Abstract
BackgroundCharacterization of intrinsic and extrinsic factors regulating the self-renewal/division and differentiation of stem cells is crucial in determining embryonic stem (ES) cell fate. ES cells differentiate into multiple hematopoietic lineages during embryoid body (EB) formation in vitro, which provides an experimental platform to define the molecular mechanisms controlling germ layer fate determination and tissue formation.Methods and FindingsThe cannabinoid receptor type 1 (CB1) and cannabinoid receptor type 2 (CB2) are members of the G-protein coupled receptor (GPCR) family, that are activated by endogenous ligands, the endocannabinoids. CB1 receptor expression is abundant in brain while CB2 receptors are mostly expressed in hematopoietic cells. However, the expression and the precise roles of CB1 and CB2 and their cognate ligands in ES cells are not known. We observed significant induction of CB1 and CB2 cannabinoid receptors during the hematopoietic differentiation of murine ES (mES)-derived embryoid bodies. Furthermore, mES cells as well as ES-derived embryoid bodies at days 7 and 14, expressed endocannabinoids, the ligands for both CB1 and CB2. The CB1 and CB2 antagonists (AM251 and AM630, respectively) induced mES cell death, strongly suggesting that endocannabinoids are involved in the survival of mES cells. Treatment of mES cells with the exogenous cannabinoid ligand Δ9-THC resulted in the increased hematopoietic differentiation of mES cells, while addition of AM251 or AM630 blocked embryoid body formation derived from the mES cells. In addition, cannabinoid agonists induced the chemotaxis of ES-derived embryoid bodies, which was specifically inhibited by the CB1 and CB2 antagonists.ConclusionsThis work has not been addressed previously and yields new information on the function of cannabinoid receptors, CB1 and CB2, as components of a novel pathway regulating murine ES cell differentiation. This study provides insights into cannabinoid system involvement in ES cell survival and hematopoietic differentiation.
Highlights
Murine embryonic stem cells, derived from the inner cell mass of preimplanted embryos, are pluripotent and retain the ability to differentiate into cells of all three germ layers of the developing mouse embryo
In vitro differentiation assays using ES cultures involve the removal of Leukemia inhibitory factor (LIF) and separation of the cells from the feeder layer under conditions that promote the formation of embryonic stem cell aggregates, termed embryoid bodies (EBs)
To examine the expression of cannabinoid receptor type 1 (CB1) and cannabinoid receptor type 2 (CB2) in murine ES (mES) cells, we performed RT-PCR analysis on control undifferentiated ES cells (Rosa26.6 and E14 ES cells) and on EBs derived from the secondary hematopoietic differentiation of these two ES cell lines at different time points as indicated
Summary
Murine embryonic stem (mES) cells, derived from the inner cell mass of preimplanted embryos, are pluripotent and retain the ability to differentiate into cells of all three germ layers of the developing mouse embryo. In vitro differentiation assays using ES cultures involve the removal of Leukemia inhibitory factor (LIF) and separation of the cells from the feeder layer under conditions that promote the formation of embryonic stem cell aggregates, termed embryoid bodies (EBs). These EBs contain a number of different cell types [1,2]. ES cells differentiate into multiple hematopoietic lineages during embryoid body (EB) formation in vitro, which provides an experimental platform to define the molecular mechanisms controlling germ layer fate determination and tissue formation. This study provides insights into cannabinoid system involvement in ES cell survival and hematopoietic differentiation
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