In Vitro Cell Surface Marker Expression on Mesenchymal Stem Cell Cultures does not Reflect Their Ex Vivo Phenotype.

Cell surface marker expression is one of the criteria for defining human mesenchymal stem or stromal cells (MSC) in vitro. However, it is unclear if expression of markers including CD73 and CD90 reflects the in vivo origin of cultured cells. We evaluated expression of 15 putative MSC markers in primary cultured cells from periosteum and cartilage to determine whether expression of these markers reflects either the differentiation state of cultured cells or the self-renewal of in vivo populations. Cultured cells had universal and consistent expression of various putative stem cell markers including > 95% expression CD73, CD90 and PDPN in both periosteal and cartilage cultures. Altering the culture surface with extracellular matrix coatings had minimal effect on cell surface marker expression. Osteogenic differentiation led to loss of CD106 and CD146 expression, however CD73 and CD90 were retained in > 90% of cells. We sorted freshly isolated periosteal populations capable of CFU-F formation on the basis of CD90 expression in combination with CD34, CD73 and CD26. All primary cultures universally expressed CD73 and CD90 and lacked CD34, irrespective of the expression of these markers ex vivo indicating phenotypic convergence in vitro. We conclude that markers including CD73 and CD90 are acquired in vitro in most 'mesenchymal' cells capable of expansion. Overall, we demonstrate that in vitro expression of many cell surface markers in plastic-adherent cultures is unrelated to their expression prior to culture.

Characterization of adult human skeletal cells in different tissues reveals a CD90+CD34+ periosteal stem/progenitor population.

The periosteum plays a crucial role in bone healing and is an important source of skeletal stem and progenitor cells. Recent studies in mice indicate that diverse populations of skeletal progenitors contribute to growth, homeostasis and healing. Information about the in vivo identity and diversity of skeletal stem and progenitor cells in different compartments of the adult human skeleton is limited. In this study, we compared non-hematopoietic populations in matched tissues from the femoral head and neck of 21 human participants using spectral flow cytometry of freshly isolated cells. High-dimensional clustering analysis indicated significant differences in marker distribution between periosteum, articular cartilage, endosteum and bone marrow populations, and identified populations that were highly enriched or unique to specific tissues. Periosteum-enriched markers included CD90 and CD34. Articular cartilage, which has very poor regenerative potential, showed enrichment of multiple markers, including the PDPN+CD73+CD164+CD146- population previously reported to represent human skeletal stem cells. We further characterized periosteal populations by combining CD90 with other strongly expressed markers. CD90+CD34+ cells sorted directly from periosteum showed significant colony-forming unit fibroblasts (CFU-F) enrichment, rapid expansion, and consistent multi-lineage differentiation of clonal populations in vitro. In situ, CD90+CD34+ cells include a perivascular population in the outer layer of the periosteum and non-perivascular cells closer to the bone surface. CD90+ cells are also highly enriched for CFU-F in bone marrow and endosteum, but not articular cartilage. In conclusion, our study indicates considerable diversity in the non-hematopoietic cell populations in different tissue compartments within the adult human skeleton, and suggests that periosteal progenitor cells reside within the CD90+CD34+ population.

Human placental vascular and perivascular cell heterogeneity differs between first trimester and term, and in pregnancies affected by foetal growth restriction.

Growth-restricted placentae have a reduced vascular network, impairing exchange of nutrients and oxygen. However, little is known about the differentiation events and cell types that underpin normal/abnormal placental vascular formation and function. Here, we used 23-colour flow cytometry to characterize placental vascular/perivascular populations between first trimester and term, and in foetal growth restriction (FGR). First-trimester endothelial cells had an immature phenotype (CD144+/lowCD36-CD146low), while term endothelial cells expressed mature endothelial markers (CD36+CD146+). At term, a distinct population of CD31low endothelial cells co-expressed mesenchymal markers (CD90, CD26), indicating a capacity for endothelial to mesenchymal transition (EndMT). In FGR, compared with normal pregnancies, endothelial cells constituted 3-fold fewer villous core cells (P < 0.05), contributing to an increased perivascular: endothelial cell ratio (2.6-fold, P < 0.05). This suggests that abnormal EndMT may play a role in FGR. First-trimester endothelial cells underwent EndMT in culture, losing endothelial (CD31, CD34, CD144) and gaining mesenchymal (CD90, CD26) marker expression. Together this highlights how differences in villous core cell heterogeneity and phenotype may contribute to FGR pathophysiology across gestation.

Differences in human placental mesenchymal stromal cells may impair vascular function in FGR.

Placentae from pregnancies with foetal growth restriction (FGR) exhibit poor oxygen and nutrient exchange, in part due to impaired placental vascular development. Placental mesenchymal stromal cells (pMSCs) reside in a perivascular niche, where they may influence blood vessel formation/function. However, the role of pMSCs in vascular dysfunction in FGR is unclear. To elucidate the mechanisms by which pMSCs may impact placental vascularisation we compared the transcriptomes of human pMSCs isolated from FGR (<5th centile) (n = 7) and gestation-matched control placentae (n = 9) using Affymetrix microarrays. At the transcriptome level, there were no statistically significant differences between normal and FGR pMSCs; however, several genes linked to vascular function exhibited notable fold changes, and thus the dataset was used as a hypothesis-generating tool for possible dysfunction in FGR. Genes/proteins of interest were followed up by real-time PCR, western blot and immunohistochemistry. Gene expression of ADAMTS1 and FBLN2 (fibulin-2) were significantly upregulated, whilst HAS2 (hyaluronan synthase-2) was significantly downregulated, in pMSCs from FGR placentae (n = 8) relative to controls (n = 7, P < 0.05 for all). At the protein level, significant differences in the level of fibulin-2 and hyaluronan synthase-2, but not ADAMTS1, were confirmed between pMSCs from FGR and control pregnancies by Western blot. All three proteins demonstrated perivascular expression in third-trimester placentae. Fibulin-2 maintains vessel elasticity, and its increased expression in FGR pMSCs could help explain the increased distensibility of FGR blood vessels. ADAMTS1 and hyaluronan synthase-2 regulate angiogenesis, and their differential expression by FGR pMSCs may contribute to the impaired angiogenesis in these placentae.

Influence of culture media on the derivation and phenotype of fetal-derived placental mesenchymal stem/stromal cells across gestation.

INTRODUCTION: Derivation of pure fetal placental mesenchymal stem/stromal cells (pMSCs) is key to understand their role in placental development. However, isolated pMSCs are often contaminated by maternal-derived decidual MSCs (dMSCs). EGM-2 medium promotes the derivation of term fetal pMSCs, but the extent of first-trimester maternal pMSC contamination remains unclear. Culture media can also affect MSC phenotype. Here, we examined the effects of culture media on maternal pMSC contamination and fetal pMSC phenotype across gestation. METHODS: pMSCs were derived from first-trimester or term placentae in advanced-DMEM/F12 medium or EGM-2 medium. Proportions of maternal (XX) and fetal (XY) cells in male pMSC cultures were determined by fluorescence in-situ hybridization. pMSC phenotype was analysed by flow cytometry, immunohistochemistry and Alamar blue proliferation assays. RESULTS: When derived in advanced-DMEM/F12, all first trimester pMSC isolates exhibited maternal contamination (>72% XX cells, n = 5), whilst 7/9 term pMSC isolates were >98% fetal. When derived in EGM-2, all first trimester (n = 4) and term (n = 9) pMSC isolates contained 95-100% fetal cells. Fetal pMSCs in EGM-2 proliferated 2-fold (first-trimester) or 4-fold (term) faster than those in advanced-DMEM/F12 (p < 0.05, n = 3). Fetal pMSCs in both media expressed the generic MSC marker profile (CD90+, CD105+, CD73+, CD31-, CD34-, CD144-). However, pMSCs transferred from EGM-2 to advanced-DMEM/F12 increased expression of smooth muscle cell markers calponin and α-smooth muscle actin, and decreased expression of the vascular cell marker VEGFR2 (n = 3). CONCLUSIONS: Deriving first-trimester pMSC in EGM-2 dramatically reduces maternal dMSC contamination. Media affects fetal pMSC phenotype, and careful consideration should be given to application specific culture conditions.

Mesenchymal Stem/Stromal Cells from the Placentae of Growth Restricted Pregnancies Are Poor Stimulators of Angiogenesis.

The extensively branched vascular network within the placenta is vital for materno-fetal exchange, and inadequate development of this network is implicated in the pregnancy disorder fetal growth restriction (FGR), where babies are born pathologically small. Placental mesenchymal stem/stromal cells (pMSCs) and placental macrophages both reside in close proximity to blood vessels within the placenta, where they are thought to promote angiogenesis via paracrine mechanisms. However, the relationship between pMSCs, macrophages and placental vascular development has not yet been examined. We aimed to determine if inadequate paracrine stimulation of placental vascular development by pMSCs and macrophages during pregnancy may contribute to the inadequate vascularisation seen in FGR. Media conditioned by MSCs from FGR placentae significantly inhibited endothelial tube formation, compared to conditioned media derived from normal pMSCs. Similarly, macrophages exposed to media conditioned by FGR pMSCs were less able to stimulate endothelial tube formation in comparison to macrophages exposed to media conditioned by normal pMSCs. While MSCs from normal placentae produce a combination of angiogenic and anti-angiogenic cytokines, there were no significant differences in the secretion of the anti-angiogenic cytokines thrombospondin-1, insulin growth factor binding protein-4, or decorin between normal and FGR pMSCs that could explain how FGR pMSCs inhibited endothelial tube formation. Together, these data suggest a dysregulation in the secretion of paracrine factors by pMSCs in FGR placentae. These findings illustrate how cross talk between pro-angiogenic cell types in the placenta may be crucial for adequate angiogenesis.