Expansion of human and murine hematopoietic stem and progenitor cells ex vivo without genetic modification using MYC and Bcl-2 fusion proteins.

The long-term repopulating hematopoietic stem cell (HSC) population can self-renew in vivo, support hematopoiesis for the lifetime of the individual, and is of critical importance in the context of bone marrow stem cell transplantation. The mechanisms that regulate the expansion of HSCs in vivo and in vitro remain unclear to date. Since the current set of surface markers only allow for the identification of a population of cells that is highly enriched for HSC activity, we will refer to the population of cells we expand as Hematopoietic Stem and Progenitor cells (HSPCs). We describe here a novel approach to expand a cytokine-dependent Hematopoietic Stem and Progenitor Cell (HSPC) population ex vivo by culturing primary adult human or murine HSPCs with fusion proteins including the protein transduction domain of the HIV-1 transactivation protein (Tat) and either MYC or Bcl-2. HSPCs obtained from either mouse bone marrow, human cord blood, human G-CSF mobilized peripheral blood, or human bone marrow were expanded an average of 87 fold, 16.6 fold, 13.6 fold, or 10 fold, respectively. The expanded cell populations were able to give rise to different types of colonies in methylcellulose assays in vitro, as well as mature hematopoietic populations in vivo upon transplantation into irradiated mice. Importantly, for both the human and murine case, the ex vivo expanded cells also gave rise to a self-renewing cell population in vivo, following initial transplantation, that was able to support hematopoiesis upon serial transplantation. Our results show that a self-renewing cell population, capable of reconstituting the hematopoietic compartment, expanded ex vivo in the presence of Tat-MYC and Tat-Bcl-2 suggesting that this may be an attractive approach to expand human HSPCs ex vivo for clinical use.

Construction and analysis of alphaherpesviruses expressing green fluorescent protein.

Over the past 10 years, the use of fluorescently tagged herpesviruses has evolved from relative obscurity into a common component in the arsenal of many molecular herpesvirology laboratories. In this chapter we provide methods for construction and analysis of recombinant alphaherpesviruses using conventional co-transfection and homologous recombination procedures. In recent years many herpesviruses have been cloned into bacterial artificial chromosomes (BACs), which has facilitated their manipulation by sophisticated bacterial molecular genetic techniques [Messerle, M., Crnkovic, I., Hammerschmidt, W., Ziegler, H., and Koszinowski, U. H. (1997) Proc Natl Acad Sci USA 94,14759-63; Smith, G. A., and Enquist, L. W. (2000) Proc Natl Acad Sci USA 97,4873-8; Tischer, B. K., von Einem, J., Kaufer, B., and Osterrieder, N. (2006) Biotechniques 40,191-7]. These technological breakthroughs have allowed for the genetic analysis of virus gene products, including those that are essential for virus replication, with unprecedented ease. The main caveat to this approach is that one requires their virus strain of interest cloned into a BAC. If the virus strain under consideration has not been introduced into a BAC, it is far from trivial to do so. While comparatively antiquated, the procedures provided in this article can be used with any strain. Here we focus on pseudorabies virus (PRV), a swine pathogen, which is the alphaherpesvirus most amenable to genetic manipulation using this transfection-based approach.