Current methods for bioprinting functional tissue lack appropriate biofabrication techniques to build complex 3D microarchitectures essential for guiding cell growth and promoting tissue maturation
. ...3D printing of central nervous system (CNS) structures has not been accomplished, possibly owing to the complexity of CNS architecture. Here, we report the use of a microscale continuous projection printing method (μCPP) to create a complex CNS structure for regenerative medicine applications in the spinal cord. μCPP can print 3D biomimetic hydrogel scaffolds tailored to the dimensions of the rodent spinal cord in 1.6 s and is scalable to human spinal cord sizes and lesion geometries. We tested the ability of µCPP 3D-printed scaffolds loaded with neural progenitor cells (NPCs) to support axon regeneration and form new 'neural relays' across sites of complete spinal cord injury in vivo in rodents
. We find that injured host axons regenerate into 3D biomimetic scaffolds and synapse onto NPCs implanted into the device and that implanted NPCs in turn extend axons out of the scaffold and into the host spinal cord below the injury to restore synaptic transmission and significantly improve functional outcomes. Thus, 3D biomimetic scaffolds offer a means of enhancing CNS regeneration through precision medicine.
Neural stem cells (NSCs) differentiate into both neurons and glia, and strategies using human NSCs have the potential to restore function following spinal cord injury (SCI). However, the time period ...of maturation for human NSCs in adult injured CNS is not well defined, posing fundamental questions about the design and implementation of NSC-based therapies. This work assessed human H9 NSCs that were implanted into sites of SCI in immunodeficient rats over a period of 1.5 years. Notably, grafts showed evidence of continued maturation over the entire assessment period. Markers of neuronal maturity were first expressed 3 months after grafting. However, neurogenesis, neuronal pruning, and neuronal enlargement continued over the next year, while total graft size remained stable over time. Axons emerged early from grafts in very high numbers, and half of these projections persisted by 1.5 years. Mature astrocyte markers first appeared after 6 months, while more mature oligodendrocyte markers were not present until 1 year after grafting. Astrocytes slowly migrated from grafts. Notably, functional recovery began more than 1 year after grafting. Thus, human NSCs retain an intrinsic human rate of maturation, despite implantation into the injured rodent spinal cord, yet they support delayed functional recovery, a finding of great importance in planning human clinical trials.
Highlights • Grafted neural stem cells extend high numbers of axons over long distances after spinal cord injury. • Graft derived axons make synaptic connections with host neurons. • Host supraspinal ...axons regenerate into and make connections with grafted neurons. • Grafted neurons serve as functional relays to improve behavioral outcomes.
Human induced pluripotent stem cells (iPSCs) from a healthy 86-year-old male were differentiated into neural stem cells and grafted into adult immunodeficient rats after spinal cord injury. Three ...months after C5 lateral hemisections, iPSCs survived and differentiated into neurons and glia and extended tens of thousands of axons from the lesion site over virtually the entire length of the rat CNS. These iPSC-derived axons extended through adult white matter of the injured spinal cord, frequently penetrating gray matter and forming synapses with rat neurons. In turn, host supraspinal motor axons penetrated human iPSC grafts and formed synapses. These findings indicate that intrinsic neuronal mechanisms readily overcome the inhibitory milieu of the adult injured spinal cord to extend many axons over very long distances; these capabilities persist even in neurons reprogrammed from very aged human cells.
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•Human iPSC-derived neural stem cells are grafted to sites of rat spinal cord injury•Human neurons extend numerous axons into host spinal cord and form synaptic contacts•Graft-derived human axons extend virtually the entire length of the rat CNS•Host axons penetrate human iPSC grafts and form synapses
Lu and colleagues report that human neural stem cells derived from iPSCs survive grafting to the injured spinal cord and extend very large numbers of axons over virtually the full length of the rat CNS.
Neural stem cells (NSCs) expressing GFP were embedded into fibrin matrices containing growth factor cocktails and grafted to sites of severe spinal cord injury. Grafted cells differentiated into ...multiple cellular phenotypes, including neurons, which extended large numbers of axons over remarkable distances. Extending axons formed abundant synapses with host cells. Axonal growth was partially dependent on mammalian target of rapamycin (mTOR), but not Nogo signaling. Grafted neurons supported formation of electrophysiological relays across sites of complete spinal transection, resulting in functional recovery. Two human stem cell lines (566RSC and HUES7) embedded in growth-factor-containing fibrin exhibited similar growth, and 566RSC cells supported functional recovery. Thus, properties intrinsic to early-stage neurons can overcome the inhibitory milieu of the injured adult spinal cord to mount remarkable axonal growth, resulting in formation of new relay circuits that significantly improve function. These therapeutic properties extend across stem cell sources and species.
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► Neural stem cells grow axons over very long distances after severe spinal cord injury ► New synaptic relays are formed, improving electrophysiological and functional outcome ► Rodent and human neural stem cells exhibit similar growth properties ► Mechanisms intrinsic to early-stage neurons overcome adult nervous system inhibition
Following severe spinal cord injury in rats, functional outcomes can be improved by the engraftment of neural stem cells embedded in a matrix that contains a growth factor cocktail. The resulting neurons extend axons over long distances and form reciprocal synaptic connections with neurons from the host.
Neural progenitor cell grafts form new relays across sites of spinal cord injury (SCI). Using a panel of neuronal markers, we demonstrate that spinal neural progenitor grafts to sites of rodent SCI ...adopt diverse spinal motor and sensory interneuronal fates, representing most neuronal subtypes of the intact spinal cord, and spontaneously segregate into domains of distinct cell clusters. Host corticospinal motor axons regenerating into neural progenitor grafts innervate appropriate pre-motor interneurons, based on trans-synaptic tracing with herpes simplex virus. A human spinal neural progenitor cell graft to a non-human primate also received topographically appropriate corticospinal axon regeneration. Thus, grafted spinal neural progenitor cells give rise to a variety of neuronal progeny that are typical of the normal spinal cord; remarkably, regenerating injured adult corticospinal motor axons spontaneously locate appropriate motor domains in the heterogeneous, developing graft environment, without a need for additional exogenous guidance.
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•Spinal neuroprogenitor grafts spontaneously segregate into motor or sensory domains•Grafted neuroprogenitor cells adopt diverse fates resembling the normal spinal cord•Corticospinal axons specifically regenerate into motor domains of grafts•Regenerating corticospinal axons avoid inappropriate sensory targets in grafts
Kumamaru et al. demonstrate that spinal cord neural progenitor cell grafts spontaneously segregate into motor and sensory domains when implanted into sites of spinal cord injury in rats and primates. Host corticospinal axons regenerating into grafts preferentially regenerate and synapse onto motor interneuron-rich domains, avoiding inappropriate sensory domains.
Previous studies have shown that neuron-specific deletion of serum response factor (SRF) results in deficits in tangential cell migration, guidance-dependent circuit assembly, activity-dependent gene ...expression, and synaptic plasticity in the hippocampus. Furthermore, SRF deletion in mouse embryonic stem cells causes cell death in vitro. However, the requirement of SRF for early neuronal development including neural stem cell homeostasis, neurogenesis, and axonal innervations remains unknown. Here, we report that SRF is critical for development of major axonal tracts in the forebrain. Conditional mutant mice lacking SRF in neural progenitor cells (Srf-Nestin-cKO) exhibit striking deficits in cortical axonal projections including corticostriatal, corticospinal, and corticothalamic tracts, and they show a variable loss of the corpus callosum. Neurogenesis and interneuron specification occur normally in the absence of SRF and the deficits in axonal projections were not due to a decrease or loss in cell numbers. Radial migration of neurons and neocortical lamination were also not affected. No aberrant cell death was observed during development, whereas there was an increase in the number of proliferative cells in the ventricular zone from embryonic day 14 to day 18. Similar axonal tract deficits were also observed in mutant mice lacking SRF in the developing excitatory neurons of neocortex and hippocampus (Srf-NEX-cKO). Together, these findings suggest distinct roles for SRF during neuronal development; SRF is specifically required in a cell-autonomous manner for axonal tract development but is dispensable for cell survival, neurogenesis, neocortical lamination, and neuronal differentiation.
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.
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