Hi,
I am looking for a protocol to differentiate mouse embryonic stem cells into primordial germ cells. Can anyone provide me with a convenient protocol? How long can the differentiated cells be propagated and is it a suitable model system for manipulation like knockdowns?
Hello
For Mouse ESC Cells Culture
cells were cultured on mitomycin-C treated mouse embryonic fibroblast (MEF) cells in DMEM (Invitrogen) containing 15% fetal calf serum (FCS, tested), sodium pyruvate (Sigma), non-essential amino acids (Invitrogen), penicillin/streptomycin (Invitrogen), β-mercaptoethanol and 1000 Uml-1 Leukaemia inhibitory factor (LIF,Chemicon). ES-Oct 4 GFP cells were cultured feeder free on pre-gelatinised tissue culture dishes. Bone morphogenetic protein 4 (BMP4) was added to the cultures at a concentration of 25 ng ml-1 to induce PGCs formation from pluripotent stem cells.
NOT :
We used only early passages (under 30) to avoid aberrant changes in cells potentially arising from later passages.
Differentiation in vitro :
For embryoid body (EB) differentiation both EG or ES cells were
dissociated with 0.25% of trypsin-EDTA (Invitrogen) and collected in EB medium (EG/ESmedium without LIF). Dissociated cells were plated in hanging drops at concentration of 500 or 800 cells per 20μl in Petri dishes (bacteriological, Sterilin). After 3 days, EBs were flushed from lids and cultured in suspension in dishes (bacteriological, Sterilin). EBs were collected after 3 to 9 days of differentiation.
For differentiation of ES/EG cells induced by all-trans retinoic acid (RA, Sigma), collected EBs were digested by collagenase IV treatment (0.01%) in order to obtain cell suspensions which were seeded onto mitotically inactive MEF in EB medium supplemented with 2μMRA for 5 days.
For Mouse iPS Cells Culture
Mouse iPS cells were cultured with the high-glucose DMEM supplemented with 15% FBS (Thermo Scientific HyClone, South Logan, UT, USA), 0.1 mM nonessential amino acids, 2 mM L-glutamine, 0.1 mM 2-mercaptoethanol, 100 U/mL penicillin, 100 mg/mL streptomycin (Invitrogen, San Diego, CA, USA), and LIF (Millipore, Billerica, MA, USA), on feeder cells in gelatinized dishes. The cells were passaged every 2-3 days from passages 16 to 30.
iPS Differentiation in vitro :
After being digested with trypsin-EDTA, ~ iPS cells were transferred to 10 cm Petri dishes containing 10 mL of the above culture medium without LIF. EBs were formed using the handing drop method and maintained in suspension. At day 3, half of the medium was changed every 2 days. In addition, 2 M retinoic acid (RA, Sigma, St. Louis, MO, USA) or 1 M testosterone (Sigma) or 2 M RA and 1 M testosterone combination was added into culture medium at day 5. Forty-eight hours later, the medium was changed, and the cells were cultured for two weeks.
Good luck
Hello
For Mouse ESC Cells Culture
cells were cultured on mitomycin-C treated mouse embryonic fibroblast (MEF) cells in DMEM (Invitrogen) containing 15% fetal calf serum (FCS, tested), sodium pyruvate (Sigma), non-essential amino acids (Invitrogen), penicillin/streptomycin (Invitrogen), β-mercaptoethanol and 1000 Uml-1 Leukaemia inhibitory factor (LIF,Chemicon). ES-Oct 4 GFP cells were cultured feeder free on pre-gelatinised tissue culture dishes. Bone morphogenetic protein 4 (BMP4) was added to the cultures at a concentration of 25 ng ml-1 to induce PGCs formation from pluripotent stem cells.
NOT :
We used only early passages (under 30) to avoid aberrant changes in cells potentially arising from later passages.
Differentiation in vitro :
For embryoid body (EB) differentiation both EG or ES cells were
dissociated with 0.25% of trypsin-EDTA (Invitrogen) and collected in EB medium (EG/ESmedium without LIF). Dissociated cells were plated in hanging drops at concentration of 500 or 800 cells per 20μl in Petri dishes (bacteriological, Sterilin). After 3 days, EBs were flushed from lids and cultured in suspension in dishes (bacteriological, Sterilin). EBs were collected after 3 to 9 days of differentiation.
For differentiation of ES/EG cells induced by all-trans retinoic acid (RA, Sigma), collected EBs were digested by collagenase IV treatment (0.01%) in order to obtain cell suspensions which were seeded onto mitotically inactive MEF in EB medium supplemented with 2μMRA for 5 days.
For Mouse iPS Cells Culture
Mouse iPS cells were cultured with the high-glucose DMEM supplemented with 15% FBS (Thermo Scientific HyClone, South Logan, UT, USA), 0.1 mM nonessential amino acids, 2 mM L-glutamine, 0.1 mM 2-mercaptoethanol, 100 U/mL penicillin, 100 mg/mL streptomycin (Invitrogen, San Diego, CA, USA), and LIF (Millipore, Billerica, MA, USA), on feeder cells in gelatinized dishes. The cells were passaged every 2-3 days from passages 16 to 30.
iPS Differentiation in vitro :
After being digested with trypsin-EDTA, ~ iPS cells were transferred to 10 cm Petri dishes containing 10 mL of the above culture medium without LIF. EBs were formed using the handing drop method and maintained in suspension. At day 3, half of the medium was changed every 2 days. In addition, 2 M retinoic acid (RA, Sigma, St. Louis, MO, USA) or 1 M testosterone (Sigma) or 2 M RA and 1 M testosterone combination was added into culture medium at day 5. Forty-eight hours later, the medium was changed, and the cells were cultured for two weeks.
Good luck
Dear Saleh Alkarim,
Thank you for the reply. As I am aware, this protocol was developed in 2008. An improved protocol (by Hayashi et al) exists now. I just want to ask if its okay to still follow the protocl that you have mentioned?
Hello
Yes, you can work protocol I mentioned that also both of protocols gives a good results, and preferably provide all optimal process conditions and precision in the application to get the best results
Best wishes
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