SummaryHuman mesenchymal stem cells (hMSCs), which conventionally are isolated based on their adherence to plastic, are heterogeneous and have poor growth and differentiation, limiting our ability to investigate their intrinsic characteristics. We report an improved prospective clonal isolation technique and reveal that the combination of three cell-surface markers (LNGFR, THY-1, and VCAM-1) allows for the selection of highly enriched clonogenic cells (one out of three isolated cells). Clonal characterization of LNGFR+THY-1+ cells demonstrated cellular heterogeneity among the clones. Rapidly expanding clones (RECs) exhibited robust multilineage differentiation and self-renewal potency, whereas the other clones tended to acquire cellular senescence via P16INK4a and exhibited frequent genomic errors. Furthermore, RECs exhibited unique expression of VCAM-1 and higher cellular motility compared with the other clones. The combination marker LNGFR+THY-1+VCAM-1hi+ (LTV) can be used selectively to isolate the most potent and genetically stable MSCs.

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ABSTRACT. Although there are several markers available for the identification of endoderm derivatives such as the lung, pancreas, liver, and intestine, there are still very few surface markers available for the prospective isolation of the definitive endoderm. Among these, CXCR4 has been used in combination with E-cadherin as a cell surface marker to identify the definitive endoderm. However, CXCR4 expression decreases in late gut epithelium. Here we report a gene, Decay Accelerating Factor (DAF1/CD55), as a candidate surface marker for the identification of the early and late definitive endoderm. Daf1 is expressed in the definitive endoderm and mesoderm in early embryo at E8.5. Flow cytometry analysis of ES cells-derived differentiated cells revealed that DAF1-expressing cells also expressed CXCR4. Moreover, DAF1 expression is maintained until differentiation day 12 in ES cell-derived definitive endoderm cells. Analysis of the Pdx1/GFP-positive cells in E9.5 embryos and ES cell-derived cells with anti-DAF1 revealed that most Pdx1-GFP cells expressed DAF1. These results suggest that DAF1, when used in combination with E-cadherin, is useful for prospective identification of the definitive endoderm cells.

Human mesenchymal stem/stromal cells (hMSCs) have garnered enormous interest as a potential resource for cell‐based therapies. However, the molecular mechanisms regulating senescence in hMSCs remain unclear. To elucidate these mechanisms, we performed gene expression profiling to compare clonal immature MSCs exhibiting multipotency with less potent MSCs. We found that the transcription factor Frizzled 5 (FZD5) is expressed specifically in immature hMSCs. The FZD5 cell surface antigen was also highly expressed in the primary MSC fraction (LNGFR+THY‐1+) and cultured MSCs. Treatment of cells with the FZD5 ligand WNT5A promoted their proliferation. Upon FZD5 knockdown, hMSCs exhibited markedly attenuated proliferation and differentiation ability. The observed increase in the levels of senescence markers suggested that FZD5 knockdown promotes cellular senescence by regulating the noncanonical Wnt pathway. Conversely, FZD5 overexpression delayed cell cycle arrest during the continued culture of hMSCs. These results indicated that the intrinsic activation of FZD5 plays an essential role in negatively regulating senescence in hMSCs and suggested that controlling FZD5 signaling offers the potential to regulate hMSC quality and improve the efficacy of cell‐replacement therapies using hMSCs.

BackgroundWe developed an efficient in vitro method to differentiate mouse ES cells into the definitive endoderm (DE) and then Pdx1-expressing pancreatic lineages using mesodermal-derived supporting cells, M15. Using this method, resulting ES cell-derived DE and Pdx1-expressing cells were isolated by cell sorting, and their gene expression profiles were investigated with DNA microarray. Genes that were specifically expressed in DE and/or in Pdx1-expressing cells were extracted and their expression patterns in normal embryonic development were studied.ResultsGenes whose expression increased in DE and Pdx1 positive cells compared to the undifferentiated ES cells were chosen and in situ hybridizations were performed. Out of 54 genes examined, 27 were expressed in the DE of E8.5 mouse embryos and 15 genes were expressed in distinct domains in the pancreatic buds of E14.5 embryos. Among those genes expressed were Foxq1, CpM, Foxp4, Pcdh1, and Zmiz1, which were previously reported in other endodermal tissues. Genes, such as Parm1, Tmem184a, Hipk2 and Sox4 were reported to be expressed during early pancreatic development. Nptx2, C2cd4b, Tcf7l2 and Kiss1r were reported to be associated with beta cell or pancreatic functions in the adult. Akr1c19, Aebp2, Pbxip1 and Creb3l1, were novel and have not been described as being expressed either in DE or the pancreas.ConclusionsWe identified 27 genes, including 4 novel genes expressed in DE and pancreatic progenitor cells during normal development using an ES cell in vitro differentiation system. These results showed that DE cells and Pdx1/GFP-expressing cells obtained from our M15 based differentiation method mimic cells during the normal developmental processes. Additionally, ES cells are an excellent model for studies of early developmental processes.