Establishment of a Novel Fetal Growth Restriction Model and Development of a Stem-Cell Therapy Using Umbilical Cord-Derived Mesenchymal Stromal Cells

Abstract

Fetal growth restriction (FGR) is a major complication of prenatal ischemic/hypoxic exposure and affects 5%-10% of pregnancies. It causes various disorders, including neurodevelopmental disabilities due to chronic hypoxia, circulatory failure, and malnutrition via the placenta, and there is no established treatment. Therefore, the development of treatments is an urgent task. We aimed to develop a new FGR rat model with a gradual restrictive load of uterus/placental blood flow and to evaluate the treatment effect of the administration of umbilical cord-derived mesenchymal stromal cells (UC-MSCs). To create the FGR rat model, we used ameroid constrictors that had titanium on the outer wall and were composed of C-shaped casein with a notch and center hole inside that gradually narrowed upon absorbing water. The ameroid constrictors were attached to bilateral ovarian/uterine arteries on the 17th day of pregnancy to induce chronic mild ischemia, which led to FGR with over 20% bodyweight reduction. After the intravenous administration of 1 × 10⁵ UC-MSCs, we confirmed a significant improvement in the UC-MSC group in a negative geotaxis test at 1 week after birth and a rotarod treadmill test at 5 months old. In the immunobiological evaluation, the total number of neurons counted via the stereological counting method was significantly higher in the UC-MSC group than in the vehicle-treated group. These results indicate that the UC-MSCs exerted a treatment effect for neurological impairment in the FGR rats.

Keywords: fetal growth restriction; mesenchymal stem cell; neurodevelopment; stem cells; umbilical cord-derived mesenchymal stromal cells.

Figure 1

Characters of the Ameroid constrictor. (A) Ameroid constrictor. An ameroid constrictor (AC) contains casein inside, has a notch and a hole in the center and is covered with a wide titanium ring. Its size is approximately 3.0 mm in diameter (a); the tool has a center hole of 0.4 mm (b) and a width of about 1.3 mm (c). This casein protein swells slowly when it absorbs water. (B) Installation of AC. The figure shows that the left and right ovarian/uterine arteries were sequentially detached from the vein, and an AC was placed in each of the four locations of the artery. (C) The change of diameter in AC. The graph shows the diameter of the center hole in an AC in a physiological saline environment of 37°C in vitro. The hole shrank to approximately 1/5 its original size in 1 day and to about 1/10 in 3 days. The photograph below shows the change of its shape over time in saline at 37°C.

Figure 2

Characters of the fetal growth restriction (FGR) model using the Ameroid constrictor. (A) The measurement area. We applied circle ROIs (blue circles) on the inside of the whole uteri and measured the four fetuses: top and bottom of the bilateral uteri before and after AC attachment on embryonic day 17 (E17) as well as E18, 19, and 20. (B) The changes in blood flow over time. The measurement of the blood flow every 24 h after the attachment of the AC in the sham group (n = 12) and the FGR group (n = 7). In the FGR group, the blood flow gradually decreased over time, and on the 20th day of pregnancy, just before birth, the blood flow had decreased to about 60% (sham, n = 12; FGR, n = 7). ***sham vs. FGR, p < 0.001. (C) The body weight change of each group. At P4 and P7 days of age, the mean body weight of the sham group was significantly higher than that of the vehicle and the umbilical cord-derived mesenchymal stromal cell (UCMSC) groups. Both the vehicle group and the UC-MSC group subsequently caught up with the sham group, and the significant difference disappeared (sham, n = 21; vehicle, n = 26; UC-MSCs, n = 23). *sham vs. vehicle, p < 0.05; ^#sham vs. UC-MSCs, p < 0.05; **sham vs. UC-MSCs, p < 0.01; ^##sham vs. UC-MSCs, p < 0.01; ***sham vs. vehicle, p < 0.001.

Figure 3

Behavioral tests. (A) Negative geotaxis. At postnatal day 9, both the vehicle group and the UC-MSC group were significantly lower scores than the sham group. At postnatal day 10, the vehicle group was significantly lower than the sham group. At postnatal day 11, the vehicle group was significantly lower than the sham group, and the UC-MSC group significantly improved over the vehicle group. Sham group is represented by a solid line, vehicle group by a dashed line and UC-MSC group by a dotted line (sham, n = 37; vehicle, n = 18; UC-MSCs, n = 15). *sham vs. UC-MSCs, p < 0.05; ^#sham vs. vehicle, p < 0.05; **UC-MSCs vs. vehicle, p < 0.01; ***sham vs. vehicle, p < 0.001; ^###sham vs. vehicle, p < 0.001. (B) Rotarod treadmill test. The average daily values were calculated. The left bar graph shows a daily average, and the right bar graph does an average of total observations. The duration was shortened in the vehicle group and prolonged in the UC-MSC group at P154-155. Sham group is represented by a solid line, vehicle group by a dashed line and UC-MSC group by a dotted line (sham, n = 7; vehicle, n = 9; UC-MSCs n = 7). *UC-MSCs vs. vehicle, p < 0.05; ^#UC-MSCs vs. vehicle, p < 0.05.

Figure 4

Effects of UC-MSC treatment on neurons in FGR. Representative photomicrographs of the hippocampus (A) and part of the cortex (B) 2 months after birth. Scale bar = 100 μm. The inset shows a higher magnification view. Scale bar = 10 μm. (C) The number of positive cells for NeuN in the hippocampus was significantly lower in the vehicle group than in the sham group, although there was no significant difference between the UC-MSC group and the sham group (sham, n = 8; vehicle, n = 3; UC-MSCs n = 4). *p < 0.05. (D) The number of positive cells for NeuN in the cortex was significantly lower in the vehicle group than that in the sham group, and a significant amelioration was observed in the UC-MSC group (sham, n = 8; vehicle, n = 3; UC-MSCs n = 4). **p < 0.01, ***p < 0.001.

Figure 5

Effects of UC-MSC treatment on astroglia and microglia in FGR. (A) There was no significant difference among the three groups in the number of S100 positive cells in the cortex. (B) The number of Iba-1 positive cells in the cortex was significantly higher in the UC-MSC group compared to the sham group (sham, n = 8; vehicle, n = 3; UC-MSCs, n = 4). *p < 0.05. The number of ED-1 and Iba-1 double-positive cells (C) and CD206 and Iba-1 double-positive cells (D) in the cortex with fluorescent immunostaining. The number of ED-1+/Iba-1+ in both the UC-MSCs and the vehicle were significantly higher than that in the sham (sham, n = 8; vehicle, n = 3; UC-MSCs, n = 4). *p < 0.05, whereas that of CD206+/Iba-1+ in the UC-MSCs was significantly higher than that in the sham (sham, n = 8; vehicle, n = 3; UC-MSCs, n = 4). *p < 0.05.