Avoidance of Maternal Cell Contamination and Overgrowth in Isolating Fetal Chorionic Villi Mesenchymal Stem Cells from Human Term Placenta

Abstract

Human placenta is rich in mesenchymal stem/stromal cells (MSC), with their origin widely presumed fetal. Cultured placental MSCs are confounded by a high frequency of maternal cell contamination. Our recent systematic review concluded that only a small minority of placental MSC publications report fetal/maternal origin, and failed to discern a specific methodology for isolation of fetal MSC from term villi. We determined isolation conditions to yield fetal and separately maternal MSC during ex vivo expansion from human term placenta. MSCs were isolated via a range of methods in combination; selection from various chorionic regions, different commercial media, mononuclear cell digest and/or explant culture. Fetal and maternal cell identities were quantitated in gender‐discordant pregnancies by XY chromosome fluorescence in situ hybridization. We first demonstrated reproducible maternal cell contamination in MSC cultures from all chorionic anatomical locations tested. Cultures in standard media rapidly became composed entirely of maternal cells despite isolation from fetal villi. To isolate pure fetal cells, we validated a novel isolation procedure comprising focal dissection from the cotyledonary core, collagenase/dispase digestion and explant culture in endothelial growth media that selected, and provided a proliferative environment, for fetal MSC. Comparison of MSC populations within the same placenta confirmed fetal to be smaller, more osteogenic and proliferative than maternal MSC. We conclude that in standard media, fetal chorionic villi‐derived MSC (CV‐MSC) do not grow readily, whereas maternal MSC proliferate to result in maternal overgrowth during culture. Instead, fetal CV‐MSCs require isolation under specific conditions, which has implications for clinical trials using placental MSC. Stem Cells Translational Medicine 2017;6:1070–1084

Anti‐fibrotic effects of different sources of MSC in bleomycin‐induced lung fibrosis in C57BL6 male mice

“…CVC exhibit a delayed replicative senescence and maintenance of telomere length; however, variations in isolation techniques may lead to varying degrees of contamination with maternal cells. 53 In fact, our study illustrates that different isolates from the same layer may have dissimilar characteristics as noted by the distinct sets of data derived from CSC and CVC for AKT and MMP-2 activation. As we did not sequence these isolates, we are unable to speculate on the potential differences between them.…”

“…Although isolation of MSC derived from placental layers including from whole chorion and chorionic villi 27,49–52 has been reported, there are few if any current clinical trials utilizing this source of MSC. CVC exhibit a delayed replicative senescence and maintenance of telomere length; however, variations in isolation techniques may lead to varying degrees of contamination with maternal cells 53 . In fact, our study illustrates that different isolates from the same layer may have dissimilar characteristics as noted by the distinct sets of data derived from CSC and CVC for AKT and MMP‐2 activation.…”

Fetal Bone Marrow‐Derived Mesenchymal Stem/Stromal Cells Enhance Humanization and Bone Formation of BMP7 Loaded Scaffolds

“…Both fPL‐MSC and fBM‐MSCs satisfied the International Society for Cellular Therapy (ISCT) specification of human MSC characterization. MSCs appeared as plastic‐adherent spindle‐shaped cells in culture and differentiated adequately along the osteogenic and adipogenic lineages . Both MSCs had the appropriate cell surface markers expression (Figure S1A and B, Supporting Information).…”

Pleiotropic Effects of Exosomes as a Therapy for Stroke Recovery

“…The theoretical strategy for cell therapies was based on replacement by differentiation of grafted MSCs or stem cells of dead neurons, but many studies showed that cell therapies promote regeneration and repair of damaged tissue due to stroke, including angiogenesis, neurogenesis, and axonal outgrowth, together with regulating corresponding molecular cascades [32]. Although the approach with these cell therapies was shown to be safe, and they passed through the BBB and reached injured tissues [50], one cannot exclude the possibility of complications after grafting cells, such as cell rejection, unpredictable immune responses, possible contamination, problems of storage of cells before their use, and inducing tumor formation [51][52][53][54]. Exosomes offer an advantage to overcome the limitations of cell therapy, such as: (1) less tumorigenicity due to their to inability to self-replicate; (2) dosing not affected by loss of transplanted cell viability; (3) not occluding the microvascular system; (4) tough lipid bilayer vesicles that can be easily stored for a long time at −80 • C and other extremes of handling, while retaining bioactivity; (5) their low immunogenicity not requiring a host immune response and a match between the donor and the recipient; (6) capable of extracting sufficient amounts from immortalized stem cells; and (7) capable of enhancing efficacy by genetic engineering of the parent cells [44,[55][56][57].…”