IntroductionThe ability of early hematopoietic progenitors to efflux certain fluorescent dyes such as rhodamine 123 and Hoechst 33342 has long been appreciated and exploited for the isolation of these cells by fluorescence-activated cell sorting (FACS). [1][2][3] More recently, one method has proven to be especially useful for the isolation of the most primitive bone marrow cells from a number of different species. 4 This method relies on incubating the target cell population with the fluorescent dye Hoechst 33342 and subsequent FACS analysis of dual-wavelength Hoechst fluorescence with gating on a specific side population displaying low red and low blue fluorescence. Hence, the isolated cells are termed side-population (SP) cells. This low-staining SP is lost after treatment with verapamil, which has led to the assumption that the MDR1-encoded adenosine triphosphate-binding cassette (ABC) transporter, P-glycoprotein (P-gp), is responsible for Hoechst dye efflux in these cells. 4 Recently, excitement has been generated by the finding that putative stem cells from solid tissues may also share this SP phenotype. [5][6][7] Moreover, the degree of efflux activity seems to correlate with the maturation state, such that cells exhibiting the highest efflux activity are the most primitive. 8 Aside from its value in stem cell purification, the dye efflux phenomenon raises 2 important questions: (1) what are the biochemical mechanisms by which it is produced in SP cells and (2) what physiologic roles do such export activities play in stem cells?The multidrug-resistance gene, MDR1, is the best-studied member of the ABC transporter super-family of genes. The products of these genes are transmembrane proteins involved in energy-dependent transport of a wide spectrum of substrates across membranes. 9,10 Currently, more than 30 members have been identified in humans. 11 To date, most drug efflux phenomena have been ascribed to expression of MDR1. 12 Due to known Hoechst transport activity of MDR1, 13 and to the expression of MDR1 in early hematopoietic progenitor cells, 14,15 the export activity in stem cells has largely been attributed to MDR1. 4,14,16,17 MDR1 is only one of many candidate transporters for Hoechst. Members of the B, C, and G families of the ABC transport super-family are all associated with multidrug resistance and exhibit overlapping substrate specificities. Of particular importance here, they possess the ability to efflux broad and overlapping ranges of lipophilic molecules, a category that includes Hoechst. 18 A careful review of the literature revealed that the efflux activity of rhodamine in murine hematopoietic stem cells is only partially blocked by the MDR1 inhibitor, verapamil. In fact, the murine long-term repopulating cells, considered to be the most primitive stem cells, are isolated on the basis of rhodamine efflux (Rho Ϫ ) in the presence of verapamil. 16 Thus, it seems probable that another transporter may contribute to the SP phenotype associated with stem cells. Materials and methods Cell cu...

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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 ␥-enolase and ␤3-tubulin; mesoderm-derived cells producing collagen IIA1, cartilage, and bone; and endoderm-derived cells expressing ␣-fetoprotein or Clara cell-specific protein.

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Gab proteins are intracellular scaffolding and docking molecules involved in signaling pathways mediated by various growth factor, cytokine, or antigen receptors. Gab3 has been shown to act downstream of the macrophage colony-stimulating factor receptor, c-Fms, and to be important for macrophage differentiation. To analyze the physiological role of Gab3, we used homologous recombination to generate mice deficient in Gab3. Gab3؊/؊ mice develop normally, are visually indistinguishable from their wild-type littermates, and are healthy and fertile. To obtain a detailed expression pattern of Gab3, we generated Gab3-specific monoclonal antibodies. Immunoblotting revealed a predominant expression of Gab3 in lymphocytes and bone marrowderived macrophages. However, detailed analysis demonstrated that hematopoiesis in mice lacking Gab3 is not impaired and that macrophages develop in normal numbers and exhibit normal function. The lack of Gab3 expression during macrophage differentiation is not compensated for by increased levels of Gab1 or Gab2 mRNA. Furthermore, Gab3-deficient mice have no major immune deficiency in T-and B-lymphocyte responses to protein antigens or during viral infection. In addition, allergic responses in Gab3-deficient mice appeared to be normal. Together, these data demonstrate that loss of Gab3 does not result in detectable defects in normal mouse development, hematopoiesis, or immune system function.

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Methods are described for isolation and culture of primary mesenchyme cells from echinoid embryos. Ninety-five percentpure primary mesenchyme cells were isolated from early gastrulae ofStrongylocentrotus purpuratus, exploiting the biological segregation of these cells within the blastocoel. When cultured, more than 90% of the isolated cells reached the differentiated state, spicule formation, in synchrony with in vivo controls. Isolated primary mesenchyme cells were cultured with and without various cellular and acellular components of normal embryos in order to study the potential involvement of these components in the morphogenesis of the primary mesenchyme. Our data indicate that: 1. primary mesenchyme cells lack the ability to form the annular pattern of the primary mesenchymal ring autonomously; 2. they autonomously produce spicules of a characteristic morphology that differs from that of embryonic spicules; 3. morphogenesis of the primary mesenchyme is not affected by association with embryonic basal lamina, blastocoel matrix, or loosely aggregated epithelial cells, or by close confinement of each set of primary mesenchyme cells within the blastocoelar space; and 4. reaggregated, tightly associated epithelial cells can promote normal primary mesenchyme ring formation, and modify the primary mesenchyme-intrinsic spicule pattern to produce more normal spicule forms.