Writing in the journal Stem Cells Translational Medicine, an international research team, led by physician-scientists at University of California San Diego School of Medicine, describe a new method for delivering neural precursor cells (NSCs) to spinal cord injuries in rats, reducing the risk of further injury and boosting the propagation of potentially reparative cells.
NSCs hold great potential for treating a variety of neurodegenerative diseases and injuries to the spinal cord.
The stem cells possess the ability to differentiate into multiple types of neural cell, depending upon their environment.
As a result, there is great interest and much effort to use these cells to repair spinal cord injuries and effectively restore related functions.
But current spinal cell delivery techniques, said Martin Marsala, MD, professor in the Department of Anesthesiology at UC San Diego School of Medicine, involve direct needle injection into the spinal parenchyma — the primary cord of nerve fibers running through the vertebral column. “As such, there is an inherent risk of (further) spinal tissue injury or intraparechymal bleeding,” said Marsala.
The new technique is less invasive, depositing injected cells into the spinal subpial space — a space between the pial membrane and the superficial layers of the spinal cord.
“This injection technique allows the delivery of high cell numbers from a single injection,” said Marsala.
“Cells with proliferative properties, such as glial progenitors, then migrate into the spinal parenchyma and populate over time in multiple spinal segments as well as the brain stem. Injected cells acquire the functional properties consistent with surrounding host cells.”
A micrograph shows effective migration of subpially injected human oligodendrocytes (a type of support cell in the central nervous system, colored here in green) into the spinal cord gray matter (left) and into the cerebellum (right) of an immunodeficient rat.
Red indicates signal for oligodendrocyte marker; blue is signal for neuronal marker. Image is credited to UC San Diego Health Sciences.
Marsala, senior author Joseph Ciacci, MD, a neurosurgeon at UC San Diego Health, and colleagues suggest that subpially-injected cells are likely to accelerate and improve treatment potency in cell-replacement therapies for several spinal neurodegenerative disorders in which a broad repopulation by glial cells, such as oligodendrocytes or astrocytes, is desired.
“This may include spinal traumatic injury, amyotrophic lateral sclerosis and multiple sclerosis,” said Ciacci.
The researchers plan to test the cell delivery system in larger preclinical animal models of spinal traumatic injury that more closely mimic human anatomy and size.
“The goal is to define the optimal cell dosing and timing of cell delivery after spinal injury, which is associated with the best treatment effect,” said Marsala.
Co-authors include: Kota Kamizato and Takahiro Tadokoro, UC San Diego and University of Ryukyus, Japan; Michael Navarro and Silvia Marsala, UC San Diego; Stefan Juhas, Jana Juhasova, Hana Studenovska and Vladimir Proks, Czech Academy of Sciences; Tom Hazel and Karl Johe, Neuralstem, Inc.; and Shawn Driscoll, Thomas Glenn and Samuel Pfaff, Salk Institute.
Disclosure: Martin Marsala is the scientific founder of Neurgain Technologies, Inc., which has licensed technologies from UC San Diego based on Marsala’s research. Marsala has equity interest in the company, and also serves as a consultant, for which he receives compensation.
Spinal cord injury (SCI) is a devastating event with sudden onset of motor and sensory dysfunction. Damage to autonomic neurons at and below the level of injury leads to bowel, bladder, and sexual functional loss.
This trauma was formerly found primarily in young patients due to high-energy accidents and contact sports, but cervical canal stenosis in elderly patients is increasing with the progress of our aging society, and elderly patients can easily injure their spinal cord from a fall.
Currently, surgical intervention and subsequent rehabilitation are the only options for SCI treatment. Although methylprednisolone is administered at the acute stage of injury, a consensus of its use has not yet been reached from the aspects of both safety and effectiveness , .
Recent developments in stem cell research have indicated that researchers are close to a breakthrough in this challenging field. Numerous preclinical studies have demonstrated the effectiveness of neural precursor cell (NPC) transplantation in animal models of SCI.
Ethical concerns remain about the use of NPCs harvested from fetal or embryonic stem cells (ESCs). However, Yamanaka and his colleagues developed induced pluripotent stem cells (iPSCs), which opened a new avenue toward the clinical application of stem cells for regenerative medicine , .
iPSCs exhibit characteristics similar to those of ESCs and can generate all three germ layers. Therefore, iPSCs can enrich ectodermal neural-lineage cells with appropriate culture induction. Because iPSCs can be derived from human somatic cells, they have the potential to overcome ethical problems.
Although the potential use of iPSCs is very attractive, artificial induction methods raise alternative problems, such as genetic and epigenetic abnormalities and subsequent tumorigenicity. Thus, solving these problems to promote the clinical application of stem cells is essential .
The purpose of this review article is to present the current status of cell therapy using NPCs, especially derived from iPSCs. In addition, we report several approaches to overcome the posttransplant tumorigenic problems and introduce interesting studies. We are currently preparing for the first human clinical trial of iPSC-NPC therapy for subacute SCI, and we are planning to start this research soon.
Provisions for safety issues in human iPSC-NPC transplantation
As we described above, histological and functional recovery were achieved after NPC transplantation if we chose the safe iPSC cell line , . However, unsafe and unstable human iPSC lines have shown tumorigenic potential after grafting . Our group has approached this critical issue and achieved significant results (Fig. 2).
First, the best method for examining which cell lines have the risk for tumor formation remains elusive. Therefore, identifying the factors that regulate the tumorigenicity of iPSC-NPCs prior to transplantation would be ideal. We focused on genetic and epigenetic mechanisms underlying the tumor pathogenesis by comprehensive DNA methylation analyses .
In this study, we clarified that distinct differences in the DNA methylation pattern were detected between safe and tumorigenic iPSC-NPCs. In particular, large differences in the DNA methylation status of several tumor suppressor genes were involved. Intriguingly, the methylation patterns were affected by differences not only in cell lines but also in passage number. Thus, the methylation profiles could be included in the criteria to choose safe iPSC-NPCs in an actual clinical setting.
Even if the NPCs are judged as safe cell lines after a quality check, the risk for tumorigenicity cannot be excluded due to the contamination of undifferentiated cells prior to transplantation. To solve this issue, one approach is to eliminate these cells using a γ-secretase inhibitor (GSI), which inhibits Notch signaling .
The status of undifferentiated NPCs is controlled by Notch signaling, and inhibition of this signaling promotes the additional maturation and neuronal differentiation of NPCs. In our study, tumorigenic human iPSC-NPCs were treated with GSI for only one day in vitro, and they exhibited neuronal differentiation, reduction in cell proliferation, and suppression of tumor-related gene expression. When transplanted into the SCI model of NOD/SCID mice, the iPSC-NPCs mainly generated mature neurons around the injury site and did not form tumors as late as 89 days after transplantation.
On the other hand, non-GSI-treated NPCs formed tumors and resulted in declining motor function. Thus, pretreatment with GSI can eliminate tumor-initiating cells in human iPSC-NPCs and promote the potential to overcome the safety issues related to tumorigenicity after cell transplantation.
Previously, oligodendrocyte progenitor cells (OPCs) have been demonstrated to be a cellular source for transplantation therapy in SCI. Keirstead and his colleagues transplanted ESC-derived human OPCs and observed robust remyelination of host neuronal axons without tumor formation .
Following the beneficial results, the Geron Corporation begun a clinical trial using ESC-OPCs in 2010, which was taken over by Asterias Biotherapeutics Inc. and validated the safety and efficacy of the cells for transplantation , . This phase I/IIa trial targets cervical SCI patients at the subacute phase, and enrollment of 25 participants has already been completed (ClinicalTrials.gov Identifier: NCT02302157).
Patients were evaluated for their neurological outcomes one year after transplantation, and the results are expected to be published soon. With respect to the oligogenic culture system, we also succeeded in establishing a protocol to enrich OPCs from human iPSC-NPCs  and grafted the cells into a rodent SCI model . Around the lesion area at the spinal cord, the frequency of graft differentiation was 40% in oligodendrocytes, which was a dramatic enhancement compared to the 3% of oligo-lineage differentiation from the original NPCs . The grafts promoted remyelination and axonal growth with synapse formation, resulting in functional and electrophysiological recovery.
Similar results were observed in another study, which established a culture protocol to generate OPCs from human iPSCs . The OPCs were transplanted into the rat spinal cord 24 h after injury, and more than 70% of the grafts differentiated into mature oligodendrocytes around the lesion site without tumorigenicity. The cells protected host axons by remyelination and reduced the cavity and glial scar area, leading to functional recovery three months after SCI. Together with the results from several studies, these observations indicate that biasing NPCs toward oligodendrogenic cells could reduce the potential for tumorigenicity by pushing immature cells to be more committed premature cells.
Moreover, transplantation of oligodendrogenic NPCs themselves may be a beneficial strategy for regenerative therapy to remyelinate damaged host axons and recover saltatory conduction, lending potential for neurological restoration.
Even if the NPCs were pretreated with GSI or pushed toward oligogenic cells, concern still remains about posttransplantation tumor formation depending on iPSC lines. Therefore, considering the provisions for tumor ablation after transplantation is critically important. We performed unique studies using a suicide gene system , .
One of the suicide genes, inducible caspase-9, was transduced into iPSC lines with potential tumorigenicity, and after NPC differentiation, the cells were transplanted into the injured spinal cord of NOD/SCID mice. When the apoptosis inducer was systematically injected into the animals, the transplanted cells disappeared histologically, and further functional loss did not occur .
However, this genomic system killed all types of transplanted cells, and ideally, only proliferating tumor cells would be eliminated, while differentiated, mature neural cells would be spared. We therefore transduced the herpes simplex virus type I thymidine kinase (HSVtk) gene into tumorigenic human iPSC-NPCs .
HSVtk phosphorylates its prodrug ganciclovir (GCV), which leads to the production of cytotoxic GCV phosphate and kills immature and/or proliferating tumor cells while sparing postmitotic mature neural cells. Upon transplantation of iPSC-NPCs transduced with HSVtk into a rodent SCI model, only the tumorigenic cells were ablated after GCV injection, and mature neuronal cells were preserved, contributing to the maintenance of the recovered locomotor function.
Because the HSVtk/GCV system has already been applied in some clinical trials without any safety problems , , this technique can also be used in the clinical setting of our iPSC project for SCI patients.