Derivation, characterization, and in vitro differentiation of canine embryonic stem cells.

Canine embryonic stem (cES) cell lines were generated to establish a large-animal preclinical model for testing the safety and efficacy of embryonic stem (ES) cell-derived tissue replacement therapy. Putative cES cell lines were initiated from canine blastocysts harvested from natural matings. Times of harvest were estimated as 12-16 days after the presumed surge in circulating levels of luteinizing hormone. Four lines established from blastocysts harvested at days 13-14 postsurge satisfied most of the criteria for embryonic stem cells, whereas lines established after day 14 did not. One line, Fred Hutchinson dog (FHDO)-7, has been maintained through 34 passages and is presented here. FHDO-7 cells are alkaline phosphatase-positive and express both message and protein for the Oct4 transcription factor. They also express message for Nanog and telomerase but do not express message for Cdx2, which is associated with trophectoderm. Furthermore, they express a cluster of pluripotency-associated microRNAs (miRs) (miR-302b, miR-302c, and miR-367) characteristic of human and mouse ES cells. The FHDO-7 cells grow on feeder layers of modified mouse embryonic fibroblasts as flat colonies that resemble ES cells from mink, a close phylogenetic relative of dog. When cultured in nonadherent plates without feeders, the cells form embryoid bodies (EBs). Under various culture conditions, the EBs give rise to ectoderm-derived neuronal cells expressing gamma-enolase and beta 3-tubulin; mesoderm-derived cells producing collagen IIA1, cartilage, and bone; and endoderm-derived cells expressing alpha-fetoprotein or Clara cell-specific protein.

Regulation of global gene expression in the bone marrow microenvironment by androgen: androgen ablation increases insulin-like growth factor binding protein-5 expression.

BACKGROUND: Prostate cancer frequently metastasizes to bone. Androgen suppression treatment is initially highly effective, but eventually results in resistant cancer cells. This study evaluates the effects of androgen suppression on the bone and bone marrow (BM). In particular we questioned whether the androgen therapy could adversely facilitate prostate cancer progression through an increase growth factor secretion by the bone microenvironment. METHODS: Global gene expression is analyzed on mPEDB DNA microarrays. Insulin-like growth factor binding protein-5 (IGFBP5) is detected by immunohistochemistry in mouse tissues and its regulation measured by qPCR and Western blotting in human BM stromal cells. Effects of extracellular matrix-associated IGFBP5 on human prostate epithelial cells are tested in an MTS cell-growth assay. RESULTS: Castration increases expression of 159 genes (including 4 secreted cytokines) and suppresses expression of 84 genes. IGFBP5 is most consistently increased and the increase in expression is reversed by testosterone administration. IGFBP5 protein is detected in vivo in osteoblasts, BM stromal cells, and endothelial cells. Primary human stromal cell cultures secrete IGFBP5. In vitro, treatment of immortalized human marrow stromal cells with charcoal-stripped serum increases IGFBP5 mRNA expression, which is reversed by androgen supplementation. IGFBP5 is incorporated into the extracellular matrix. Further, IGFBP5 immobilized on extracellular matrices of stromal cells enhances the growth of immortalized prostate epithelial cells. CONCLUSIONS: Androgen suppressive therapy increases IGFBP5 in the BM microenvironment and thereby may facilitate the progression of prostate cancer.

Monocyte-derived CXCL7 peptides in the marrow microenvironment.

The marrow microenvironment consists of several different interacting cell types, including hematopoietic-derived monocyte/macrophages and nonhematopoietic-derived stromal cells. Gene-expression profiles of stromal cells and monocytes cultured together differ from those of each population alone. Here, we report that CXCL7 gene expression, previously described as limited to the megakaryocyte lineage, is expressed by monocytes cocultured with stromal cells. CXCL7 gene expression was confirmed by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), and secretion of protein was detected by enzyme-linked immunosorbent assay (ELISA) and Western blot. At least 2 stromal-derived activities, one yet to be identified, were required for optimal expression of CXCL7 by monocytes. NAP-2, the shortest form of CXCL7 detected in the coculture media, was confirmed to decrease the size and number of CFU-Meg colonies. The propeptide LDGF, previously reported to be mitogenic for fibroblasts, was not secreted by stimulated monocytes. The recombinant form of LDGF produced in a prokaryotic expression system did not have biologic activity in our hands. The monocytic source of CXCL7 was also detected by immunohistochemistry in normal bone marrow biopsies, indicating an in vivo function. We conclude that stromal-stimulated monocytes can serve as an additional source for CXCL7 peptides in the microenvironment and may contribute to the local regulation of megakaryocytopoiesis.

Human marrow stromal cells activate monocytes to secrete osteopontin, which down-regulates Notch1 gene expression in CD34+ cells.

The hematopoietic microenvironment, approximated in vitro by long-term marrow cultures (LTCs), consists of both nonhematopoietic-derived stromal elements and hematopoietic-derived monocyte/macrophages. To better understand the consequences of monocyte-stroma interactions, we compared gene expression profiles of CD14+ peripheral blood monocytes and HS-27a stromal cells cultured alone and together in cocultures. Results from 7 separate experiments revealed 22 genes were significantly up- or down-regulated in the cocultures, with osteopontin (OPN) up-regulated more than 15-fold. The microarray OPN data were confirmed by Northern blot, real-time polymerase chain reaction (PCR), and by detection of OPN protein. High levels of OPN gene expression were also detected in 2- to 3-week-old primary LTCs. Using Transwells we determined that stromal cells were secreting a factor that up-regulated OPN gene expression in CD14+ cells. When CD34+ cells were cultured in the presence of purified OPN, tyrosine phosphorylation of a 34-kDa molecule was increased 2- to 3-fold, an effect that was diminished in the presence of an OPN neutralizing monoclonal antibody. In addition, Notch1 gene expression was decreased 5-fold in OPN-treated CD34+ cells. We conclude that interactions between stroma and monocytes can result in activities that limit the role of Notch signaling in hematopoietic regulation.

Functional interleukin-7 receptors (IL-7Rs) are expressed by marrow stromal cells: binding of IL-7 increases levels of IL-6 mRNA and secreted protein.

DNA spotted microarrays were used to compare gene expression profiles from 2 functionally distinct human marrow stromal cell lines: HS-27a, which supports cobblestone area formation by early hematopoietic progenitors, and HS-5, which secretes multiple cytokines that support the proliferation of committed progenitors. One unexpected result was the high level of interleukin-7 receptor (IL-7R) gene expression in HS-27a stromal cells. Northern blot analysis confirmed the IL-7R RNA expression, and Western blots for the IL-7R protein detected both a full-length (90-kd) IL-7R and a smaller 30-kd fragment in both HS-27a cells and primary stromal cell cultures, whereas only the 90-kd receptor protein was detected in peripheral blood mononuclear cells. Biotinylated IL-7 was shown to bind to HS-27a cells under physiologic conditions, and this binding was inhibited by blocking anti-IL-7 antibodies. Tyrosine phosphorylation of several proteins (55 kd, 30 kd, and 24 kd) in HS-27a cells was rapidly increased after incubation with recombinant IL-7. One of the phosphorylated proteins proved to be the 30-kd IL-7R fragment. Exposure of HS-27a cells to IL-7 resulted in a 10-fold increase in secretion of IL-6 into culture supernatants but no increase in the cytokines stromal cell-derived factor 1, macrophage inflammatory protein 1 alpha, or IL-1 beta. The up-regulation of IL-6 secretion is associated with a rapid but transient increase in detectable levels of IL-6 messenger RNA. These data suggest that IL-7 may function to regulate the milieu of the microenvironment by modulating IL-6 secretion by the IL-7R-expressing stromal elements.

Size, shape and orientation of cells in budding hydra and regulation of regeneration in cell aggregates.

Changes in the number, shape, volume, orientation and vacuolization of cells involved in the budding of hydra were measured in histological sections. Before evagination, a group of about 800 epithelial cells are visibly recruited for the bud to be produced and this number increases to about 5,000 within a day. Thereafter, bud size increases mainly by proliferation of the cells within the bud. Upon recruitment for budding, the epithelial cells assume a columnar shape, with a smaller contact area facing the mesoglea, accompanied by a decrease in volume which is mostly accounted for by devacuolization. In later stages, cells progressively resume the form typical for non-budding areas of hydra. Evagination proceeds without reorientation of epithelio-muscular fibers, whereas elongation of the bud is accompanied by fiber reorientation.The process of sorting out and regeneration in aggregates of previously dissociated hydra cells was followed using various ratios of endodermal to ectodermal epithelial cells. From different initial compositions, the ratio in the regenerate rapidly approaches 1∶1, the ratio found in normal hydra tissue.The experimental findings are discussed in the context of theoretical notions on pattern formation, evagination, elongation and stability of layered structures.