Enhanced isolation of adult thymic epithelial cell subsets for multiparameter flow cytometry and gene expression analysis
School of Natural Sciences, University of California, Merced, 5200 North Lake Road, Merced, CA 95340, USA.Journal of immunological methods (Impact Factor: 1.82). 02/2011; 367(1-2):85-94. DOI: 10.1016/j.jim.2011.02.008
The epithelial cells (TECs) are microenvironmental niche cells which support T lymphocyte development in the thymus. Most studies of TEC biology have focused on TEC at the fetal stage of development, whereas the biology of adult-stage TECs is not as well-understood. Delineating the molecular mechanisms that control adult TEC differentiation has implications for the success of T-lymphocyte based therapies for autoimmune diseases and induction of immunological tolerance to stem cell-derived tissues. Detailed analysis of adult TECs is technically challenging due to their rarity, their diminishing numbers with age, and the limited number of markers to distinguish between unique TEC subpopulations. Here, we have devised an improved isolation protocol for adult mouse TECs and combined it with six-color multiparameter flow cytometry. Using these techniques, we have identified four distinct subsets of CD45- EpCAM+ TECs in adult mice: a) UEA1(low) CDR1(low) (UC(low)); b) UEA1(high) CDR1(high)(UC(high)); c) UEA1(low) CDR1(high) MHC(high) (cTEC); and d) UEA1(high)CDR1(low) MHC(int/high) (mTEC). PCR analysis verified that these TEC subsets differentially expressed known TEC genes. TEC subsets were further analyzed using high-throughput quantitative PCR arrays to reveal novel genes that could be important for TEC subset maintenance. Intracellular staining for keratin-5 and keratin-8 can also be added, but our results suggest that keratin expression alone cannot be used to distinguish adult TEC subsets. Our enhanced isolation allows for detailed analysis of rare TEC subpopulations in the adult mouse at the cellular and molecular levels.
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ABSTRACT: Embryonic stem cells (ESC) can potentially be manipulated in vitro to differentiate into cells and tissues of all three germ layers. This pluripotent feature is being exploited to use ESC-derived tissues as therapies for degenerative diseases and replacement of damaged organs. Although their potential is great, the promise of ESC-derived therapies will be unfulfilled unless several challenges are overcome. For example, inefficient production of ESC-derived tissues before transplantation, inability of ESC-derived tissues to integrate well into the adult microenvironments due to developmental stage incompatibility, or active immune rejection of the ESC-derived graft are all potential challenges to successful ESC-derived therapies. One way to induce immunological tolerance to allogeneic tissues is via the establishment of mixed hematopoietic chimerism in which the host and donor cells are educated to recognize each other as "self". Proof of principle that in vitro cultured ESC-derived hematopoietic progenitors can be transplanted and induce immunological tolerance to allogeneic tissues exists in mouse models. In this review, we discuss the challenges to in vitro development of a bona fide ESC-derived hematopoietic stem cell and their differentiation fate in vivo, and provide suggestions to predict the immunogenicity of specific ESC-derived hematopoietic populations before transplantation that could be used to prevent their rejection after transplantation into an adult host.Current topics in medicinal chemistry 03/2011; 11(13):1621-37. DOI:10.2174/156802611796117702 · 3.40 Impact Factor
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ABSTRACT: The reproducible isolation and accurate characterization of thymic epithelial cell (TEC) subsets is of critical importance to the ongoing study of thymopoiesis and its functional decline with age. The study of adult TEC, however, is significantly hampered due to the severely low stromal to hematopoietic cell ratio. Non-biased digestion and enrichment protocols are thus essential to ensure optimal cell yield and accurate representation of stromal subsets, as close as possible to their in vivo representation. Current digestion protocols predominantly involve diverse, relatively impure enzymatic variants of crude collagenase and collagenase/dispase (col/disp) preparations, which have variable efficacy and are often suboptimal in their ability to mediate complete digestion of thymus tissue. To address these issues we compared traditional col/disp preparations with the latest panel of Liberase products that contain a blend of highly purified collagenase and neutral protease enzymes. Liberase enzymes revealed a more rapid, complete dissociation of thymus tissue; minimizing loss of viability and increasing recovery of thymic stromal cell (TSC) elements. In particular, the recovery and viability of TEC, notably the rare cortical subsets, were significantly enhanced with Liberase products containing medium to high levels of thermolysin. The improved stromal dissociation led to numerically increased TEC yield and total TEC RNA isolated from pooled digests of adult thymus. Furthermore, the increased recovery of TEC enhanced resolution and quantification of TEC subsets in both adult and aged mice, facilitating flow cytometric analysis on a per thymus basis. We further refined the adult TEC phenotype by correlating surface expression of known TEC markers, with expression of intracellular epithelial lineage markers, Keratin 5 and Keratin 8. The data reveal more extensive expression of K8 than previously recognized and indicates considerable heterogeneity still exists within currently defined adult TEC subsets.Journal of immunological methods 08/2012; 385(1-2):23-34. DOI:10.1016/j.jim.2012.07.023 · 1.82 Impact Factor
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ABSTRACT: Recent evidence suggests that head and neck squamous cell carcinomas (HNSCCs) harbor a small subpopulation of highly tumorigenic cells, designated cancer stem cells. A limiting factor in cancer stem cell research is the intrinsic difficulty of expanding cells in an undifferentiated state in vitro. Here, we describe the development of the orosphere assay, a method for the study of putative head and neck cancer stem cells. An orosphere is defined as a nonadherent colony of cells sorted from primary HNSCCs or from HNSCC cell lines and cultured in 3-dimensional soft agar or ultralow attachment plates. Aldehyde dehydrogenase activity and CD44 expression were used here as stem cell markers. This assay allowed for the propagation of head and neck cancer cells that retained stemness and self-renewal. The orosphere assay is well suited for studies designed to understand the pathobiology of head and neck cancer stem cells. © 2012 Wiley Periodicals, Inc. Head Neck, 2013Head & Neck 07/2013; 35(7). DOI:10.1002/hed.23076 · 2.64 Impact Factor
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