Submitted October 17, 2008
Accepted January 21, 2009
Forward RNAi screens in primary human hematopoietic stem/progenitor cells
Nicole Ali, Christine Karlsson, Marie Aspling, Guang Hu, Nir Hacohen, David T Scadden*, and Jonas Larsson
Center for Regenerative Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA, United States
Molecular Medicine and Gene Therapy, Lund Stem Cell Center, Lund University, Lund, Sweden
Harvard Stem Cell Institute, Harvard University, Cambridge, MA, United States
Howard Hughes Medical Institute and Department of Genetics, Center for Genetics and Genomics, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, United States
Broad Institute of MIT and Harvard, Cambridge, MA, United States
Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, United States
* Corresponding author; email: scadden.david{at}mgh.harvard.edu.
The mechansisms regulating key fate decisions such as self-renewal and differentiation in hematopoietic stem and progenitor cells (HSPC) remain poorly understood. We report here a screening strategy developed to assess modulators of human hematopoiesis using a lentiviral shRNA library transduced into cord blood-derived stem/progenitor cells. To screen for modifiers of self-renewal/differentiation, we used the limited persistence of HSPCs under ex vivo culture conditions as a baseline for functional selection of shRNAs conferring enhanced maintenance or expansion of the stem/progenitor potential. This approach enables complex, pooled screens in large numbers of cells. Functional selection identified novel specific gene targets (Exostoses 1) or shRNA constructs capable of altering human hematopoietic progenitor differentiation or stem cell expansion respectively, thereby demonstrating the potential of this forward screening approach in primary human stem cell populations.
