al. reported that iPSC-derived NS/PCs exhibit remarkable axonal outgrowth in the injured spinal cord, even when the cells are derived from an elderly person [59]. Ruzicka et al. reported the superiority of the therapeutic effects of iPSC derived neural progenitors compared with bone marrow-derived MSCs and fetus-derived neural progenitors in a rat SCI model [60]. Strnadel et al. reported that transient immunosuppression was sufficient following transplantation of allogenic iPSC-NS/PC in a porcine model [61]. Fujimoto et al. generated human iPSC-derived long-term self-renewing neuroepithelial-like stem cells, and reported the effectiveness of these cells in a subacute contusive SCI model in immunodeficient mice [62]. All these reports support the utility of iPSCs as a cell source for studies of cell transplantation in SCI.

Cell Transplantation Therapy and Rehabilitation for Chronic SCI

The development of an effective therapy for chronic SCI is a key goal in SCI research. Many groups have conducted studies aimed at uses of cell transplantation, including iPSCs-derived NS/PCs for chronic SCI. Recently, the first human trial for chronic SCI (n = 4) using fetal NS/PCs was reported [25], which primarily focused on transplantation safety. However, previous studies have indicated that functional recovery by cell transplantation alone is not clear for SCI at chronic stage. For example, Kumamaru et al. used whole transcriptome analyses to show that, although grafted NS/PCs produce many regenerative/neurotrophic molecules and the chronically injured spinal cord exhibits the potential to permit NS/PCs to differentiate into neurons and oligodendrocytes, NS/PCs did not improve locomotor function in the chronic stage. In this regard, the authors highlight the importance of environmental modulation [22]. Consistent to Kumamaru's report, our group also conducted similar experiments and reported that there was no functional recovery in the chronic phase (42 dpi), whereas there was no significant difference in the transplants’ viability or differentiation rate between the subacute (9 dpi) and chronic phase (42 dpi) [23]. We speculated that within the damaged spinal cord microenvironment, the glial scar formation and inflammatory phenotype may be the most efficient targets to modify to achieve functional recovery by NS/PC transplantation at the chronic phase.

To gain more insight into chronic contuse SCI, we referred to analyses of transplantation of Neurotrophin-3 (NT-3)-expressing rat fetus-derived NS/PCs into the contusive injured spinal cord of rat at the chronic phase. Although there was no improvement in motor function in transplantation of normal NS/PCs, NT-3-expressing NS/PCs enhanced motor function recovery and remyelination in a rat thoracic SCI model, even at the chronic stage (42 dpi; Basso, Beattie and Bresnahan scale (BBB) subscore at 84 dpi showed significant recovery) [63]. It has been reported that NT-3 expression is significantly increased in the injured spinal cord (rat hemisection model) at early-chronic phase (28 dpi) following walking training [64]. We suggested that NS/PC-transplantation combined with walking training might produce synergistic effects on motor function recovery in chronic SCI. However, there are very few reports on the combination of NS/PCs-transplantation and rehabilitation. There have, however, been several reports on combination use of rehabilitation and cell transplantation other than NS/PCs. In 2011, Takeoka et al. reported the effects of combination of rehabilitation with olfactory ensheathing glia (OEG) grafts. They transplanted OEGs immediately after transection injury of rat thoracic spinal cord and started treadmill bipedal exercise with partial weight support, and found that this promoted a fourfold increase in regenerating axons within the caudal stump of transected spinal cords, and consequently improved hindlimb function and electrophysiological states [65]. Sun et al. transplanted both OEGs and Schwann cells into a moderate contusion-injured rat spinal cord at 14 dpi, and reported that, in combination with treadmill training, this contributed to enhanced motor function recovery (BBB score: 13) compared with the controls (BBB score: 10) [66]. In a study of combined NS/PCs-transplantation and rehabilitation, Hwang et al. transplanted NS/PCs into contused rat spinal cord at 7 days after injury, followed by treadmill training with partial weight support for 8 weeks. The combination approach promoted motor function recovery (BBB score: 16) compared with the control (BBB score: 9), enhanced tissue protection, and preserved more myelinated area. The grafted NS/PCs differentiated into more neurons and oligodendrocytes (30% βIII tubulin+ neurons and 25% CC1+ oligodendrocytes) compared with the control group (20% neurons and oligodendrocytes), and the number of Nestin+ undifferentiated cells decreased (control: 25% and treadmill: 15%) at 9 weeks postinjury, which the authors attributed to the reduction of stress caused by active oxygen or active nitrogen through IGF-1 signaling [67].

Thus, our understanding of the synergistic effects of cell transplantation combined with rehabilitation at the chronic phase continues to expand. However, the pathology of chronic SCI remains to be elucidated. We analyzed the mechanisms underlying spasticity, which is a major problem in chronic SCI patients, and found that in a contuse SCI rat model, the expression of BDNF was increased at the lumbar enlargement of spinal cord after treadmill training, resulting in increased expression of KCC2 and suppression of spasticity [67]. We also studied the combination of treadmill training and transplantation of mouse fetus-NS/PCs for mice thoracic cord contusion injury from 7 weeks after injury (chronic stage), and found improved hind limb motor function in the combined group (NS/PCs + treadmill). This superior functional recovery, even at the chronic stage, was enabled by the combined effects of the improvement of injured spinal cord electrical conduction, newly developed fibers and synapses within the central pattern generator at the lumbar enlargement, and appropriate motor control [46]. In a study of treadmill training in chronic SCI, we confirmed improvement of hind limb motor function by concurrent use of chondroitinase ABC (C-ABC) via intrathecal continuous administration (BBB score: 6 at 14 weeks postinjury, compared with the control BBB score: 3) for severe rat thoracic chronic contusion injury model. The mechanism of functional recovery in this system was attributed to an increase in serotonergic nerve fibers at the lesion site [68].

Conclusion

In this review, we described the preparation for first-in-human transplantation of iPSC-NS/PCs for subacute-complete SCI patients. After confirmed safety and further efficacy of this treatment, there would still be a long way to put this technology into practical use in general. Meanwhile, there are various situations such as incomplete injury or transection injury in the state of spinal cord injury, and some additional approach seems to be necessary to treat them. Further development of future treatment is expected.

Future Prospects and Assignments

In this review, we have outlined studies on the pathology and treatment of subacute to chronic SCI. Overcoming chronic SCI remains the most formidable outstanding challenge for SCI researchers. Several important findings on stem cell transplantation and medication approaches for chronic SCI have been reported in recent years, but none promises to fully restore function. The trinity of stem cell transplantation, medication, and rehabilitation thus appears to be the way forward. We need to aim at the most effective reconstruction of organized neural circuits by iPSCs-based cell therapy after chemical suppression of axon growth inhibitory factors at the lesion site, although pursuing further rehabilitation, including future applications such as robotic engineering, functional electrical stimulation, and their combination.

Acknowledgments

We thank all of the members of 5S7, Dr. Hideyuki Okano's, Dr. Masaya Nakamura's, and Dr. Shinya Yamanaka's laboratories for their encouragement and generous support. We are grateful to Drs. Akio Iwanami, Satoshi Nori, Yoshiomi Kobayashi, Soraya Nishimura, Tsunehiko Konomi, Morito Takano, Akimasa Yasuda, Masahiro Ozaki, Yuichiro Nishiyama, Soya Kawabata, Kohei Matsubayashi, Shinjiro Kaneko, and Kyoko Miura for their significant contributions to the series of our studies, and to Drs. Francois Renault-Mihara and Shinsuke Shibata for their invaluable scientific and technical advice. We also thank the Center for iPS Cell Research and Application, Kyoto University, for the human iPSC clones, and Osaka National Hospital, National Hospital Organization for the human iPSC-NSPC clones, described in the original works cited in the present paper. This study was supported by the Research Center Network for Realization of Regenerative Medicine (Centers for Clinical Application Research on Specific Disease/Organ); Grant No. [18bm0204001h0006]) and Grant No. [18bk01045h003] from Research Project for Practical Applications of Regenerative Medicine, Japan Agency for Medical Research and Development (AMED).

Author Contributions

O.T., K.S., H.O.: manuscript writing; M.N., H.O.: administrative support; O.T., N.N., J.K., R.Y., K.S., T.I., T.O., M.I., T.S., K.F., S.T., N.N., M.S., K.F., K.Y., and S.Y.: played major roles in the designing and conducting a wide range of studies needed to justify a first-in-human of iPSCs-based cell therapy for SCI. All of the authors have approved of the final manuscript.

Disclosure of Potential Conflicts of Interest

H.O. receives financial compensation as a founding scientist of SanBio Co., Ltd. and K-Pharma, Inc. M.N. is a founding scientist of K-Pharma, Inc. R.Y. and M.I. declared leadership position with Sumitomo Dainippon Pharma Co., Ltd. S.Y. declared consulting, research funding from iPS Academia Japan. The other authors indicated no potential conflicts of interest.

References

Shingu

Ohama

Ikata

et al.

A nationwide epidemiological survey of spinal cord injuries in Japan from January 1990 to December 1992

Paraplegia

1995

;

183

–

188

Furlan

Sakakibara

Miller

et al.

Global incidence and prevalence of traumatic spinal cord injury

Can J Neurol Sci

2013

;

456

–

464

Ahuja

Shroeder

Vaccaro

et al.

Spinal cord injury-what are controversies?

J Orthop Trauma

2017

;

Suppl 4

–

S13

Iwanami

Kaneko

Nakamura

et al.

Transplantation of human neural stem cells for spinal cord injury in primates

J Neurosci Res

2005

;

182

–

190

McDonald

Liu

et al.

Transplanted embryonic stem cells survive, differentiate and promote recovery in injured rat spinal cord

Nat Med

1999

;

1410

–

1412

Guest

Santamaria

Benavides

Clinical translation of autologous Schwann cell transplantation for the treatment of spinal cord injury

Curr Opin Organ Transplant

2013

;

682

–

689

Neirinckx

Cantinieaux

Coste

et al.

Concise review: Spinal cord injuries: How could adult mesenchymal and neural crest stem cells take up the challenge?

Stem Cells

2014

;

829

–

843

Mackay-Sim

St John

Olfactory ensheathing cells from the nose: Clinical application in human spinal cord injuries

Exp Neurol

2011

;

229

174

–

180

Tsuji

Miura

Okada

et al.

Therapeutic potential of appropriately evaluated safe-induced pluripotent stem cells for spinal cord injury

Proc Natl Acad Sci USA

2010

;

107

12704

–

12709

Nori

Okada

Yasuda

et al.

Grafted human-induced pluripotent stem-cell-derived neurospheres promote motor functional recovery after spinal cord injury in mice

Proc Natl Acad Sci USA

2011

;

108

16825

–

16830

Kobayashi

Okada

Itakura

et al.

Pre-evaluated safe human iPSC-derived neural stem cells promote functional recovery after spinal cord injury in common marmoset without tumorigenicity

PLoS ONE

2012

;

e52787

Kim

Efe

Zhu

et al.

Direct reprogramming of mouse fibroblasts to neural progenitors

Proc Natl Acad Sci USA

2011

;

108

7838

–

7843

Lujan

Chanda

Ahlenius

et al.

Direct conversion of mouse fibroblasts to self-renewing, tripotent neural precursor cells

Proc Natl Acad Sci USA

2012

;

109

2527

–

2532

Matsui

Takano

Yoshida

et al.

Neural stem cells directly differentiated from partially reprogrammed fibroblasts rapidly acquire gliogenic competency

Stem Cells

2012

;

1109

–

1119

Kitamura

Fujiyoshi

Yamane

et al.

Human hepatocyte growth factor promotes functional recovery in primates after spinal cord injury

PLoS ONE

2011

;

e27706

Thompson

Zurko

Hanna

et al.

The therapeutic role of interleukin-10 after spinal cord injury

J Neurotrauma

2013

;

1311

–

1324

Inada

Takahashi

Yamazaki

et al.

Multicenter prospective nonrandomized controlled clinical trial to prove neurotherapeutic effects of granulocyte colony-stimulating factor for acute spinal cord injury: Analyses of follow-up cases after at least 1 year

Spine

2014

;

213

–

219

Kaneko

Iwanami

Nakamura

et al.

A selective Sema3A inhibitor enhances regenerative responses and functional recovery of the injured spinal cord

Nat Med

2006

;

1380

–

1389

Zorner

Schwab

Anti-Nogo on the go: From animal models to a clinical trial

Ann N Y Acad Sci

2010

;

1198

(

Suppl 1

E22

–

E34

Kopp

Liebscher

Watzlawick

et al.

SCISSOR-spinal cord injury study on small molecule-derived Rho inhibition: A clinical study protocol

BMJ Open

2016

;

e010651

Bradbury

Moon

Popat

et al.

Chondroitinase ABC promotes functional recovery after spinal cord injury

Nature

2002

;

416

636

–

640

Kumamaru

Saiwai

Kubota

et al.

Therapeutic activities of engrafted neural stem/precursor cells are not dormant in the chronically injured spinal cord

Stem Cells

2013

;

1535

–

1547

Nishimura

Yasuda

Iwai

et al.

Time-dependent changes in the microenvironment of injured spinal cord affects the therapeutic potential of neural stem cell transplantation for spinal cord injury

Mol Brain

2013

;

Ghobrial

Anderson

Dididze

et al.

Human neural stem cell transplantation in chronic cervical spinal cord injury: Functional outcomes at 12 months in a phase II clinical trial

Neurosurgery

2017

;

–

Curtis

Martin

Gabel

et al.

A first-in-human, phase I study of neural stem cell transplantation for chronic spinal cord injury

Cell Stem Cell

2018

;

941.e6

–

950.e6

Priest

Manley

Denham

et al.

Preclinical safety of human embryonic stem cell-derived oligodendrocyte progenitors supporting clinical trials in spinal cord injury

Regen Med

2015

;

939

–

958

Vaquero

Zurita

Rico

et al.

Intrathecal administration of autologous mesenchymal stromal cells for spinal cord injury: Safety and efficacy of the 100/3 guideline

Cytotherapy

2018

;

806

–

819

Satti

Waheed

Ahmed

et al.

Autologous mesenchymal stromal cell transplantation for spinal cord injury: A phase I pilot study

Cytotherapy

2016

;

518

–

522

Shin

Kim

Yoo

et al.

Clinical trial of human fetal brain-derived neural stem/progenitor cell transplantation in patients with traumatic cervical spinal cord injury

Neural Plast

2015

;

2015

630932

Zhu

Poon

Liu

et al.

Phase I-II clinical trial assessing safety and efficacy of umbilical cord blood mononuclear cell transplant therapy of chronic complete spinal cord injury

Cell Transplant

2016

;

1925

–

1943

Takahashi

Yamanaka

Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors

Cell

2006

;

126

663

–

676

Mandai

Watanabe

Kurimoto

et al.

Autologous induced stem-cell-derived retinal cells for macular degeneration

N Engl J Med

2017

;

376

1038

–

1046

Okita

Matsumura

Sato

et al.

A more efficient method to generate integration-free human iPS cells

Nat Methods

2011

;

409

–

412

Gourraud

Gilson

Girard

et al.

The role of human leukocyte antigen matching in the development of multiethnic “haplobank” of induced pluripotent stem cell lines

Stem Cells

2012

;

180

–

186

Sugita

Iwasaki

Makabe

et al.

Successful transplantation of retinal pigment epithelial cells from MHC homozygote iPSCs in MHC-matched models

Stem Cell Rep

2016

;

635

–

648

Konomi

Tobita

Kimura

et al.

New Japanese initiatives on stem cell therapies

Cell Stem Cell

2015

;

350

–

352

Sipp

Okano

Japan strengthens regenerative medicine oversight

Cell Stem Cell

2018

;

153

–

156

Ogawa

Sawamoto

Miyata

et al.

Transplantation of in vitro-expanded fetal neural progenitor cells results in neurogenesis and functional recovery after spinal cord contusion injury in adult rats

J Neurosci Res

2002

;

925

–

933

Okada

Ishii

Yamane

et al.

In vivo imaging of engrafted neural stem cells: Its application in evaluating the optimal timing of transplantation for spinal cord injury

FASEB J

2005

;

1839

–

1841

Kumagai

Okada

Yamane

et al.

Roles of ES cell-derived gliogenic neural stem/progenitor cells in functional recovery after spinal cord injury

PLoS ONE

2009

;

e7706

Miura

Okada

Aoi

et al.

Variation in the safety of induced pluripotent stem cell lines

Nat Biotechnol

2009

;

743

–

745

Nagoshi

Okano

iPSC-derived neural precursor cells: Potential for cell transplantation therapy in spinal cord injury

Cell Mol Life Sci

2018

;

989

–

1000

Wang

Graham

et al.

Long-distance growth and connectivity of neural stem cells after severe spinal cord injury

Cell

2012

;

150

1264

–

1273

Barnabe-Heider

Frisen

Stem cells for spinal cord repair

Cell Stem Cell

2008

;

–

Nakamura

Okano

Cell transplantation therapies for spinal cord injury, focusing on induced pluripotent stem cells

Cell Res

2013

;

–

Tashiro

Nishimura

Iwai

et al.

Functional recovery from neural stem/progenitor cell transplantation combined with treadmill training in mice with chronic spinal cord injury

Sci Rep

2016

;

30898

Nori

Okada

Nishimura

et al.

Long-term safety issues of iPSC-based cell therapy in a spinal cord injury model: Oncogenic transformation with epithelial–mesenchymal transition

Stem Cell Rep

2015

;

360

–

373

Iida

Iwanami

Sanosaka

et al.

Whole-genome DNA methylation analyses revealed epigenetic instability in tumorigenic human iPS cell-derived neural stem/progenitor cells

Stem Cells

2017

;

1316

–

1327

Sugai

Fukuzawa

Shofuda

et al.

Pathological classification of human iPSC-derived neural stem/progenitor cells towards safety assessment of transplantation therapy for CNS diseases

Mol Brain

2016

;

Isoda

Kohyama

Iwanami

et al.

Robust production of human neural cells by establishing neuroepithelial-like stem cells from peripheral blood mononuclear cell-derived feeder-free iPSCs under xeno-free conditions

Neurosci Res

2016

;

110

–

Okubo

Iwanami

Kohyama

et al.

Pretreatment with a gamma-secretase inhibitor prevents tumor-like overgrowth in human iPSC-derived transplants for spinal cord injury

Stem Cell Rep

2016

;

649

–

663

Ogura

Morizane

Nakajima

et al.

Gamma-secretase inhibitors prevent overgrowth of transplanted neural progenitors derived from human-induced pluripotent stem cells

Stem Cells Dev

2013

;

374

–

382

Itakura

Kobayashi

Nishimura

et al.

Controlling immune rejection is a fail-safe system against potential tumorigenicity after human iPSC-derived neural stem cell transplantation

PLoS ONE

2015

;

e0116413

Itakura

Kawabata

Ando

et al.

Fail-safe system against potential tumorigenicity after transplantation of iPSC derivatives

Stem Cell Rep

2017

;

673

–

684

Turner

Leslie

Martin

et al.

Toward the development of a global induced pluripotent stem cell library

Cell Stem Cell

2013

;

382

–

384

Fujimori

Matsumoto

Kisa

et al.

Escape from pluripotency via inhibition of TGF-beta/BMP and activation of Wnt signaling accelerates differentiation and aging in hPSC progeny cells

Stem Cell Rep

2017

;

1675

–

1691

Okano

Yamanaka

iPS cell technologies: Significance and applications to CNS regeneration and disease

Mol Brain

2014

;

Khazaei

Ahuja

Fehlings

Induced pluripotent stem cells for traumatic spinal cord injury

Front Cell Dev Biol

2016

;

152

Woodruff

Wang

et al.

Long-distance axonal growth from human induced pluripotent stem cells after spinal cord injury

Neuron

2014

;

789

–

796

Ruzicka

Machova-Urdzikova

Gillick

et al.

A comparative study of three different types of stem cells for treatment of rat spinal cord injury

Cell Transplant

2017

;

585

–

603

Strnadel

Carromeu

Bardy

et al.

Survival of syngeneic and allogeneic iPSC-derived neural precursors after spinal grafting in minipigs

Sci Transl Med

2018

;

eaam6651

Fujimoto

Abematsu

Falk

et al.

Treatment of a mouse model of spinal cord injury by transplantation of human induced pluripotent stem cell-derived long-term self-renewing neuroepithelial-like stem cells

Stem Cells

2018

;

1063

–

1073

Kusano

Enomoto

Hirai

et al.

Transplanted neural progenitor cells expressing mutant NT3 promote myelination and partial hindlimb recovery in the chronic phase after spinal cord injury

Biochem Biophys Res Commun

2010

;

393

812

–

817

Ying

Roy

Edgerton

et al.

Exercise restores levels of neurotrophins and synaptic plasticity following spinal cord injury

Exp Neurol

2005

;

193

411

–

419

Takeoka

Jindrich

Munoz-Quiles

et al.

Axon regeneration can facilitate or suppress hindlimb function after olfactory ensheathing glia transplantation

J Neurosci

2011

;

4298

–

4310

Sun

Zhang

et al.

Cotransplantation of olfactory ensheathing cells and Schwann cells combined with treadmill training promotes functional recovery in rats with contused spinal cords

Cell Transplant

2013

;

(

Suppl 1

S27

–

S38

Hwang

Shin

Kwon

et al.

Survival of neural stem cell grafts in the lesioned spinal cord is enhanced by a combination of treadmill locomotor training via insulin-like growth factor-1 signaling

J Neurosci

2014

;

12788

–

12800

Shinozaki

Iwanami

Fujiyoshi

et al.

Combined treatment with chondroitinase ABC and treadmill rehabilitation for chronic severe spinal cord injury in adult rats

Neurosci Res

2016

;

113

–

Sugai

Nishimura

Kato-Negishi

et al.

Neural stem/progenitor cell-laden microfibers promote transplant survival in a mouse transected spinal cord injury model

J Neurosci Res

2015

;

1826

–

1838

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Concise Review: Laying the Groundwork for a First-In-Human Study of an Induced Pluripotent Stem Cell-Based Intervention for Spinal Cord Injury (original) (raw)

Cite

Abstract

Introduction

iPSCs-Based Cell Therapy and Japan's New Law Systems on Regenerative Medicine

Preparing the First Human Trial of iPSCs-Based Cell Therapy for SCI of Subacute Phase

Cell Transplantation Therapy and Rehabilitation for Chronic SCI

Conclusion

Future Prospects and Assignments

Acknowledgments

Author Contributions

Disclosure of Potential Conflicts of Interest

References

Citations

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Concise Review: Laying the Groundwork for a First-In-Human Study of an Induced Pluripotent Stem Cell-Based Intervention for Spinal Cord Injury (original) (raw)

Cite

Abstract

Introduction

iPSCs-Based Cell Therapy and Japan's New Law Systems on Regenerative Medicine

Preparing the First Human Trial of iPSCs-Based Cell Therapy for SCI of Subacute Phase

Cell Transplantation Therapy and Rehabilitation for Chronic SCI

Conclusion

Future Prospects and Assignments

Acknowledgments

Author Contributions

Disclosure of Potential Conflicts of Interest

References

Citations

Views

Altmetric

Email alerts

Citing articles via

Latest

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