Methods for use of neural stem cell compositions for treatment of central nervous system lesions
Inventors
Assignees
US Department of Veterans Affairs • University of California San Diego UCSD
Publication Number
US-9649358-B2
Publication Date
2017-05-16
Expiration Date
2032-12-10
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Abstract
Methods for inducing non-embryonic lesioned central nervous system neurons to survive, integrate, extend axons over long distances, induce intra-lesion ingrowth of neurons into the lesion from host tissue and form synapses in vivo. Pluripotent neural stem cells are grafted into the lesioned CNS tissue within a tissue adhesive suspension, optionally in the presence of growth factors. No modification of the neuronal regenerative inhibitory environment of the CNS is necessary.
Core Innovation
The invention provides methods for inducing survival, integration, and extensive axonal growth of non-embryonic lesioned central nervous system neurons. These neurons extend axons over long distances, induce intra-lesion ingrowth of neurons into the lesion from host tissue, and form functional synapses in vivo. The methods involve grafting pluripotent neural stem cells into lesioned CNS tissue within a tissue adhesive suspension, optionally in the presence of growth factors, without the need to modify the inhibitory regenerative environment of the CNS.
This approach challenges the conventional understanding that modification of the inhibitory CNS milieu is necessary to achieve axonal regeneration and synapse formation after CNS injury. Contrary to prior beliefs, neuron-intrinsic mechanisms are sufficient to support extensive corticospinal regeneration and synapse formation when neural stem cells at an early, undifferentiated stage of development (N1 or earlier) are grafted appropriately and evenly distributed within the lesion using a transplantation matrix such as fibrin glue.
The invention further optionally enhances stem cell survival and axonal growth by co-delivering growth factors directly at the graft site, including brain-derived neurotrophic factor (BDNF) and others. Importantly, applying growth factors to the surrounding CNS environment or modifying the inhibitory environment is not required. The grafted neural stem cells differentiate into neurons, astrocytes, and oligodendrocytes, extend axons through the lesion site into host tissue over long distances, including the brain and spinal cord segments caudal to the lesion, and form synaptic contacts with host circuitry. Host axons also penetrate the grafts and form synapses, demonstrating reciprocal connectivity.
Claims Coverage
The patent contains two independent claims covering methods for treating lesions in post-embryonic spinal nerves using grafting of human pluripotent neural stem cells distributed with a fibrin transplantation matrix and brain-derived neurotrophic factor (BDNF). The inventive features focus on the cell stage, distribution, growth factor use, functional outcomes, and synapse formation.
Grafting of human pluripotent neural stem cells with growth factor and matrix into lesion
The method involves grafting human pluripotent neural stem cells evenly distributed throughout a transplantation matrix comprising fibrin together with brain-derived neurotrophic factor (BDNF) into the lesion or no more than 1 mm away, by injection or infusion.
Use of early-stage (N1 or earlier) pluripotent neural stem cells
The pluripotent neural stem cells used are at the N1 stage or earlier of development to achieve effective axonal growth and functional restoration.
Use of embryonic or induced pluripotent mammalian origin cells
The pluripotent neural stem cells grafted are of embryonic mammalian origin or induced pluripotent cells derived from post-natal mammalian tissue.
Concentration ranges for stem cells and BDNF
The neural stem cell concentration in grafts ranges from 10,000 to 500,000 cells per microliter of transplantation matrix. The total concentration of BDNF administered ranges from 1 fg protein/ml to 1 mg protein/ml of pharmaceutically acceptable composition or is expressed in situ via recombinant vectors at equivalent concentrations.
Filling lesion fully with stem cells and matrix
The lesion is fully filled by the neural stem cells and transplantation matrix to ensure even distribution and support of regrowth.
Formation of new synaptic junctions and functional restoration
New synaptic junctions form at termini of growing axons, and the method leads to clinically significant restoration of locomotor function of spinal nerves comparable to improvements measured by the Basso, Beattie, and Bresnahan (BBB) scale.
Overall, the independent claims cover methods of treating spinal cord lesions by grafting early-stage human pluripotent neural stem cells in a fibrin matrix with BDNF applied directly at or near the lesion, achieving extensive axonal growth, synapse formation, and significant functional locomotor recovery without modifying the CNS inhibitory environment.
Stated Advantages
The invention achieves extensive, long-distance CNS axonal regeneration and formation of new synaptic junctions without needing to modify the inhibitory CNS environment.
It provides a clinically useful protocol that results in survival, integration, and differentiation of grafted neural stem cells within the lesion site.
Grafted neurons extend axons over remarkably long distances into the host spinal cord and brainstem and into ventral roots, with axons often myelinated and expressing mature markers.
The method induces functional synaptic contacts between graft-derived axons and host neurons and reciprocal host axonal ingrowth into grafts, facilitating host-graft connectivity.
Co-administration of growth factor BDNF significantly increases the density of axonal growth and terminal bouton clustering, improving regenerative outcomes.
The method leads to significant improvements in locomotor function in spinal cord injured animals as measured by behavioral scales such as the BBB.
Documented Applications
Treatment of central nervous system lesions in mammals, including spinal cord injuries resulting from trauma, disease, or degeneration.
Use of neural stem cells to induce corticospinal regeneration and formation of functional synaptic junctions in the spinal cord.
Grafting of pluripotent neural stem cells in lesion sites of the mammalian spinal cord to induce axonal extension through white matter tracts extending to brainstem and caudal spinal cord.
Human therapy applications using human induced pluripotent stem cell-derived neural stem cells for spinal cord injury repair.
Assessment of motor function recovery in treated subjects using locomotor function tests such as the Basso, Beattie, and Bresnahan (BBB) scale and grid-walking tasks.
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