**4. Materials and Methods**

### *4.1. Ethical Approval and Sample Collection*

The study with identification number Dnr 2014/191 was approved on 24 April 2014 by the Lund Regional Ethics Committee Review Board (EPN) for studies on human subjects at Lund University and Skåne University Hospital, Lund, Sweden. Collection of UCB from normotensive and PE pregnancies was performed following both Caesarean and vaginal deliveries at Skåne University Hospital, after written informed consent from patients. All the experiments were performed in accordance with relevant guidelines and regulations. Preeclampsia was defined as blood pressure ≥140/90 mmHg and proteinuria ≥300 mg/<sup>L</sup> according to ISSHP definition [2]. A summary of the clinical condition of the patients included in this study and the subsequent experiments performed is available in Table 2 (details in Supplementary Table S4). The UCB was collected in flasks containing 10 mL Dulbecco's Modified Eagle's Medium, 10% fetal bovine serum (FBS), 100 IU/mL penicillin, 100 μg/mL streptomycin (Gibco ®, Stockholm, Sweden) and 25 IU/mL heparin (Vianex S.A., Athens, Greece). The samples were stored at 4 ◦C and processed within 4 h after sampling.




**Table 2.** *Cont*.

#### *4.2. Mononuclear Cell Isolation from the UCB*

Total mononuclear cells (MNCs) were isolated from UCB using the density centrifugation media Ficoll-Paque PLUS (GE Healthcare Life Sciences, Uppsala, Sweden) according to the manufacturer's protocol. In brief, the total UCB was mixed 1:1 (*w*/*v*) with wash buffer containing 1× phosphate buffered saline (PBS), 2% FBS and 2 mM EDTA. Each sample was carefully laid upon Ficoll-Paque PLUS before centrifugation at 400× *g* for 30 min at room temperature (RT). After the centrifugation, the interphase layer containing the MNCs was retrieved and mixed with ice-cold Iscoves Modified Dulbecco's Medium (IMDM), 10% FBS in 1:2 (*w*/*v*) (Gibco®). The MNC suspension was centrifuged and rinsed in wash buffer for erythroid profile analysis or CD34+ cell isolation.

### *4.3. Isolation of UCB CD34*+ *Cells*

Using the human CD34 MicroBead Kit (Miltenyi Biotec, Lund, Sweden), the UCB CD34+ cells were isolated according to the manufacturer's protocol. In summary, the MNCs were incubated with FcR Blocking Reagent and CD34 MicroBeads at 4 ◦C for 30 min followed by rinse and centrifugation at 300× *g* at 4 ◦C for 10 min. The cell suspension was filtered at 40 μm and CD34+ cells were magnetically selected on LS columns (Miltenyi Biotec, Lund, Sweden). The CD34+ cells were either resuspended in 10% Dimethyl sulfoxide (DMSO) freezing medium, stored at −80 ◦C for at least 24 h and transferred to liquid nitrogen tank for later in vitro cell culture assays, or were stained and analyzed for surface adhesion molecules using flow cytometry.

#### *4.4. Flow Cytometric Analysis of SAM Expression on UCB HSPCs*

The UCB HSPCs were detected by flow cytometry as CD34+ CD45+ cells and the expression of 6 different SAMs was analyzed using phycoerythrin-conjugated mouse anti-human antibodies (Table 1) [49,94–97]. Six separate suspensions of CD34+-enriched cells were prepared and each was incubated for 30 min on ice with appropriate amounts of antibodies (1:25) specific for CD34 (CD34-phycoerythrin/Cy7) and CD45 (CD45-FITC) in combination with one of the SAMs. After rinse in wash buffer, the cells were analyzed using a BD FACSAria™ I. Spectral compensation was carried out using VersaComp Antibody Capture Beads kit (Beckman Coulter, Bromma, Sweden) and the gates were set based on unstained and fluorescent minus one controls. 7AAD at 10 μg/mL (Sigma Aldrich, Stockholm, Sweden) was used as a viability marker. All viable CD34+ CD45+ cells from each sample were sorted into a tube and used for RNA extraction. Data analysis was performed using FlowJo (V.10.0.8. Ashland, OR, US).

#### *4.5. Flow Cytometric Analysis of UCB Stem Cells and Colony Formation Assay*

Frozen CD34+ enriched cells were thawed and stored with FcR Blocking Reagent (Miltenyi Biotec) at 4 ◦C for 30 min. The cells were then stained as described above, with the following antibodies: CD34-phycoerythrin/Cy7, CD38-APC, CD45RA-FITC, and CD90-BV421 (1:25, BD Biosciences, San Jose, CA, US). After performing spectral compensation and setting the gates as mentioned earlier, the UCB HSC population was analyzed by flow cytometry (Table 1) and a total of 15000 viable CD34+ cells were collected from each sample using fluorescent activated cell sorting (FACS). The cells were mixed with Cell Resuspension Solution (R&D Systems, Oxon, Sweden) and Human Methylcellulose Complete Media containing EPO, Granulocyte macrophage colony-stimulating factor (GM-CSF), Interlukin-3 (IL-3), and Stem Cell Factor (SCF) (HSC003, R&D Systems). 500 cells/well were plated in triplicate in 6-well plates and placed in a humid chamber incubated at 37 ◦C with 5% CO2. Burst forming units-erythroid (BFU-Es) were counted in each well after 14 days of culture as indicated by R&D Systems (https://www.rndsystems.com/resources/protocols/human-colony-forming-cell-cfcassay-using-methylcellulose-based-media).

#### *4.6. RNA Extraction and cDNA Subtractive Hybridization*

The sorted viable CD34+ CD45+ cells from aforementioned SAM expression analysis were used for total RNA extraction was performed by lysing the cells in Trizol (Ambion, Naugatuck, CT, US). Following addition of chloroform to the lysate, the aqueous phase was mixed with 70% ethanol and transferred to RNeasy Mini spin columns (Qiagen, Hilden, Germany). Total RNA preparation was completed according to the manufacturer's protocol. Poly A<sup>+</sup> RNA purification was performed using Oligotex Direct mRNA mini kit (Qiagen) following the manufacturer's instructions. Subtractive hybridization was carried out using the Clontech ® PCR-Select ™ Di fferential Screening kit (Takara Bio USA, Inc., Mountain View, CA 94043, USA) according to the manufacturer's protocol. The poly A<sup>+</sup> RNA from PE and normotensive groups were respectively used as tester and driver for cDNA synthesis. Enriched tester-specific amplicons from the second round of subtraction were ligated into pGEM-T Easy Vector System I (Promega, Madison, WI, US) for insert sequencing (Beckman Coulter Genomics, Bishop's Stortford, United Kingdom). All retrieved sequences representing genes unique to the tester (PE) population compared to the normotensive population were blasted using BLAST analysis on NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi).
