IntroductionHematopoiesis in bone marrow (BM) occurs in distinct microenvironmental niches. Discrete extracellular matrix (ECM) microenvironments within the BM help to separate endosteum, an interface between bone and BM, from the central marrow. Methods for studying hematopoiesis include clonal culture systems in semisolid media, 1 short-term 2 and long-term 3,4 liquid cultures, and tissue culture systems where hematopoietic cells grow on feeder layers of BM stromal cells. 5,6 However, cell culture systems involving growth on the surface of tissue culture plastic do not accurately represent tissue architecture 7 or the complex interactions between cells and their micro-environment. Recently, a stromal spheroid coculture model 8 and various scaffolds 9,10 have been developed to recreate the three-dimensional (3-D) environment of the BM, but these models fail to recapitulate the physiologic conditions of the BM. To adequately study B-cell development, 11 pathogenesis, 12,13 and neoplasia, 14 a culture system that places BM cells within their physiologic environment is required. For BM-localized malignancies, more effective culture systems must incorporate all compartments of the malignant clone, including cancer stem and progenitor cells, to identify their therapeutic vulnerabilities.Multiple myeloma (MM), an incurable cancer with 3-to 5-year survival despite the development of potent new therapies, 15 is characterized by monoclonal immunoglobulin (Ig), lytic bone lesions, 16 Here we present a robust 3-D tissue culture model in which the human BM microenvironment is reconstructed in vitro. In 3-D, the MM clone expands within its native microenvironment providing a valuable preclinical model within which conventional (melphalan) and novel (bortezomib) therapeutics selectively kill their target cells. In 3-D cultures, nonproliferating cells from MM BM concentrate at a reconstructed endosteummarrow junction (rEnd). Purified nonproliferating MM BM cells include MM-CSCs, as defined by their ability in a secondary culture to generate B/PC progeny harboring the unique MM clonotypic signature. Three-dimensional cultures of BM or mobilized blood autografts (MBAs) offer a preclinical model within which new therapies can be tested for their impact on all compartments of the MM clone, as well as providing access to the MM-CSC that underlie disease progression. An Inside Blood analysis of this article appears at the front of this issue.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 USC section 1734. For personal use only. on May 8, 2018. by guest www.bloodjournal.org From Methods MaterialsAfter approval from the Health Research Board (University of Alberta) and the Alberta Cancer Board, and after informed consent was obtained in accordance with the Declaration of Helsinki, BM samples (n ϭ 48) were provided from patients undergoing BM biopsies at the Cross Cancer Institute. Al...

We are investigating the expression and linkage of major histocompatibility complex (MHC) class I genes in the duck ( Anas platyrhynchos) with a view toward understanding the susceptibility of ducks to two medically important viruses: influenza A and hepatitis B. In mammals, there are multiple MHC class I loci, and alleles at a locus are polymorphic and co-dominantly expressed. In contrast, in lower vertebrates the expression of one locus predominates. Southern-blot analysis and amplification of genomic sequences suggested that ducks have at least four loci encoding MHC class I. To identify expressed MHC genes, we constructed an unamplified cDNA library from the spleen of a single duck and screened for MHC class I. We sequenced 44 positive clones and identified four MHC class I sequences, each sharing approximately 85% nucleotide identity. Allele-specific oligonucleotide hybridization to a Northern blot indicated that only two of these sequences were abundantly expressed. In chickens, the dominantly expressed MHC class I gene lies adjacent to the transporter of antigen processing ( TAP2) gene. To investigate whether this organization is also found in ducks, we cloned the gene encoding TAP2 from the cDNA library. PCR amplification from genomic DNA allowed us to determine that the dominantly expressed MHC class I gene was adjacent to TAP2. Furthermore, we amplified two alleles of the TAP2 gene from this duck that have significant and clustered amino acid differences that may influence the peptides transported. This organization has implications for the ability of ducks to eliminate viral pathogens.

DNA mismatch repair (MMR) proteins are integral to the maintenance of genomic stability and suppression of tumorigenesis due to their role in repair of post-replicative DNA errors. Recent data also support a role for MMR proteins in cellular responses to exogenous DNA damage that does not involve removal of DNA adducts. We have demonstrated previously that both Msh2- and Msh6-null primary mouse embryonic fibroblasts are significantly less sensitive to UVB (ultraviolet B)-induced cytotoxicity and apoptosis than wild-type control cells. In order to ascertain the physiological relevance of the data we have exposed MMR-deficient mice to acute and chronic UVB radiation. We found that MMR-deficiency was associated with reduced levels of apoptosis and increased residual UVB-induced DNA adducts in the epidermis 24-h following acute UVB exposure. Moreover, Msh2-null mice developed UVB-induced skin tumors at a lower level of cumulative UVB exposure and with a greater severity of onset than wild-type mice. The Msh2-null skin tumors did not display microsatellite instability, suggesting that these tumors develop via a different tumorigenic pathway than tumors that develop spontaneously. Therefore, we propose that dysfunctional MMR promotes UVB-induced tumorigenesis through reduced apoptotic elimination of damaged epidermal cells.

A small percentage of cases of Waldenstrom macroglobulinemia (WM) present with biclonality, defined here as the rearrangement of two distinct VDJ gene segments. Here we investigated the expansion of two clones from a patient with WM expressing molecularly detectable clonotypic gene rearrangements, one V(H)3 and one V(H)4. Biclonality was determined in blood and bone marrow mononuclear cells using real-time quantitative PCR (RQ-PCR). V(H)4 expressing cells but not V(H)3 expressing cells underwent clonal expansion in 3-D culture of reconstructed WM bone marrow. After 3-D culture, secondary culture in a colony forming unit assay, and RQ-PCR, only the V(H)4 clone was shown to harbor a subpopulation with characteristics of cancer stem cells, including proliferative quiescence, self-regeneration, and the ability to generate clonotypic progeny, suggesting that the V(H)4, but not the V(H)3, clone is clinically significant. Enrichment of potential WM stem cells in 3-D cultures holds promise for monitoring their response to treatment and for testing new therapies.