Human embryonic stem cells (hESCs), derived from the inner cell mass (ICM) of the blastocyst, have the capacity for longterm undifferentiated growth in culture and the theoretical potential to differentiate into all somatic cell types. [1] These hESCs offer not only a model system for human development, but also a potentially unlimited source of graft material for transplantation-based therapies. Long-term hESCs can be supported using feeder-fibroblast conditioned medium (CM), [2] or high concentrations of basic FGF, [3] PEDF, [4] or TGF-b/activin/Nodal proteins. [5] Recently, small molecules that can support mouse embryonic stem cell self-renewal and hESC self-renewal have been reported. [6][7][8][9][10][11][12] Unfortunately, none of the reported small molecules is able to support longterm hESC self-renewal (five or more passages) under defined culture conditions without bFGF or TGF-b/activin/ Nodal proteins. Consequently, unbiased cellular screens for small molecules that regulate long-term hESC self-renewal may provide new insights into stem cell biology, and also facilitate practical applications of ES cells in research and therapy. Herein, we report the identification of a small molecule that promotes long-term hESC self-renewal.
To screen for small-molecules that promote self-renewal, H9 hESCs were seeded into matrigel coated 384-well plates at 2000 cells well ร1 in UM medium (DMEM/F12 + 1X N2/B27 supplement) and screened against a diverse chemical library of 50 000 heterocyclic compounds. Compounds were added a day after plating at a final concentration of 2 mm in 0.1 % DMSO. After treatment with compound for 7 days, the cells were fixed and stained with OCT-4 antibodies (OCT-4 is highly expressed in ES cells and downregulated upon differentiation), [1] and analyzed using an Opera high-content confocal image system. Of 11 confirmed screen hits (Supporting Information Figure S1), dorsomorphin, (Figure 1 a), an inhibitor of bone morphogenic protein (BMP) type I receptors (ALK2, ALK3, and ALK6), [13] maintained the highest