Fig 1.
AT-MSC characterizations and Transduction efficiency.
In freshly isolated cells major two subpopulations were present: (A) Hematopoietic (P1—CD45 positive) and non-hematopoietic cells (events outside P1, CD45 negative). Non-hematopoietic cells were analyzed in (B) and (C). Pre-adipocytes were detected in B (P2—CD146 negative and CD34 positive) and in C (P2—CD31 negative and CD34 positive). MSC were detected in B (P5—CD146 positive, CD34 negative). Endothelial progenitors were detected in C (P5—CD31 positive, CD34 positive). (D—F) After seeding, AT-MSC from monolayer was still negative for CD45 and positive for CD34 and CD146. (G-I) AT-MSC were also positive for CD105, CD73 and CD90, which are all mesenchymal surface markers in vitro. (J—L) AT-MSC are multipotent for adipogenic (Oil Red O staining), osteogenic (Alizarin staining) and chondrogenic (Pellet culture, safranin staining) lineages, respectively. (M) Transduction efficiency in AT-MSC of the lentivirus containing Tk-GFP is 80%.
Fig 2.
Bystander effect of transduced AT-MSC-Tk in U-87 cells.
U-87 cells were co-cultured in each well with the indicated AT-MSC cells at the same proportion. GCV 25 and GCV 50 indicate 25 μM and 50 μM of GCV, respectively; CONTROL indicates co-culture of U-87 and AT-MSC-GFP without GCV; GFP and TK-GFP indicate AT-MSC-GFP and AT-MSC-GFP-Tk cells, respectively. (A) Cell images were acquired, using an inverted microscope 8 days after GCV addition. (B) AT-MSC-Tk + GCV (25 and 50 μM) on the 15th day of culture. Bar = 50 μm.
Fig 3.
Tumor areas after gene therapy.
Ten days after AT-MSC or PBS injection, mice were sacrificed to determine tumor area by histology. Tissue samples were stained with hematoxylin. All mice received U-87 via intracranial injection and treated with:(A): PBS (IP); (B): AT-MSC-Tk (IC) and PBS (IP); (C): AT-MSC-Tk (IC) and GCV (IP). (D): a sample of tumor demarcation using Image J software to determine tumor area. (E) Tumor areas determined from all mice. * p = 0.0136 (group A vs group C). IC: intracranial injection; IP: intraperitoneal injection.
Fig 4.
Co-localization of tumor and AT-MSC-Tk by immunohistochemistry.
U-87 tumor and AT-MSC were labeled with human nuclei monoclonal antibody conjugated to FITC (green) and AT-MSC-Tk-GFP labeled with anti-GFP antibody conjugated to Alexa Fluor 594 (red). Therefore, AT-MSC double labeled is in yellow. (A-C) U-87 + AT-MSC-Tk + GCV (200 X); (A) anti-Hu; (B) Anti-GFP; (C) Merge; (D-F) U-87 + AT-MSC-Tk no GCV (100 X); (D) anti-Hu; (E) Anti-GFP; (F) Merge;(G-I) Brain slices from the contralateral hemisphere (200 X); (G) anti-Hu; (H) Anti-GFP; (I) Merge of G and I under visible light. (J-L) human brain tissue (100 X); (J) anti-Hu; (K) Anti-GFP; (L) Merge; (M) Digital amplification of the square area of the Fig 4C; (N) Digital amplification of the square area of the Fig 4F. White arrows indicate areas where tumor cells died by treatment, supposedly. Bar = 50 μm.