Proof of Concept

MSCalvary (our modified MSCs) is a set of MSCs engineered with specific chemokine receptors and marking proteins. This summer vacation, we constructed the following devices which were testified both in vivo and in vitro.

Figure 3.1.1

Figure 3.1.2

With the purpose of increasing homing efficiency of MSCs and illustrating their distribution, we focused on three aspects of great importance for proof of our concept:
- Whether our devices express as expected;
- Whether our parts properly exert their function;
- Whether MSCs are negatively influenced by our system.

Expression

1 Chemokine receptors
To figure out whether our devices express properly, mRNA and protein levels of CXCR4 and CXCR5 of MSCs and our modified MSCs were semi-quantified and detected by qPCR and western blot, respectively. mRNA and protein levels of CXCR4 and CXCR5 all elevated in our modified MSCs.

Figure 3.2.1	Figure 3.2.2	Figure 3.2.3

2 Marking proteins
- eGFP
As for expression of eGFP, transfected 293T cells (Figure 3.2.4A: EF-1α-CXCR5-IRES-eGFP; Figure 3.2.4B: EF-1α-CXCR4-T2A-luciferase-IRES-eGFP) and MSCs (Figure 3.2.5A: EF-1α-CXCR5-IRES-eGFP; Figure 3.2.5B: EF-1α-CXCR4-T2A-luciferase-IRES-eGFP) were observed under fluorescent microscope. As a result, eGFP were expressed.

Figure 3.2.4

Figure 3.2.5

- Luciferase
Under IVIS Spectrum, MSCs with degraded luciferin could be clearly observed.

Figure 3.2.6

Function

1 Elevated homing ability

In order to verify whether engineered MSCs successfully acquired elevated chemotaxis, in vivo and in vitro experiments were conducted.

- In vitro verification
Chemotaxis of engineered MSCs were examined against CXCL12 and CXCL13 respectively. Results were reported as number of cells migrated. Chemotaxis capacity of our engineered MSCs all improved.

Figure 3.3.1	Figure 3.3.2
Figure 3.3.3

- In vivo verification

· Under IVIS Spectrum, MSCsCXCR4 exhibited enhanced capacities for targeted migration to the bowels in IBD model.

Figure 3.3.4
· The inflamed ears were collected from each group and subjected to in situ immunofluorescence staining. Our results revealed that MSCCXCR5 exhibited enhanced capacities for targeted migration to the ears in DTH model.

Figure 3.3.5
2 Locating
· Under IVIS Spectrum, MSCsCXCR4 could be detected and located in IBD mice.

Figure 3.3.6

· The inflamed ears were collected from each group, subjected to in situ immunofluorescence staining and observed under fluorescence microscope. Our results revealed MSCCXCR5 could be located in the inflamed ears of DTH mice.

Figure 3.3.7

Identification

Our modified MSCs still remained their own characteristics, including phenotypes and Immunoregulatory function.
1 Phenotypes of MSCs
Our modified MSCs were still positive for CD105, CD90, CD29 and CD73 and negative for CD34 (Figure 3.4.1 A-E), and were able to differentiate to osteoblasts, adipocytes, and chondroblasts under standard in vitro differentiating conditions (Figure 3.4.2 F-K). (Meet the standard of International Society for Cellular Therapy (ISCT))

Figure 3.4.1	Figure 3.4.2

2 Immunoregulatory function of MSCs
For MSCsCXCR4
· Colon sampling for quantitative PCR analysis. Comparing with TNBS+MSC group, levels of the anti-inflammatory cytokine (IL-10) elevated in TNBS+MSCCXCR4 group while pro-inflammatory cytokines (IL-6, IL-1β, TNF-α) levels decreased in TNBS+ MSCCXCR4 group. In a word, MSCsCXCR4 have greater immunoregulatory function.

Figure 3.4.3	Figure 3.4.4	Figure 3.4.5	Figure 3.4.6

· After injecting with MSCsCXCR4, 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. In a word, MSCsCXCR4 have greater immunoregulatory function.

Figure 3.4.7	Figure 3.4.8	Figure 3.4.9	Figure 3.4.10

· Photographs of hematoxylin/eosin-stained colon cross-sections 2 days after injecting MSCs. MSCsCXCR4 displayed better treatment efficacy than TNBS+MSC group and TNBS+saline group, characterized by their abilities to decrease leukocyte infiltration. In a word, MSCsCXCR4 have greater immunoregulatory function.

Figure 3.4.11

For MSCsCXCR5
· Ear sampling for quantitative PCR analysis. Comparing with DTH+MSCs group, levels of the anti-inflammatory cytokines (IL-10), elevated in DTH+MSCsCXCR5 group, while levels of pro-inflammatory cytokines (IL-4, TNF-α, IFN-γ, IL-6 and IL-17) decreased in DTH+MSCsCXCR5 group. In a word, MSCsCXCR5 have greater immunoregulatory function.

Figure 3.4.12

· After injection, MSCsCXCR5 displayed better treatment efficacy than DTH+MSCeGFP group, characterized by their abilities to decrease ear thickness and leukocyte infiltration. MSCsCXCR5 significantly attenuated DTH as early as 24 hours post-injection, and had even greater effects at 48 hours post-injection. In a word, MSCsCXCR5 have greater immunoregulatory function.

Figure 3.4.13

Figure 3.4.14