Longitudinal Magnetic Resonance Imaging Tracking of Transplanted Neural Progenitor Cells in the Spinal Cord Utilizing the Bright-Ferritin Mechanism.

Human neural progenitor cells (hNPCs) hold promise for treating spinal cord injury. Studies to date have focused on improving their regenerative potential and therapeutic effect. Equally important is ensuring successful delivery and engraftment of hNPCs at the injury site. Unfortunately, no current imaging solution for cell tracking is compatible with long-term monitoring in vivo. The objective of this study was to apply a novel bright-ferritin magnetic resonance imaging (MRI) mechanism to track hNPC transplants longitudinally and on demand in the rat spinal cord. We genetically modified hNPCs to stably overexpress human ferritin. Ferritin-overexpressing (FT) hNPCs labeled with 0.2 mM manganese provided significant T1-induced bright contrast on in vitro MRI, with no adverse effect on cell viability, morphology, proliferation, and differentiation. In vivo, 2 M cells were injected into the cervical spinal cord of Rowett nude rats. MRI employed T1-weighted acquisitions and T1 mapping on a 3 T scanner. Conventional short-term cell tracking was performed using exogenous Mn labeling prior to cell transplantation, which displayed transient bright contrast on MRI 1 day after cell transplantation and disappeared after 1 week. In contrast, long-term cell tracking using bright-ferritin allowed on-demand signal recall upon Mn supplementation and precise visualization of the surviving hNPC graft. In fact, this new cell tracking technology identified 7 weeks post-transplantation as the timepoint by which substantial hNPC integration occurred. Spatial distribution of hNPCs on MRI matched that on histology. In summary, bright-ferritin provides the first demonstration of long-term, on-demand, high-resolution, and specific tracking of hNPCs in the rat spinal cord.

Human Spinal Oligodendrogenic Neural Progenitor Cells Enhance Pathophysiological Outcomes and Functional Recovery in a Clinically Relevant Cervical Spinal Cord Injury Rat Model.

Traumatic spinal cord injury (SCI) results in the loss of neurons, oligodendrocytes, and astrocytes. Present interventions for SCI include decompressive surgery, anti-inflammatory therapies, and rehabilitation programs. Nonetheless, these approaches do not offer regenerative solutions to replace the lost cells, fiber tracts, and circuits. Neural stem/progenitor cell (NPC) transplantation is a promising strategy that aims to encourage regeneration. However, NPC differentiation remains inconsistent, thus, contributing to suboptimal functional recovery. As such, we have previously engineered oligodendrogenically biased NPCs (oNPCs) and demonstrated their efficacy in a thoracic model of SCI. Since the majority of patients with SCI experience cervical injuries, our objective in the current study was to generate human induced pluripotent stem cell-derived oNPCs (hiPSC-oNPCs) and to characterize these cells in vitro and in vivo, utilizing a clinically relevant rodent model of cervical SCI. Following transplantation, the oNPCs engrafted, migrated to the rostral and caudal regions of the lesion, and demonstrated preferential differentiation toward oligodendrocytes. Histopathological evaluations revealed that oNPC transplantation facilitated tissue preservation while diminishing astrogliosis. Moreover, oNPC transplantation fostered remyelination of the spared tissue. Functional analyses indicated improved forelimb grip strength, gait, and locomotor function in the oNPC-transplanted rats. Importantly, oNPC transplantation did not exacerbate neuropathic pain or induce tumor formation. In conclusion, these findings underscore the therapeutic potential of oNPCs in promoting functional recovery and histopathological improvements in cervical SCI. This evidence warrants further investigation to optimize and advance this promising cell-based therapeutic approach.

How can clinical safety and efficacy concerns in stem cell therapy for spinal cord injury be overcome?

INTRODUCTION: Spinal cord injury (SCI) can lead to severe neurological dysfunction. Despite scientific and medical advances, clinically effective regenerative therapies including stem cells are lacking for SCI. AREAS COVERED: This paper discusses translational challenges related to the safe, effective use of stem cells for SCI, with a focus on mesenchymal stem cells (MSCs), neural stem cells (NSCs), Schwann cells (SCs), olfactory ensheathing cells (OECs), oligodendrocyte precursor cells (OPCs), embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs). We discuss approaches to enhance the efficacy of cell-based strategies by i) addressing patient heterogeneity and enhancing patient selection; ii) selecting cell type, cell source, cell developmental stage, and delivery technique; iii) enhancing graft integration and mitigating immune-mediated graft rejection; and iv) ensuring availability of cells. Additionally, we review strategies to optimize outcomes including combinatorial use of rehabilitation and discuss ways to mitigate potential risks of tumor formation associated with stem cell-based strategies. EXPERT OPINION: Basic science research will drive translational advances to develop stem cell-based therapies for SCI. Genetic, serological, and imaging biomarkers may enable individualization of cell-based treatments. Moreover, combinatorial strategies will be required to enhance graft survival, migration and functional integration, to enable precision-based intervention.

Cell-Cell Contact Mediates Gene Expression and Fate Choice of Human Neural Stem/Progenitor Cells.

Transplantation of Neural Stem/Progenitor Cells (NPCs) is a promising regenerative strategy to promote neural repair following injury and degeneration because of the ability of these cells to proliferate, migrate, and integrate with the host tissue. Precise in vitro control of NPC proliferation without compromising multipotency and differentiation ability is critical in stem cell maintenance. This idea was highlighted in recent clinical trials, where discrepancies in NPC culturing protocols produced inconsistent therapeutic benefits. Of note, cell density plays an important role in regulating the survival, proliferation, differentiation, and fate choice of stem cells. To determine the extent of variability produced by inconsistent culturing densities, the present study cultured human-induced pluripotent NPCs (hiPSC-NPCs) at either a low or high plating density. hiPSC-NPCs were then isolated for transcriptomic analysis or differentiation in vitro. Following sequencing analysis, genes involved in cell-cell contact-mediated pathways, including Hippo-signaling, NOTCH, and WNT were differentially expressed. Modulation of these pathways was highly associated with the regulation of pro-neuronal transcription factors, which were also upregulated in response to higher-density hiPSC-NPC culture. Moreover, higher plating density translated into a greater neuronal and less astrocytic differentiation in vitro. This study highlights the importance of precisely controlling culture conditions during the development of NPC transplantation therapies.

Administration of C5a Receptor Antagonist Improves the Efficacy of Human Induced Pluripotent Stem Cell-Derived Neural Stem/Progenitor Cell Transplantation in the Acute Phase of Spinal Cord Injury.

Human-induced pluripotent stem cell-derived neural stem/progenitor cell (hiPSC-NS/PC) transplantation during the acute phase of spinal cord injury (SCI) is not effective due to the inflammatory response occurring immediately after SCI, which negatively impacts transplanted cell survival. Therefore, we chose to study the powerful chemoattractant complement C5a as a method to generate a more favorable transplantation environment. We hypothesized that suppression of the inflammatory response immediately after SCI by C5a receptor antagonist (C5aRA) would improve the efficacy of hiPSC-NS/PCs transplantation for acute phase SCI. Here, we evaluated the influence of C5aRA on the inflammatory reaction during the acute phase after SCI, and observed significant reductions in several inflammatory cytokines, macrophages, neutrophils, and apoptotic markers. Next, we divided the SCI mice into four groups: 1) phosphate-buffered saline (PBS) only; 2) C5aRA only; 3) PBS + transplantation (PBS+TP); and 4) C5aRA + transplantation (C5aRA+TP). Immediately after SCI, C5aRA or PBS was injected once a day for 4 consecutive days, followed by hiPSC-NS/PC transplantation or PBS into the lesion epicenter on Day 4. The C5aRA+TP group had better functional improvement compared with the PBS only group. The C5aRA+TP group also had a significantly higher cell survival rate compared with the PBS+TP group. This study demonstrates that administration of C5aRA can suppress the inflammatory response during the acute phase of SCI, while improving the survival rate of transplanted hiPSC-NS/PCs, as well as enhancing motor functional restoration. Human-induced pluripotent stem cell-derived neural stem/progenitor cell transplantation with C5aRA is a promising treatment during the acute injury phase for SCI patients.

Regenerative replacement of neural cells for treatment of spinal cord injury.

Introduction: Traumatic Spinal Cord Injury (SCI) results from primary physical injury to the spinal cord, which initiates a secondary cascade of neural cell death. Current therapeutic approaches can attenuate the consequences of the primary and secondary events, but do not address the degenerative aspects of SCI. Transplantation of neural stem/progenitor cells (NPCs) for the replacement of the lost/damaged neural cells is suggested here as a regenerative approach that is complementary to current therapeutics.Areas Covered: This review addresses how neurons, oligodendrocytes, and astrocytes are impacted by traumatic SCI, and how current research in regenerative-NPC therapeutics aims to restore their functionality. Methods used to enhance graft survival, as well as bias progenitor cells towards neuronal, oligodendrogenic, and astroglia lineages are discussed.Expert Opinion: Despite an NPC's ability to differentiate into neurons, oligodendrocytes, and astrocytes in the transplant environment, their potential therapeutic efficacy requires further optimization prior to translation into the clinic. Considering the temporospatial identity of NPCs could promote neural repair in region specific injuries throughout the spinal cord. Moreover, understanding which cells are targeted by NPC-derived myelinating cells can help restore physiologically-relevant myelin patterns. Finally, the duality of astrocytes is discussed, outlining their context-dependent importance in the treatment of SCI.

The leading edge: Emerging neuroprotective and neuroregenerative cell-based therapies for spinal cord injury.

Spinal cord injuries (SCIs) are associated with tremendous physical, social, and financial costs for millions of individuals and families worldwide. Rapid delivery of specialized medical and surgical care has reduced mortality; however, long-term functional recovery remains limited. Cell-based therapies represent an exciting neuroprotective and neuroregenerative strategy for SCI. This article summarizes the most promising preclinical and clinical cell approaches to date including transplantation of mesenchymal stem cells, neural stem cells, oligodendrocyte progenitor cells, Schwann cells, and olfactory ensheathing cells, as well as strategies to activate endogenous multipotent cell pools. Throughout, we emphasize the fundamental biology of cell-based therapies, critical features in the pathophysiology of spinal cord injury, and the strengths and limitations of each approach. We also highlight salient completed and ongoing clinical trials worldwide and the bidirectional translation of their findings. We then provide an overview of key adjunct strategies such as trophic factor support to optimize graft survival and differentiation, engineered biomaterials to provide a support scaffold, electrical fields to stimulate migration, and novel approaches to degrade the glial scar. We also discuss important considerations when initiating a clinical trial for a cell therapy such as the logistics of clinical-grade cell line scale-up, cell storage and transportation, and the delivery of cells into humans. We conclude with an outlook on the future of cell-based treatments for SCI and opportunities for interdisciplinary collaboration in the field.

GDNF rescues the fate of neural progenitor grafts by attenuating Notch signals in the injured spinal cord in rodents.

Neural progenitor cell (NPC) transplantation is a promising strategy for the treatment of spinal cord injury (SCI). In this study, we show that injury-induced Notch activation in the spinal cord microenvironment biases the fate of transplanted NPCs toward astrocytes in rodents. In a screen for potential clinically relevant factors to modulate Notch signaling, we identified glial cell-derived neurotrophic factor (GDNF). GDNF attenuates Notch signaling by mediating delta-like 1 homolog (DLK1) expression, which is independent of GDNF's effect on cell survival. When transplanted into a rodent model of cervical SCI, GDNF-expressing human-induced pluripotent stem cell-derived NPCs (hiPSC-NPCs) demonstrated higher differentiation toward a neuronal fate compared to control cells. In addition, expression of GDNF promoted endogenous tissue sparing and enhanced electrical integration of transplanted cells, which collectively resulted in improved neurobehavioral recovery. CRISPR-induced knockouts of the DLK1 gene in GDNF-expressing hiPSC-NPCs attenuated the effect on functional recovery, demonstrating that this effect is partially mediated through DLK1 expression. These results represent a mechanistically driven optimization of hiPSC-NPC therapy to redirect transplanted cells toward a neuronal fate and enhance their integration.

Examining the fundamental biology of a novel population of directly reprogrammed human neural precursor cells.

BACKGROUND: Cell reprogramming is a promising avenue for cell-based therapies as it allows for the generation of multipotent, unipotent, or mature somatic cells without going through a pluripotent state. While the use of autologous cells is considered ideal, key challenges for their clinical translation include the ability to reproducibly generate sufficient quantities of cells within a therapeutically relevant time window. METHODS: We performed transfection of three distinct human somatic starting populations of cells with a non-integrating synthetic plasmid expressing Musashi 1 (MSI1), Neurogenin 2 (NGN2), and Methyl-CpG-Binding Domain 2 (MBD2). The resulting directly reprogrammed neural precursor cells (drNPCs) were examined in vitro using RT-qPCR, karyotype analysis, immunohistochemistry, and FACS at early and late time post-transfection. Electrophysiology (patch clamp) was performed on drNPC-derived neurons to determine their capacity to generate action potentials. In vivo characterization was performed following transplantation of drNPCs into two animal models (Shiverer and SCID/Beige mice), and the numbers, location, and differentiation profile of the transplanted cells were examined using immunohistochemistry. RESULTS: Human somatic cells can be directly reprogrammed within two weeks to neural precursor cells (drNPCs) by transient exposure to Msi1, Ngn2, and MBD2 using non-viral constructs. The drNPCs generate all three neural cell types (astrocytes, oligodendrocytes, and neurons) and can be passaged in vitro to generate large numbers of cells within four weeks. drNPCs can respond to in vivo differentiation and migration cues as demonstrated by their migration to the olfactory bulb and contribution to neurogenesis in vivo. Differentiation profiles of transplanted cells onto the corpus callosum of myelin-deficient mice reveal the production of oligodendrocytes and astrocytes. CONCLUSIONS: Human drNPCs can be efficiently and rapidly produced from donor somatic cells and possess all the important characteristics of native neural multipotent cells including differentiation into neurons, astrocytes, and oligodendrocytes, and in vivo neurogenesis and myelination.

Generation of Definitive Neural Progenitor Cells from Human Pluripotent Stem Cells for Transplantation into Spinal Cord Injury.

In this chapter, we first describe two interchangeable protocols optimized in our lab for deriving definitive neuronal progenitor cells from human pluripotent stem cells (hPSCs). The resultant NPCs can then be propagated and differentiated to produce differing proportions of neurons, oligodendrocytes, and astrocytes as required for in vitro cell culture studies or in vivo transplantation. Following these protocols, we explain the method for transplanting these cells into the rat model of spinal cord injury (SCI).

Human Oligodendrogenic Neural Progenitor Cells Delivered with Chondroitinase ABC Facilitate Functional Repair of Chronic Spinal Cord Injury.

Treatment of chronic spinal cord injury (SCI) is challenging due to cell loss, cyst formation, and the glial scar. Previously, we reported on the therapeutic potential of a neural progenitor cell (NPC) and chondroitinase ABC (ChABC) combinatorial therapy for chronic SCI. However, the source of NPCs and delivery system required for ChABC remained barriers to clinical application. Here, we investigated directly reprogrammed human NPCs biased toward an oligodendrogenic fate (oNPCs) in combination with sustained delivery of ChABC using an innovative affinity release strategy in a crosslinked methylcellulose biomaterial for the treatment of chronic SCI in an immunodeficient rat model. This combinatorial therapy increased long-term survival of oNPCs around the lesion epicenter, facilitated greater oligodendrocyte differentiation, remyelination of the spared axons by engrafted oNPCs, enhanced synaptic connectivity with anterior horn cells and neurobehavioral recovery. This combinatorial therapy is a promising strategy to regenerate the chronically injured spinal cord.

Severe-combined immunodeficient rats can be used to generate a model of perinatal hypoxic-ischemic brain injury to facilitate studies of engrafted human neural stem cells.

Cerebral palsy (CP) encompasses a group of non-progressive brain disorders that are often acquired through perinatal hypoxic-ischemic (HI) brain injury. Injury leads to a cascade of cell death events, resulting in lifetime motor and cognitive deficits. There are currently no treatments that can repair the resulting brain damage and improve functional outcomes. To date, preclinical research using neural precursor cell (NPC) transplantation as a therapy for HI brain injury has shown promise. To translate this treatment to the clinic, it is essential that human-derived NPCs also be tested in animal models, however, a major limitation is the high risk of xenograft rejection. A solution is to transplant the cells into immune-deficient rodents, but there are currently no models of HI brain injury established in such a cohort of animals. Here, we demonstrate that a model of HI brain injury can be generated in immune-deficient Prkdc knockout (KO) rats. Long-term deficits in sensorimotor function were similar between KO and wildtype (WT) rats. Interestingly, some aspects of the injury were more severe in KO rats. Additionally, human induced pluripotent stem cell derived (hiPSC)-NPCs had higher survival at 10 weeks post-transplant in KO rats when compared to their WT counterparts. This work establishes a reliable model of neonatal HI brain injury in Prkdc KO rats that will allow for future transplantation, survival, and long-term evaluation of the safety and efficacy of hiPSC-NPCs for neonatal brain damage. This model will enable critical preclinical translational research using human NPCs.

Human Spinal Oligodendrogenic Neural Progenitor Cells Promote Functional Recovery After Spinal Cord Injury by Axonal Remyelination and Tissue Sparing.

Cell transplantation therapy utilizing neural precursor cells (NPCs) is a conceptually attractive strategy for traumatic spinal cord injury (SCI) to replace lost cells, remyelinate denuded host axons and promote tissue sparing. However, the number of mature oligodendrocytes that differentiate from typical NPCs remains limited. Herein, we describe a novel approach to bias the differentiation of directly reprogrammed human NPCs (drNPCs) toward a more oligodendrogenic fate (oNPCs) while preserving their tripotency. The oNPCs derived from different lines of human NPCs showed similar characteristics in vitro. To assess the in vivo efficacy of this approach, we used oNPCs derived from drNPCs and transplanted them into a SCI model in immunodeficient Rowett Nude (RNU) rats. The transplanted cells showed significant migration along the rostrocaudal axis and proportionally greater differentiation into oligodendrocytes. These cells promoted perilesional tissue sparing and axonal remyelination, which resulted in recovery of motor function. Moreover, after transplantation of the oNPCs into intact spinal cords of immunodeficient NOD/SCID mice, we detected no evidence of tumor formation even after 5 months of observation. Thus, biasing drNPC differentiation along an oligodendroglial lineage represents a promising approach to promote tissue sparing, axonal remyelination, and neural repair after traumatic SCI. Stem Cells Translational Medicine 2018;7:806-818.

Exogenous Neural Precursor Cell Transplantation Results in Structural and Functional Recovery in a Hypoxic-Ischemic Hemiplegic Mouse Model.

Cerebral palsy (CP) is a common pediatric neurodevelopmental disorder, frequently resulting in motor and developmental deficits and often accompanied by cognitive impairments. A regular pathobiological hallmark of CP is oligodendrocyte maturation impairment resulting in white matter (WM) injury and reduced axonal myelination. Regeneration therapies based on cell replacement are currently limited, but neural precursor cells (NPCs), as cellular support for myelination, represent a promising regeneration strategy to treat CP, although the transplantation parameters (e.g., timing, dosage, mechanism) remain to be determined. We optimized a hemiplegic mouse model of neonatal hypoxia-ischemia that mirrors the pathobiological hallmarks of CP and transplanted NPCs into the corpus callosum (CC), a major white matter structure impacted in CP patients. The NPCs survived, engrafted, and differentiated morphologically in male and female mice. Histology and MRI showed repair of lesioned structures. Furthermore, electrophysiology revealed functional myelination of the CC (e.g., restoration of conduction velocity), while cylinder and CatWalk tests demonstrated motor recovery of the affected forelimb. Endogenous oligodendrocytes, recruited in the CC following transplantation of exogenous NPCs, are the principal actors in this recovery process. The lack of differentiation of the transplanted NPCs is consistent with enhanced recovery due to an indirect mechanism, such as a trophic and/or "bio-bridge" support mediated by endogenous oligodendrocytes. Our work establishes that transplantation of NPCs represents a viable therapeutic strategy for CP treatment, and that the enhanced recovery is mediated by endogenous oligodendrocytes. This will further our understanding and contribute to the improvement of cellular therapeutic strategies.

Generation of Oligodendrogenic Spinal Neural Progenitor Cells From Human Induced Pluripotent Stem Cells.

This unit describes protocols for the efficient generation of oligodendrogenic neural progenitor cells (o-NPCs) from human induced pluripotent stem cells (hiPSCs). Specifically, detailed methods are provided for the maintenance and differentiation of hiPSCs, human induced pluripotent stem cell-derived neural progenitor cells (hiPS-NPCs), and human induced pluripotent stem cell-oligodendrogenic neural progenitor cells (hiPSC-o-NPCs) with the final products being suitable for in vitro experimentation or in vivo transplantation. Throughout, cell exposure to growth factors and patterning morphogens has been optimized for both concentration and timing, based on the literature and empirical experience, resulting in a robust and highly efficient protocol. Using this derivation procedure, it is possible to obtain millions of oligodendrogenic-NPCs within 40 days of initial cell plating which is substantially shorter than other protocols for similar cell types. This protocol has also been optimized to use translationally relevant human iPSCs as the parent cell line. The resultant cells have been extensively characterized both in vitro and in vivo and express key markers of an oligodendrogenic lineage. © 2017 by John Wiley & Sons, Inc.

Induced Pluripotent Stem Cells for Traumatic Spinal Cord Injury.

Spinal cord injury (SCI) is a common cause of mortality and neurological morbidity. Although progress had been made in the last decades in medical, surgical, and rehabilitation treatments for SCI, the outcomes of these approaches are not yet ideal. The use of cell transplantation as a therapeutic strategy for the treatment of SCI is very promising. Cell therapies for the treatment of SCI are limited by several translational road blocks, including ethical concerns in relation to cell sources. The use of iPSCs is particularly attractive, given that they provide an autologous cell source and avoid the ethical and moral considerations of other stem cell sources. In addition, different cell types, that are applicable to SCI, can be created from iPSCs. Common cell sources used for reprogramming are skin fibroblasts, keratinocytes, melanocytes, CD34+ cells, cord blood cells and adipose stem cells. Different cell types have different genetic and epigenetic considerations that affect their reprogramming efficiencies. Furthermore, in SCI the iPSCs can be differentiated to neural precursor cells, neural crest cells, neurons, oligodendrocytes, astrocytes, and even mesenchymal stromal cells. These can produce functional recovery by replacing lost cells and/or modulating the lesion microenvironment.

The Potential for iPS-Derived Stem Cells as a Therapeutic Strategy for Spinal Cord Injury: Opportunities and Challenges.

Spinal cord injury (SCI) is a devastating trauma causing long-lasting disability. Although advances have occurred in the last decade in the medical, surgical and rehabilitative treatments of SCI, the therapeutic approaches are still not ideal. The use of cell transplantation as a therapeutic strategy for the treatment of SCI is promising, particularly since it can target cell replacement, neuroprotection and regeneration. Cell therapies for treating SCI are limited due to several translational roadblocks, including ethical and practical concerns regarding cell sources. The use of iPSCs has been particularly attractive, since they avoid the ethical and moral concerns that surround other stem cells. Furthermore, various cell types with potential for application in the treatment of SCI can be created from autologous sources using iPSCs. For applications in SCI, the iPSCs can be differentiated into neural precursor cells, neurons, oligodendrocytes, astrocytes, neural crest cells and mesenchymal stromal cells that can act by replacing lost cells or providing environmental support. Some methods, such as direct reprogramming, are being investigated to reduce tumorigenicity and improve reprogramming efficiencies, which have been some of the issues surrounding the use of iPSCs clinically to date. Recently, iPSCs have entered clinical trials for use in age-related macular degeneration, further supporting their promise for translation in other conditions, including SCI.