High Proliferative Placenta-Derived Multipotent Cells Express Cytokeratin 7 at Low Level

The purpose of this study was to investigate the immunophenotypes and gene expression profile of high proliferative placenta-derived multipotent cells (PDMCs) population at different stages of culture. We demonstrated that the colonies resulting from single cells were either positive or negative for CK7, whereas only PDMC clones with weak CK7 expression (CK7low-clones) were highly proliferative. Interestingly, vimentin positive (Vim+) placental stromal mesenchymal cells did not express CK7 in situ, but double CK7+Vim+ cells detection in tissue explants and explants outgrowth indicated CK7 inducible expression in vitro. PCNA presence in CK7+Vim+ cells during placental explants culturing confirmed belonging of these cells to proliferative subpopulation. Transcription factors CDX2 and EOMES were expressed in both CK7low-clones and subset of stromal mesenchymal cells of first-trimester placental tissue in situ. Meanwhile, CK7low -clones and stromal mesenchymal cells of full-term placental tissue in situ expressed ERG heterogeneously. SPP1, COL2A1, and PPARG2 mesodermal-related genes expression by CK7low-clones additionally confirms their mesenchymal origin. Inherent stem cell-related gene expression (IFTM3, POU5F1, and VASA) in CK7low-clones might indicate their enrichment for progenitors. Finally, in CK7low-clones we observed expression of such trophoblast-associated genes as CGB types I and II, fusogenic ERVW-1, GCM1, and GATA3. Thus, our results indicate that PDMCs acquired the representative immunophenotype signature under culture conditions.

The medium was replaced twice weekly. After 21 days of incubation in the adipogenic induction medium, this medium was aspirated and the cells were washed with PBS. Cells were fixed with 4% paraformaldehyde (PFA) and washed with sterile water. Next, the cells were incubated with 60% isopropanol at RT for 5 min and dried. The cultures were incubated with Oil Red O (Sigma, USA) solution (0.5% vol/vol in isopropanol) for 20 min at RT. After staining, the cells were washed with distilled water before microscopy. In addition, PDMCs were pelleted, and total RNA was collected after 21 days for adipogenic gene expression analysis by qRT-PCR.
qRT-PCR reactions were carried out as described below with the specific primers for PPARG2 (Table S1), normalized to endogenous ACTB and compared to basal expression levels in non-differentiated cells.
Differentiation control PDMCs were cultured in complete culture medium for 21 days and estimated for adipogenic differentiation by staining with Oil Red O (Sigma, USA) and qRT-PCR as described above.

Osteogenic differentiation
For osteogenic differentiation, cells were treated with osteogenic differentiation medium containing DMEM high glucose (HyClone), 10% FBS (Gibco), 2 mМ L-glutamine (Biomedicals), 5 mМ НЕРЕS (Biomedicals), 0.1 mM ascorbic acid 2-phosphate (Sigma, USA), 0.1 µM dexamethasone (Sigma, USA), 10 mM β-glycerophosphate (Sigma, USA), 100 U/ml penicillin and 50 µg/ml streptomycin (Sigma, USA). The cells were placed on 6-well plates. When the cells were of 80-90% confluence in a monolayer, the culture medium was changed to osteogenic differentiation medium, and the cells were incubated for 21 days. The differentiation medium was changed twice a week. Alizarin Red S staining was performed at day 21 to assess extracellular calcium deposition. For Alizarin Red S staining, the cells were washed with PBS and fixed with ice-cold 70% ethanol for 5 min at RT. Next, the cells were rinsed three to four times with distilled water and stained with 0.5% Alizarin Red S (pH 4.2, Sigma, USA) for 10 min at RT. The cells were rinsed with distilled water to remove excess dye before observing under the microscope for imaging. PDMCs were pelleted, and RNA was collected after 21 days for osteogenic gene expression analysis byqRT-PCR. qRT-PCR reactions were carried out as described below using specific primers for osteopontin (SPP1) (Table   S1), normalized to endogenous ACTB and compared to basal expression levels in non-differentiated cells. As differentiation control, PDMCs were cultured in complete culture medium for 21 days and estimated for osteogenic differentiation by staining with Alizarin Red S (Sigma, USA) and qRT-PCR as described above.

RNA extraction and RT-PCR
Total RNA was isolated using TRI Reagent (Sigma, USA) and stored at -80 °C. Trace amounts of genomic DNA were removed by RNAse-free DNAse I treatment (Thermo Scientific, USA). Then, cDNA was synthesized from 1 µg of total RNA with oligo(dT) 18 primers and RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Scientific, USA) according to the manufacturer's instructions. Two percent of the first-strand cDNA was amplified by sequence-specific primers (Table   S1)