Synaptic transmission of neural stem cells seeded in 3-dimensional PLGA scaffolds.

To explore therapeutic potential of engineered neural tissue, we combined genetically modified neural stem cells (NSCs) and poly(lactic acid-co-glycolic acid) (PLGA) polymers to generate an artificial neural network in vitro. NSCs transfected with either NT-3 or its receptor TrkC gene were seeded into PLGA scaffold. The NSCs were widely distributed and viable in the scaffold after culturing for 14 days. Immunoreactivity against Map2 was detected in >70% of these grafted cells, suggesting a high rate of differentiation toward neurons. Immunostaining of synapsin-I and PSD95 revealed formation of synaptic structures, which was also observed under electron microscope. Furthermore, using FM1-43 dynamic imaging, synapses in these differentiated neurons were found to be excitable and capable of releasing synaptic vesicles. Taken together, our artificial PLGA construct permits NSCs to differentiate toward neurons, establish connections and exhibit synaptic activities. These findings provide a biological basis for future application or transplantation of this artificial construct in neural repair.

[1]  J. Barker,et al.  CNS stem and progenitor cell differentiation into functional neuronal circuits in three-dimensional collagen gels , 2004, Experimental Neurology.

[2]  Andrés Hurtado,et al.  Poly (D,L-lactic acid) macroporous guidance scaffolds seeded with Schwann cells genetically modified to secrete a bi-functional neurotrophin implanted in the completely transected adult rat thoracic spinal cord. , 2006, Biomaterials.

[3]  Xuebao Zhang,et al.  Co-transplantation of neural stem cells and NT-3-overexpressing Schwann cells in transected spinal cord. , 2007, Journal of neurotrauma.

[4]  K. Kang,et al.  NPC1 Gene Deficiency Leads to Lack of Neural Stem Cell Self‐Renewal and Abnormal Differentiation Through Activation of p38 Mitogen‐Activated Protein Kinase Signaling , 2006, Stem cells.

[5]  D. Riche,et al.  Maturation of Fetal Human Neural Xenografts in the Adult Rat Brain , 1997, Experimental Neurology.

[6]  Robert Langer,et al.  Functional recovery following traumatic spinal cord injury mediated by a unique polymer scaffold seeded with neural stem cells , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[7]  Nobuko Uchida,et al.  Human neural stem cells differentiate and promote locomotor recovery in spinal cord-injured mice. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[8]  Y. Ding,et al.  Cotransplant of neural stem cells and NT-3 gene modified Schwann cells promote the recovery of transected spinal cord injury , 2007, Spinal Cord.

[9]  M. Chao,et al.  Neurotrophins and their receptors: A convergence point for many signalling pathways , 2003, Nature Reviews Neuroscience.

[10]  James R. Woodgett,et al.  Phosphorylation of c-jun mediated by MAP kinases , 1991, Nature.

[11]  Kwideok Park,et al.  Quantitative Analysis of Temporal and Spatial Variations of Chondrocyte Behavior in Engineered Cartilage during Long-Term Culture , 2007, Annals of Biomedical Engineering.

[12]  Seeram Ramakrishna,et al.  Manufacture of PLGA multiple-channel conduits with precise hierarchical pore architectures and in vitro/vivo evaluation for spinal cord injury. , 2009, Tissue engineering. Part C, Methods.

[13]  Wenlin Huang,et al.  Recombinant adenovirus vector-mediated functional expression of neurotropin-3 receptor (TrkC) in neural stem cells , 2007, Experimental Neurology.

[14]  Zhuan Zhou,et al.  “Kiss-and-Run” Glutamate Secretion in Cultured and Freshly Isolated Rat Hippocampal Astrocytes , 2005, The Journal of Neuroscience.

[15]  Jiasong Guo,et al.  Co-Transplantation of Schwann Cells Promotes the Survival and Differentiation of Neural Stem Cells Transplanted into the Injured Spinal Cord , 2005, Developmental Neuroscience.

[16]  A. Faden.  Experimental neurobiology of central nervous system trauma. , 1993, Critical reviews in neurobiology.

[17]  M. Oudega,et al.  Freeze-dried poly(D,L-lactic acid) macroporous guidance scaffolds impregnated with brain-derived neurotrophic factor in the transected adult rat thoracic spinal cord. , 2004, Biomaterials.

[18]  D. Lackland,et al.  Traumatic spinal cord injury mortality, 1981-1998. , 2009, The Journal of trauma.

[19]  Feng Yang,et al.  Distinct Mechanisms for Neurotrophin-3-Induced Acute and Long-Term Synaptic Potentiation , 2005, The Journal of Neuroscience.

[20]  R. Misra,et al.  Biomaterials , 2008 .

[21]  Peter Sterling,et al.  Synaptic Ca2+ in Darkness Is Lower in Rods than Cones, Causing Slower Tonic Release of Vesicles , 2007, The Journal of Neuroscience.

[22]  T. Zahir,et al.  Biodegradable Polymer Composite Grafts Promote the Survival and Differentiation of Retinal Progenitor Cells , 2005, Stem cells.

[23]  F. Kirchhoff,et al.  Astroglial processes show spontaneous motility at active synaptic terminals in situ , 2004, The European journal of neuroscience.

[24]  S. Goldman.  Stem and progenitor cell–based therapy of the human central nervous system , 2005, Nature Biotechnology.

[25]  F. Gage,et al.  Mammalian neural stem cells. , 2000, Science.

[26]  D. Anderson,et al.  Stem Cells and Pattern Formation in the Nervous System The Possible versus the Actual , 2001, Neuron.

[27]  M. Tuszynski,et al.  Neural stem cells constitutively secrete neurotrophic factors and promote extensive host axonal growth after spinal cord injury , 2003, Experimental Neurology.

[28]  Yaniv Ziv,et al.  Synergy between immune cells and adult neural stem/progenitor cells promotes functional recovery from spinal cord injury , 2006, Proceedings of the National Academy of Sciences.

[29]  E. Snyder,et al.  A Novel, Immortal, and Multipotent Human Neural Stem Cell Line Generating Functional Neurons and Oligodendrocytes , 2007, Stem cells.

[30]  Krista L. Niece,et al.  Selective Differentiation of Neural Progenitor Cells by High-Epitope Density Nanofibers , 2004, Science.

[31]  Young Ha Kim,et al.  In vivo conjunctival reconstruction using modified PLGA grafts for decreased scar formation and contraction. , 2003, Biomaterials.

[32]  M. J. Moore,et al.  Multiple-channel scaffolds to promote spinal cord axon regeneration. , 2006, Biomaterials.

[33]  W. Huttner,et al.  Isolation of neural stem cells from the postnatal cerebellum , 2005, Nature Neuroscience.

[34]  Feng Yang,et al.  Neurotrophin 3 induces structural and functional modification of synapses through distinct molecular mechanisms , 2006, The Journal of cell biology.

[35]  Petti T. Pang,et al.  The yin and yang of neurotrophin action , 2005, Nature Reviews Neuroscience.

[36]  P. Stieg,et al.  Neural stem cells may be uniquely suited for combined gene therapy and cell replacement: Evidence from engraftment of Neurotrophin-3-expressing stem cells in hypoxic–ischemic brain injury , 2006, Experimental Neurology.