In this video, we demonstrate the isolation of mouse blastocysts and the derivation of trophoblast stem cells from blastocysts. We also describe conditions for maintenance of the stem cell property as well as induction of differentiation in culture.
In this protocol, we describe the preparation of mouse blastocysts and the establishment of TS cell lines. General mouse manipulations prior to the collection of blastocysts, including the set up for natural mating and the induction of super ovulation, basically follow the standard protocol illustrated by Nagy et al. 2003 (pp146-150).
Derivation and maintenance of TS cells
Freezing TS cells
Thawing TS cells
TS cell differentiation into TGC
Differentiation of TS cells into trophoblast giant cells (TGCs) can be induced by the withdrawal of MEF-CM, FGF4 and heparin. The TGC phenotype (large nuclei) can be detected four days after the induction (Tanaka et al., 1998 and Chiu et al. 2008). Immunostaining of p450scc detect the expression of this TGC marker in the differentiated culture (Figure 4A). Flow cytometric analysis of PI (propidium iodide) stained cells further shows the presence of differentiated cells containing higher DNA content (Figure 4B). These results suggest that TS cells undergo endoreduplication to become polyploid TGCs.
Representative Results
Figure 1. Dissection of mouse uteri. (A) Location of the uteri is indicated by arrows. (B) Cut across the junction of uterus and cervix as shown. (C) Cut the uterus just below the oviduct (OD) underneath the ovary (OV). (D) Image of the uteri.
Figure 2. Representative pictures illustrate mouse blastocyst, blastocyst outgrowth and trophoblast colony. (A) A mouse blastocyst collected at E3.5 is shown. ICM, inner cell mass; MT, mural trophectoderm; PT, polar trophectoderm. (B) A trophoblast outgrowth on MEF feeders is shown. TS, trophoblast stem cell. (C) A trophoblast stem cell colony shows a clear edge on MEF feeders. Arrows indicate the cell boundary.
Figure 3. Identification of TS cells by immunostaining analysis. TS (A-D) and ES (control, E-H) cells are subject to marker identification. Cells are immunostained with antibodies to recognize Oct4 (A, E) or Cdx2 (B, F), and counterstained by DAPI (C, G) presented in merged images (D, H). Scale bar, 50 μm.
Figure 4. Differentiation of TS cells in vitro. (A) Undifferentiated (Undiff) or differentiated (Diff) cells in culture were examined for the expression of a TGC marker, p450scc (green), counterstained with DAPI (red). (B) Flow cytometric analysis of the differentiated cells, stained with PI, measures the DNA contents (M1, two to four copies; M2, more than four copies). The M2 population represents the polyploid trophoblast cells.
In this video, we demonstrate the process to collect E3.5 blastocysts from uteri and experimental procedures to establish TS cell lines. We also describe the condition to maintain the stemness of TS cells and to induce their differentiation into differentiated cells. Two critical steps to obtain pure TS cell lines are the timing for outgrowth disaggregation (step 9) and processing the first passage (step 11). It has been reported that primitive endoderm-derived cells can arise in the culture after extensive blastocyst outgrow in step 9 or overgrowth of TS colonies (>50% confluency) in step 11 (Kunath et al., 2005). Although primitive endoderm-derived cells can be identified by their distinctive morphology with a rounded cell shape and reduced cell-cell contact compared to the TS cells, it is difficult to remove them from the culture because they grow well in the TS medium and do not seem to require MEF feeders or FGF4.
The differentiated TGCs may arise constantly in the TS culture even in the presence of MEF-CM and FGF4. If a pure TS population is desired, these differentiated cells may be separated on the basis of the differential adherent rates. Similar to MEFs, TGCs attach to culture plates faster than TS cells, providing a procedure possible to acquire a pure TS population as described in step 13. Alternatively, because TGCs are highly adherent and more resistant to trypsinization, a pure TS population also may be obtained by quick trypisinization (3 min), followed by collection of the suspended TS cells.
The authors have nothing to disclose.
The authors thank Janet Rossant for the original protocol. This study was supported by National Institute of Health grant CA106308 to WH.
Reagents
TS medium:
RPMI-1640, 20% FBS, 1 mM sodium pyruvate, 100 mM beta-mercaptoethanol and 100 mg/ml penicillin-streptomycin.
Mitomycin C:
Dissolve 2 mg mitomycin C (Sigma M-0503) in 2 ml PBS and add this 2 ml mixture into 200 ml TS medium (10 ng/ml). Make 20 aliquots (10 ml) and store at -20°C until use. Add one aliquot per 100 mm plate.
Mouse embryonic fibroblast-condition medium (MEF-CM):
Plate MEFs in 100 mm plates (1 x 106/plate). Next day, add 10 ml TS medium per dish and continue to culture for 48 hours. Collect the culture medium, filter them (0.45 mm) and store at -20°C in 35 ml aliquots. Thaw each aliquot when needed which can be stored at 4°C. The MEFs can be used to prepare for two more batches of CM before they become confluent.
PBS/BSA:
Dissolve BSA, fraction V (Sigma A3311, 0.1% (w/v)) in PBS (10 ml), filter through a 0.45 mm syringe filter and make 1 ml aliquots in 1.5 ml tubes. Store at -70°C and thaw one tube when needed to prepare FGF4.
FGF4 (1000x):
Resuspend lyophilized FGF4 (Sigma F8424, 25 mg) with 1 ml of the PBS/BSA solution. Mix well and make 10 aliquots (100 ml) into 1.5 ml tubes and store at -70°C. Thaw each tube when needed and store the remaining at 4°C, do not re-freeze. Dilute 1000 times in TS medium to obtain 1x FGF4 (25 ng/ml).
Heparin (1000x):
Resuspend heparin (Sigma H3149, 10,000 units) in PBS to a final concentration of 1 mg/ml (1000x). Make 100 ml aliquots into 1.5 ml tubes and store at -70°C. Thaw aliquots when needed and store the remaining at 4°C, do not re-freeze. Dilute 1000 time in TS medium to obtain 1x heparin (1 ng/ml).