Application Of Biodegradable Synthetic Polymer To Neural Stem Cells

Although neural stem cells appear to have the capacity to repopulate HI-injured brain, particularly in the penumbra, their ability to reform structural and functional neural connections may be limited by the vast amount of brain parenchymal loss.

The core of the infarct changes rapidly to a cystic cavity. Even the most capable stem cell may need intrinsic organization and a template to guide restructuring. Furthermore, large volumes of cells will not survive if located greater than a few 100 |im from the nearest capillary. To address this need, a pilot experiment was performed.55 Polyglycolic acid (PGA) is a synthetic biodegradable polymer used widely in clinical medicine.56 Highly hydrophilic, PGA loses its mechanical strength rapidly over 2-4 weeks in the body. We hypothesized that three-dimensional highly porous "scaffolds" composed of PGA, if co-transplanted with neural stem cells into the infarction cavity might facilitate reformation of structural and functional circuits, particularly if the cells had been engineered ex vivo to also express factors that might attract ingrowth of host fibers. The scaffold might initially provide a matrix to guide cellular organization and growth, allow diffusion of nutrients to the transplanted cells, become vascularized, and then disappear, obviating concerns over long-term biocompatibility.

To test this hypothesis, clone C17.2 neural stem cells were seeded onto PGA scaffolds in culture. The cells grew robustly, migrated readily throughout the structure, and differentiated spontaneously into neurons and glias, adhering to the polymer fibers. Immunostaining showed that most cells robustly differentiated into neurons, sending out long neurofilament (NF)+ processes. They extended long axon-like processes along the fibers and developed small, complex dendrite-like processes. After 4-6 days, in vitro, the cell-polymer unit was implanted into the evolving cystic infarct cavity of mice subjected 1 week prior to unilateral HI brain injury. After 2-6 weeks, the cells had, indeed, completely impregnated the polymer, and the polymer/stem cell unit refilled the infarction cavity, becoming incorporated into animal's cerebrum and even becoming vascularized by the host (Figure 3.3). The NSCs seeded on polymers displayed robust engraftment, foreign gene (lacZ)

figure 3.3 Implantation of PGA (polyglycolic acid) polymers-neural stem cells complex into the infracted region of a mouse brain subjected to unilateral focal hypoxic-ischemic (HI) injury. This mouse was subjected to right hypoxic-ischemic injury on postnatal day 7 (P7). Seven days later (P14), the animal received a transplant of clone C17.2 neural stem cells-PGA matrix complex, initially generated in vitro, into HI-generated infarction cavity. The animal was analyzed at maturity. A representative coronal section is shown. NSCs-polymer complex can fill that cavity (arrow in A), appear to be incorporated into the infracted cerebrum (A), and even support neovascularization (arrows in B point to host blood vessels in the stem cellpolymer unit) (arrowheads in A and B point to dark fibers from the polymer).

figure 3.3 Implantation of PGA (polyglycolic acid) polymers-neural stem cells complex into the infracted region of a mouse brain subjected to unilateral focal hypoxic-ischemic (HI) injury. This mouse was subjected to right hypoxic-ischemic injury on postnatal day 7 (P7). Seven days later (P14), the animal received a transplant of clone C17.2 neural stem cells-PGA matrix complex, initially generated in vitro, into HI-generated infarction cavity. The animal was analyzed at maturity. A representative coronal section is shown. NSCs-polymer complex can fill that cavity (arrow in A), appear to be incorporated into the infracted cerebrum (A), and even support neovascularization (arrows in B point to host blood vessels in the stem cellpolymer unit) (arrowheads in A and B point to dark fibers from the polymer).

expression and differentiation into neurons and glia within the region of HI injury, virtually repopulating the injured brains. Many long neuronal processes of host and donor-derived neurons enwrapped the polymer fibers and ran along the length of the fibers, often interconnecting with each other. Donor-derived neurons appeared to extend many exceedingly long, complex processes along the length of the disappearing matrix, extending ultimately into host parenchyma apparently as far as the opposite intact hemisphere. Host neuronal processes, in a reciprocal manner, appeared to enter the matrix, possibly making contact with donor-derived neurons. In order to confirm the ability of engrafted NSCs-polymer complex to establish longdistance neuronal connections, both the anterograde and retrograde label DiI, and the anterograde label "biotinylated dextran amine conjugated with fluorescein" (BDA-FITC) was stereotactically injected into NSCs-polymer complex transplant site or contralateral intact cortex 8 weeks after transplantation. Indeed, neuronal tracing studies showed that long-distance neuronal circuitry between donor-derived and host neurons in both cerebral hemispheres may have been reformed through the corpus callosum in some instances55. Although these findings are still preliminary, it appears feasible to implant NSCs-polymer complexes in order to augment reparative responses, to facilitate even further the differentiation of host and donor neurons, and to enhance the ingrowth/outgrowth of such cells in an effort literally to facilitate reformation of structural/functional cortical tissue and promote neuronal connectivity.

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