Avoidance of maternal cell contamination and overgrowth in isolating fetal chorionic villi mesenchymal stem cells from human term placenta

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 hybridiza-tion. 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

Isolation and expansion of mesenchymal stem/stromal cells derived from human placenta tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use

Modelling retrial-upon-conflict systems with product-form stochastic Petri nets

In this paper we consider a particular class of stochastic Petri nets that admits a product-form stationary distribution under general conditions (independent exponentially distributed firing time). We show that the structure of the these stochastic Petri nets is appropriate for modelling systems in which several components compete for a resource and conflicts may arise during the contention that require to newly perform the operation after a recovery time. Examples of these systems are wireless stations competing for a channel or processes operating on the same database concurrently. We derive the performance indices as functions of the model parameters, i.e., firing rates, probabilities of conflicts, number of competing components

Manufacture of clinical grade human placenta-derived multipotent mesenchymal stromal cells

Clinical grade human mesenchymal stem cells (MSC) are manufactured and used in clinical trials for a range of regenerative and inflammatory diseases. Human MSC have now been derived from tissues other than bone marrow, such as placenta, as described in this laboratory protocol. It provides instructions for clinical grade MSC manufacturing according to the Code of Good Manufacturing Practice (cGMP) principles and according to policies and procedures of our internal Quality Management System (QMS), which is based on the International Organization for Standardization (ISO) standard requirements. Relevant organizational structure and QMS elements are presented

Isolation and expansion of mesenchymal stem/stromal cells derived from human placenta tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use

Transcriptional ontogeny of first trimester human fetal and placental mesenchymal stem cells: gestational age versus niche

Early fetal and placental MSCs have translationally-advantageous characteristics compared to later pregnancy MSCs. During the first trimester, the fetus and placenta grow rapidly with divergent developmental requirements, but studies comparing mesenchymal stem cells (MSCs) from different origins have paid little attention to the effect of gestational age over this temporal window. Here we present the transcriptome through first trimester development of MSC isolated from fetal bone marrow (BM) or placental structures. Samples were collected weekly from 8 to 12 weeks. The raw microarray data are available on the ArrayExpress database (www.ebi.ac.uk/arrayexpress) under accession number E-TABM-1224. Additionally, the data have been integrated into the stem cell collaboration platform www.Stemformatics.org. These data provide a valuable resource for developmental biology and stem cell investigation

Multipotent human stromal cells isolated from cord blood, term placenta and adult bone marrow show distinct differences in gene expression pattern

Multipotent mesenchymal stromal cells derived from human placenta (pMSCs), and unrestricted somatic stem cells (USSCs) derived from cord blood share many properties with human bone marrow-derived mesenchymal stromal cells (bmMSCs) and are currently in clinical trials for a wide range of clinical settings. Here we present gene expression profiles of human cord blood-derived unrestricted somatic stem cells (USSCs), human placental-derived mesenchymal stem cells (hpMSCs), and human bone marrow-derived mesenchymal stromal cells (bmMSCs), all derived from four different donors. The microarray data are available on the ArrayExpress database (www.ebi.ac.uk/arrayexpress) under accession number E-TABM-880. Additionally, the data has been integrated into a public portal, www.stemformatics.org. Our data provide a resource for understanding the differences in MSCs derived from different tissues