Neural Progenitor Cell Grafts Research Articles

Optical coherence tomography enables longitudinal evaluation of cell graft-directed remodeling in stroke lesions.

Stem cell grafting can promote glial repair of adult stroke injuries during the subacute wound healing phase, but graft survival and glial repair outcomes are perturbed by lesion severity and mode of injury. To better understand how stroke lesion environments alter the functions of cell grafts, we employed optical coherence tomography (OCT) to longitudinally image mouse cortical photothrombotic ischemic strokes treated with allogeneic neural progenitor cell (NPC) grafts. OCT angiography, signal intensity, and signal decay resulting from optical scattering were assessed at multiple timepoints across two weeks in mice receiving an NPC graft or an injection of saline at two days after stroke. OCT scattering information revealed pronounced axial lesion contraction that naturally occurred throughout the subacute wound healing phase that was not modified by either NPC or saline treatment. By analyzing OCT signal intensity along the coronal plane, we observed dramatic contraction of the cortex away from the imaging window in the first week after stroke which impaired conventional OCT angiography but which enabled the detection of NPC graft-induced glial repair. There was moderate, but variable, NPC graft survival at photothrombotic strokes at two weeks which was inversely correlated with acute stroke lesion sizes as measured by OCT prior to treatment, suggesting a prognostic role for OCT imaging and reinforcing the dominant effect of lesion size and severity on graft outcome. Overall, our findings demonstrate the utility of OCT imaging for both tracking and predicting natural and treatment-directed changes in ischemic stroke lesion cores.

Clickable Granular Hydrogel Scaffolds for Delivery of Neural Progenitor Cells to Sites of Spinal Cord Injury.

Spinal cord injury (SCI) is a serious condition with limited treatment options. Neural progenitor cell (NPC) transplantation is a promising treatment option, and the identification of novel biomaterial scaffolds that support NPC engraftment and therapeutic activity is a top research priority. The objective of this study is to evaluate in situ assembled poly (ethylene glycol) (PEG)-based granular hydrogels for NPC delivery in a murine model of SCI. Microgel precursors are synthesized by using thiol-norbornene click chemistry to react four-armed PEG-amide-norbornene with enzymatically degradable and cell adhesive peptides. Unreacted norbornene groups are utilized for in situ assembly into scaffolds using a PEG-di-tetrazine linker. The granular hydrogel scaffolds exhibit good biocompatibility and do not adversely affect the inflammatory response after SCI. Moreover, when used to deliver NPCs, the granular hydrogel scaffolds supported NPC engraftment, do not adversely affect the immune response to the NPC grafts, and successfully support graft differentiation toward neuronal or astrocytic lineages as well as axonal extension into the host tissue. Collectively, these data establish PEG-based granular hydrogel scaffolds as a suitable biomaterial platform for NPC delivery and justify further testing, particularly in the context of more severe SCI.

Ischemic and hemorrhagic stroke lesion environments differentially alter the glia repair potential of neural progenitor cell and immature astrocyte grafts

Using cell grafting to direct glia-based repair mechanisms in adult CNS injuries represents a potential therapeutic strategy for supporting functional neural parenchymal repair. However, glia repair directed by neural progenitor cell (NPC) grafts is dramatically altered by increasing lesion size, severity, and mode of injury. To address this, we studied the interplay between astrocyte differentiation and cell proliferation of NPC in vitro to generate proliferating immature astrocytes (ImA) using hysteretic conditioning. ImA maintain proliferation rates at comparable levels to NPC but showed robust immature astrocyte marker expression including Gfap and Vimentin. ImA demonstrated enhanced resistance to myofibroblast-like phenotypic transformations upon exposure to serum enriched environments in vitro compared to NPC and were more effective at scratch wound closure in vitro compared to quiescent astrocytes. Glia repair directed by ImA at acute ischemic striatal stroke lesions was equivalent to NPC but better than quiescent astrocyte grafts. While ischemic injury environments supported enhanced survival of grafts compared to healthy striatum, hemorrhagic lesions were hostile towards both NPC and ImA grafts leading to poor survival and ineffective modulation of natural wound repair processes. Our findings demonstrate that lesion environments, rather than transcriptional pre-graft states, determine the survival, cell-fate, and glia repair competency of cell grafts applied to acute CNS injuries.

The Emerging Role of Biological Sex in Cell Therapy for Spinal Cord Injury.

Neural progenitor cell (NPC) transplantation is a promising potential therapy for replacing spinal cord neurons and glial cells following spinal cord injury (SCI). Despite the rapid advancement of NPC transplantation to SCI clinical trials, we still lack understanding of fundamental biology underlying how NPC grafts interact with the injured host nervous system. Our recent study demonstrated a potent effect of biological sex mismatch between donor and host on graft immune rejection. Here we discuss the implications of this study in the context of clinical trials for SCI, and important topics for future research in SCI cell transplantation.

Rehabilitation combined with neural progenitor cell grafts enables functional recovery in chronic spinal cord injury.

We reported previously that neural progenitor cell (NPC) grafts form neural relays across sites of subacute spinal cord injury (SCI) and support functional recovery. Here, we examine whether NPC grafts after chronic delays also support recovery and whether intensive rehabilitation further enhances recovery. One month after severe bilateral cervical contusion, rats received daily intensive rehabilitation, NPC grafts, or both rehabilitation and grafts. Notably, only the combination of rehabilitation and grafting significantly improved functional recovery. Moreover, improved functional outcomes were associated with a rehabilitation-induced increase in host corticospinal axon regeneration into grafts. These findings identify a critical and synergistic role of rehabilitation and neural stem cell therapy in driving neural plasticity to support functional recovery after chronic and severe SCI.

Injured adult neurons regress to an embryonic transcriptional growth state.

Grafts of spinal-cord-derived neural progenitor cells (NPCs) enable the robust regeneration of corticospinal axons and restore forelimb function after spinal cord injury1; however, the molecular mechanisms that underlie this regeneration are unknown. Here we perform translational profiling specifically of corticospinal tract (CST) motor neurons in mice, to identify their 'regenerative transcriptome' after spinal cord injury and NPC grafting. Notably, both injury alone and injury combined with NPC grafts elicit virtually identical early transcriptomic responses in host CST neurons. However, in mice with injury alone this regenerative transcriptome is downregulated after two weeks, whereas in NPC-grafted mice this transcriptome is sustained. The regenerative transcriptome represents a reversion to an embryonic transcriptional state of the CST neuron. The huntingtin gene (Htt) is a central hub in the regeneration transcriptome; deletion of Htt significantly attenuates regeneration, which shows that Htt has a key role in neural plasticity after injury.

Regeneration of Corticospinal Axons into Neural Progenitor Cell Grafts After Spinal Cord Injury.

Spinal cord injuries leave patients with lifelong paralysis. To date, there are no therapies that promote the critical step required for the recovery of voluntary motor function: corticospinal axon regeneration. Spinal cord-derived neural progenitor cell (NPC) grafts integrate into the injured host spinal cord, enable robust corticospinal axon regeneration, and restore forelimb function following spinal cord injury in rodents. Consequently, engineered stem cell differentiation and transplantation techniques harbor promising potential for the design and implementation of therapies promoting corticospinal axon regeneration. However, in order to optimize the outcome of clinical trials, it is critical to fully understand the cellular and molecular mechanisms underlying this regeneration. Our recent study highlights the unexpected intrinsic potential of corticospinal neurons to regenerate and allows us to investigate new hypotheses exploiting this newly discovered potential.

Origins of Neural Progenitor Cell-Derived Axons Projecting Caudally after Spinal Cord Injury.

SummaryNeural progenitor cells (NPCs) transplanted into sites of spinal cord injury (SCI) extend large numbers of axons into the caudal host spinal cord. We determined the precise locations of neurons in the graft that extend axons into the caudal host spinal cord using AAV9-Cre-initiated retrograde tracing into floxed-TdTomato-expressing NPC grafts. 7,640 ± 630 grafted neurons extended axons to a single caudal host spinal cord site located 2 mm beyond the lesion, 5 weeks post injury. While caudally projecting axons arose from neurons located in all regions of the graft, the majority of caudally projecting graft neurons (53%) were located within the caudal one-third of the graft. Numerous host corticospinal axons formed monosynaptic projections onto caudally projecting graft neurons; however, we find that the majority of host axonal neuronal projections formed by neural progenitor cell interneuronal “relays” across sites of SCI are likely polysynaptic in nature.

Activation of Intrinsic Growth State Enhances Host Axonal Regeneration into Neural Progenitor Cell Grafts.

SummaryAxonal regeneration after spinal cord injury (SCI) can be enhanced by activation of the intrinsic neuronal growth state and, separately, by placement of growth-enabling neural progenitor cell (NPC) grafts into lesion sites. Indeed, NPC grafts support regeneration of all host axonal projections innervating the normal spinal cord. However, some host axons regenerate only short distances into grafts. We examined whether activation of the growth state of the host injured neuron would elicit greater regeneration into NPC grafts. Rats received NPC grafts into SCI lesions in combination with peripheral “conditioning” lesions. Six weeks later, conditioned host sensory axons exhibited a significant, 9.6-fold increase in regeneration into the lesion/graft site compared with unconditioned axons. Regeneration was further enhanced 1.6-fold by enriching NPC grafts with phenotypically appropriate sensory neuronal targets. Thus, activation of the intrinsic host neuronal growth state and manipulation of the graft environment enhance axonal regeneration after SCI.

Injured adult motor and sensory axons regenerate into appropriate organotypic domains of neural progenitor grafts

Neural progenitor cell (NPC) transplantation has high therapeutic potential in neurological disorders. Functional restoration may depend on the formation of reciprocal connections between host and graft. While it has been reported that axons extending out of neural grafts in the brain form contacts onto phenotypically appropriate host target regions, it is not known whether adult, injured host axons regenerating into NPC grafts also form appropriate connections. We report that spinal cord NPCs grafted into the injured adult rat spinal cord self-assemble organotypic, dorsal horn-like domains. These clusters are extensively innervated by regenerating adult host sensory axons and are avoided by corticospinal axons. Moreover, host axon regeneration into grafts increases significantly after enrichment with appropriate neuronal targets. Together, these findings demonstrate that injured adult axons retain the ability to recognize appropriate targets and avoid inappropriate targets within neural progenitor grafts, suggesting that restoration of complex circuitry after SCI may be achievable.

Rodent Neural Progenitor Cells Support Functional Recovery after Cervical Spinal Cord Contusion.

Previously, we and others have shown that rodent neural progenitor cells (NPCs) can support functional recovery after cervical and thoracic transection injuries. To extend these observations to a more clinically relevant model of spinal cord injury, we performed unilateral midcervical contusion injuries in Fischer 344 rats. Two-weeks later, E14-derived syngeneic spinal cord-derived multi-potent NPCs were implanted into the lesion cavity. Control animals received either no grafts or fibroblast grafts. The NPCs differentiated into all three neural lineages (neurons, astrocytes, oligodendrocytes) and robustly extended axons into the host spinal cord caudal and rostral to the lesion. Graft-derived axons grew into host gray matter and expressed synaptic proteins in juxtaposition with host neurons. Animals that received NPC grafts exhibited significant recovery of forelimb motor function compared with the two control groups (analysis of variance p < 0.05). Thus, NPC grafts improve forelimb motor outcomes after clinically relevant cervical contusion injury. These benefits are observed when grafts are placed two weeks after injury, a time point that is more clinically practical than acute interventions, allowing time for patients to stabilize medically, simplifying enrollment in clinical trials, and enhancing predictability of spontaneous improvement in control groups.

Myelination of axons emerging from neural progenitor grafts after spinal cord injury

Neural progenitor cells (NPCs) grafted to sites of spinal cord injury (SCI) extend numerous axons over long distances and form new synaptic connections with host neurons. In the present study we examined the myelination of axons emerging from NPC grafts. Rat embryonic day 14 (E14) multipotent NPCs constitutively expressing GFP were grafted into adult C5 spinal cord hemisection lesions; 3months later we examined graft-derived axonal diameter and myelination using transmission electron microscopy. 104 graft-derived axons were characterized. Axon diameter ranged from 0.15 to 1.70μm, and 24% of graft-derived axons were myelinated by host oligodendrocytes caudal to the lesion. The average diameter of myelinated axons (0.72±0.3μm) was significantly larger than that of non-myelinated axons (0.61±0.2μm, p<0.05). Notably, the G-ratio of myelinated graft-derived axons (0.77±0.01) was virtually identical to that of the normal, intact spinal cord described in published reports. These findings indicate that axons emerging from early stage neural grafts into the injured spinal cord recapitulate both the small/medium size range and myelin thickness of intact spinal cord axons.

Comprehensive Monosynaptic Rabies Virus Mapping of Host Connectivity with Neural Progenitor Grafts after Spinal Cord Injury.

SummaryNeural progenitor cells grafted to sites of spinal cord injury have supported electrophysiological and functional recovery in several studies. Mechanisms associated with graft-related improvements in outcome appear dependent on functional synaptic integration of graft and host systems, although the extent and diversity of synaptic integration of grafts with hosts are unknown. Using transgenic mouse spinal neural progenitor cell grafts expressing the TVA and G-protein components of the modified rabies virus system, we initiated monosynaptic tracing strictly from graft neurons placed in sites of cervical spinal cord injury. We find that graft neurons receive synaptic inputs from virtually every known host system that normally innervates the spinal cord, including numerous cortical, brainstem, spinal cord, and dorsal root ganglia inputs. Thus, implanted neural progenitor cells receive an extensive range of host neural inputs to the injury site, potentially enabling functional restoration across multiple systems.

Optimization of trophic support for neural stem cell grafts in sites of spinal cord injury

Previously we utilized fibrin matrices and a cocktail of nine growth factors and a cell death inhibitor to promote survival and fill of neural stem cell (NSC) or neural progenitor cell (NPC) grafts to sites of spinal cord injury (SCI). In the current study, we examined whether the number of growth factors in a supportive matrix could be reduced to a more clinically practical number while retaining extensive NPC survival and fill of a spinal cord lesion site. NPCs derived from embryonic day 14 Fischer 344 rat spinal cords expressing green fluorescent protein (GFP) were embedded in fibrin matrices containing a defined growth factor cocktail: one to four factors were tested among nine different groups. Grafts were placed into C5 lateral hemisection lesion sites two weeks post-injury, and graft survival and fill was assessed two weeks later. We found that a four growth factor cocktail consisting of brain-derived neurotrophic factor (BDNF), basic-fibroblastic growth factor (bFGF), vascular endothelial growth factor (VEGF), and MDL28170, a cell death inhibitor, resulted in consistent graft survival, neuronal differentiation, and fill of the lesion site. Extensive stem cell-derived axonal outgrowth from the lesion site occurred, consistent with previous reports. Fewer than four growth factors resulted in suboptimal NPC fill of the lesion site. Collectively, these findings indicate that a simplified, four-component cocktail can support neural progenitor cell engraftment to a spinal cord lesion site to the same extent as a 10-component cocktail.

Natural killer cell-activating receptor NKG2D mediates innate immune targeting of allogeneic neural progenitor cell grafts

Cell replacement therapy holds promise for a number of untreatable neurological or psychiatric diseases but the immunogenicity of cellular grafts remains controversial. Emerging stem cell and reprogramming technologies can be used to generate autologous grafts that minimize immunological concerns but autologous grafts may carry an underlying genetic vulnerability that reduces graft efficacy or survival. Healthy allogeneic grafts are an attractive and commercially scalable alternative if immunological variables can be controlled. Stem cells and immature neural progenitor cells (NPC) do not express major histocompatibility complex (MHC) antigens and can evade adaptive immune surveillance. Nevertheless, in an experimental murine model, allogeneic NPCs do not survive and differentiate as well as syngeneic grafts, even when traditional immunosuppressive treatments are used. In this study, we show that natural killer (NK) cells recognize the lack of self-MHC antigens on NPCs and pose a barrier to NPC transplantation. NK cells readily target both syngeneic and allogeneic NPC, and killing is modulated primarily by NK-inhibiting "self" class I MHC and NK-activating NKG2D-ligand expression. The absence of NKG2D signaling in NK cells significantly improves NPC-derived neuron survival and differentiation. These data illustrate the importance of innate immune mechanisms in graft outcome and the potential value of identifying and targeting NK cell-activating ligands that may be expressed by stem cell derived grafts.

Intraspinal transplantation of GABAergic neural progenitors attenuates neuropathic pain in rats: A pharmacologic and neurophysiological evaluation

Dysfunctional γ-aminobutyric acid (GABA)-ergic inhibitory neurotransmission is hypothesized to underlie chronic neuropathic pain. Intraspinal transplantation of GABAergic neural progenitor cells (NPCs) may reduce neuropathic pain by restoring dorsal horn inhibition. Rat NPCs pre-differentiated to a GABAergic phenotype were transplanted into the dorsal horn of rats with unilateral chronic constriction injury (CCI) of the sciatic nerve. GABA signaling in antinociceptive effects of NPC grafts was tested with the GABAA receptor antagonist bicuculline (BIC), GABAB receptor antagonist CGP35348 (CGP) and GABA reuptake inhibitor SKF 89976A (SKF). NPC-treated animals showed decreased hyperalgesia and allodynia 1–3week post-transplantation; vehicle-injected CCI rats continued displaying pain behaviors. Intrathecal application of BIC or CGP attenuated the antinociceptive effects of the NPC transplants while SKF injection induced analgesia in control rats. Electrophysiological recordings in NPC treated rats showed reduced responses of wide dynamic range (WDR) neurons to peripheral stimulation compared to controls. A spinal application of BIC or CGP increased wind-up response and post-discharges of WDR neurons in NPC treated animals. Results suggest that transplantation of GABAergic NPCs attenuate pain behaviors and reduce exaggerated dorsal horn neuronal firing induced by CCI. The effects of GABA receptor inhibitors suggest participation of continuously released GABA in the grafted animals.

Growth factor-expressing human neural progenitor cell grafts protect motor neurons but do not ameliorate motor performance and survival in ALS mice

Neural progenitor cells (NPs) have shown several promising benefits for the treatment of neurological disorders. To evaluate the therapeutic potential of human neural progenitor cells (hNPs) in amyotrophic lateral sclerosis (ALS), we transplanted hNPs or growth factor (GF)-expressing hNPs into the central nervous system (CNS) of mutant Cu/Zn superoxide dismutase (SOD1(G93A)) transgenic mice. The hNPs were engineered to express brain-derived neurotrophic factor (BDNF), insulin-like growth factor-1 (IGF-1), VEGF, neurotrophin-3 (NT-3), or glial cell-derived neurotrophic factor (GDNF), respectively, by adenoviral vector and GDNF by lentiviral vector before transplantation. Donor-derived cells engrafted and migrated into the spinal cord or brain of ALS mice and differentiated into neurons, oligodendrocytes, or glutamate transporter-1 (GLT1)-expressing astrocytes while some cells retained immature markers. Transplantation of GDNF- or IGF-1-expressing hNPs attenuated the loss of motor neurons and induced trophic changes in motor neurons of the spinal cord. However, improvement in motor performance and extension of lifespan were not observed in all hNP transplantation groups compared to vehicle-injected controls. Moreover, the lifespan of GDNF-expressing hNP recipient mice by lentiviral vector was shortened compared to controls, which was largely due to the decreased survival times of female animals. These results imply that although implanted hNPs differentiate into GLT1-expressing astrocytes and secrete GFs, which maintain dying motor neurons, inadequate trophic support could be harmful and there is sexual dimorphism in response to GDNF delivery in ALS mice. Therefore, additional therapeutic approaches may be required for full functional recovery.

Transplantation and Magnetic Resonance Imaging of Canine Neural Progenitor Cell Grafts in the Postnatal Dog Brain

Cellular transplantation in the form of bone marrow has been one of the primary treatments of many lysosomal storage diseases (LSDs). Although bone marrow transplantation can help central nervous system manifestations in some cases, it has little impact in many LSD patients. Canine models of neurogenetic LSDs provide the opportunity for modeling central nervous system transplantation strategies in brains that more closely approximate the size and architectural complexity of the brains of children. Canine olfactory bulb-derived neural progenitor cells (NPCs) isolated from dog brains were expanded ex vivo and implanted into the caudate nucleus/thalamus or cortex of allogeneic dogs. Canine olfactory bulb-derived NPCs labeled with micron-sized superparamagnetic iron oxide particles were detected by magnetic resonance imaging both in vivo and postmortem. Grafts expressed markers of NPCs (i.e. nestin and glial fibrillary acidic protein), but not the neuronal markers Map2ab or beta-tubulin III. The NPCs were from dogs with the LSD mucopolysaccharidosis VII, which is caused by a deficiency of beta-glucuronidase. When mucopolysaccharidosis VII canine olfactory bulb-NPCs that were genetically corrected with a lentivirus vector ex vivo were transplanted into mucopolysaccharidosis VII recipient brains, they were detected histologically by beta-glucuronidase expression in areas identified by antemortem magnetic resonance imaging tracking. These results demonstrate the potential for ex vivo stem cell-based gene therapy and noninvasive tracking of therapeutic grafts in vivo.

Instructive cross‐talk between neural progenitor cells and gliomas

Gliomas are the most common primary brain tumors and offer a poor prognosis in patients because of their infiltrative and treatment-resistant nature. The median survival time after diagnosis is approximately 11-12 months. There is a strong need for novel treatment modalities in targeting gliomas, and recent advances use neural progenitor cells as delivery systems for different therapeutic strategies. In this study, we show that rat embryonic neural progenitor cell (NPC) lines, transplanted at a distant site from a 3-day-preestablished glioma in the striatum, were able to migrate toward and colocalize with tumor isles without general spread into the brain parenchyma. Upon encounter with tumor, neural progenitor cells changed phenotype and became vimentin positive. These results demonstrate that transplanted neural progenitor cells respond to queues from a tumor and home to and exert an antitumor effect on the preestablished glioma, significantly decreasing the tumor volume with approximately 67% compared with control tumors after 1-2 weeks. Moreover, these early effects could be translated into increased survival times of animals treated with neural progenitor cell grafts 3 days after intrastriatal tumor inoculation. In contrast, there was no activation or migration of endogenous subventricular zone (SVZ) neuroblasts in response to an intrastriatal syngeneic tumor. In conclusion, NPC possess the ability to influence tumor growth as well as respond to queues from the tumor or tumor microenvironment, demonstrating a cross-talk between the cells.

Failure of Schwann cells as supporting cells for adult neural progenitor cell grafts in the acutely injured spinal cord

Adult neural progenitor cells (NPC) co-grafted with fibroblasts replace cystic lesion defects and promote cell-contact-mediated axonal regeneration in the acutely injured spinal cord. Fibroblasts are required as a platform to maintain NPC within the lesion; however, they are suspected to create an inhospitable milieu for regenerating central nervous system (CNS) axons. Therefore, we thought to replace fibroblasts by primary Schwann cells, which might serve as a superior scaffold to maintain NPC within the lesion and might further enhance axon regrowth and remyelination following spinal cord injury. Adult rats underwent a cervical dorsal column transection immediately followed by transplantation of either NPC/Schwann cell or NPC/Schwann cell/fibroblast co-grafts. Animals receiving Schwann cell or fibroblast grafts alone, or Schwann cell/fibroblast co-grafts served as controls. At 3 weeks after injury/transplantation, histological analysis revealed that only fibroblast-containing grafts were able to replace the cystic lesion defect. In both co-cultures and co-grafts, Schwann cells and NPC were segregated. Almost all NPC migrated out of the graft into the adjacent host spinal cord. As a consequence, only peripheral-type myelin, but no CNS-type myelin, was detected within co-grafts containing NPC/Schwann cells. Corticospinal axon regeneration into Schwann-cell-containing co-grafts was reduced. Taken together, Schwann cells within NPC grafts contribute to remyelination. However, Schwann cells fail as a supporting platform to maintain NPC within the graft and impair CNS axon regeneration; this makes them an unfavorable candidate to support/augment NPC grafts following spinal cord injury.