In vitro hematopoietic differentiation of murine embryonic stem cells

In recent years, the field of stem cells has become one of the most rapidly growing areas in biological and medical sciences. Embryonic stem (ES) cells differentiate efficiently in vitro and give rise to many different somatic cell types. The ability to generate a wide spectrum of differentiated cell types from ES cells in culture offers a powerful approach for studying lineage induction and specification and a promising source of progenitors for cell replacement therapy. Hematopoietic progenitors present within ES cell-derived embryoid bodies (EB) can be assayed by directly replating EB cells or by replating sorted cell populations into semisolid media with hematopoietic growth factors. The developmental kinetics of various hematopoietic lineage precursors within EBs and molecular and cellular studies of these cells have suggested that the sequence of events leading to the onset of hematopoiesis within EB is similar to that found within the mouse embryo. Thus, the in vitro differentiation model of ES cells to hematopoietic cells provides a unique opportunity to study onset mechanisms involved in hematopoietic development and to characterize hematopoietic lineage-specific gene expression. In this chapter, we attempt to be as comprehensive as possible and yet focus on what we perceive to be the most widely used protocols for maintenance of murine ES cells, in vitro hematopoietic differentiation of ES cells, and clonal assays of hematopoietic progenitors.