Lynn Graf

The Human Homolog of Rat Jagged1Expressed by Marrow Stroma Inhibits Differentiation of 32D Cells through Interaction with Notch1

A cDNA clone encoding the human homolog of rat Jagged1 was isolated from normal human marrow. Analyses of human stromal cell lines indicate that this gene, designated hJagged1, is expressed by marrow stromal cells typified by the cell line HS-27a, which supports the long-term maintenance of hematopoietic progenitor cells. G-CSF-induced differentiation of 32D cells expressing Notch1 was inhibited by coculturing with HS-27a. A peptide corresponding to the Delta/Serrate/LAG-2 domain of hJagged1 and supernatants from COS cells expressing a soluble form of the extracellular portion of hJagged1 were able to mimic this effect. These observations suggest that hJagged1 may function as a ligand for Notch1 and play a role in mediating cell fate decisions during hematopoiesis.

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 γ‐enolase and β3‐tubulin; mesoderm‐derived cells producing collagen IIA1, cartilage, and bone; and endoderm‐derived cells expressing α‐fetoprotein or Clara cell‐specific protein.

Dissecting the Marrow Microenvironment

Abstract: Cloned human stromal cell lines representing functionally distinct cellular components of the marrow microenvironment were generated to serve as tools for identifying gene products that regulate hematopoiesis. Oligonucleotide arrays, or “gene chips” were used to provide a comprehensive comparison of gene expression among the cell lines. One line, designated HS‐5, was found to secrete large amounts of cytokines, and conditioned media from this line was found to support the ex vivo expansion of both immature and mature progenitors. In contrast, a second line, designated HS‐27a, does not secrete known cytokines but does support cobblestone area formation by CD34+/38lo cells. HS‐27a, but not HS‐5, was also found to express hJagged1, a ligand for Notch1, which may function to influence cell fate decisions of hematopoietic precursors. Both cell lines are currently being used to identify other gene products that regulate hematopoiesis and to generate reagents that will allow more formal evaluation of the putative role of hJagged1 in hematopoietic cell fate decisions.

Stromal Inhibition of Myeloid Differentiation: A Possible Role for hJagged1

Cloned human stromal cell lines, HS-27a and HS-23, were established in our laboratories as previously described.1 HS-27a, unlike HS-23, supports the formation of cobblestone areas from immature CD34+/38− cells. Recently we reported that HS27a expresses a human homolog for rat Jagged1 (hJagged1), a ligand for Notch1, which may play a role in cell-fate decisions that prevent differentiation and allow cobblestone area formation.2 The purpose of the current study was to develop a model for investigating whether hJagged1 can prevent myeloid differentiation. Towards this end we used U937 cells, which can be induced to differentiate by exposure to all trans retinoic acid (ATRA). This model was attractive because the induction of differentiation could be easily followed by the expression of CD11b on the surface of the differentiating cells. Critical to the model was the observation that U937 cells do express considerable amounts of mRNA for Notch1 (data not shown), suggesting that the receptor may be present on these cells. Based on the observations that Notch/Jagged interactions induce several genes that have the recombination binding protein Jκ (RBPJκ) consensus sequence, we first used electrophoretic mobility shift assays (EMSA) to detect the induction of nuclear protein binding to this consensus sequence in U937 cells before and after they were cultured on either hJagged1-expressing HS-27a cells or hJagged1-negative HS23 cells. The data shown in FIGURE 1 indicate that the EMSA assay is specific for the DNA binding protein and, further, that nuclear protein binding to the RBPJκ consensus sequence was increased when Notch-expressing cells were cultured with HS27a, but not after coculturing with HS-23. This strongly suggested that Notch/Jagged signaling occurs in the U937 cells, although it is possible that some other, as yet undefined, molecule on HS-27a cells is responsible for inducing the increase in DNA binding protein. We next hypothesized that if Notch/Jagged signaling prevented U937 from differentiating in response to ATRA, this block would result in the failure of U937 cells to express CD11b, which is uniformly induced during differentiation. To test this hypothesis, U937 cells were cultured in ATRA alone or in the presence of HS-23 or HS-27a cells. After 4 days the nonadherent and adherent U937 cells were harvested separately and analyzed by fluorescence-activated cell sorter (FACS) for expression of CD11b. FIGURE 2 shows that U937 cells exposed to ATRA alone or in the pres-