Characterization of TEC Progenitors and their Capacity to Reconstitute Thymic Function
Mark Pezzano, PhD and Derek Sant’Angelo, PhD
Hematopoietic stem cell transplantation (HSCT) can cure many forms of blood-derived cancers. Unfortunately, particularly in adult patients, HSCT is associated with a period of immune incompetence due to a loss of capacity to generate functional T cells. The structural and functional changes in thymic epithelial cells (TECs), induced by HSCT, directly impact the capacity of the thymus to facilitate T cell development. Clinical treatment strategies that enhance T cell reconstitution could significantly improve the survival of HSCT recipients through reduced incidence of fatal infectious complications and enhanced T-cell mediated tumor activity. The goal of this study will be to identify thymic epithelial stem cells/progenitors and test the capacity of grafting of these populations to speed the recovery of thymic function after HSCT. Aim 1 of this proposal will be to purify and characterize thymic epithelial progenitor cells. Due to their slow turnover rate, stem cells in a variety of tissues have been identified by their capacity to retain labeled nucleotides, so called “label retaining cells” (LRCs). A novel transgenic model, which utilizes a histone2B-GFP fusion protein (H2B-GFP), under control of a tetracycline response element driven by a K5 promoter, was used by Elaine Fuch’s group at Rockefeller to identify epithelial stem cells in the hair follicle bulge . This model is unique because it allows sorting of viable stem cells, in the absence of defining cell surface markers. Since putative thymic epithelial progenitors also express K5, we have obtained this transgenic model and will apply the method to identify thymic epithelial stem/progenitor populations. Further separation of putative stem cell populations will be performed using previously characterized epithelial stem cell surface characteristics. Sorted subsets will be assayed for progenitor potential based on their capacity to reform a functional thymus after transfer under the kidney capsule of nude mice. Aim 2 will assess the ex vivo growth potential of thymic epithelial progenitors and their subsequent capacity to enhance immune reconstitution following hematopoietic stem cell transplantation (HSCT). Optimal culture conditions for expansion and maintenance of sorted TEC progenitors will be assessed. Maintenance of a stem cell phenotype in expanded TEC progenitors will then be assayed by testing their capacity to reconstitute a full thymus after grafting under the nude mice kidney capsule. The capacity of similarly prepared stem cell populations to enhance thymic recovery and reconstitution of immune functionin mice following HSCT will be tested using both intrathymic injection and kidney capsule grafting ofsynegeneic TEC stem cells, followed by quantitative and functional T cell analysis.
Osada M, Jardine L, Misir R, Andl T, Millar SE, et al. (2010) DKK1 Mediated Inhibition of Wnt Signaling in Postnatal Mice Leads to Loss of TEC Progenitors and Thymic Degeneration. PLoS ONE 5(2): e9062. doi:10.1371/journal.pone.0009062
Osada M, Jardine L, Misir R, Andl T, Millar SE, et al. DKK1 Mediated Inhibition of Wnt Signaling in Postnatal Mice Leads to Loss of TEC Progenitors and Thymic Degeneration. May 2010: AAI Conference