Developing international open science collaborations: Funder reflections on the Open Science Prize.

The Open Science Prize was established with the following objectives: first, to encourage the crowdsourcing of open data to make breakthroughs that are of biomedical significance; second, to illustrate that funders can indeed work together when scientific interests are aligned; and finally, to encourage international collaboration between investigators with the intent of achieving important innovations that would not be possible otherwise. The process for running the competition and the successes and challenges that arose are presented.

Characterization of ectopic colonies that form in widespread areas of the nervous system with neural stem cell transplants into the site of a severe spinal cord injury.

We reported previously the formation of ectopic colonies in widespread areas of the nervous system after transplantation of fetal neural stem cells (NSCs) into spinal cord transection sites. Here, we characterize the incidence, distribution, and cellular composition of the colonies. NSCs harvested from E14 spinal cords from rats that express GFP were treated with a growth factor cocktail and grafted into the site of a complete spinal cord transection. Two months after transplant, spinal cord and brain tissue were analyzed histologically. Ectopic colonies were found at long distances from the transplant in the central canal of the spinal cord, the surface of the brainstem and spinal cord, and in the fourth ventricle. Colonies were present in 50% of the rats, and most rats had multiple colonies. Axons extended from the colonies into the host CNS. Colonies were strongly positive for nestin, a marker for neural precursors, and contained NeuN-positive cells with processes resembling dendrites, GFAP-positive astrocytes, APC/CC1-positive oligodendrocytes, and Ki-67-positive cells, indicating ongoing proliferation. Stereological analyses revealed an estimated 21,818 cells in a colony in the fourth ventricle, of which 1005 (5%) were Ki-67 positive. Immunostaining for synaptic markers (synaptophysin and VGluT-1) revealed large numbers of synaptophysin-positive puncta within the colonies but fewer VGluT-1 puncta. Continuing expansion of NSC-derived cell masses in confined spaces in the spinal cord and brain could produce symptoms attributable to compression of nearby tissue. It remains to be determined whether other cell types with self-renewing potential can also form colonies.

Grafted neural progenitors integrate and restore synaptic connectivity across the injured spinal cord.

Transplantation of neural progenitor cells (NPC) is a promising therapeutic strategy for replacing neurons lost after spinal cord injury, but significant challenges remain regarding neuronal integration and functional connectivity. Here we tested the ability of graft-derived neurons to reestablish connectivity by forming neuronal relays between injured dorsal column (DC) sensory axons and the denervated dorsal column nuclei (DCN). A mixed population of neuronal and glial restricted precursors (NRP/GRP) derived from the embryonic spinal cord of alkaline phosphatase (AP) transgenic rats were grafted acutely into a DC lesion at C1. One week later, BDNF-expressing lentivirus was injected into the DCN to guide graft axons to the intended target. Six weeks later, we observed anterogradely traced sensory axons regenerating into the graft and robust growth of graft-derived AP-positive axons along the neurotrophin gradient into the DCN. Immunoelectron microscopy revealed excitatory synaptic connections between regenerating host axons and graft-derived neurons at C1 as well as between graft axons and DCN neurons in the brainstem. Functional analysis by stimulus-evoked c-Fos expression and electrophysiological recording showed that host axons formed active synapses with graft neurons at the injury site with the signal propagating by graft axons to the DCN. We observed reproducible electrophysiological activity at the DCN with a temporal delay predicted by our relay model. These findings provide the first evidence for the ability of NPC to form a neuronal relay by extending active axons across the injured spinal cord to the intended target establishing a critical step for neural repair with stem cells.

Spatial and temporal changes in promoter activity of the astrocyte glutamate transporter GLT1 following traumatic spinal cord injury.

After traumatic spinal cord injury (SCI), there is an opportunity for preserving function by attenuating secondary cell loss. Astrocytes play crucial roles in the adult CNS and are responsible for the vast majority of glutamate buffering, potentially preventing excitotoxic loss of neurons and oligodendrocytes. We examined spatial and temporal changes in gene expression of the major astrocyte glutamate transporter GLT1 following moderate thoracic contusion SCI using transgenic BAC-GLT1-eGFP promoter reporter mice. In dorsal column white matter, total intensity of GLT1-eGFP expression per region was significantly reduced following SCI at both lesion epicenter and at rostral and caudal areas where no tissue loss occurred. This regional decrease in GLT1 expression was due to significant loss of GLT1-eGFP(+) cells, partially accounted for by apoptosis of eGFP(+) /GFAP(+) astrocytes in both white and gray matter. There were also decreased numbers of GLT1-eGFP-expressing cells in multiple gray matter regions following injury; nevertheless, there was sustained or even increased regional GLT1-eGFP expression in gray matter as a result of up-regulation in astrocytes that continued to express GLT1-eGFP. Although there were increased numbers of GFAP(+) cells both at the lesion site and in surrounding intact spinal cord following SCI, the majority of proliferating Ki67(+) /GFAP(+) astrocytes did not express GLT1-eGFP. These findings demonstrate that spatial and temporal alterations in GLT1 expression observed after SCI result from both astrocyte death and gene expression changes in surviving astrocytes. Results also suggest that following SCI a significant portion of astrocytes lacks GLT1 expression, possibly compromising the important role of astrocytes in glutamate homeostasis.

Promoting directional axon growth from neural progenitors grafted into the injured spinal cord.

Spinal cord injury (SCI) is a devastating condition characterized by disruption of axonal connections, failure of axonal regeneration, and loss of motor and sensory function. The therapeutic promise of neural stem cells has been focused on cell replacement, but many obstacles remain in obtaining neuronal integration following transplantation into the injured CNS. This study investigated the neurotransmitter identity and axonal growth potential of neural progenitors following grafting into adult rats with a dorsal column lesion. We found that using a combination of neuronal and glial restricted progenitors (NRP and GRP) produced graft-derived glutamatergic and GABAergic neurons within the injury site, with minimal axonal extension. Administration of brain-derived neurotrophic factor (BDNF) with the graft promoted modest axonal growth from grafted cells. In contrast, injecting a lentiviral vector expressing BDNF rostral into the injured area generated a neurotrophin gradient and promoted directional growth of axons for up to 9 mm. Animals injected with BDNF lentivirus (at 2.5 and 5.0 mm) showed significantly more axons and significantly longer axons than control animals injected with GFP lentivirus. However, only the 5.0-mm-BDNF group showed a preference for extension in the rostral direction. We concluded that NRP/GRP grafts can be used to produce excitatory and inhibitory neurons, and neurotrophin gradients can guide axonal growth from graft-derived neurons toward putative targets. Together they can serve as a building block for neuronal cell replacement of local circuits and formation of neuronal relays.

Repair of spinal cord injury with neuronal relays: From fetal grafts to neural stem cells.

Spinal cord injury (SCI) disrupts the long axonal tracts of the spinal cord leading to devastating loss of function. Cell transplantation in the injured spinal cord has the potential to lead to recovery after SCI via a variety of mechanisms. One such strategy is the formation of neuronal relays between injured long tract axons and denervated neurons. The idea of creating a neuronal relay was first proposed over 25 years ago when fetal tissue was first successfully transplanted into the injured rodent spinal cord. Advances in labeling of grafted cells and the development of neural stem cell culturing techniques have improved the ability to create and refine such relays. Several recent studies have examined the ability to create a novel neuronal circuit between injured axons and denervated targets. This approach is an alternative to long-distance regeneration of damaged axons that may provide a meaningful degree of recovery without direct recreation of lost pathways. This brief review will examine the contribution of fetal grafting to current advances in neuronal grafting. Of particular interest will be the ability of transplanted neurons derived from fetal grafts, neural precursor cells and neural stem cells to reconnect long distance motor and sensory pathways of the injured spinal cord. This article is part of a Special Issue entitled SI: Spinal cord injury.

Preparation of neural stem cells and progenitors: neuronal production and grafting applications.

Neural stem cells (NSC) are not only a valuable tool for the study of neural development and function, but an integral component in the development of transplantation strategies for neural disease. NSC can be used to study how neurons acquire distinct phenotypes and how the reciprocal interactions between neurons and glia in the developing nervous system shape the structure and function of the central nervous system (CNS). In addition, neurons prepared from NSC can be used to elucidate the molecular basis of neurological disorders as well as potential treatments. Although NSC can be derived from different species and many sources, including embryonic stem cells, induced pluripotent stem cells, adult CNS, and direct reprogramming of non-neural cells, isolating primary NSC directly from rat fetal tissue is the most common technique for preparation and study of neurons with a wealth of data available for comparison. Regardless of the source material, similar techniques are used to maintain NSC in culture and to differentiate NSC toward mature neural lineages. This chapter will describe specific methods for isolating multipotent NSC and neural precursor cells (NPC) from embryonic rat CNS tissue (mostly spinal cord). In particular, NPC can be separated into neuronal and glial restricted precursors (NRP and GRP, respectively) and used to reliably produce neurons or glial cells both in vitro and following transplantation into the adult CNS. This chapter will describe in detail the methods required for the isolation, propagation, storage, and differentiation of NSC and NPC isolated from rat spinal cords for subsequent in vitro or in vivo studies.