Figure 1.
In vitro luciferase reporter activity in F3-effLuc cells.
(A) Retroviral construct that contains the effLuc gene and Thy1.1 (CD90.1), linked with an IRES (internal ribosomal entry site). (B) Magnetic-activated cell sorting (MACS) was performed to collect the F3-effLuc cells. Flow activated cell sorting (FACS) analysis showed that more than 90% of cells were successively transfected with the effLuc vector. (C) The luciferase activity (n = 3) of F3-effLuc cells cultured in a 96-well plate were measured using an IVIS-100 optical imaging device. Firefly luciferase activity continuously increased in F3-effLuc cells in proportion to cell number, and (D) quantitative analysis showed a linear relationship between the cell number and bioluminescence signals.
Figure 2.
Validation of stem cell characteristics in F3 cells and F3-effLuc cells.
Flow cytometric analysis showed F3 and F3-effLuc cells are positive for the stem cell surface marker, (a) CD44, and the intracellular marker s, (b) Nestin, (c) Ki67, (d) Sox1, and (e) Sox2.
Figure 3.
In vitro proliferative effect in F3-effLuc cells within the PLLA scaffold.
(A) Scanning electron microscope (SEM) analysis was conducted to confirm adhesion of F3-effLuc cells to the PLLA scaffold. SEM images showed that F3-effLuc cells were stably attached onto the microfibers of the PLLA scaffold. (B) The luciferase intensity was quantified after F3-effLuc cells were incubated with the sterile PLLA scaffold. F3-effLuc cells incorporated within the PLLA scaffold were stably proliferated at 10 days.
Figure 4.
Schematic representation of the procedure for in vivo optical imaging.
(A) The protocol is for in vivo monitoring of F3-effLuc cells implanted in a corticectomized rat model. The motor cortex region of the Sprague-Dawley rat brain was surgically removed at the given coordinates and after 7 days, the rats were transplanted with F3-effLuc cells alone or F3-effLuc/PLLA scaffold complexes, and then administered cyclosporine A everyday. The grafted cells were monitored at 0, 1, 3, 5, 8, 11, and 14 days using a bioluminescence-imaging device. At the end of the implant period, histological analyses were performed using hematoxylin and eosin (H&E) staining and immunohistochemistry. (B) Behavior tests were performed 7 days after motor cortex ablation. The traumatic brain injury (TBI) models were evaluated by forelimb placing tests and whisker tactile tests in normal and corticectomized rats (n = 10).
Figure 5.
In vivo bioluminescence imaging of the implanted F3-effLuc/PLLA scaffold in a corticectomized rat model.
(A) After F3-effLuc cells were incubated within the PLLA scaffold for 2 hr, the cell/scaffold complex was implanted into the ablated motor cortex area of the rat brain. Firefly luciferase bioluminescence imaging was performed over 14 days. The prolonged luminescence signals in F3-effLuc cells within the PLLA scaffold were clearly visualized in the ablated area. (B) Quantitative ROI analysis showed significantly enhanced survival duration for F3-effLuc cells within the PLLA scaffold (n = 6). P value, * <0.005.
Figure 6.
Immunohistochemistry results for PLLA-encapsulated F3-effLuc cells implanted in a corticectomized rat brain.
(A) Hematoxylin and eosin (H&E) staining was performed on the corticectomized rat brains transplanted with the F3-effLuc/PLLA scaffold complex. Slices of the fixed brain were stained with hematoxylin and eosin to examine the presence of transplanted cells on the PLLA microfibers. (B) Confocal fluorescence images of the transplanted region (purple) revealed luciferase expression (green) in F3-effLuc cells was partially co-localized with the expression of Tuj1, a neuron-specific marker (red). Nuclei were visualized with DAPI (blue). Scale bars represent 20 µm. No luciferase expression was observed in F3 cell only-transplanted rat model.