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Expression and function of cannabinoid receptors CB1 and CB2 and their cognate cannabinoid ligands in murine embryonic stem cells.

Jiang S, Fu Y, Williams J, Wood J, Pandarinathan L, Avraham S, Makriyannis A, Avraham S, Avraham HK - PLoS ONE (2007)

Bottom Line: We observed significant induction of CB1 and CB2 cannabinoid receptors during the hematopoietic differentiation of murine ES (mES)-derived embryoid bodies.This 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.

View Article: PubMed Central - PubMed

Affiliation: Division of Experimental Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT

Background: Characterization 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 findings: The 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 Delta(9)-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.

Conclusions: This 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.

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Related in: MedlinePlus

Effects of Δ9-THC and cannabinoid inhibitors (AM251 and AM630) on Rosa ES cell survival.Rosa ES cells were either untreated (as control) or treated with Δ9-THC, control DMSO (0.01%), control methanol (0.01%), or with the inhibitors AM251 (for the CB1 receptor) or AM630 (for the CB2 receptor), as indicated. After 48 hours, the cells were analyzed for their viability by light microscopy. This is a representative experiment out of three experiments.
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pone-0000641-g005: Effects of Δ9-THC and cannabinoid inhibitors (AM251 and AM630) on Rosa ES cell survival.Rosa ES cells were either untreated (as control) or treated with Δ9-THC, control DMSO (0.01%), control methanol (0.01%), or with the inhibitors AM251 (for the CB1 receptor) or AM630 (for the CB2 receptor), as indicated. After 48 hours, the cells were analyzed for their viability by light microscopy. This is a representative experiment out of three experiments.

Mentions: To analyze the effects of Δ9-THC on the survival of Rosa ES cells, the Rosa ES cells were untreated or treated with Δ9-THC (1 µM) or with the specific inhibitors for CB1 (AM251) or CB2 (AM630) (in the absence of Δ9-THC) for 48 hours. In addition, Rosa ES cells were treated with DMSO (0.01%) or with methanol (0.01%) as vehicle controls. After 48 hours, cells were analyzed for viability. As seen in Figure 5, no effects on Rosa ES cell viability were observed upon treatment with DMSO or methanol as compared to the cannabinoid-treated ES cells. Δ9-THC also had no apoptotic effects on the Rosa ES cells. However, both inhibitors (AM251 and AM630) induced significant cell death in the absence of Δ9-THC (Fig. 5). These results suggest that endocannabinoids, either secreted by ES cells and/or by the Primary Embryonic Fibroblast (PEF) feeder cells, are important for the survival of ES cells and that specific inhibition of these endogenous ligands by inhibitors for CB1 and CB2 results in cell apoptosis.


Expression and function of cannabinoid receptors CB1 and CB2 and their cognate cannabinoid ligands in murine embryonic stem cells.

Jiang S, Fu Y, Williams J, Wood J, Pandarinathan L, Avraham S, Makriyannis A, Avraham S, Avraham HK - PLoS ONE (2007)

Effects of Δ9-THC and cannabinoid inhibitors (AM251 and AM630) on Rosa ES cell survival.Rosa ES cells were either untreated (as control) or treated with Δ9-THC, control DMSO (0.01%), control methanol (0.01%), or with the inhibitors AM251 (for the CB1 receptor) or AM630 (for the CB2 receptor), as indicated. After 48 hours, the cells were analyzed for their viability by light microscopy. This is a representative experiment out of three experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1919431&req=5

pone-0000641-g005: Effects of Δ9-THC and cannabinoid inhibitors (AM251 and AM630) on Rosa ES cell survival.Rosa ES cells were either untreated (as control) or treated with Δ9-THC, control DMSO (0.01%), control methanol (0.01%), or with the inhibitors AM251 (for the CB1 receptor) or AM630 (for the CB2 receptor), as indicated. After 48 hours, the cells were analyzed for their viability by light microscopy. This is a representative experiment out of three experiments.
Mentions: To analyze the effects of Δ9-THC on the survival of Rosa ES cells, the Rosa ES cells were untreated or treated with Δ9-THC (1 µM) or with the specific inhibitors for CB1 (AM251) or CB2 (AM630) (in the absence of Δ9-THC) for 48 hours. In addition, Rosa ES cells were treated with DMSO (0.01%) or with methanol (0.01%) as vehicle controls. After 48 hours, cells were analyzed for viability. As seen in Figure 5, no effects on Rosa ES cell viability were observed upon treatment with DMSO or methanol as compared to the cannabinoid-treated ES cells. Δ9-THC also had no apoptotic effects on the Rosa ES cells. However, both inhibitors (AM251 and AM630) induced significant cell death in the absence of Δ9-THC (Fig. 5). These results suggest that endocannabinoids, either secreted by ES cells and/or by the Primary Embryonic Fibroblast (PEF) feeder cells, are important for the survival of ES cells and that specific inhibition of these endogenous ligands by inhibitors for CB1 and CB2 results in cell apoptosis.

Bottom Line: We observed significant induction of CB1 and CB2 cannabinoid receptors during the hematopoietic differentiation of murine ES (mES)-derived embryoid bodies.This 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.

View Article: PubMed Central - PubMed

Affiliation: Division of Experimental Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT

Background: Characterization 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 findings: The 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 Delta(9)-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.

Conclusions: This 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.

Show MeSH
Related in: MedlinePlus