Anti-apoptotic effect of sphingosine-1-phosphate and platelet-derived growth factor in human embryonic stem cells.

Human embryonic stem (hES) cells hold great promise for use in regenerative medicine. However, technologies first need to be established to maintain hES cells efficiently in vitro. Understanding the signaling networks involved in hES cell maintenance will prove to be essential to the development of such culture systems. Previously, we described a serum-free medium capable of supporting prolonged hES cell maintenance using sphingosine-1-phosphate (S1P) and platelet-derived growth factor (PDGF). Here, we describe an anti-apoptotic effect of S1P and PDGF in hES cells and demonstrate a direct effect of S1P in preventing hES cell apoptosis. Western blot analysis shows that S1P stimulates the phosphorylation of the mitogen-activated protein (MAP) kinases Erk1/2 but not of Akt, whereas PDGF stimulates both Erk1/2 and Akt phosphorylation. Moreover, our study suggests that the Erk1/2 and PI3K/Akt signaling pathways act independently of each other. Furthermore, neither S1P nor PDGF modify intracellular calcium concentration ([Ca(2+)]( i )) and Smad2 phosphorylation. Using pharmacological inhibitors of Erk1/2 and PI3K, our results demonstrate a critical role of the Erk1/2 and PI3K/Akt signaling pathways in mediating the anti-apoptotic effect of S1P and PDGF on hES cells. However, inhibition of the mammalian target of rapamycin (mTOR), a common downstream effector of Erk1/2 and PI3K/Akt, has no effect on hES cell apoptosis.

CD30 is a survival factor and a biomarker for transformed human pluripotent stem cells.

The application of human embryonic stem (hES) cells in regenerative medicine will require rigorous quality control measures to ensure the safety of hES cell-derived grafts. During propagation in vitro, hES cells can acquire cytogenetic abnormalities as well as submicroscopic genetic lesions, such as small amplifications or deletions. Many of the genetic abnormalities that arise in hES cell cultures are also implicated in human cancer development. The causes of genetic instability of hES cells in culture are poorly understood, and commonly used cytogenetic methods for detection of abnormal cells are capable only of low-throughput analysis on small numbers of cells. The identification of biomarkers of genetic instability in hES cells would greatly facilitate the development of culture methods that preserve genomic integrity. Here we show that CD30, a member of the tumor necrosis factor receptor superfamily, is expressed on transformed but not normal hES cells, and that CD30 expression protects hES cells against apoptosis.

Essential roles of sphingosine-1-phosphate and platelet-derived growth factor in the maintenance of human embryonic stem cells.

Human embryonic stem cells (hESCs) have great potential for use in research and regenerative medicine, but very little is known about the factors that maintain these cells in the pluripotent state. We investigated the role of three major mitogenic agents present in serum--sphingosine-1-phosphate (S1P), lysophosphatidic acid (LPA), and platelet-derived growth factor (PDGF)--in maintaining hESCs. We show here that although LPA does not affect hESC growth or differentiation, coincubation of S1P and PDGF in a serum-free culture medium successfully maintains hESCs in an undifferentiated state. Our studies indicate that signaling pathways activated by tyrosine kinase receptors act synergistically with those downstream from lysophospholipid receptors to maintain hESCs in the undifferentiated state. This study is the first demonstration of a role for lysophospholipid receptor signaling in the maintenance of stem cell pluri-potentiality.

Elf5 is essential for early embryogenesis and mammary gland development during pregnancy and lactation.

Elf5 is an epithelial-specific ETS factor. Embryos with a null mutation in the Elf5 gene died before embryonic day 7.5, indicating that Elf5 is essential during mouse embryogenesis. Elf5 is also required for proliferation and differentiation of mouse mammary alveolar epithelial cells during pregnancy and lactation. The loss of one functional allele led to complete developmental arrest of the mammary gland in pregnant Elf5 heterozygous mice. A quantitative mRNA expression study and Western blot analysis revealed that decreased expression of Elf5 correlated with the downregulation of milk proteins in Elf5(+/-) mammary glands. Mammary gland transplants into Rag(-/-) mice demonstrated that Elf5(+/-) mammary alveolar buds failed to develop in an Elf5(+/+) mammary fat pad during pregnancy, demonstrating an epithelial cell autonomous defect. Elf5 expression was reduced in Prolactin receptor (Prlr) heterozygous mammary glands, which phenocopy Elf5(+/-) glands, suggesting that Elf5 and Prlr are in the same pathway. Our data demonstrate that Elf5 is essential for developmental processes in the embryo and in the mammary gland during pregnancy.