SUMMARY 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 novel relay circuits that significantly improve function. These therapeutic properties extend across stem cell sources and species.

Current methods for bioprinting functional tissue lack appropriate biofabrication techniques to build complex 3D microarchitectures essential for guiding cell growth and promoting tissue maturation1. 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 rodents1,2. 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.

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 central nervous system. 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.

Spinal cord neural stem cells (NSCs) have great potential to reconstitute damaged spinal neural circuitry, but they have yet to be generated in vitro. We now report the derivation of spinal cord NSCs from human pluripotent stem cells (hPSCs). Our observations show that these spinal cord NSCs differentiate into a diverse population of spinal cord neurons occupying multiple positions along the dorso-ventral axis, and can be maintained for prolonged time periods. Grafts into injured spinal cords were rich with excitatory neurons, extended large numbers of axons over long distances, innervated their target structures, and enabled robust corticospinal regeneration. The grafts synaptically integrated into multiple host intraspinal and supraspinal systems, including the corticospinal projection, and improved functional outcomes after injury. hPSC-derived spinal cord NSCs could enable a broad range of biomedical applications for in vitro disease modeling and constitute an improved clinically translatable cell source for 'replacement' strategies in several spinal cord disorders.

Abstract. The leukocyte adhesion molecule L-selectin mediates binding to lymph node high endothelial venules (HEV) and contributes to leukocyte rolling on endothelium at sites of inflammation. Previously, it was shown that truncation of the L-selectin cytoplasmic tail by 11 amino acids abolished binding to lymph node HEV and leukocyte rolling in vivo, but the molecular basis for that observation was not determined. This study examined potential interactions between L-selectin and cytoskeletal proteins. We found that the cytoplasmic domain of L-selectin interacts directly with the cytoplasmic actin-binding protein ot-actinin and forms a complex with vinculin and possibly talin. Solid phase binding assays using the full-length L-selectin cytoplasmic domain bound to microtiter wells demonstrated direct, specific, and saturable binding of purified ot-actinin to L-selectin (Kd = 550 nM), but no direct binding of purified talin or vinculin. Interestingly, talin potentiated binding of ot-actinin to the L-selectin cytoplasmic domain peptide despite the fact that direct binding of talin to L-selectin could not be measured. Vinculin binding to the L-selectin cytoplasmic domain peptide was detectable only in the presence of ot-actinin. L-selectin coprecipitated with a complex of cytoskeletal proteins including ct-actinin and vinculin from cells transfected with L-selectin, consistent with the possibility that ot-actinin binds directly to L-selectin and that vinculin associates by binding to ot-actinin in vivo to link actin filaments to the L-selectin cytoplasmic domain. In contrast, a deletion mutant of L-selectin lacking the COOH-terminal 11 amino acids of the cytoplasmic domain failed to coprecipitate with ot-actinin or vinculin. Surprisingly, this mutant L-selectin localized normally to the microvillar projections on the cell surface. These data suggest that the COOH-terminal 11 amino acids of the L-selectin cytoplasmic domain are required for mediating interactions with the actin cytoskeleton via a complex of ct-actinin and vinculin, but that this portion of the cytoplasmic domain is not necessary for proper localization of L-selectin on the cell surface. Correct L-selectin receptor positioning is therefore insufficient for leukocyte adhesion mediated by L-selectin, suggesting that this adhesion may also require direct interactions with the cytoskeleton. YMPHOCYTE migration through lymphoid organs and leukocyte traffic into sites of inflammation are related processes that are both regulated principally at the level of leukocyte interactions with the lumenal surface of postcapillary venules. A number of leukocyte and endothelial cell surface molecules that participate in this interaction have been identified, including members of at least three Please address all correspondence to E M. Pavalko;

Cardiac valve calcification often results in obstruction of blood flow, which eventually leads to valve replacement. The molecular mechanisms resulting in valve calcification are unknown. Collagen and specific bone matrix proteins are thought to provide the framework for ectopic tissue calcification. This investigation was performed to determine whether the bone matrix protein osteopontin was present in calcified human aortic valves. Proteins extracted from human aortic valve tissue were subjected to polyacrylamide gel electrophoresis followed by Western blotting, using polyclonal antibodies directed against osteopontin. Fresh frozen tissue sections were also screened for osteopontin and macrophages using immunohistochemical techniques. Osteopontin was present in both heavily and minimally calcified aortic valves and absent in noncalcified purely regurgitant or normal aortic valves by both radioimmunoassay (n = 16) and immunohistochemical techniques (n = 8). Osteopontin colocalized with valvular calcific deposits, and macrophages were identified in the vicinity of osteopontin. These results, in addition to showing that osteopontin is present in calcified human aortic valves, suggest that osteopontin is a regulatory protein in pathological calcification. Identification of the cells producing osteopontin in abnormal cardiac valves and of proximate stimuli for its secretion may lead to novel therapeutic strategies to prevent and/or reverse calcific valve disease.

We subjected rats to either partial mid-cervical or complete upper thoracic spinal cord transections and examined whether combinatorial treatments support motor axonal regeneration into and beyond the lesion. Subjects received cAMP injections into brainstem reticular motor neurons to stimulate their endogenous growth state, bone marrow stromal cell grafts in lesion sites to provide permissive matrices for axonal growth, and brain-derived neurotrophic factor (BDNF) gradients beyond the lesion to stimulate distal growth of motor axons. Findings were compared to several control groups. Combinatorial treatment generated motor axon regeneration beyond both C5 hemisection and complete transection sites. Yet despite formation of synapses with neurons below the lesion, motor outcomes worsened after partial cervical lesions and spasticity worsened after complete transection. These findings highlight the complexity of spinal cord repair, and the need for additional control and shaping of axonal regeneration.

Olfactory ensheathing cells (OECs) have been reported to migrate long distances and to bridge lesion sites, guiding axonal regeneration after spinal cord injury (SCI). To understand mechanisms of OEC migration and axonal guidance, we injected lamina propria OECs 1 mm rostral and caudal to C4 SCI sites. One month later, OECs formed an apparent migrating cell tract continuously extending from the injection site through the lesion, physically bridging the lesion. Confocal immunolabeling demonstrated that, whereas this cell tract displaced host astrocytes, descending or ascending long tract axons did not preferentially extend into the cell tract and OECs failed to support bridging of corticospinal axons. Notably, the "bridging" tract of OECs formed within 1 h of cell injection, raising the possibility that cells passively spread from the pressure injection site rather than actively migrating. Control injections of bone marrow stromal cells (MSCs) or fibroblasts 1 mm from the lesion site also rapidly dispersed into the lesion cavity. Cell tracts extending into the lesion site were not seen when cells were injected either at low volumes, into spinal cord gray matter, or 3 d before or 9 d after SCI. OECs proliferated in injection sites, cell tracts, and lesion sites, indicating that OECs can also accumulate through cell proliferation. Thus, OECs do not appear to exhibit significant migratory properties when grafted to the spinal cord, exhibit no detectable difference in promoting axon growth into a SCI site compared with MSCs or fibroblasts, and do not support bridging of corticospinal axons beyond a dorsal column lesion.