TSC Derivation, Cx

(protocol from Rossant lab via Celeste Simon)
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Trophoblast Stem Cell Protocols

The protocols and reagents described here are for the maintenance and derivation of trophoblast stem (TS) cell lines (1). Slight variations from the procedures or the suppliers of reagents are probably acceptable. If you have any questions or concerns contact Tilo Kunath by e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it. If you have made improvements or simplifications to the protocols please let us know about these too.

The following reagents should be prepared before attempting to culture or derive TS cell lines.
 
1000X FGF4 (25mg/ml) Human recombinant FGF4 (Sigma, cat# F2278, 25µg)2
Resuspend lyophilized FGF4 in its vial with 1.0ml of PBS/0.1%(w/v) BSA (fraction V)3.
Mix well with a P200 set at 100ml and make 10 aliquots (10 X 100ml) into 1.5ml- microfuge tubes and freeze at –80˚C. Thaw each aliquot as needed and store it at 4˚C; do not re-freeze.
 
1000X Heparin (1.0mg/ml)4
Heparin (Sigma, cat# H3149,  10,000 units)
Resuspend heparin in PBS to a final concentration of 1.0mg/ml (1000X) and store at – 80˚C. Thaw each aliquot as needed and store it at 4˚C.

TS medium

TS medium (650ml) is prepared by adding the following reagents to RPMI 1640 + antibiotics* (500 ml).  
  Final Stock Supplier
   Volume Conc. Conc.  
RPMI 1640 + antibiotics* 500ml      
Fetal bovine serum 130ml 20% 100% GibcoBRL
Sodium pyruvate 6.5ml 1mM 100mM GibcoBRL
b-mercaptoethanol 6.5ml 100mM 10mM Sigma
L-glutamine 6.5ml 2mM 200mM GibcoBRL
 *antibiotics = penicillin and streptomycin at 50 µg/ml each

Feeder-CM

Feeder-conditioned medium (CM) is used to culture TS cells in the absence of EMFIs. Prepare mitomycin-treated primary embryonic fibroblasts (EMFIs) in 100mm dishes (2 x 106cells; 2 x 105cells/ml) and culture in TS medium (10.0 – 10.5 ml per dish). Culture for at least 72h (3 days) and then collect the medium. Spin to remove floating cells and debris, filter (0.45mm), and store at –20˚C in 10ml aliquots. Thaw each aliquot as needed and store it at 4˚C; do not re-freeze. Use the EMFIs to prepare two more batches of feeder-CM, then discard the cells. EMFIs are only used up to 10 days after the mitomycin treatment.

Culturing TS Cells

TS cells may be grown on (A) EMFIs or (B) regular tissue culture plastic (gelatin-coating is not required).

A. Culturing TS cells on EMFIs

Plate mitomycin-treated EMFIs at half the density (1 x 105cells/ml) used to culture embryonic stem (ES) cells and for the preparation of feeder-CM. TS cells may be plated on the EMFIs after they have adhered or co-plated with the EMFIs.
 
Prepare fresh TS+F4H medium: e.g.
10 ml TS medium
10 µl 1000X FGF4 (25ng/ml)
10 µl 1000X heparin 10ml (1mg/ml)
 
Culture TS cells in the above medium in a standard tissue culture incubator (37˚C/5%CO2). The medium is normally changed every two days and the cells are passaged (1 in 10 to 1 in 20) every 4th day or when the culture has reached approximately 80% confluency. Passaging TS cells at higher densities (i.e. 1 in 3 or 1 in 5) may lead to precocious differentiation. The cells are trypsinized to very small clumps with some single cells. A complete single-cell suspension is not required and it may even be detrimental to the culture. Trypsinizing for 3-4 min at 37˚C with some pipetting up and down is usually sufficient.

B. Culturing TS cells on tissue culture plastic

TS cells grow well on standard tissue culture dishes if the medium is supplemented with feeder-CM. The dishes do not need to be gelatin-coated as mentioned in Tanaka et al. (1). The percentage of feeder-CM used is 70% and the rest is TS medium (30%).
 
Prepare fresh 70cond+F4H medium: e.g.
7 ml feeder-CM (70%)
3 ml TS medium (30%)
10 µl 1000X FGF4 (25ng/ml)
10 µl 1000X heparin (1mg/ml)
Culture the TS cells in the above medium; feed and passage the cells as above.

C. Freezing TS cells

Prepare 2X freezing medium and cool to 4˚C:
50% FBS
30% TS medium
20% DMSO

Lift and pellet TS cells. Resuspend the cells in TS medium and add an equal volume of 2X freezing medium. Freeze the cells slowly at -80˚C overnight and then transfer to liquid nitrogen after 24-48 hours. A confluent 100mm dish has enough cells for about 5 vials.

D. Thawing TS cells

Hand-thaw the vial and put the contents over TS medium (1.0ml) and pellet. Resuspend the cells in TS + F4H medium (5ml) and plate over EMFIs (half-density) in a 60mm plate. Change the medium after 8 hours to get rid of the non-adherent cells. Feed two days later.

E. Removing EMFIs from TS cell cultures

When switching from EMFI cells to EMFI-CM it may be desirable to get rid of the EMFI cells immediately. The different adherence rates of EMFI cells (fast) and TS cells (slow) can be used to obtain a pure TS cell population. Passage the cells to a new plate and incubate the culture for 1.5h at 37oC/5%CO2. Remove the supernatant and plate onto another dish. This population of cells should consist almost entirely of TS cells. Since some TS cells do adhere along with the EMFIs, the desired passage density may be increased a bit (e.g. 1 in 14 instead of 1 in 16).

Derivation of TS cell lines from Blastocysts

The derivation of TS cell lines from 3.5dpc mouse blastocysts is similar to the derivation of embryonic stem (ES) cell lines (5, 6). However, the success rate is considerably higher and less expertise is required to recognize pluripotent TS cell colonies.
  1. Set up matings (natural or super-ovulated) between the mice of interest.
  2. Prepare 4-well plates of EMFIs (5 x 104cells; 500ml of 1 x 105cells/ml per well) in TS medium the day before flushing (7). This is the same density used to culture TS cells.
  3. Replace the TS medium with TS+F4H medium (500ml per well) in the morning of the flushing (Day 1).
  4. Flush and collect 3.5dpc blastocysts (6). In sterile conditions, place one blastocyst per well in the 4-well plates containing TS+F4H medium and culture at 37˚C/5%CO2.
  5. The blastocysts should hatch and attach to the wells in 24h-36h (Day 2).
  6. On Day 3, a small outgrowth is formed from each embryo. Feed each culture with TS + F4H medium (500ml).
  7. Day 4; this is the day the outgrowth is usually disaggregated. However, this will depend on its size; it is smaller than the size of the outgrowth disaggregated for ES cell line derivation. The ideal size for TS cell line derivation is illustrated on page 87, Figure 4b of (5) or on page 267, Figure 2B of (6). Larger outgrowths will also work, but with less efficiency.
  8. Once suitable outgrowths have been chosen, they may be disaggregated by different means. The microdrop technique described in (5) and (6) may be used. However, we perform the disaggregation directly in the wells they were cultured in. Remove the medium and wash the cells with PBS (500ml). Aspirate the PBS, add 0.1% trypsin/EDTA (100ml), and incubate for 5min at 37˚C/5%CO2. Using a P2 pipetteman or a drawn Pasteur pipette disaggregate the clump by pipetting up and down vigorously until the outgrowth is reduced to small clumps of cells. Immediately stop the trypsinization by adding 70cond+1.5X F4H (400ml) and returning to the incubator.
  9. Change the medium 8h after the disaggregation (70cond+1.5X F4H, 500ml).
  10. Day 6; feed each culture (70cond+F4H, 500ml) and continue to re-feed every two days.
  11. Between Day 6 and Day 10 (highly variable) TS cell colonies will begin to appear. They look like flat, epithelial sheets with a distinctive colony boundary (1). They are likely identical to the colonies described as “epitheloid cells” on page 91, Figure 8d of (5) or the “epithelial-like cells” on page 269-270, Figure 3D of (6).
  12. Continue to feed the cultures until TS cell colonies get sufficiently large to cover about 50% of the well. Some differentiation will be observed at the edges of colonies. This is normal; they are most often giant cells and other unidentified cell-types that may be between a stem cell and giant cell phenotype.
  13. Passage the half-confluent well of TS cells to a 6-well plate or 35mm dish of pre-plated EMFIs (1 x 105cells/ml) (Day 15 to Day 20). Aspirate the medium and wash TS cells with PBS (500ml). Aspirate the PBS, add trypsin/EDTA (100ml), and incubate for 5min at 37˚C/5%CO2. Stop the trypsinization by adding TS+1.5X F4H (400ml) and pipetting up and down to get a near single cell suspension. Transfer the cells to a 6-well plate or 35mm dish of TS+1.5X F4H medium (2.5ml) on EMFIs. This first passage is crucial; this is the most likely time for the culture to differentiate.
  14. Change the medium 8h after passage (TS+1.5X F4H, 3ml).
  15. Feed the cells every two days (TS+F4H, 3ml). Follow the “Culturing TS cell lines” guidelines above. After one or two more passages on EMFIs, TS cells may be cultured without them in the presence of 70cond+F4H (see 7).

References & Footnotes

  1. Tanaka, S., Kunath, T., Hadjantonakis, A.-K., Nagy, A., and Rossant, J. (1998) Promotion of Trophoblast Stem Cell Proliferation by FGF4. Science 282:2072-2075.
  2. Other sources of FGF4 would likely work as well. We found that aFGF (Sigma, cat# F5542) and bFGF (Sigma, cat#F0291) also maintained TS cells in culture at 25ng/ml. However, derivation of TS cell lines with these other FGFs have not been attempted.
  3. PBS/0.1%(w/v) BSA (10ml) is prepared by dissolving BSA, fraction V (10mg, Sigma, cat# A3311) in PBS without Ca2+/Mg2+ (10ml), filtering (0.45mm), and aliquoting (1.05ml-1.10ml) to 9 – 1.5ml microfuge tubes. Store at –80˚C and thaw when a vial of FGF4 is to be reconstituted.
  4. Heparin can also be prepared as a 10,000X (10mg/ml) stock in PBS without Ca2+/Mg2+ and stored at –80˚C. This can be used multiple times to make batches of 1000X heparin.
  5. Robertson, E.J. (1987). Embryo-derived stem cell lines. In Teratocarcinoma and Embryonic Stem Cells: A Practical Approach (ed. E.J. Robertson). Oxford, UK: IRL Press.
  6. Hogan, B., Beddington, R., Costantini, F., Lacy, E. (1994) Manipulating the Mouse Embryo. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
  7. TS cell lines have been derived from blastocysts in the absence of EMFI cells, but in the presence of 70% EMFI-CM.