Methods in Molecular Biology (2016) 1341: 425–435 DOI 10.1007/7651_2014_126 © Springer Science+Business Media New York 2014 Published online: 23 November 2014
Osteogenic Differentiation from Embryonic Stem Cells Yanhong Yu, Carlos Pilquil, and Michal Opas Abstract Embryonic stem (ES) cells have been widely studied due to their pluripotency and their potential of self-renewal. Murine ES cells are useful in investigating the molecular pathways underlying their differentiation to various mature cell types in the body. This chapter describes the maintenance of murine ES cells in culture and a routine ES cell osteogenic differentiation protocol utilized in our laboratory. Keywords: Murine embryonic stem cells, Routine culture, Hanging drop, Embryoid bodies, Osteogenesis
1
Introduction Since their first isolation by two independent laboratories (1) two decades ago, murine embryonic stem (ES) cells have been extensively studied. This work in turn has revolutionized the fields of regenerative medicine, genetics, and developmental biology. This tremendous interest in the field of ES cells is due to their potential to differentiate to all types of mature cells in the body and their ability of unlimited self-renewal in vitro. ES cells are isolated from the inner cell mass of blastocysts in the pre-implanted stage. Under certain culture conditions, they can differentiate and give rise to the three germ layers: the endoderm, mesoderm, and ectoderm. ES cells are commonly propagated on a layer of mitomycin-treated, and thus division-incompetent mouse fibroblasts, which have been shown to be critical in maintaining the undifferentiated state of ES cells (2). A soluble factor, leukemia inhibitory factor (LIF), is also added to prevent the spontaneous in vitro differentiation of ES cells. The withdrawal of LIF from culture and growth in suspension causes differentiation and embryoid body (EB) formation (3). EBs are a unique in vitro model which allows for the study of early embryonic development such as the generation of the three germ layers. They are three-dimensional aggregates of differentiating stem cells. There are many different ways one can generate EBs: growth in suspension culture using low-adherence vessels or bacteriological dishes, bioreactor/spinner flask techniques, culture using methylcellulose media, and hanging drops (4). The hanging drop
425
426
Yanhong Yu et al.
a
P0-P2
b
Days P0-P2 0-2 3-5 5-9 10-21
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Medium Composition 10% FBS, LIF 20% FBS 20% FBS, RA 20% FBS, B-Gly, AA 20% FBS, B-Gly, AA, Dex
Fig. 1 ES cell maintenance and osteogenic differentiation. (a) Murine ES cells are maintained on mouse embryonic fibroblast feeder cells for two passages. At passage 2, ES cells form EBs via the hanging drop method. From days 3 to 5, they grow in suspension. They are attached on gelatinized tissue culture dishes on day 6 and grow for the remainder of the 21 days. (b) Different medium composition is required for different stages of ES cell maintenance and osteogenic differentiation. (Abbreviations: ES embryonic stem, EB embryoid body, FBS fetal bovine serum, LIF leukemia inhibitory factor, RA retinoic acid, B-Gly β-glycerophosphate; AA ascorbic acid, Dex dexamethasone)
method is one of the more widely utilized protocols that have successfully generated various mature cell types (cardiomyocytes, neuronal cells, adipocytes, osteoblasts, etc.) (5–8). In brief, 20–30 μL of solution containing ES cells is pipetted on Petri dish lids. These lids are then inverted and placed over the Petri dish. Due to gravity, the ES cells in solution then collect at the bottom of the drops and aggregate to form EBs. In this chapter, we describe routine maintenance and propagation of murine ES cells in vitro, and illustrate in detail the hanging drop differentiation method. As an example of differentiation, we demonstrate the osteogenic induction and differentiation of ES cells (Fig. 1).
2 2.1
Materials Tissue Culture
2.1.1 Reagents and Equipments
1. 10 cm Tissue culture dish, 100 20 mm, polystyrene (Falcon, cat. no. 353003). 2. 6 cm Tissue culture dish, 60 15 mm, polystyrene (Falcon, cat. no. 353002). 3. Hanging drop petri dish, 150 15 mm, polystyrene (VWR, cat. no. 25384-326). 4. Bacteriological plates (VWR, cat. no. 25389-328).
Osteogenic Differentiation from Murine ES Cells
427
5. 15 mL Centrifuge tube, polypropylene (FroggaBio, cat. no. TB15-25). 6. Phosphate-buffered saline (PBS): 10 stock, 76.5 g sodium chloride (NaCl) (Bioshop, cat. no. SOD002.205), 7.25 g sodium phosphate dibasic anhydrous (Na2HPO4) (SigmaAldrich, cat. no. RES20908), 2.12 g potassium phosphate dibasic anhydrous (KH2PO4) (Sigma-Aldrich, cat. no. P0662), 1 L distilled water (H2O). For 1 PBS, dilute stock 1:10 with distilled H2O. Autoclave and store at room temperature. 7. 0.1 % gelatin: 0.5 g gelatin (Anachemia, cat. no. AC-4622) in 500 mL PBS. Autoclave and store at room temperature. 8. Trypsin/EDTA: 0.25 % trypsin 2.21 mM EDTA tetrasodium (Multicell, Wisent, cat. no. 325-043-CL). Store at 20 C. 9. Freezing vial: CryoELITE Cryogenic vials (Wheaton, cat. no. W985862). 10. Mytomycin C (Sigma-Aldrich, cat. no. M4287-2mg), stock 1 mg/mL, store at 20 C. 11. Leukemia inhibitory factor (LIF): stock 10 μg/mL. Store at 20 C. 12. 0.4 % Trypan blue solution (Life technologies, cat. no. 15250061). 13. Quick-Read Precision Cell, with 18 counting circles, 10 tests/ slide (Globe Scientific Inc., cat. no. 010G18). 14. 8-Channel pipette (Rainin, 20–200 μL, cat. no. L8-200XLS+). 15. Retinoic acid (Sigma-Aldrich, cat. no. R625): 10 mM stock, store at 20 C. 16. β-Glycerophosphate (Sigma-Aldrich, cat. no. G9422): stock 2 M, store at 20 C. 17. Ascorbic acid (Sigma-Aldrich, cat. no. A4403): stock 5 mg/ mL, store at 20 C. 18. Dexamethasone (Sigma-Aldrich, cat. no. D4902): stock 0.1 mM, store at 20 C. 2.1.2 Medium Preparation
1. 10 % FBS medium: 500 mL DMEM with sodium pyruvate and L-glutamine (Multicell, Wisent, cat. no. 319-005-ES), 50 mL Fetal Bovine Serum (FBS) (Multicell, Winsent, cat. no. 080150), 5 mL MEM nonessential amino acids 100 (Gibco, Life Technologies, cat. no. 11140-050), 500 μL diluted 2-mercaptoethanol, 35 μL 2-mercaptoethanol (Bioshop, cat. no. CAS60-24-2) in 5 mL autoclaved distilled H2O. 2. Freezing medium: 8 mL 10 % FBS medium, 1 mL FBS, 1 mL dimethyl sulfoxide (DMSO) (Sigma-Aldrich, cat. no. D2650). 3. 17 % FBS medium: preparation same as 10 % FBS medium except for the addition of 100 mL FBS.
428
2.2
Yanhong Yu et al.
Von Kossa Assay
1. Formaldehyde solution, 36.5–38 % in water (Sigma-Aldrich, cat. no. F8775): store at room temperature. 2. Silver nitrate (Bioshop, cat. no. SIL222). 3. Na2HPO4 (Sigma-Aldrich, cat. no. RES20908). 4. Sodium phosphate monobasic monohydrate (Na2HPO4·H2O) (Sigma-Aldrich, cat. no. S9638).
2.3 RNA Isolation, Reverse Transcription, Real-Time Polymerase Chain Reaction (PCR)
1. RNeasy Plus Mini Kit (Qiagen, cat. no. 74134). 2. 1.5 mL Eppendorf tube (Axygen, cat. no. PMI11006v2). 3. Oligo(dt)18 Primer 0.5 μg/μL: (Thermo Scientific, cat. no. SO132). Store at 20 C. 4. dNTP mix, 10 mM (Invitrogen. cat. no. 18427-013). Store at 30 C. 5. UltraPure Distilled water, DNase, RNase, free (Invitrogen, cat. no. 10977-015). 6. 5 First strand buffer (Invitrogen. cat. no. P/N y02321). Store at 20 C. 7. 0.1 M DTT (Invitrogen. cat. no. P/N y00147). Store at 20 C. 8. RNaseOUT Recombinant Ribonuclease Inhibitor, 40 U/μL (Invitrogen. cat. no. P/N 100000840). Store at 20 C. 9. SuperScript II Reverse Transcriptase, 200 U/μL (Invitrogen. cat. no 18064-014). Store at 20 C. 10. Power SYBR Green PCR Master Mix (Applied Biosystems, cat. no. 4367659). For short-term storage, keep in 2–8 C. For long-term storage, store at 20 C. 11. 384 well plate, MicroAmp Optical Reaction Plate with Barcode (Applied Biosystems by Life Technologies, cat. no. 4309849). 12. Sealing tape, optically clear (Sarstedt, cat. no. 95.1994). 13. Osteogenic markers primers (Invitrogen): Osterix (OSX): forward primer 50 -GCAACTGGCTAGGTGGTGGTC-30 and reverse primer 50 -GCAAAGTCAGATGGGTAAGTAGGC-30 . Collagen 1 (Col 1): forward primer 50 -GAACGGTCCACGATTGCATG-30 and reverse primer 50 -GGCATGTTGCTAGGCACGAAG-30 . Stock 250 nM. Store at 20 C. 14. CFX384 TouchTM Detection System (Biorad, cat. no. 1855485).
Osteogenic Differentiation from Murine ES Cells
3 3.1
429
Methods Cell Culture
3.1.1 Thawing of Mouse Embryonic Fibroblasts (Feeders) Cells
1. Warm cells quickly in 37 C incubator or water bath. 2. Add 2 mL of 10 % FBS medium in a 15 mL Falcon tube, and add the thawed cell solution in the same tube. 3. Centrifuge at 700 g for 3 min. 4. In the meanwhile, add 2 mL of 0.1 % gelatin in a 6 cm tissue culture dish, cover the bottom of the dish evenly with gelatin, and aspirate gelatin. Set dish aside for later use (see Note 1). 5. Aspirate the supernatant and add fresh 2 mL 10 % FBS medium to resuspend the cell pellet in the tube (see Note 2). 6. Transfer the resuspended cell solution to the gelatinized dish, and add 2 mL 10 % FBS medium to the dish. Move dish around gently for even distribution of cells in the dish. 7. Change medium every other day. Add 4 mL of fresh 10 % FBS medium per 6 cm dish.
3.1.2 Preparation of Feeders for ES cells and Plating ES Cells
1. When feeders are at 65 % confluency, treat cells with 10 μL/mL medium of mytomycin C for 2 h to inactivate their ability to undergo mitosis. 2. After 2 h, aspirate mitomycin C, rinse cells twice with fresh 10 % FBS medium. Add 4 mL of fresh 10 % FBS medium. 3. Quickly thaw ES cells using steps 1–3 from Section 3.1.1. 4. Aspirate the supernatant and add 1 mL 10 % FBS medium to resuspend the ES cells pellet. Transfer the cell solution to the dish containing the mitomycin C-treated feeders. 5. To maintain pluripotency, add 1 μL leukemia inhibitory factor (LIF) for 1 mL medium. 6. Change medium every other day.
3.1.3 Maintenance of ES cells
1. When ES cells are at about 65 % confluency (approximately 2 days after last passage), aspirate medium, and add 1 mL trypsin/EDTA per 6 cm tissue culture dish. Incubate for 3 min. Pipette up and down a few times to break up cell clumps into single cell suspension. 2. Add 1 mL fresh 10 % FBS medium to a 15 mL Falcon tube, transfer the cell solution with trypsin into this tube. 3. Centrifuge at 700 g for 3 min. 4. In the meanwhile, add 2 mL of 0.1 % gelatin in a 6 cm tissue culture dish, cover the bottom of the dish evenly with gelatin, and aspirate gelatin. Set dish aside for later use.
430
Yanhong Yu et al.
5. Carefully aspirate the supernatant from the 15 mL Falcon tube. Resuspend ES cell pellet in 4 mL 10 % FBS medium. Transfer this cell solution to the gelatinized dish. Add 4 μL LIF into the dish, and gently move the dish for even distribution of cells and LIF. 3.1.4 Freezing of Cells
This protocol is applicable to both feeders and ES cells. 1. Repeat steps 1–3 in Section 3.1.3. For a 10 cm tissue culture dish, use 3 mL trypsin/EDTA and add 3 mL 10 % FBS medium into the 15 mL Falcon tube. 2. Aspirate the supernatant from the 15 mL Falcon tube, Add 1 mL freezing medium, resuspend cell pellet, and transfer cells to freezing vial. 3. Store the cells at 80 C for a day, and then keep them in liquid nitrogen for long term.
3.1.5 Preparing of EBs for Osteogenic Differentiation (Days 0–2)
1. Add 7 mL autoclaved PBS into the hanging drop petri dishes. PBS is added to prevent drying out of the hanging drops. 2. When passage 2 ES cells are at 65 % confluency, prepare for making EBs by steps 1–3 in Section 3.1.3. 3. Carefully aspirate the supernatant from the 15 mL Falcon tube. Resuspend cell pellet in 6 mL 17 % FBS medium. Gently pipette up and down to mix. Add 50 μL of cell solution to an equal volume 0.4 % trypan blue. Pipette to mix well. Let stand for 1 min. 4. Transfer 50 μL of the solution containing ES cells and trypan blue to a well on a Quick-Read Precision Cell. Count number of cells within a group of nine circles. Count cells in the another group of nine circles. Take the average of the 2 numbers, multiply by 2 to account for dilution with trypan blue, and multiply by 104. This is the number of cells present in 1 mL original cell solution. Repeat with another well on the same Precision Cell slide. Take the average of the number of cells calculated. 5. For osteogenic differentiation, our laboratory discovered that the optimal initial seeding number of cells per EB is approximately 250 cells. Each hanging drop dish requires approximately 3.6 mL cell solution (144 drops per dish 25 μL medium per drop). Each dish will require 36,000 cells per dish (144 drops per dish 250 cells per drop). Add appropriate number of cells into the medium for the desired number of hanging drop dishes. Pipette gently to mix. 6. Use an 8-channel pipette to dispense 25 μL drops of cell solution onto the lids of the Petri dishes. Carefully invert the lid and place it over the bottom of the dish. Transfer to incubator.
Osteogenic Differentiation from Murine ES Cells
431
Fig. 2 Images of EBs at different stages of differentiation. A light microscope image of (a) a day-4 EB growing in suspension and (b) a day-6 EB growing on attached gelatinized tissue culture dish
7. ES cells will aggregate at the bottom of each drop and form EBs. Leave hanging drop dishes undisturbed in the incubator for 3 days. 3.1.6 Growth of EBs in Suspension
1. On day 3, transfer the EBs to bacteriological dishes. Prepare 17 % FBS and add 1 μL RA stock per 100 mL medium for a final concentration of 0.1 μM. 2. Pipette 3 mL of medium with RA onto the lids of the hanging drop dish to wash down the EBs. Collect and transfer the EBs of one hanging drop dish to one bacteriological dish. Wash the lid three times with medium. 3. On the next day (day 4) (Fig. 2a), replace the medium with fresh 17 % FBS medium with 0.1 μM RA (Fig. 1b).
432
Yanhong Yu et al.
3.1.7 Growth of EBs in Attachment Phase
1. On day 5, EBs are plated on 10 cm tissue culture dish and they remain attached for the rest of the 21-day differentiation period (Fig. 2b). 2. Add 4 mL of 0.1 % gelatin in a 10 cm tissue culture dish, cover the bottom of the dish evenly with gelatin, and aspirate gelatin. Set dish aside for later use. 3. Add 9 mL 17 % FBS medium in each dish, and transfer 25 EBs to each 10 cm dish. Gently move the dish around for even distribution of EBs. 4. On day 6, per mL 17 % FBS medium, add 5 μL of β-glycerophosphate (for a final concentration of 10 mM) and 5 μL of ascorbic acid (for a final concentration of 50 μg/mL). β-Glycerophosphate and ascorbic acid are kept throughout differentiation (Fig. 1b). Change medium every other day. 5. On day 10, dexamethasone (100 nM) is added and kept throughout the differentiation. Add 1 μL dexamethasone per mL medium (Fig. 1b).
3.1.8 Detection of Osteogenic Lineage Cells: von Kossa Stain
von Kossa stain is a routine assay which detects the presence of calcium deposits in culture. Mature osteoblasts secrete calcium and phosphate ions on a matrix composed mainly of collagen 1 and other organic components. The silver ions present in the von Kossa stain displace calcium ions, and the silver ions are subsequently visualized (Fig. 3a). 1. Prepare 10 % neutral formalin buffer solution by adding 100 mL 36.5 % formaldehyde to 16 g Na2HPO4, 4 g Na2HPO4·H2O and 1 L distilled H2O. 2. Prepare 2.5 % silver nitrate solution by dissolving 2.5 g silver nitrate into distilled H2O. 3. On day 21, carefully aspirate medium from the dish, wash cells three times with distilled H2O. 4. Fix cells in 10 % neutral formalin buffer for 2 h. 5. Rinse cells with distilled H2O three times. 6. Stain cells with 2.5 % silver nitrate solution for 30 min. 7. Aspirate all silver nitrate solution and rinse three times with distilled H2O. Cells are now ready to be examined for mineral deposits (appears black).
3.1.9 Detection of Osteogenic Lineage Cells: Osteogenic Marker Expression RNA Isolation and Reverse Transcription
1. RNA is isolated using the Qiagen RNeasy Plus Mini Kit according to the manufacturer’s instruction. RNA should be stored at 80 C (see Notes 3 and 4). 2. In a 1.5 mL Eppendorf tube, add 4 μL Oligo (dt)18 primer, 4 μg RNA, 4 μL 10 mM dNTP, and 36 μL UltraPure Distilled H2O. 3. Heat mixture to 65 C for 5 min.
Osteogenic Differentiation from Murine ES Cells
433
Fig. 3 Detection of osteogenic lineage cells. (a) von Kossa stain of differentiated day-21 nodules. Cells grew in the presence of osteogenic differentiation medium (Osteogenic) had more mineral deposition compared to those grew in 17 % FBS medium (Control). (b) Expression of bone markers in the nodules detected by RealTime PCR. Cells cultured in the presence of osteogenic medium (Osteogenic) had higher abundance of Osterix and Collagen 1 compared to those cultured in the presence of only 17 % FBS medium (Control) (experiments were repeated at least three times, data presented are means SD)
4. Quickly chill contents on ice. 5. In each tube, add 16 μL 5 first strand buffer, 8 μL 0.1 M DTT, and 4 μL RNaseOUT Recombinant Ribonuclease Inhibitor.
434
Yanhong Yu et al.
6. Incubate at 42 C for 2 min. 7. Add 4 μL SuperScript II Reverse Transcriptase to each tube. 8. Incubate at 42 C for 50 min. Then incubate at 70 C for 15 min. 9. Store cDNA at 80 C. Real-Time PCR
1. Using a 384-well plate, in each well, add 2 μL forward primer stock, 2 μL reverse primer stock, 10 μL SYBR green PCR Master Mix, and 1 μL 10 ng/ μL cDNA. For each sample, do triplicates. 2. Amplify cDNA using the Bio-Rad’s CFX384 TouchTM Detection System. Program the system as the following: step 1: 94 C for 6 min, step 2: 94 C for 1 min, step 3: 56 C for 1 min, step 4: 72 C for 1 min, step 5: go to step 2 45 times, step 6: 72 C for 7 min, step 7: 4 C for 5 h. 3. Normalize gene expression to a housekeeping gene (i.e., L32) (Fig. 3b).
4
Notes 1. For all liquids used in tissue culture such as PBS, 0.1 % gelatin, and distilled H2O, use only after they have been autoclaved. 2. Use autoclaved pipette tips for all work in tissue culture. 3. Use UltraPure RNAse DNAse Distilled H2O for steps in reverse transcription and real-time PCR. 4. Use sterile, DNase- and RNase-free, filtered pipette tips and DNase- and RNase-free gloves when working with RNA and cDNA.
Acknowledgement This work is supported by grants from CIHR MOP-102549 and MOP-106461 to M.O. References 1. Evans MJ, Kaufman MH (1981) Establishment in culture of pluripotential cells from mouse embryos. Nature 292:154–156 2. Khillan JS, Chen L (2010) Feeder-independent culture of mouse embryonic stem cells using vitamin A/retinol 7. Methods Mol Biol 652:75–83 3. Niwa H, Ogawa K, Shimosato D, Adachi K (2009) A parallel circuit of LIF signalling
pathways maintains pluripotency of mouse ES cells. Nature 460:118–122 4. Kurosawa H (2007) Methods for inducing embryoid body formation: in vitro differentiation system of embryonic stem cells. J Biosci Bioeng 103:389–398 5. Szabo E, Qiu Y, Baksh S, Michalak M, Opas M (2008) Calreticulin inhibits commitment to adipocyte differentiation. J Cell Biol 182:103–116
Osteogenic Differentiation from Murine ES Cells 6. Szabo E, Feng T, Dziak E, Opas M (2009) Cell adhesion and spreading affect adipogenesis from ES cells: the role of calreticulin. Stem Cells 27:2092–2102 7. Papp S, Dziak E, Opas M (2009) Embryonic stem cell derived cardiomyogenesis: a novel role for calreticulin as a regulator. Stem Cells 27:1507–1515
435
8. Hwang YS, Randle WL, Bielby RC, Polak JM, Mantalaris A (2006) Enhanced derivation of osteogenic cells from murine embryonic stem cells after treatment with HepG2-conditioned medium and modulation of the embryoid body formation period: application to skeletal tissue engineering. Tissue Eng 12:1381–1392
Osteogenic Differentiation from Embryonic Stem Cells Yanhong Yu, Carlos Pilquil, and Michal Opas Abstract Embryonic stem (ES) cells have been widely studied due to their pluripotency and their potential of self-renewal. Murine ES cells are useful in investigating the molecular pathways underlying their differentiation to various mature cell types in the body. This chapter describes the maintenance of murine ES cells in culture and a routine ES cell osteogenic differentiation protocol utilized in our laboratory. Keywords: Murine embryonic stem cells, Routine culture, Hanging drop, Embryoid bodies, Osteogenesis
1
Introduction Since their first isolation by two independent laboratories (1) two decades ago, murine embryonic stem (ES) cells have been extensively studied. This work in turn has revolutionized the fields of regenerative medicine, genetics, and developmental biology. This tremendous interest in the field of ES cells is due to their potential to differentiate to all types of mature cells in the body and their ability of unlimited self-renewal in vitro. ES cells are isolated from the inner cell mass of blastocysts in the pre-implanted stage. Under certain culture conditions, they can differentiate and give rise to the three germ layers: the endoderm, mesoderm, and ectoderm. ES cells are commonly propagated on a layer of mitomycin-treated, and thus division-incompetent mouse fibroblasts, which have been shown to be critical in maintaining the undifferentiated state of ES cells (2). A soluble factor, leukemia inhibitory factor (LIF), is also added to prevent the spontaneous in vitro differentiation of ES cells. The withdrawal of LIF from culture and growth in suspension causes differentiation and embryoid body (EB) formation (3). EBs are a unique in vitro model which allows for the study of early embryonic development such as the generation of the three germ layers. They are three-dimensional aggregates of differentiating stem cells. There are many different ways one can generate EBs: growth in suspension culture using low-adherence vessels or bacteriological dishes, bioreactor/spinner flask techniques, culture using methylcellulose media, and hanging drops (4). The hanging drop
425
426
Yanhong Yu et al.
a
P0-P2
b
Days P0-P2 0-2 3-5 5-9 10-21
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Medium Composition 10% FBS, LIF 20% FBS 20% FBS, RA 20% FBS, B-Gly, AA 20% FBS, B-Gly, AA, Dex
Fig. 1 ES cell maintenance and osteogenic differentiation. (a) Murine ES cells are maintained on mouse embryonic fibroblast feeder cells for two passages. At passage 2, ES cells form EBs via the hanging drop method. From days 3 to 5, they grow in suspension. They are attached on gelatinized tissue culture dishes on day 6 and grow for the remainder of the 21 days. (b) Different medium composition is required for different stages of ES cell maintenance and osteogenic differentiation. (Abbreviations: ES embryonic stem, EB embryoid body, FBS fetal bovine serum, LIF leukemia inhibitory factor, RA retinoic acid, B-Gly β-glycerophosphate; AA ascorbic acid, Dex dexamethasone)
method is one of the more widely utilized protocols that have successfully generated various mature cell types (cardiomyocytes, neuronal cells, adipocytes, osteoblasts, etc.) (5–8). In brief, 20–30 μL of solution containing ES cells is pipetted on Petri dish lids. These lids are then inverted and placed over the Petri dish. Due to gravity, the ES cells in solution then collect at the bottom of the drops and aggregate to form EBs. In this chapter, we describe routine maintenance and propagation of murine ES cells in vitro, and illustrate in detail the hanging drop differentiation method. As an example of differentiation, we demonstrate the osteogenic induction and differentiation of ES cells (Fig. 1).
2 2.1
Materials Tissue Culture
2.1.1 Reagents and Equipments
1. 10 cm Tissue culture dish, 100 20 mm, polystyrene (Falcon, cat. no. 353003). 2. 6 cm Tissue culture dish, 60 15 mm, polystyrene (Falcon, cat. no. 353002). 3. Hanging drop petri dish, 150 15 mm, polystyrene (VWR, cat. no. 25384-326). 4. Bacteriological plates (VWR, cat. no. 25389-328).
Osteogenic Differentiation from Murine ES Cells
427
5. 15 mL Centrifuge tube, polypropylene (FroggaBio, cat. no. TB15-25). 6. Phosphate-buffered saline (PBS): 10 stock, 76.5 g sodium chloride (NaCl) (Bioshop, cat. no. SOD002.205), 7.25 g sodium phosphate dibasic anhydrous (Na2HPO4) (SigmaAldrich, cat. no. RES20908), 2.12 g potassium phosphate dibasic anhydrous (KH2PO4) (Sigma-Aldrich, cat. no. P0662), 1 L distilled water (H2O). For 1 PBS, dilute stock 1:10 with distilled H2O. Autoclave and store at room temperature. 7. 0.1 % gelatin: 0.5 g gelatin (Anachemia, cat. no. AC-4622) in 500 mL PBS. Autoclave and store at room temperature. 8. Trypsin/EDTA: 0.25 % trypsin 2.21 mM EDTA tetrasodium (Multicell, Wisent, cat. no. 325-043-CL). Store at 20 C. 9. Freezing vial: CryoELITE Cryogenic vials (Wheaton, cat. no. W985862). 10. Mytomycin C (Sigma-Aldrich, cat. no. M4287-2mg), stock 1 mg/mL, store at 20 C. 11. Leukemia inhibitory factor (LIF): stock 10 μg/mL. Store at 20 C. 12. 0.4 % Trypan blue solution (Life technologies, cat. no. 15250061). 13. Quick-Read Precision Cell, with 18 counting circles, 10 tests/ slide (Globe Scientific Inc., cat. no. 010G18). 14. 8-Channel pipette (Rainin, 20–200 μL, cat. no. L8-200XLS+). 15. Retinoic acid (Sigma-Aldrich, cat. no. R625): 10 mM stock, store at 20 C. 16. β-Glycerophosphate (Sigma-Aldrich, cat. no. G9422): stock 2 M, store at 20 C. 17. Ascorbic acid (Sigma-Aldrich, cat. no. A4403): stock 5 mg/ mL, store at 20 C. 18. Dexamethasone (Sigma-Aldrich, cat. no. D4902): stock 0.1 mM, store at 20 C. 2.1.2 Medium Preparation
1. 10 % FBS medium: 500 mL DMEM with sodium pyruvate and L-glutamine (Multicell, Wisent, cat. no. 319-005-ES), 50 mL Fetal Bovine Serum (FBS) (Multicell, Winsent, cat. no. 080150), 5 mL MEM nonessential amino acids 100 (Gibco, Life Technologies, cat. no. 11140-050), 500 μL diluted 2-mercaptoethanol, 35 μL 2-mercaptoethanol (Bioshop, cat. no. CAS60-24-2) in 5 mL autoclaved distilled H2O. 2. Freezing medium: 8 mL 10 % FBS medium, 1 mL FBS, 1 mL dimethyl sulfoxide (DMSO) (Sigma-Aldrich, cat. no. D2650). 3. 17 % FBS medium: preparation same as 10 % FBS medium except for the addition of 100 mL FBS.
428
2.2
Yanhong Yu et al.
Von Kossa Assay
1. Formaldehyde solution, 36.5–38 % in water (Sigma-Aldrich, cat. no. F8775): store at room temperature. 2. Silver nitrate (Bioshop, cat. no. SIL222). 3. Na2HPO4 (Sigma-Aldrich, cat. no. RES20908). 4. Sodium phosphate monobasic monohydrate (Na2HPO4·H2O) (Sigma-Aldrich, cat. no. S9638).
2.3 RNA Isolation, Reverse Transcription, Real-Time Polymerase Chain Reaction (PCR)
1. RNeasy Plus Mini Kit (Qiagen, cat. no. 74134). 2. 1.5 mL Eppendorf tube (Axygen, cat. no. PMI11006v2). 3. Oligo(dt)18 Primer 0.5 μg/μL: (Thermo Scientific, cat. no. SO132). Store at 20 C. 4. dNTP mix, 10 mM (Invitrogen. cat. no. 18427-013). Store at 30 C. 5. UltraPure Distilled water, DNase, RNase, free (Invitrogen, cat. no. 10977-015). 6. 5 First strand buffer (Invitrogen. cat. no. P/N y02321). Store at 20 C. 7. 0.1 M DTT (Invitrogen. cat. no. P/N y00147). Store at 20 C. 8. RNaseOUT Recombinant Ribonuclease Inhibitor, 40 U/μL (Invitrogen. cat. no. P/N 100000840). Store at 20 C. 9. SuperScript II Reverse Transcriptase, 200 U/μL (Invitrogen. cat. no 18064-014). Store at 20 C. 10. Power SYBR Green PCR Master Mix (Applied Biosystems, cat. no. 4367659). For short-term storage, keep in 2–8 C. For long-term storage, store at 20 C. 11. 384 well plate, MicroAmp Optical Reaction Plate with Barcode (Applied Biosystems by Life Technologies, cat. no. 4309849). 12. Sealing tape, optically clear (Sarstedt, cat. no. 95.1994). 13. Osteogenic markers primers (Invitrogen): Osterix (OSX): forward primer 50 -GCAACTGGCTAGGTGGTGGTC-30 and reverse primer 50 -GCAAAGTCAGATGGGTAAGTAGGC-30 . Collagen 1 (Col 1): forward primer 50 -GAACGGTCCACGATTGCATG-30 and reverse primer 50 -GGCATGTTGCTAGGCACGAAG-30 . Stock 250 nM. Store at 20 C. 14. CFX384 TouchTM Detection System (Biorad, cat. no. 1855485).
Osteogenic Differentiation from Murine ES Cells
3 3.1
429
Methods Cell Culture
3.1.1 Thawing of Mouse Embryonic Fibroblasts (Feeders) Cells
1. Warm cells quickly in 37 C incubator or water bath. 2. Add 2 mL of 10 % FBS medium in a 15 mL Falcon tube, and add the thawed cell solution in the same tube. 3. Centrifuge at 700 g for 3 min. 4. In the meanwhile, add 2 mL of 0.1 % gelatin in a 6 cm tissue culture dish, cover the bottom of the dish evenly with gelatin, and aspirate gelatin. Set dish aside for later use (see Note 1). 5. Aspirate the supernatant and add fresh 2 mL 10 % FBS medium to resuspend the cell pellet in the tube (see Note 2). 6. Transfer the resuspended cell solution to the gelatinized dish, and add 2 mL 10 % FBS medium to the dish. Move dish around gently for even distribution of cells in the dish. 7. Change medium every other day. Add 4 mL of fresh 10 % FBS medium per 6 cm dish.
3.1.2 Preparation of Feeders for ES cells and Plating ES Cells
1. When feeders are at 65 % confluency, treat cells with 10 μL/mL medium of mytomycin C for 2 h to inactivate their ability to undergo mitosis. 2. After 2 h, aspirate mitomycin C, rinse cells twice with fresh 10 % FBS medium. Add 4 mL of fresh 10 % FBS medium. 3. Quickly thaw ES cells using steps 1–3 from Section 3.1.1. 4. Aspirate the supernatant and add 1 mL 10 % FBS medium to resuspend the ES cells pellet. Transfer the cell solution to the dish containing the mitomycin C-treated feeders. 5. To maintain pluripotency, add 1 μL leukemia inhibitory factor (LIF) for 1 mL medium. 6. Change medium every other day.
3.1.3 Maintenance of ES cells
1. When ES cells are at about 65 % confluency (approximately 2 days after last passage), aspirate medium, and add 1 mL trypsin/EDTA per 6 cm tissue culture dish. Incubate for 3 min. Pipette up and down a few times to break up cell clumps into single cell suspension. 2. Add 1 mL fresh 10 % FBS medium to a 15 mL Falcon tube, transfer the cell solution with trypsin into this tube. 3. Centrifuge at 700 g for 3 min. 4. In the meanwhile, add 2 mL of 0.1 % gelatin in a 6 cm tissue culture dish, cover the bottom of the dish evenly with gelatin, and aspirate gelatin. Set dish aside for later use.
430
Yanhong Yu et al.
5. Carefully aspirate the supernatant from the 15 mL Falcon tube. Resuspend ES cell pellet in 4 mL 10 % FBS medium. Transfer this cell solution to the gelatinized dish. Add 4 μL LIF into the dish, and gently move the dish for even distribution of cells and LIF. 3.1.4 Freezing of Cells
This protocol is applicable to both feeders and ES cells. 1. Repeat steps 1–3 in Section 3.1.3. For a 10 cm tissue culture dish, use 3 mL trypsin/EDTA and add 3 mL 10 % FBS medium into the 15 mL Falcon tube. 2. Aspirate the supernatant from the 15 mL Falcon tube, Add 1 mL freezing medium, resuspend cell pellet, and transfer cells to freezing vial. 3. Store the cells at 80 C for a day, and then keep them in liquid nitrogen for long term.
3.1.5 Preparing of EBs for Osteogenic Differentiation (Days 0–2)
1. Add 7 mL autoclaved PBS into the hanging drop petri dishes. PBS is added to prevent drying out of the hanging drops. 2. When passage 2 ES cells are at 65 % confluency, prepare for making EBs by steps 1–3 in Section 3.1.3. 3. Carefully aspirate the supernatant from the 15 mL Falcon tube. Resuspend cell pellet in 6 mL 17 % FBS medium. Gently pipette up and down to mix. Add 50 μL of cell solution to an equal volume 0.4 % trypan blue. Pipette to mix well. Let stand for 1 min. 4. Transfer 50 μL of the solution containing ES cells and trypan blue to a well on a Quick-Read Precision Cell. Count number of cells within a group of nine circles. Count cells in the another group of nine circles. Take the average of the 2 numbers, multiply by 2 to account for dilution with trypan blue, and multiply by 104. This is the number of cells present in 1 mL original cell solution. Repeat with another well on the same Precision Cell slide. Take the average of the number of cells calculated. 5. For osteogenic differentiation, our laboratory discovered that the optimal initial seeding number of cells per EB is approximately 250 cells. Each hanging drop dish requires approximately 3.6 mL cell solution (144 drops per dish 25 μL medium per drop). Each dish will require 36,000 cells per dish (144 drops per dish 250 cells per drop). Add appropriate number of cells into the medium for the desired number of hanging drop dishes. Pipette gently to mix. 6. Use an 8-channel pipette to dispense 25 μL drops of cell solution onto the lids of the Petri dishes. Carefully invert the lid and place it over the bottom of the dish. Transfer to incubator.
Osteogenic Differentiation from Murine ES Cells
431
Fig. 2 Images of EBs at different stages of differentiation. A light microscope image of (a) a day-4 EB growing in suspension and (b) a day-6 EB growing on attached gelatinized tissue culture dish
7. ES cells will aggregate at the bottom of each drop and form EBs. Leave hanging drop dishes undisturbed in the incubator for 3 days. 3.1.6 Growth of EBs in Suspension
1. On day 3, transfer the EBs to bacteriological dishes. Prepare 17 % FBS and add 1 μL RA stock per 100 mL medium for a final concentration of 0.1 μM. 2. Pipette 3 mL of medium with RA onto the lids of the hanging drop dish to wash down the EBs. Collect and transfer the EBs of one hanging drop dish to one bacteriological dish. Wash the lid three times with medium. 3. On the next day (day 4) (Fig. 2a), replace the medium with fresh 17 % FBS medium with 0.1 μM RA (Fig. 1b).
432
Yanhong Yu et al.
3.1.7 Growth of EBs in Attachment Phase
1. On day 5, EBs are plated on 10 cm tissue culture dish and they remain attached for the rest of the 21-day differentiation period (Fig. 2b). 2. Add 4 mL of 0.1 % gelatin in a 10 cm tissue culture dish, cover the bottom of the dish evenly with gelatin, and aspirate gelatin. Set dish aside for later use. 3. Add 9 mL 17 % FBS medium in each dish, and transfer 25 EBs to each 10 cm dish. Gently move the dish around for even distribution of EBs. 4. On day 6, per mL 17 % FBS medium, add 5 μL of β-glycerophosphate (for a final concentration of 10 mM) and 5 μL of ascorbic acid (for a final concentration of 50 μg/mL). β-Glycerophosphate and ascorbic acid are kept throughout differentiation (Fig. 1b). Change medium every other day. 5. On day 10, dexamethasone (100 nM) is added and kept throughout the differentiation. Add 1 μL dexamethasone per mL medium (Fig. 1b).
3.1.8 Detection of Osteogenic Lineage Cells: von Kossa Stain
von Kossa stain is a routine assay which detects the presence of calcium deposits in culture. Mature osteoblasts secrete calcium and phosphate ions on a matrix composed mainly of collagen 1 and other organic components. The silver ions present in the von Kossa stain displace calcium ions, and the silver ions are subsequently visualized (Fig. 3a). 1. Prepare 10 % neutral formalin buffer solution by adding 100 mL 36.5 % formaldehyde to 16 g Na2HPO4, 4 g Na2HPO4·H2O and 1 L distilled H2O. 2. Prepare 2.5 % silver nitrate solution by dissolving 2.5 g silver nitrate into distilled H2O. 3. On day 21, carefully aspirate medium from the dish, wash cells three times with distilled H2O. 4. Fix cells in 10 % neutral formalin buffer for 2 h. 5. Rinse cells with distilled H2O three times. 6. Stain cells with 2.5 % silver nitrate solution for 30 min. 7. Aspirate all silver nitrate solution and rinse three times with distilled H2O. Cells are now ready to be examined for mineral deposits (appears black).
3.1.9 Detection of Osteogenic Lineage Cells: Osteogenic Marker Expression RNA Isolation and Reverse Transcription
1. RNA is isolated using the Qiagen RNeasy Plus Mini Kit according to the manufacturer’s instruction. RNA should be stored at 80 C (see Notes 3 and 4). 2. In a 1.5 mL Eppendorf tube, add 4 μL Oligo (dt)18 primer, 4 μg RNA, 4 μL 10 mM dNTP, and 36 μL UltraPure Distilled H2O. 3. Heat mixture to 65 C for 5 min.
Osteogenic Differentiation from Murine ES Cells
433
Fig. 3 Detection of osteogenic lineage cells. (a) von Kossa stain of differentiated day-21 nodules. Cells grew in the presence of osteogenic differentiation medium (Osteogenic) had more mineral deposition compared to those grew in 17 % FBS medium (Control). (b) Expression of bone markers in the nodules detected by RealTime PCR. Cells cultured in the presence of osteogenic medium (Osteogenic) had higher abundance of Osterix and Collagen 1 compared to those cultured in the presence of only 17 % FBS medium (Control) (experiments were repeated at least three times, data presented are means SD)
4. Quickly chill contents on ice. 5. In each tube, add 16 μL 5 first strand buffer, 8 μL 0.1 M DTT, and 4 μL RNaseOUT Recombinant Ribonuclease Inhibitor.
434
Yanhong Yu et al.
6. Incubate at 42 C for 2 min. 7. Add 4 μL SuperScript II Reverse Transcriptase to each tube. 8. Incubate at 42 C for 50 min. Then incubate at 70 C for 15 min. 9. Store cDNA at 80 C. Real-Time PCR
1. Using a 384-well plate, in each well, add 2 μL forward primer stock, 2 μL reverse primer stock, 10 μL SYBR green PCR Master Mix, and 1 μL 10 ng/ μL cDNA. For each sample, do triplicates. 2. Amplify cDNA using the Bio-Rad’s CFX384 TouchTM Detection System. Program the system as the following: step 1: 94 C for 6 min, step 2: 94 C for 1 min, step 3: 56 C for 1 min, step 4: 72 C for 1 min, step 5: go to step 2 45 times, step 6: 72 C for 7 min, step 7: 4 C for 5 h. 3. Normalize gene expression to a housekeeping gene (i.e., L32) (Fig. 3b).
4
Notes 1. For all liquids used in tissue culture such as PBS, 0.1 % gelatin, and distilled H2O, use only after they have been autoclaved. 2. Use autoclaved pipette tips for all work in tissue culture. 3. Use UltraPure RNAse DNAse Distilled H2O for steps in reverse transcription and real-time PCR. 4. Use sterile, DNase- and RNase-free, filtered pipette tips and DNase- and RNase-free gloves when working with RNA and cDNA.
Acknowledgement This work is supported by grants from CIHR MOP-102549 and MOP-106461 to M.O. References 1. Evans MJ, Kaufman MH (1981) Establishment in culture of pluripotential cells from mouse embryos. Nature 292:154–156 2. Khillan JS, Chen L (2010) Feeder-independent culture of mouse embryonic stem cells using vitamin A/retinol 7. Methods Mol Biol 652:75–83 3. Niwa H, Ogawa K, Shimosato D, Adachi K (2009) A parallel circuit of LIF signalling
pathways maintains pluripotency of mouse ES cells. Nature 460:118–122 4. Kurosawa H (2007) Methods for inducing embryoid body formation: in vitro differentiation system of embryonic stem cells. J Biosci Bioeng 103:389–398 5. Szabo E, Qiu Y, Baksh S, Michalak M, Opas M (2008) Calreticulin inhibits commitment to adipocyte differentiation. J Cell Biol 182:103–116
Osteogenic Differentiation from Murine ES Cells 6. Szabo E, Feng T, Dziak E, Opas M (2009) Cell adhesion and spreading affect adipogenesis from ES cells: the role of calreticulin. Stem Cells 27:2092–2102 7. Papp S, Dziak E, Opas M (2009) Embryonic stem cell derived cardiomyogenesis: a novel role for calreticulin as a regulator. Stem Cells 27:1507–1515
435
8. Hwang YS, Randle WL, Bielby RC, Polak JM, Mantalaris A (2006) Enhanced derivation of osteogenic cells from murine embryonic stem cells after treatment with HepG2-conditioned medium and modulation of the embryoid body formation period: application to skeletal tissue engineering. Tissue Eng 12:1381–1392
