Results

Major achievements:

1. Our modified MSCs still remained their own characteristics.
2. CCR7/CXCR4/CXCR5 were successfully overexpressed on our modified MSCs.
3. Chemotaxis of our engineered MSCs improved.
4. Marking proteins (eGFP/dTomato/Luciferase) worked.
5. Therapeutic effects of our engineered MSCs for IBD (inflammatory bowel disease) improved.
6. Therapeutic effects of our engineered MSCs for DTH (delayed type hypersensitivity) improved.
7. The switch element worked.

Details:

1. Our modified MSCs still remained their own characteristics.

Figure 2.1.1	Figure 2.1.2

Our modified MSCs were positive for CD105, CD90, CD29 and CD73 and negative for CD34 (Figure 2.1.1 A-E), and were able to differentiate to osteoblasts, adipocytes, and chondroblasts under standard in vitro differentiating conditions (Figure 2.1.2 F-K). (Meet the standard of International Society for Cellular Therapy (ISCT))

2. CCR7/CXCR4/CXCR5 were successfully overexpressed on our modified MSCs.

Figure 2.2.1	Figure 2.2.2	Figure 2.2.3

Figure 2.2.4

mRNA and protein levels of CCR7, CXCR4 and CXCR5 of MSCs and our modified MSCs (EF-1α-CCR7, EF-1α-CXCR4-T2A-Luciferase-IRES-eGFP, EF-1α-CXCR5-IRES-eGFP, respectively) (BBa_K1993001, BBa_K1993009, BBa_K1993005, respectively) were semi-quantified and detected by qPCR and western blot, respectively. mRNA and protein levels of CCR7, CXCR4 and CXCR5 all elevated in our modified MSCs.

3. Chemotaxis of our engineered MSCs improved.

Figure 2.3.1	Figure 2.3.2	Figure 2.3.3

Figure 2.3.4

Chemotaxis of our engineered MSCs (EF-1α-CCR7, EF-1α-CXCR4-T2A-Luciferase-IRES-eGFP, EF-1α-CXCR5-IRES-eGFP, respectively) (BBa_K1993001, BBa_K1993009, BBa_K1993005, respectively) were examined against CCL19, CXCL12 and CXCL13, respectively. Results were reported as number of cells migrated. Chemotaxis capacity of our engineered MSCs all improved.

4. Marking proteins (eGFP/dTomato) worked.

Figure 2.4.1

4.1 2 days later, after transduction into 293FT cells, marking proteins (eGFP/dTomato) expressed.
Figure A: 293FT cells (BBa_K1993011: EF-1α-CXCR4-IRES-Luciferase-T2A-dTomato-T2A- hFTH)
Figure B: 293FT cells (BBa_K1993008: EF-1α-CXCR5-IRES-eGFP)
Figure C: 293FT cells (BBa_K1993009: EF-1α-CXCR4-T2A-Luciferase-IRES-eGFP)
Figure D: 293FT cells (BBa_K1993008: EF-1α-Luciferase-IRES-eGFP)

Figure 2.4.2

4.2 4 days later, after transduction into MSCs, marking proteins (eGFP/dTomato) expressed.
Figure A: MSCs (BBa_K1993011: EF-1α-CXCR4-IRES-Luciferase-T2A-dTomato-T2A-hFTH)
Figure B: MSCs (BBa_K1993008: EF-1α-CXCR5-IRES-eGFP)
Figure C: MSCs (BBa_K1993009: EF-1α-CXCR4-T2A-Luciferase-IRES-eGFP)
Figure D: MSCs (BBa_K1993008: EF-1α-Luciferase-IRES-eGFP)

Figure 2.4.3

4.3 After intraperitoneal injection of Luciferin, MSCs engineered with GFP and Luciferase were administrated intravenously to caudal vein. Oxidized Luciferin was observed under IVIS spectrum system. As a result, mice administered with engineered MSCs （BBa_K1993008: EF-1α-Luciferase-IRES-eGFP）
5. Therapeutic effects of our engineered MSCs for IBD (inflammatory bowel disease) improved.

Our MSCs were modified by BBa_K1993009 (EF-1α-CXCR4-T2A-Luciferase-IRES-eGFP).

Figure 2.5.1.1	Figure 2.5.1.2
Figure 2.5.1.3	Figure 2.5.1.4

5.1 We established murine 2,4,6-trinitro benzene sulfonic acid (TNBS)-colitis (IBD animal model). After injecting with MSCs^CXCR4, survival rate, body weight, length of colon and DAI score of IBD mice were improved significantly comparing to TNBS+MSC group and TNBS+saline group.

Figure 2.5.2

5.2 Photographs of hematoxylin/eosin-stained colon cross-sections 2 days after injecting MSCs. MSCs^CXCR4 displayed better treatment efficacy than TNBS+MSC group and TNBS+saline group, characterized by their abilities to decrease leukocyte infiltration.

Figure 2.5.3.1	Figure 2.5.3.2
Figure 2.5.3.3	Figure 2.5.3.4

5.3 Colon sampling for quantitative PCR analysis. Comparing with TNBS+MSC group, levels of the anti-inflammatory cytokine (IL-10) elevated in TNBS+MSC^CXCR4 group while pro-inflammatory cytokines (IL-6, IL-1β, TNF-α) levels decreased in TNBS+ MSC^CXCR4 group.

Figure 2.5.4.1

5.4 Under IVIS Spectrum, MSCs^CXCR4 exhibited enhanced capacities for targeted migration to the bowels in inflammatory condition in vivo.

6. Therapeutic effects of our engineered MSCs for DTH (delayed type hypersensitivity) improved.

Our MSCs were modified by BBa_K1993005 (EF-1α-CXCR5-IRES-eGFP).

Figure 2.6.1.1

Figure 2.6.1.2

6.1 MSCs^CXCR5 infusion dramatically ameliorated DTH. MSCs^CXCR5 displayed better treatment efficacy than DTH+MSC^eGFP group, characterized by their abilities to decrease ear thickness and leukocyte infiltration. MSCs^CXCR5 significantly attenuated DTH as early as 24 hours post-injection, and had even greater effects at 48 hours post-injection.

Figure 2.6.2

6.2 Ear sampling for quantitative PCR analysis. Comparing with DTH+MSCs group, levels of the anti-inflammatory cytokines (IL-10), elevated in DTH+MSCs^CXCR5 group, while levels of pro-inflammatory cytokines (IL-4, TNF-α, IFN-γ, IL-6 and IL-17) decreased in DTH+MSCs^CXCR5 group.

Figure 2.6.3

6.3 The inflamed ears were collected from each group on Day2 post-injection, and subjected to in situ immunofluorescence staining. Our results revealed that MSCs^eGFP were almost undetectable, whereas MSCs^CXCR5 were highly accumulated in the inflamed ears of DTH mice.

7. The switch element worked.

Figure 2.7

Transduction of our switch plasmid into MSCs. After administering TGF-β1 (7ng/mL), α-SMA was expressed and green fluorescence could be detected in MSCs (BBa_K1993014: pMSA-eGFP worked), which indicated our switch was running.