Post natal bone repair elicits a regenerative mechanism that restores the injured tissue to its pre-injury cellular composition and structure and is believed to recapitulate the embryological processes of bone formation. Prior studies showed that Nanog, a central epigenetic regulator associated with the maintenance of embryonic stem cells (ESC) was transiently expressed during fracture healing, Bais et al. [1] In In this study, we show that murine bone marrow stromal cells (MSCs) before they are induced to undergo osteogenic differentiation express similar to 50x the background levels of Nanog seen in murine embryonic fibroblasts (MEFs) and the W20-17 murine marrow stromal cell line stably expresses Nanog at similar to 80x the MEF levels. Nanog expression in this cell line was inhibited by BMP7 treatment and Nanog lentivrial shRNA knockdown induced the expression of the terminal osteogenic gene osteocalcin. Lentivrial shRNA knockdown or lentiviral overexpression of Nanog in bone MSCs had inverse effects on proliferation, with knockdown decreasing and overexpression increasing MSC cell proliferation. Surgical marrow ablation of mouse tibia by medullary reaming led to a similar to 3-fold increase in Nanog that preceded osteogenic differentiation during intramembranous bone formation. Lentiviral shRNA knockdown of Nanog after surgical ablation led to an initial overexpression of osteogenic gene expression with no initial effect on bone formation but during subsequent remodeling of the newly formed bone a similar to 50% decrease was seen in the expression of terminal osteogenic gene expression and a similar to 50% loss in trabecular bone mass. This loss of bone mass was accompanied by an increased similar to 2- to 5-fold adipogenic gene expression and observed increase of fat cells in the marrow space. In summary these data show that Nanog is expressed during surgically induced marrow bone formation and is functionally involved in post natal marrow stromal cell maintenance and differentiation. (C) 2011 Elsevier Inc. All rights reserved.