Coseeded Schwann cells myelinate neurites from differentiated neural stem cells in neurotrophin-3-loaded PLGA carriers

Abstract

Biomaterials and neurotrophic factors represent promising guidance for neural repair. In this study, we combined poly-(lactic acid-co-glycolic acid) (PLGA) conduits and neurotrophin-3 (NT-3) to generate NT-3-loaded PLGA carriers in vitro. Bioactive NT-3 was released stably and constantly from PLGA conduits for up to 4 weeks. Neural stem cells (NSCs) and Schwann cells (SCs) were coseeded into an NT-releasing scaffold system and cultured for 14 days. Immunoreactivity against Map2 showed that most of the grafted cells (>80%) were differentiated toward neurons. Double-immunostaining for synaptogenesis and myelination revealed the formation of synaptic structures and myelin sheaths in the coculture, which was also observed under electron microscope. Furthermore, under depolarizing conditions, these synapses were excitable and capable of releasing synaptic vesicles labeled with FM1-43 or FM4-64. Taken together, coseeding NSCs and SCs into NT-3-loaded PLGA carriers increased the differentiation of NSCs into neurons, developed synaptic connections, exhibited synaptic activities, and myelination of neurites by the accompanying SCs. These results provide an experimental basis that supports transplantation of functional neural construction in spinal cord injury.

Keywords: NT-3; PLGA; Schwann cells; myelin sheath; neural stem cells.

Figure 1

Schwann cells (SCs) and neural stem cells (NSCs) culture and identification. (A) SCs (arrow) were viewed under light microscopy; (B) Cell bodies (arrow) were stained with antibodies against S100 and nuclei were labeled by DAPI; (C) Neurospheres were stained with antibodies against nestin, a marker of NSCs; (D) Nuclei in the neurosphere were labeled by DAPI. Scale bar = 20 μm in (A–B) and 10 μm in (C–D); (E) SEM of a transverse section of PLGA scaffold shows one of the tubes (arrow). There are numerous pores with variable diameters between tubes; (F) A longitudinal section of PLGA was imaged under high SEM. Note: The arrow points to the radial channel that extends from the scaffold tube. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; SEM, scanning electron microscope; PLGA, poly-(lactic acid-co-glycolic acid).

Figure 2

Secretion of NT-3 from NT-3-PLGA carriers loaded with three different SF solutions groups for at least 4 weeks. Daily release of NT-3 was examined by ELISA in (A) the 3% SF solution group; (B) the 1% SF solution group; (C) the 6% SF solution group, respectively; and (D) There was cumulative release of NT-3 across the three different groups after at least 4 weeks. Note: Error bars represent mean ± SD (n = 3 for each group). Abbreviations: NT-3, neurotrophin-3; PLGA, poly-(lactic acid-co-glycolic acid); SF, silk fibroin; ELISA, enzyme-linked immunosorbent assay.

Figure 3

Survival of cultured cells in the scaffold. NSCs and SCs were cultured in the NT-3-PLGA carriers for 14 days, and stained with calcein-AM and EthD-1. Viable cells were labeled green and dead cells were marked by red fluorescence. The arrows point to dead cells in (A) PLGA only; (B) PLGA-SF; and (C) the PLGA-SF-NT-3 group. The stars mark living cells; (D) Percentage of dead cells in the periphery and center of PLGA slices. Note: Scale bar = 20 μm in A–C. Abbreviations: NSCs, neural stem cells; SCs, Schwann cells; EthD, ethidium homodimer; NT-3, neurotrophin-3; PLGA, poly-(lactic acid-co-glycolic acid); SF, silk fibroin.

Figure 4

Differentiation detection of grafted NSCs. Cells were immunostained with markers for neurons in PLGA only. (A) PLGA-SF; (B) PLGA-SF-NT-3; (C) group (Map2; arrows in (A–C) green), astrocytes (GFAP; stars in A–C = red). The nuclei were labeled by DAPI (=blue); (D) Compared with other groups, cells expressing Map2 in the PLGA-SF-NT-3 group were significantly higher than any other groups (P < 0.05; n = 3 for each group). Cells positive for GFAP were much less. By contrast, astrocytes were abundant in both the PLGA and PLGA-SF groups. Scale bar = 20 μm in (A–C). Cells collected from the slices were lysed, and proteins were extracted for Western blotting analysis; (E) Levels of TuJ-1 expression appeared higher in the PLGA-SF-NT-3 group than those in the PLGA, or PLGA-SF groups. Proteins extracted from the rat brain were used as a positive control (brain); (F) Quantification of TuJ-1 blot normalized against GAPDH. (P < 0.05; n = 3 for each group); (G) Western blotting against GFAP; (H) Quantitative analysis showed that the level of GFAP in the PLGA-SF-NT-3 was lower than that of any other groups (P < 0.05; n = 3 for each group). Stars in (D), (F), and (H) indicate P < 0.05 when PLGA-SF-NT-3 versus PLGA or PLGA-SF. Abbreviations: NSCs, neural stem cells; PLGA, poly-(lactic acid-co-glycolic acid); SF, silk fibroin; NT-3, neurotrophin-3; Map2, microtubule-associated protein 2; GFAP, glial fibrillary acidic protein; DAPI, 4′,6-diamidino-2-phenylindole; TuJ-1, neuron-specific class III beta-tubulin; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase.

Figure 5

Synaptic formation of culture cells in the NT-3-PLGA carriers. Cells were immunostained with antibodies against pre- and postsynaptic markers (PSD95 versus synapsin). Nuclei were labeled by DAPI (blue). (A) Only a few cells expressed synapsin (stars in A–C = green) and PSD95 (arrows in A–C = red) in the PLGA group. PSD95 was localized in the cell body; whereas synapsin was expressed in both the cell body and neurite; (B) Synapsin- or PSD95-positive cells were sparse in the PLGA-SF group; (C) In the PLGA-SF-NT3 group, the cells expressed (1) cells positive for either PSD95 or synapsin; (2) cells positive for both PSD95 and synapsin; (3) cells with positive PSD95-staining in the body, but receiving synapsin-positive neurites extending from another cell; (D) Cells with staining of PSD95 and synapsin were manually counted. Compared with other groups, positively stained cells (Synapsin⁺ or PSD95⁺) in the PLGA-SF-NT3 group were significantly higher than any other groups (P < 0.05; the star in D indicates P < 0.05 when PLGA-SF-NT-3 versus PLGA or PLGA-SF); (E) Under transmission electron microscopy (TEM), a neuron was identified in PLGA-SF-NT-3 group. Note the neurite extending from the soma of one cell and connecting to a neutrite of another cell. (F) High magnification TEM of synapse formation in cultured cells, revealing specialized high-density pre- and postsynaptic membrane, synaptic cleft, and vesicles in its terminal. Note: Scale bar = 20 μm in A–C, 200 nm in F. Abbreviations: NT-3, neurotrophin-3; PLGA, poly-(lactic acid-co-glycolic acid); PSD95, postsynaptic density-95; DAPI, 4′,6-diamidino-2-phenylindole; SF, silk fibroin.

Figure 6

Release of synaptic vesicles during depolarization by high [K⁺]. (A) In the PLGA-SF-NT-3 group, FM1-43 fluorescent puncta were particularly concentrated at sites where neurites crossed over (see arrows) under stimulation of high [K⁺] (50 mM); (B) After 3-minute stimulation by the high [K⁺], fluorescence intensity of these puncta was weakened or disappeared; (C) By contrast, uptake of the fluorescent signal into the cells in the NSCs group was only minimal, and showed weak fluorescence prior to three-minute stimulation; (D) Fluorescence intensity remained unchanged after the stimulation period; (E) FM4-64 fluorescent puncta before; and (F) after stimulation of high [K⁺]; (G). FM1-43 fluorescence was tracked on the cells in the PLGA-SF-NT-3 group, which was continuously depolarized for 5 minutes. Recording from the cytoplasm was used as a control, and showed no significant change over the 5-minute period (arrow in G). Abbreviations: PLGA, poly-(lactic acid-co-glycolic acid); SF, silk fibroin; NT-3, neurotrophin-3; NSCs, neural stem cells; FM1-43, [(N-3-triethylammonmpropyl)-4- (4-(dibutylamino)styryl)]; FM4-64, [N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl) hexatrienyl)pyridiniumdibromide].

Figure 7

Formation of myelin sheath in the coculture system. NSCs and SCs in the cells/PLGA were cultured for 14 days, and immunostained with antibodies against neuron and myelin sheath markers (Map2 = red versus MBP = green). Nuclei were labeled by DAPI (blue). (A) Arrow indicates segments of neuronal axons wrapped by (MBP-positive) myelin sheath in PLGA; (B) the PLGA-SF group; and (C) PLGA-SF-NT-3 group, respectively; (D) Abundance of MBP-positive fibers in the PLGA-SF-NT-3 group compared with other groups (P < 0.05; n = 3 for each group); (E) TEM showing formation of the myelin sheath in the PLGA-SF-NT-3 group; (F) Typical, characteristic sheath composed of several layers wrapped around axons. Note: Scale bar = 20 μm in (A–C), 200 nm in (F). Abbreviations: NSCs, neural stem cells; SCs, Schwann cells; PLGA, poly-(lactic acid-co-glycolic acid); SF, silk fibroin; NT-3, neurotrophin-3; Map2, microtubule-associated protein 2; MBP, myelin basic protein; TEM, transmission electron microscopy.