Figure 1.
After culturing in hematopoietic induction medium for one week, human ADSCs were photographed and then harvested for RT-PCR analysis. (A) The induced ADSCs detached from plastic surface and formed sphere clusters. (B) RT-PCR analysis showed that induced ADSCs expressed CD34, CD45, and KDR mRNA at much higher levels than un-induced ADSCs. (C) Flow cytometric analysis showed that induced ADSCs expressed CD34, CD45, and KDR cell surface antigens at much higher levels than un-induced ADSCs. Note that red lines indicate negative isotype controls for the respective antibodies. Each experimental and control group contained three replicates and each experiment was performed three times independently.
Figure 2.
E4BP4 and NK cell marker expression.
(A) Western blot analysis showed that ADSC had no detectable level of E4BP4 while ADSC-NK and ADSC-NKE had increasing levels of E4BP4. (B) Flow cytometric analysis for NK cell markers in ADSC, ADSC-NK, and ADSC-NKE. Note that red lines indicate negative isotype controls for the respective antibodies. Each experimental and control group contained three replicates and each experiment was performed three times independently.
Figure 3.
Cytotoxicity toward cancer cells.
(A) ADSC, ADSC-NK, ADSC-NKE, and NKL were used as effector cells while prostate cancer cell DU-145 as target. Each effector was mixed with the target at the indicated ratio. The next day, each mixture was analyzed by flow cytometry to determine the ratio of dead versus live cells. * P<0.05, ADSC-NK versus ADSC; # P<0.05, ADSC-NKE versus ADSC-NK. (B) ADSC, ADSC-NK, ADSC-NKE, and NKL were used as effector cells while the indicated prostate cancer cell lines and MCF7 as targets. Each effector was mixed with the target at a ratio of 25∶1. The next day, each mixture was analyzed by flow cytometry to determine the ratio of dead versus live cells. * P<0.05, ADSC-NK versus ADSC; # P<0.05, ADSC-NKE versus ADSC-NK; Ω P<0.05, NKL versus ADSC-NKE. Each experimental and control group contained three replicates and each experiment was performed three times independently.
Figure 4.
Cancer cell-induced activation.
(A) ADSC-NKE cells were untreated (Negative control), treated with PMA/ionomycin (Positive control), or mixed with K562 cells at a ratio of 10∶1. Six hours later, the cell samples were stained for CD107a (green) and with DAPI (blue, for nuclei). (B) ADSC, ADSC-NKE, and NKL cells were untreated (Negative control), treated with PMA/ionomycin (Positive control), or mixed with K562 or PC3 cells at a ratio of 10∶1. Six hours later, the cell samples were analyzed for CD107a expression by flow cytometry. In the resulting histograms red lines indicate negative isotype control for anti-CD107a antibody. Each experimental and control group contained three replicates and each experiment was performed three times independently.
Figure 5.
Cytolytic activities against cancer and normal cells.
(A) ADSC, ADSC-NKE, and NKL were used as effector cells while prostate cancer cell PC3 as target. Each effector was mixed with the target (labeled with CFSE) at a ratio of 25∶1. After overnight incubation, each mixture was observed and photographed microscopically with phase-contrast and fluorescent settings. The phase-contrast and fluorescent images were then superimposed digitally. (B) From 60 randomly selected images for each cell preparation, intact and lysed CFSE-labeled cells were counted. The number of lysed cells was divided by the number of CFSE-labeled cells to generate the percentage of lysed cells. * P<0.05, ADSC-NKE versus ADSC; # P<0.05, NKL versus ADSC-NKE. (C) ADSC, ADSC-NKE, and NKL were used as effector cell while cancer cell PC3 and non-cancer cells (HCSMC, HCEC, and HUVEC) as targets. Each effector was mixed with each target at a ratio of 25∶1. The next day, each mixture was analyzed by flow cytometry to determine the ratio of dead versus live cells. Unmixed target cells served as controls. * P<0.05, ADSC-NKE versus ADSC; # P<0.05, NKL versus ADSC-NKE. Each experimental and control group contained three replicates and each experiment was performed three times independently.
Figure 6.
(A) Each of 12 male nude mice was subcutaneously injected with 1×106 PC3 cells. One week later, 6 of these mice were each injected with 1×107 ADSC-NKE cells via tail vein. All 12 mice were also each intraperitoneally injected with 10,000 U of IL-2 and 10 ng of IL-15. Afterward, each mouse received IL-15 injection daily for one week and IL-2 injection every third day for the entire course. Tumor size was determined at the indicated time points. (B) Same as in A except (1) 20 female nude mice were used, with 10 each serving as experimental and control animals, (2) MCF7 cells were used in place of PC3 cells, and (3) the ADSC-NKE and interleukin injections were initiated at 2 weeks after MCF7 injection.
Figure 7.
Survival and distribution of ADSC-NKE cells in immunocompetent recipients.
EdU-labeled ADSC-NKE cells were injected subcutaneously into 30 Sprague-Dawley rats at 1×106 cells per rat. At each indicated time point, 6 rats were sacrificed for tracking of EdU+ cells (red) in the indicated tissues as shown in the representative images (400×, with blue stains indicating cell nuclei).