Bcl2 enhances induced hematopoietic differentiation of murine embryonic stem cells

Abstract

Bcl2 is a potent antiapoptotic gene that can increase resistance of adult bone marrow hematopoietic progenitor cells to lethal irradiation, and thereby preserve their ability to differentiate. However, the effect of Bcl2 on murine embryonic stem (ES) cells induced to undergo hematopoietic differentiation in the absence of a toxic stress is not known. To test this, murine CCE-ES cells that can be induced to undergo hematopoietic differentiation in a two-step process that results in upregulation of Bcl2 were used. Upregulation of Bcl2 precedes formation of hematopoietic embryoid bodies (EB) and their further differentiation into hematopoietic colony-forming units, when plated as single cells in methylcellulose. ES cells stably expressing a Bcl2 siRNA plasmid to "knock-down" endogenous expression or cells expressing wild-type (WT) Bcl2 or phosphomimetic Bcl2 mutants were examined. ES cells expressing the Bcl2 siRNA or those expressing a dominant-negative, nonphosphorylatable Bcl2 display a strikingly reduced capacity to form hematopoietic EBs and colony-forming units compared to cells expressing WT or phosphomimetic Bcl2 that demonstrate an increased capacity. Bcl2's effect on induced-hematopoietic differentiation of ES cells does not result from either decreased apoptosis or a reduced number of cells. Rather, Bcl2-enhances hematopoietic differentiation of ES cells by upregulating p27, which results in retardation of the cell cycle at G1/G 0. Thus siRNA silencing of p27 reverts Bcl2's enhancement phenotype in a manner similar to that of Bcl2 "silencing" or expression of a nonphosphorylable Bcl2. In addition to Bcl2's well-described antiapoptotic and cell-cycle retardant effect on somatic cells, Bcl2 may also function to enhance induced hematopoietic cell differentiation of murine ES cells. These findings may have potential relevance for expanding hematopoietic stem/progenitor cell numbers from an ES cell source for stem cell transplantation applications.