Cannabis Research A-Z

Expression of endocannabinoids in mES cells and embryoid bodies derived from mES cells at days 7 and 14

To examine whether mES cells as well as EBs derived from mES cells express endocannabinoids, mES cells were analyzed for the expression of various fatty acids and their ethanolamide and monoglyceride derivatives using LC-APCI-MS analysis. As shown in Figure 3, derivations of the endocannabinoids were detected and quantitated in mES cells and EBs at days 7 and 14. The level of anandamide (AEA) expression in the mES cells was much lower as compared to that of 2-AG, and AEA was not detected at all in EBs at days 7 and 14. The expression levels of: 2-AG, docosahexaenoic acid (DHA), arachidonic acid (AA), 2-oleoyl glycerol (2-OG), eicosapentaenoic acid (EPA), 2-docosahexaenoyl glycerol (2-DHG) and 2-eicosapentaenoyl glycerol (2-EPG), were abundant in mES cells, and EBs at days 7 and 14. Endocannabinoid levels in the embryonic stem cells were correlated to the number of mES cells (data not shown). These analyses showed that mES cells abundantly express endocannabinoids, specifically 2-AG which might be important for their survival. Furthermore, since both EBs at days 7 and 14 express endocannabinoids, this could suggest that endocannabinoids may play a role in the hematopoietic differentiation of mES cells.

Figure 3

Comparison of endocannabinoid levels in mES cells and EBs at days 7 and 14, when the number of cells in each group is normalized to 10⁷ (=1e⁷).

Effects of exogenous and endogenous cannabinoid ligands on the chemotaxis of mES cells

A major function of the 2-AG endocannabinoid is the stimulation of migration in B lymphocytes. Since CXCR4 and its cognate ligand SDF-1α are involved in hematopoietic stem cell chemotaxis, migration and homing, and since CXCR4, CB1 and CB2 are members of the GPCR family, we therefore studied whether cannabinoid ligands act as chemotactic or chemokinetic agents for ES cells. We analyzed the effects of the endogenous cannabinoid ligand 2-AG, the exogenous ligand Δ⁹-THC and the specific CB2 receptor agonist, JWH-015, on the chemotaxis of undifferentiated ES cells as well as day 10 EBs derived from secondary hematopoietic differentiation.

The chemotaxis assays were performed using Costar Transwells (Corning-Costar, Cambridge, MA). As shown in Figure 4, chemotaxis was observed with differentiated EBs at day 10 in the presence of the Δ⁹-THC, 2-AG and JWH-015 cannabinoid ligands, while the chemotaxis of undifferentiated ES cells was very low. This chemotaxis was inhibited by the CB1 and CB2 specific inhibitors, AM251 and AM630, respectively. Thus, cannabinoid ligands, such as 2-AG, exogenous Δ⁹-THC and JWH-015 induce the chemotaxis of hematopoietic differentiated ES-derived EB cells, mediated through both the CB1 and CB2 receptors.

Figure 4

Effects of cannabinoid ligands on the chemotaxis of ES cells and hematopoietic differentiated ES-derived EB cells (EBs-day 10).

Effects of cannabinoid inhibitors on the survival of Rosa ES cells

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.

Figure 5

Effects of Δ⁹-THC and cannabinoid inhibitors (AM251 and AM630) on Rosa ES cell survival.

RT-PCR analysis of CB1 and CB2 expression

RNA from total mES cells was extracted using the RNeasy Mini Kit (Qiagen, Valencia, CA) following the manufacturer's protocol. A QIAshredder spin column and DNase digestion were included in the isolation procedure to limit the possibility of PCR amplification of CB1 and CB2 from genomic DNA. cDNA and PCR amplification were performed with the BD Biosciences TITANIUM One-Step RT-PCR Kit using 200 ng of RNA as a template for first-strand synthesis. CB1 was amplified using primers: 5′-CGT GGG CAG CCT GTT CCT CA-3′ and 5′-CAT GCG GGC TTG GTC TGG-3′, which yield a product of 403 bp. CB2 was amplified using: 5′-CCG GAA AAG AGG ATG GCA ATG AAT-3′ and 5-CTG CTG AGC GCC CTG GAG AAC-3′, which yield a product of 479 bp. GAPDH was used as a positive control with primers: 5′-CTC ACT GGC ATG GCC TTC CG-3′ and 5′-ACC ACC CTG TTG CTG TAG CC-3′, which yield a product of 292 bp. The template was first denatured at 94°C for 2 min followed by 35 cycles (94°C for 30 sec, 58°C for 30 sec and 68°C for 1 min), followed by 68°C for 2 min in a myCycler Personal Thermal Cycler (Bio-Rad Laboratories, Inc). Aliquots (20 ml) of the PCR products were run on a 1.2% agarose gel containing 0.5 mg/ml ethidium bromide.

Origination of embryoid bodies from ES cells

The Rosa26.6 ES cell line was obtained from Dr. Stuart Orkin (Children's Hospital, Harvard Medical School); The E14 and GFP-E14 cell lines were obtained from Dr. Bing Lim (Beth Israel Deaconess Medical Center, Boston). Culture and maintenance of ES cells in an undifferentiated state were performed as described previously. Briefly, ES cells were maintained on a mouse PEF feeder cell line in ES medium containing Dulbecco's modified Eagle's medium (DMEM) with high glucose, 10 ng/ml murine leukemia inhibitory factor (mLIF; Chemicon International, Temecula, CA), 15% fetal calf serum (FCS; Hyclone, Logan, UT), 1 mM sodium pyruvate, 2 mM glutamine, 0.1 mM nonessential amino acid, 100 µM monothioglycerol (MTG; Sigma), 50 U/ml penicillin, and 10 µg/ml streptomycin. The ES cell lines were regularly analyzed, by using an ES cell characterization kit (Chemicon), for determination of alkaline phosphatase activity and detection of surface markers and transcription factors that are expressed by undifferentiated ES cells, such as Oct-4, Rex-1, SSEA-1 and Genesis (Fox D-3).

In vitro hematopoietic differentiation of ES cells was performed as described, essentially according to the protocol of StemCell Technologies. The embryoid body (EB) method involves two steps: first, spherical cell aggregates (termed embryoid bodies=EBs) are generated that contain ectodermal, mesodermal and endodermal derivatives (=Primary Differentiation); second, these aggregates are selected for hematopoietic precursors and expanded with growth factors such as IL-3 and IL-6 (=Secondary Hematopoietic Differentiation). Briefly, EBs were generated in 1% methylcellulose cultures (1×10⁴ ES cells per 35-mm Petri dish). To promote primary differentiation into EBs, ES cells were cultured in ES differentiation medium containing Iscove's modified Dulbecco's medium (IMDM), 15% FCS (StemCell Technologies), 2 mM glutamine, 150 µM MTG, and 40 ng/ml murine stem cell factor (mSCF). After 8 days of differentiation, the EBs were collected and washed. 1×10⁴ of single cells were seeded on 1% methylcellulose from the secondary hematopoietic differentiation medium. 15% FBS, 2 mM L-glutamate, 150 µM MTG, 20% BIT (10% BSA, 10 µg/ml insulin, 200 µg/ml transferrin), 150 ng/ml mSCF, 30 µg/ml IL-3, 30 µg/ml IL-6 and 3 U/ml Epo were added to the culture to promote hematopoietic differentiation. Cells were processed for Wright-Giemsa staining, RT-PCR and Western blot analyses at different times of EB culture differentiation, as indicated.

To determine the characteristics of various types of hematopoietic progenitors present during ES cell differentiation, EBs from ES cell lines were collected from the cultures at days 8 and 11 (from the day of replating) to obtain the hematopoietic progenitors. Cytospin preparation of these cells was stained with Wright-Giemsa and examined under a light microscope. Undifferentiated ES cells have a large nucleus, minimal cytoplasm, and one or more prominent dark nucleoli. Hematopoietic progenitors found in EB-day 14 cultures were identified by the morphology of erythroids, megakaryocytes, monocytes/macrophages, granulocytes and mast cells, as analyzed by field microscopy.

Chemotaxis assays

The chemotaxis assays were performed using 5 µm-pore size and 6.5 mm-diameter Costar Transwells (Corning-Costar, Cambridge, MA), as previously described. Cells were washed twice with Hank's balanced salt solution (HBSS) medium, resuspended in 100 µl medium [Iscove's Modified Dulbecco's Medium (IMDM) plus 0.5% BSA] and placed in the upper chamber of the Transwells. In the lower chamber, 600 µl of medium with or without ligand was placed, as indicated. After 4 hours of incubation at 37°C and 5% CO₂, the upper chamber was removed and the number of migrated cells was determined using a CASY/TTC cell counter. The ligand Δ⁹-THC (Δ⁹-Tetrahydrocannabinol) and the endogenous ligand 2-AG (Cayman Chemical, Ann Arbor, MI, Catalog #62165) were added at 1 µM concentrations in IMDM media. The specific CB2 receptor agonist JWH-015 (Tocris Catalog number #1341) was also tested at a 1 µM concentration. The CB1 specific inhibitor AM251 (1 µM) (Tocris Catalog number #1117) and the CB2 specific inhibitor AM630 (1 µM) (Tocris Catalog number #1120) were used to block the effects of cannabinoid ligands on ES cell chemotaxis. For the inhibition studies, cells were preincubated with the inhibitor agonists for 30 min as indicated. SDF-1 alpha (25 ng/ml) was used as a positive control (PeproTech Inc., Catalog number #250-20A).

Survival assays

2×10⁴ Rosa ES cells (per well of 96 wells), CB1 and CB2 specific ligands as well as inhibitors were added to the cell culture as indicated. A 1 µM final concentration was used for CP55940 (CB1 and CB2 agonists), ACEA (CB1 ligand) and JWH133 (CB2 ligand). A 1 µM final concentration of both AM251 (CB1 inhibitor) and AM630 (CB2 inhibitor) was used, as indicated. Cells were incubated for two days in a humidified CO₂ atmosphere. The MTT assay was performed according to the Promega manual (Promega Cat# G5421), and the absorbance at 490 nm was then recorded.

Endocannabinoid levels in embryonic stem cells

The extraction procedure for the calibration standards was performed as described. Cells (mES cells, EBs at day 7 and EBs at day 14), at various concentrations as indicated, were homogenized in cold acetone:PBS, pH 7.4 (31). The homogenates were sonicated for 30 seconds prior to centrifugation at 20,800 g for 5 minutes. The acetone from the resulting supernatants was removed under nitrogen. To the remaining supernatant, 50 µl PBS, one volume of methanol and two volumes of chloroform were added for liquid-liquid phase extraction of the lipids. The two phases were separated by centrifugation and the bottom organic layer was evaporated under nitrogen. The cell samples were reconstituted in 50 µl ethanol.

The system used for analysis was a TSQ Quantum Ultra triple quadrupole mass spectrometer (Thermo Electron, San Jose, CA) with an Agilent 1100 HPLC on the front end (Agilent Technologies, Wilmington, DE). The mobile phase consisted of 10 mM ammonium acetate (pH 7.3 using ammonium hydroxide; A) and 100% methanol (B). Separation of each analyte was achieved using a Zorbax SB-CN 2.1×50mm, 5 µm, 80Å, column (Agilent Technologies) and gradient elution; the autosampler was kept at 4°C to prevent analyte degradation. Eluted peaks were ionized via atmospheric pressure chemical ionization (APCI) and detected by each analyte's SRM transition.

We thank Dr. Radoslaw Zagozdzon for his critical reading and input on this manuscript, Wei Fu and Chiung Pei Wang for typing assistance and Janet Delahanty for editing the manuscript.