A total of 38 pregnant women were considered eligible for this prospective cohort study conducted at the University Hospital “San Giovanni di Dio e Ruggi d’Aragona”, Salerno, Italy, and at the Hospital “San Giuseppe Moscati”, Avellino, Italy, and 35 of them were enrolled (Table 1). Study protocol and written informed consent were reviewed and approved by local Institutional Review Board (Ethics Committee “Campania Sud”, Brusciano, Naples, Italy; prot./SCCE n. 24988). Subjects have given their written informed consent before sampling. 10 of them were diagnosed with placental syndromes: 6 with preeclampsia and 4 with IUGR. 28 patients had physiological pregnancies. Inclusion criteria for the pathologic group were an antepartum diagnosis of preeclampsia or IUGR.

Human WJ-MSCs were isolated from fresh human umbilical cords placed in 0.9% NaCl physiological solution supplemented with 1 g of ampicillin (Sigma Aldrich, Milan, Italy) and 500 mg of sulbactam (Sigma Aldrich, Milan, Italy), stored at 4 °C, and processed within 4 h after collection. Briefly, samples were cut in small segments, washed in fresh medium to remove blood and debris, cut open lengthwise with sterile scissors, and arteries and veins were removed. Each piece was then transferred to a 175 cm${}^2$ tissue culture flask (BD Falcon, Bedford, MA, USA) containing alpha-MEM medium (Corning Cellgro, Manassas, VA, USA) supplemented with 10% fetal bovine serum (FBS, Corning Cellgro, Manassas, VA, USA), Glutagro${}^{\text{TM}}$ (Corning Cellgro, Manassas, VA, USA) and Penicillin-Streptomycin (Sigma Aldrich, Milan, Italy), and incubated at 37 °C in a humidified atmosphere containing 5% CO${}_2$. Cell growth was daily monitored, culture medium was changed twice a week, and cells were detached using 0.05% trypsin (Sigma Aldrich, Milan, Italy) and 0.53 mM ethylenediaminetetraacetic acid (EDTA) (Corning Cellgro, Manassas, VA, USA) upon reaching 100% confluence. Subsequently, cells were re-seeded at a density of 4 $\times$ 10${}^3$ cells/cm${}^2$ and cultured until reaching a plateau in the growth curve.

After vaginal or caesarean delivery, umbilical cord was clamped and disinfected; and 35 mL of blood was collected in sterile BD Vacutainer Z (no Additive) collection tubes (BD Diagnostic, Oxford, UK) containing 1 mL of citrate-phosphate dextrose as anticoagulant (MacoPharma, Tourcoing, France). Fresh whole blood was centrifuged for 15 minutes at 1900 rpm at 20 °C, and then the buffy coat interphase layer was collected, placed in a 15 mL falcon tube (BD Biosciences, Oxford, UK), and mixed with an equal volume of 0.9% NaCl physiological solution, and mononuclear cells were isolated by Ficoll-Paque density gradient centrifugation as per manufacturer’s instructions. Cells were then seeded at a density of 5 $\times$ 10${}^5$ cells/cm${}^2$ in a 35 $\times$ 10 mm dish (BD Biosciences, Oxford, UK) in complete culture medium consisting of alpha-Minimum Essential Medium (MEM) supplemented with 20% FBS, glutamine (Sigma Aldrich, Milan, Italy), and Penicillin-Streptomycin Solution, and incubated at 37 °C in a humidified atmosphere containing 5% CO${}_2$ with complete culture medium change twice a week. At 90% confluence, cells were detached using 0.05% trypsin and 0.53 mM EDTA, washed with phosphate buffer saline (PBS; Corning Cellgro, Manassas, VA, USA), re-seeded at a density of 4 $\times$10${}^3$ cells/cm${}^2$, and cultured until reaching a plateau in the growth curve. Adherent cells that did not form colonies after 40 days were eliminated.

Flow Cytometry immunophenotype of WJ- and CB-MSCs was performed on cells at first culture passage. Briefly, samples were incubated with the following mouse anti-human antibodies: cluster of differentiation (CD)90-fluorescein (FITC), CD45-Phycoerythrin (PE)-Cyanine7 (PC7), CD105-PE, CD56-PE-Cyanine5 (PC5), CD14-PC7, CD45-PC7, human leukocyte antigen (HLA)-DR-FITC, CD34-PE (Beckman Coulter, BC, Fullerton, CA, USA), and CD73-APC (Miltenyi Biotec, Gladbach, Germany), as described elsewhere [15]. Sample acquisition was carried out using a BD FACSVerse flow cytometer (BD Biosciences) equipped with blue (488 nm) and red lasers (628 nm) and BD FACSuite software (BD Biosciences, Oxford, UK). PMT voltages setting, and compensation were performed using single-color controls for each fluorochrome and an unstained sample as negative control. All samples were run with the same PMT voltages, and a minimum of 10,000 events were recorded.

Population doubling was calculated for WJ-MSCs and CB-MSCs using the following equation: population doubling (PD) = log${}_{10}$(N)/log${}_{10}$(2); where N is the number of cells harvested at the end of the culture or the number of seeded cells. To calculate expansion potential, cumulative population doublings (CPD) were calculated by recording cell counts and cellular dilution factors at each passage (P). Cell counting was performed using Trypan Blue to discriminate dead cells.

Data were analyzed using SPSS software (v.2.0; SPSS Inc., Chicago, IL, USA) and Prism (v.9.3.1; GraphPad Software, San Diego, CA, USA).

A power calculation was performed to determine an appropriate sample size for this noninferiority study. We set the mean population doubling numbers at 10. The standard deviation was set as 1. This power calculation indicated that 32 patients in each group would be necessary to detect a 15% difference in with a power of 80% at a 0.5% level of significance.

For flow cytometry data, results are presented as percentage of positive cells, and expression of each marker on single cells is also reported as histograms and using unstained samples as negative controls. For characterization of mesenchymal cells, percentage of positive cells. Shapiro-Wilk’s test for parametrical distribution and t-test were employed for statistical comparisons between groups. A p 0.05 was considered statistically significant.

From the 35 patients enrolled in this prospective cohort study, a total of 28 umbilical cord samples and 31 cord blood samples were obtained. Of the 28 umbilical cord blood samples, 11 resulted in WJ-MSC growth: (i) 10 from physiological pregnancies; (ii) 1 from pathological pregnancy, specifically, from IUGR. Of the 31 collected cord blood samples, 5 resulted in CB-MSC growth: (i) 3 from physiological pregnancies; (ii) 2 from pathological pregnancy, specifically, 1 from a preeclampsia and 1 from IUGR. The overall growth percentage of CB- and WJ-MSCs was 16.1% and 39.2%, respectively. Percentage of cord blood and umbilical cord samples that failed in cellular growth for any reason was compared to that of samples that resulted in MSC colonies by Chi square test. Umbilical cord samples tended to have increased ability to result in MSC colonies compared to cord blood.

Next, cellular growth rates of MSCs obtained from cord blood or umbilical cord samples were analyzed. Of the 5 cord blood samples that originated o MSC colonies, CB-MSCs from pathological placenta showed a slower growth rate compared to those obtained from physiological pregnancies (Fig. 1 and Table 2). Moreover, CB-MSCs from pathological samples, ultimately resulted in cell death. Conversely, WJ-MSCs from the pathological group showed faster growth rate compared to that observed in the physiological group (Fig. 2 and Table 2). To confirm our result, we divided physiological pregnancies into two groups based on the newborn’s weight (2.5 kg - namely physiological small for gestational age (SGA) infants or $>$2.5 kg): WJ-MSCs from the physiological group with SGA newborns have a lower growth rate compared to that observed in the physiological group with newborns weighted $>$2.5 kg (Fig. 2).

Fig. 1.

Cord blood derived mesenchymal stem cell (CB-MSC) growth curves. Each CB-MSC growth curve from every healthy pregnancy is reported as shades of blue curves, while each CB-MSC growth curve for each pathological pregnancy is shown as shades of red curves. IUGR, intrauterine growth restriction.

Fig. 2.

Wharton’s Jelly mesenchymal stem cell (WJ-MSC) growth curves. Each WJ-MSC growth curve from every healthy pregnancy is reported as shades of blue curves, while each CB-MSC growth curve for each pathological pregnancy is shown as shades of red curves. IUGR, intrauterine growth restriction.

Finally, immunophenotype of CB- and WJ-MSCs was evaluated by flow cytometry (Fig. 3). Of the 5 cord blood samples that originated MSC colonies, flow cytometry immunophenotype was performed on all three CB-MSCs from healthy pregnancies, while no pathological samples were analyzed due to premature death of cultured cells. Of the 11 UC-MSCs, flow cytometry analysis was not performed on one sample of the control group and one of the pathological groups, because of earlier cell death that did not allow further immunophenotype analysis. Both CB- and WJ-MSCs showed a mesenchymal phenotype with positivity for CD90, CD73, and CD105, and negativity for CD45, HLA-DR, CD34, CD14, and CD56, and no differences were described between the two sources of stem cells and between healthy and pathological pregnancies.

Fig. 3.

Cord blood derived (CB-MSCs) and Wharton’s Jelly-derived mesenchymal stem cell (WJ-MSCs) immunophenotype. Flow cytometry analysis was performed on CB- and WJ-MSCs, and cells were positive for mesenchymal markers, such as CD90, CD73, and CD105, and negative for lineage-specific markers, such as HLA-DR, CD45, CD34, CD14, and CD56. SSC-A, side scatter area; FSC-A, forward side scatter-area; CD, cluster of differentiation.