BMSCs were successfully identificated with flow cytometry method in wistar rats before transplantation
Four kinds of BMSC surface markers at P0-6 were detected by flow cytometry. The results showed that CD73 and CD90 were positive, CD34 and CD45 were negative (Figure 1A). Among them, CD73 and CD90 were fibrin and ribosomal receptors on the surface of BMSC, which were non-hematopoietic cell origin. CD34 and CD45 were the surface receptors of endothelial cells and hematopoietic cells, which indicated that BMSCs from wistar rats have been successfully isolated.
BMSCs proliferation activity were determined by CCK-8 test and the STAT3 plasmid was successfully transfected before transplantation
At 4, 8, 12, 16, 20, 24, 48 and 72 hours as time nodes, BMSCs proliferation activity at P0, P1-3, P4-6 in blank control group, negative control group and experimental group was detected before transplantation. The results showed that there was no significant difference in the number of living cells among the three groups at the time point (P > 0.05), which suggested that lentivirus transfection has no significant effect on the proliferation of BMSCs ( Figure 1B, C and D). STAT3 mRNA expression in experimental group increased more statistically than that in blank control group and negative control group (P < 0.05), which demonstrated that STAT3 overexpression plasmid transfection was successfully constructed ( Figure 1E, F and G).
Survival of rat fetuses in each group after BMSCs transplantation
220 fetuses of 72 pregnant rats were BMSCs transplanted, 5 pregnant rats died after transplantation on the 18th day, the survival rate of transplanted pregnant rats was 93% (67/72). The pregnant, transplanted rats were killed on E18, E19, E19.5 and E20, then transplanted fetuses with SBA were harvested for lumbosacral spinal sample collection. 70 cases arised from MSCs without recombinant lentivirus transfection (blank control group); 74 cases from MSCs with STAT3 overexpression plasmid transfection (experimental group), 76 cases with only recombinant lentivirus transfection (negative control group).
BMSCs combined with STAT3 overexpression plasmid transfection can improve the success rate of transplantation
Before transplantation, The purity of BMSCs was about enriched from 35±1.02% at P0, 34±1.16% at P1-3, and 33±1.22% from P4 to P6 in blank control group and negative control group. However, as BMSCs combined with STAT3 overexpression plasmid transfection processed as above. The purity of BMSCs was enriched from 75±1.13% at P0, 87±1.29% from P1 to P3, and 73±1.07% from P4 to P6 when CD73 and CD90 expression was also tested, which was statistically significant (P= 0.031, 0.023 and 0.037, compared with blank control group and negative control group, respectively) (Figure 2A). P1-3 BMSCs combined with STAT3 overexpression plasmid transfection has a better purity than P0 and P4-6. Then we transplanted P1-3 BMSCs into each group of rat fetuses on E15, E16 and E17 to examine whether STAT3 overexpression plasmid transfection may affect cell survival ratio. Green fluorescent protein (GFP) was labeled to BMSCs and positive cell numbers were counted on E19 after transplantation. The overall survival rates of BMSCs in experimental group was found to be 36±1.08% on E15, 46 ±0.72% on E16 and 35±0.93% on E17, which was a little higher than that of blank control group and negative control group, and statistically significant (P= 0.029, 0.021 and 0.033). Better survival after STAT3 overexpression plasmid transfection was achieved in the experimental groups performed on later development stages (E16) as compared with E15 and E17 group, but it did not reach a statistical significance (P =0.65 E15 versus E16; P=0.32 E17 versus E16) (Figure 2B). Secondly, we wanted to detect whether different BMSC passages transplanted after STAT3 overexpression plasmid transfection could interpose survival ratio, and three groups were divided on E16 transplanation from rat fetuses: P0, P1-3 and to P4-6. BMSCs survival was found to be P0 (42.36±1.08%), P1-3 (44.06±1.1%) and P4-6 (41.09±1.2%) in experimental group from E19 transplanted fetuses, which showed a notable increase compared with blank control group and negative control group, again the differences reached a statistical significance (P= 0.023, 0.021 and 0.026, respectively) (Figure 2C). Thirdly, we evaluated the effects of injected cell number on survival ratio after P1-3 BMSCs with STAT3 overexpression plasmid transfection on E16 transplanation from rat fetuses. Medium injection group (2000–4000 cells/injection/spinal cord) was remarkably different from the change that occurred in low injection group (1000–1999 cells/injection/spinal cord) and high injection group (4001–6000 cells/injection /spinal cord) on E19 transplanted fetuses, which reached a statistical significance (P=0.026, 0.022 and 0.029, respectively) (Figure 2D).
CCK-8 test was used to detect cell proliferation activity after BMSCs transplantation again
At 4, 8, 12, 16, 20 and 24 hours as time nodes, the proliferation activity of P0-6 BMSCs in blank control group, negative control group and experimental group was detected, and OD value of living cells in each group was measured. The results showed that OD value in experimental group was the highest at 8 hours on E19 after E16 transplantation, and then gradually decreased, which was significantly different from blank control group and negative control group (P < 0.05) (Figure 3A-C). However, there was no significant difference in OD peak time and value between blank control group and negative control group.
Analysis of STAT3 and pSTAT3 expression in spinal cord of each group after BMSCs transplantation
The developmental change in pSTAT3 expression of experimental group from P1-3 BMSCs transplantation was notably different from the change that occurred in blank control group and negative control group on E18, E19, E19.5 and E20, especially at embryonic day 19 (E19). Both pSTAT3 mRNA and protein levels were significantly increased in experimental group from E18 to E20, and E19 reached the peak (Figure 4A, B and C). Overall, the expression of STAT3 mRNA and protein levels in experimental group gradually decreased with embryonic development between E18 and E19, E19 dropped down to bottom. Then From E18 to E20, pSTAT3 and STAT3 in mRNA and protein levels kept steady level in blank control group and negative control group, which statistical decreased than that in experimental group (P < 0.05) (Figure 4D, E and F).
Analysis of GFAP,NSE,NF and Nestin expression in spinal cord of each group after BMSCs transplantation
The relative expression of GFAP mRNA in blank control group, negative control group and experimental group was statistically different from P1-3 BMSCs transplantation on E16. The level of GFAP mRNA in experimental group was significantly higher than that in blank control group and negative control group on E19 transplanted fetuses (P < 0.05). At the same time, the relative expression trend of NSE, NF and Nestin mRNA in each group was basically consistent with above (Figure 5A). In addition, the protein levels of GFAP, NSE, NF and Nestin using Western Blot were identical with real-time PCR (Figure 5B,C).
Analysis of Caspase-8 and Bcl-2 expression in spinal cord of each group after MSCs transplantation
Caspase-8 and Bcl-2 as apoptosis gene play an important role in the development of spina bifida aperta. Next we studied the apoptosis of whole spinal column after BMSCs transplantation with STAT3 overexpression plasmid transfection from caspase-8 and Bcl-2 expression. Compared with blank control group and negative control group at P1-3 BMSCs on E16 transplantation , both caspase-8 and Bcl-2 in mRNA and protein levels were significantly decreased in experimental group with a statistical significance on E19 (Figure 5D,E and F).