High level in vitro expansion of murine hematopoietic stem cells

Development of strategies to extensively expand hematopoietic stem cells (HSCs) in vitro will be a major factor in enhancing the success of a range of transplant-based therapies for malignant and genetic disorders. In addition to potential clinical applications, the ability to increase the number of HSCs in culture will facilitate investigations into the mechanisms underlying self-renewal. In this unit, we describe a robust strategy for consistently achieving over 1000-fold net expansion of HSCs in short-term in vitro culture by using novel engineered fusions of the N-terminal domain of nucleoporin 98 (NUP98) and the homeodomain of the hox transcription factor, HOXA10 (so called NUP98-HOXA10hd fusion). We also provide a detailed protocol for monitoring the magnitude of HSC expansion in culture by limiting dilution assay of competitive lympho-myeloid repopulating units (CRU Assay). These procedures provide new possibilities for achieving significant numbers of HSCs in culture, as well as for studying HSCs biochemically and genetically.