Enforced Bcl-2 expression overrides serum and feeder cell requirements for mouse embryonic stem cell self-renewal

Abstract

Leukemia inhibitory factor (LIF) is required, but not sufficient, for pluripotent mouse embryonic stem (ES) cell expansion in vitro in the absence of serum or a feeder cell layer, suggesting that additional signals are provided by serum or feeders that are necessary to support self-renewal. Here we show that transgenic ES cell lines expressing Bcl-2, an antiapoptotic protein, continue to self-renew in serum-and feeder-free conditions when supplemented with LIF; even in the absence of bone morphogenic proteins. Bcl-2-expressing clones sustain the characteristics of undifferentiated, pluripotent ES cells during long-term culture, and maintain their potential to differentiate into mature cell types. These results suggest that LIF and Bcl-2 overexpression are sufficient to expand these mouse pluripotent stem cells in vitro.leukemia inhibitory factor ͉ antiapoptotic protein ͉ pluripotency S tem cells are defined as cells that, at the single-cell level, are capable of self-renewal and differentiation to specialized cell types (1). ES cells are pluripotent stem cells derived from the inner cell mass of blastocysts (2) and can self-renew indefinitely in vitro in the presence of leukemia inhibitory factor (LIF) and FBS or mouse feeder layer cells, resulting in daughter cells that maintain their potential for multilineage differentiation (3, 4). When ES cells are maintained in serum-and feeder-free conditions, the number of undifferentiated cells quickly reaches a plateau and begins to decline after only a couple of passages (5), and cells with a non-ES cell morphology quickly arise in culture (6) despite the presence of LIF. Thus, additional factors provided by serum or feeders appear to be required to fully support the self-renewal of mouse ES cells.Bone morphogenic proteins (BMPs) have been implicated as the factor contained in serum or provided by feeder layers that acts in concert with LIF to maintain undifferentiated mouse ES cells in vitro (7). It was recently suggested that BMPs can replace serum and feeder cell requirements in ES cell culture by activating the Smad pathway and inducing expression of the Id gene, a common target of Smad signaling (8) that appears to block differentiation by negatively regulating basic helix-loophelix proteins (5). Although the exact mechanism by which BMP promotes self-renewal of ES cells is not certain, recent work suggests that it might also inhibit the mitogen-activated protein kinase (MAPK) pathway independent of Smads (7). Importantly, inhibition of p38 MAPK facilitates derivation of ES cells from blastocysts lacking Alk-3 (BMPRIA) (7), and ES cells can be derived from blastocysts lacking Smad4 (the common partner of all Smads; ref. 9), supporting the hypothesis that BMP acts by means of different mechanisms depending on the presence or absence of serum and feeders.Considering the possibility that serum and feeder cells provide cell survival signals manifest as growth factors and cytokines (10) and that extrinsic survival signals are especially critical in low cell density co...

Overexpression of BCL2 enhances survival of human embryonic stem cells during stress and obviates the requirement for serum factors

“…We have previously shown that transgenic mESCs expressing human BCL2 continue to self-renew in serum-and feeder-free conditions when supplemented with leukemia inhibitory factor (21). These studies suggest that leukemia inhibitory factor and BCL2 overexpression are sufficient to maintain and expand mESCs in culture.…”

“…Enforced expression of the 26-kDa BCL2 antiapoptotic protein has been reported to result in enhanced survival and proliferation in several different systems (16,19,20). We demonstrated that mouse embryonic stem-cell (mESC) lines expressing the human BCL2 transgene could self-renew continuously under serum-free and feeder cell-free conditions (21), and improved efforts to identify and isolate mouse ES-derived HSC (22).…”

“…We have previously shown that mESCs that express BCL2 are able to survive in media lacking serum or feeders, the primary sources of these prosurvival signals (21). To determine whether BCL2 overexpression can similarly compensate for the absence of prosurvival signals in hESCs, parental and BCL2-hESC cells were cultured in feeder-free conditions (Matrigel) and with decreasing concentrations of KOSR.…”

Bcl2, a transcriptional target of p38α, is critical for neuronal commitment of mouse embryonic stem cells

“…In addition, when overexpressed in pluripotent cells under serum-free conditions, these proteins synergize with LIF and maintain cell pluripotency. 41,42 Altogether, these data shed light on the pleiotropic effects of genes whose functions rely exclusively on cell context. In addition, our results emphasize the key potential role of p38a to control Ids gene expression at specific stages of neuronal maturation.…”

How One Thing Led to Another

“…We thought if we generated a mouse whose granulocytes do not die on time and crossed it to CF mice, we might get clues as to how to ameliorate the disease and perhaps how to treat it with HSC transplantation from healthy donors. We cloned the upstream region of mrp8 and used it to drive human Bcl2 in transgenic mice (209 (212,213), but also triggered a variety of studies in the lab (152,201,(214)(215)(216)(217)(218)(219)(220)(221)(222)(223). For example, we demonstrated a number of developmental events wherein cytokine instructions acted by keeping a cell type alive to follow its intrinsic developmental progression, and the signal transduction pathway, e.g., monocytes to macrophages and osteoclasts in M-CSF-deficient osteopetrotic mice, could be replaced by stage-specific expression of bcl2 (52,214).…”