Fetal Blood

Experiments were approved by the Ethical Review Committee of the University of Cambridge and were carried out under the UK Animals (Scientific Procedures) Act 1986.
Wistar rat pregnancies were established as described [13] , [28] (link). On day 6 of pregnancy, rats were randomly divided into 4 groups (n = 20 per group): control and hypoxic pregnancy, with and without vitamin C treatment (5 mg.ml⁻¹ maternal drinking water freshly prepared every day). Pregnant rats subjected to hypoxia were placed inside a chamber, which combined a PVC isolator with a nitrogen generator [13] , [28] (link). Pregnancies undergoing hypoxia were maintained at a constant inspired fraction of oxygen of 13% from day 6 to 20 of gestation (term is ca. 21 days). At day 20 of gestation, one set of dams (n = 10) from each group was anesthetised with isoflurane and then maintained by a mixture of ketamine (40 mg•kg⁻¹) and xylazine (5 mg•kg⁻¹) injected intraperitoneally. A maternal blood sample (1 ml in EDTA plus 0.5 ml in metaphosphoric acid) for measurement of ascorbic acid was taken by cardiac puncture, the pregnant uterus was exposed via a mid-line incision and the anesthetised pups were killed via spinal transection. Dams which had been housed in the hypoxic chamber underwent the procedure while being spontaneously ventilated with 13% O₂ via a small cone. All fetuses and associated placentas were isolated and weighed. Additional maternal and fetal blood was taken for measurement of haematocrit in duplicate. In all pups, the ano-genital distance was measured with digital callipers for determination of sex. Only cardiovascular tissues associated with one male pup per litter per measured outcome variable were used to control for sex and within-litter variation. Therefore, the fetal thorax (containing heart and aorta) was immersion fixed from one male per litter per group (n = 8) and the fetal heart was frozen from a littermate male per litter per group (n = 8) for subsequent stereological, histological or molecular analyses. The remaining 10 litters per group were allowed to deliver. Following determination of birth weight, litters were culled to 4 males and 4 females to standardise nutritional access and maternal care [13] . At weaning, only male offspring were raised to adulthood. At 4 months, following weighing, 1 male from each litter per outcome variable underwent euthanasia and tissues were either perfusion fixed for stereological and histological analyses (n = 8 per group), or frozen for molecular analysis (n = 8 per group), or dissected for the isolated organ preparations (n = 8 per group).

Samples (0.3 ml) of ascending and descending aortic fetal as well as descending aortic maternal blood were taken daily for measurement of fetal and maternal blood gas, acid base and metabolic status. Arterial blood gas and acid base values were measured using an ABL5 blood gas analyser (Radiometer, Copenhagen, Denmark; maternal measurements corrected to 38 °C, fetal measurements corrected to 39.5 °C). Values for percentage saturation of haemoglobin with oxygen (SatHb) and for the concentration of haemoglobin in blood ([Hb]) were determined using a haemoximeter (OSM3; Radiometer). Blood glucose and lactate concentrations were measured using an automated analyser (Yellow Springs 2300 Stat Plus Glucose/Lactate Analyser; YSI Ltd, Farnborough, UK). Values for haematocrit were obtained in duplicate using a microhaematocrit centrifuge (Hawksley, Lancing, UK). An additional 1 ml of maternal arterial and fetal arterial blood was taken during baseline (24 h prior to chronic hypoxia) and at +1 h, +6 h, +1 day, +5 days and +10 days of chronic hypoxia and at +2 days following chronic hypoxia or at equivalent times in normoxic animals for determination of plasma vitamin C and urate concentrations.

We measured the temporal changes in intrauterine blood flow under isoflurane anesthesia (2.0% isoflurane) using laser speckle flowmetry (Omegazone, Omegawave Inc., Tokyo, Japan) at three time points: before stenosis, 1 h, and 3 days after stenosis on both ovarian and vaginal sides (Fig. 1). To quantify blood flow in all fetuses or placentas, regions of interest were determined in two sham and two MIUH rats (Fig. 1A,B).

Placentas were bisected along the midline and one half fixed in 4% paraformaldehyde for histology, the other half snap-frozen for RNA extraction. Fixed tissues were processed for routine paraffin histology, and serial 7 µm sections produced using a Leica rotary microtome. Sections through the midline of the placenta were identified by the insertion of the umbilical cord. For assessment of placental layers and fetal/maternal blood space areas, at least three sections, positioned 70 µm apart (i.e., 10 consecutive sections apart), were analyzed per placenta. In situ hybridizations using a probe against Tpbpa were performed as previously described^{8 (link),56 (link)}. For immunostaining, sections were deparaffinised in xylene and processed through an ethanol series to PBS. Antigen retrieval was performed by boiling in 10 mM Na-citrate pH 6.0 buffer in a pressure cooker followed by blocking in PBS, 0.5% BSA, 0.1% Tween-20. Antibodies used were against MCT1 (1:200, Sigma AB1286-I), MCT4 (1:200, Sigma AB3314P), RXRa (1:100, Abcam ab125001) and phospho-histone H3S10 (1:200, Millipore-Sigma #06-570). Primary antibodies were detected with appropriate AlexaFluor488- or AlexaFluor568-conjugated secondary antibodies (1:500, ThermoFisher). Counterstaining was performed with 4′,6-diamidino-2-phenylindole (Sigma D9542), slides were mounted with Fluoromount-G (SouthernBiotech) mounting medium and imaged at a Leica DMRE epifluorescence microscope. In the case of the immunofluorescence stainings for beta-galactosidase (LacZ) and MCT1/4, tissue was fixed in 4% paraformaldehyde, embedded in OCT, and cryosectioned. To avoid cross-reactivity with the MCT1 and MCT4 antibodies, two different antibodies against beta-galactosidase were used: Cell Signaling #27198 (1:200) and Abcam ab9361 (1:200). For LacZ/MCT4 double staining, MCT4 was detected with a horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:100, ThermoFisher 31460) followed by color reaction with DAB substrate (ThermoFisher 34002) and counterstaining with nuclear fast red (Sigma N3020). Image analyses of area and perimeter measurements were performed in ImageJ (1.53). Statistical differences were calculated in GraphPad Prism software (9.4.1). All p-values are provided in Supplementary Data 1.

We used ultracentrifugation to collect extracellular vesicles from the culture supernatant of hucMSCs (3rd–5th passage cells). When cell density reached 70%–80%, we removed the medium, washed the cells thrice with 1×PBS, and added serum-free DMEM/F12 for 48 h to exclude the influence of EV from fetal blood serum. We collected the culture supernatant and collected EV immediately or froze them at −80°C. The culture supernatant was centrifuged at 10 000 × g for 45 min at 4°C to remove unwanted cells and cell debris. To obtain higher-purity EV, the collected supernatant was filtered through a 0.22-μm filter (SLGP033RB-0.22; Merck Millipore, United States) and ultracentrifuged (JXN-30; Beckman, United States) at 108 000 × g for 70 min at 4°C (Optima L-90K; Beckman, United States). The supernatant was discarded, and the pellet was resuspended in 1×Dulbecco’s phosphate-buffered saline (DPBS) to remove unwanted proteins. The samples were ultracentrifuged at 108 000 × g for 70 min at 4°C and resuspended in 200 μL of 1×DPBS to obtain high-density, pure EV.