Isolation of Primordial Germ Cells

Primordial germ cells can be obtained from different stages of embryos. Subheading 3.1.1. will describe how to dissect early embryos (8.5 d pc) in order to obtain early migratory PGCs, whereas Subheading 3.1.2. will describe how to isolate the genital ridge from later embryos (11.5 d pc and 15.5 d pc) in order to obtain later PGCs. Subheading 3.1.3. describes how these isolated tissues should be processed for in vitro culture. Further information about the numbers of PGCs and their location in the embryo at various times can be found in refs. 18 and 19.

Since the ultimate goal of these dissections is to generate PGCs for in vitro culture, care should be taken to maintain sterility at all times. Dissections are done using sterile PBS with 0.1% BSA (0.1 g/100 mL) and sterilized instruments.

3.1.1. Dissection of 5-8 Somite Stage Embryos

In the 8.5 d pc embryo, most of the PGCs are located in the posterior third of the embryo at the base of the allantois. After the embryo is removed from the uterus, decidual tissue, and yolk sac, it will appear as in Fig. 1A. Details of this

Drawing Shadows

Fig. 1. (A,B) A 8.5-d pc embryo dissected free of the uterus, decidual tissue, and extraembryonic membranes. At this stage, the majority of the PGCs are located at the base of the allantois. In order to isolate the PGC-containing tissue, the embryo is cut at the position of the dashed lines and the indicated tissue processed further. (C) An embryo at 10.5 d pc with the position of the genital ridges indicated by the box. (D) An isolated 10.5-d pc genital ridge showing the position of the mesonephros relative to the developing gonad. (E) An embryo at 15.5 d pc with the positions of the developing ovary or testis (depending on the sex of the embryo) illustrated by the boxes. (F) Isolated gonads from 15.5-d pc embryos. Note the well-differentiated tubules in the developing testes. Abbreviations: al, allantois; gr, genital ridge; hf, head folds; ov, ovary; ts, testes.

Fig. 1. (A,B) A 8.5-d pc embryo dissected free of the uterus, decidual tissue, and extraembryonic membranes. At this stage, the majority of the PGCs are located at the base of the allantois. In order to isolate the PGC-containing tissue, the embryo is cut at the position of the dashed lines and the indicated tissue processed further. (C) An embryo at 10.5 d pc with the position of the genital ridges indicated by the box. (D) An isolated 10.5-d pc genital ridge showing the position of the mesonephros relative to the developing gonad. (E) An embryo at 15.5 d pc with the positions of the developing ovary or testis (depending on the sex of the embryo) illustrated by the boxes. (F) Isolated gonads from 15.5-d pc embryos. Note the well-differentiated tubules in the developing testes. Abbreviations: al, allantois; gr, genital ridge; hf, head folds; ov, ovary; ts, testes.

dissection can be found in ref. 20. Briefly, the embryos are removed from the uterus, and the broader end of the decidua is cut away. The embryo must then be gently shelled out of the remaining decidual tissue and yolk sac. In order to isolate large numbers of PGCs, the anterior two-thirds of the embryo and the allantois are discarded, as indicated in Fig. 1A and B. The remaining tissue is then processed as in Subheading 3.1.3.

3.1.2. Dissection of Genital Ridges and Embryonic Gonads

Alternatively, if later PGCs are desired, they can be obtained from the developing genital ridge. After the 8.5 d pc stage, the PGCs become incorporated into the hindgut of the embryo. They subsequently migrate into the dorsal mesentery and then the genital ridge by 10.5-11.5 d pc. By this stage of embryogenesis, the genital ridges are attached to the dorsal body wall on either side of the spinal column. Their position inside the embryo is illustrated in Fig. 1C. At 10.5 d pc, it is often difficult to differentiate between a developing testis and a developing ovary in the light microscope as shown in Fig. 1D. In order to remove the genital ridges, the embryo is dissected out of the uterus and the extraembryonic tissues are removed. The embryo should be killed by pinching off the head with forceps. Next, the intestines and liver are removed from the body cavity. The genital ridges lie attached to the dorsal wall of the body cavity, adhered tightly to the developing mesonephros. A genital ridge from a 10.5-d pc embryo is shown in Fig. 1D. The genital ridge and the mesonephros are gently teased apart with forceps, and the genital ridge is processed as in Subheading 3.1.3. Be careful to avoid disrupting the integrity of the genital ridge, since PGCs can flow out if it is poked or torn.

Using a similar approach, germ cells can be isolated from later developing gonads. Shown in Fig. 1F are testes and ovaries from 15.5-d pc embryos. At this stage, it is now easy to distinguish between a testis and an ovary, since the tubules in the testes are well differentiated. Again, the embryo should be sacrificed by removing the head, and then the intestines and liver are removed in order to isolate the gonads. Testes will be located in the caudal region of the pelvis (above the hips), but ovaries will be located further rostrally near the kidneys. Their location is illustrated in Fig. 1E.

3.1.3. Isolating Germ Cells for Culture

Depending on the experiment, PGC-containing tissues are either pooled at this stage or treated individually. Isolated tissues are kept on ice while the remaining dissections are completed. Using a mouth pipet (see Note 6), the isolated tissue is rinsed through at least two 30-^L drops of PBS, and then placed into a drop of trypsin, being careful to minimize the amount of PBS transferred so as not to dilute the trypsin. The tissue is mechanically disrupted by tearing with forceps, and the sample is incubated at 37°C for 5 min. Then the tissue is again mechanically disrupted using a finely pulled Pasteur pipet. The internal diameter of the pipet should be approx 75% smaller than the tissue that is being treated. It is often necessary to use a series of pipets, decreasing in diameter, as the tissue is disrupted. The internal diameter of the pipets range between 200 pm for the largest genital ridges down to 30-50 pm for single cells. Depending on how well the cells are disrupted, the plate can be returned to the 37°C incubator for an additional 5 min. This is repeated until the tissue has been reduced to a single-cell suspension. This should be fairly easy for a 8.5-d pc embryo and a 11.5-d pc genital ridge. However, the size and extent of development of a 15.5-d pc genital ridge makes more vigorous enzymatic treatment necessary. In this case, a mixture of collagenase and dispase can be used at 37°C. Again, it is best to disrupt the tissue mechanically as much as possible before enzymatic treatment, and also to check the tissue at 5-min intervals, incubating for up to 30 min total. Despite these efforts, it is often difficult to dissociate the testes tubules, and any remaining large clumps of intact tissue should not be plated out in the next step. However, plenty of germ cells will be released from the tubules and can be collected with a mouth pipet (see Note 7).

Once the tissue is primarily a single cell suspension of somatic cells and PGCs, the cells are plated onto mitotically inactivated feeder layers. One kind of feeder cells that can be used is Sl4m220 fibroblasts. Alternatively, a mouse embryonic fibroblast line, STO, can also be used (see Note 3). Feeder cells are mitotically inactivated with either mitomycin C or irradiation (see Note 4) and plated onto gelatin coated tissue culture dishes at a density of 1.5 x 105 cells for each well of a 24-well dish (1 cm in diameter). For convenience, feeder layers are usually prepared the day prior to the dissection. However, most feeder cells have adhered by 2-4 h after plating, so the feeder plates could be prepared the same day as the dissection.

At this stage of the procedure, cells from the equivalent of one 8.5-d pc embryo are plated into each well. Similarly, 1/10 of a 11.5-d pc or 15.5-d pc genital ridge is placed into each well. Growth factors are added as desired. If the goal is to produce EG cell lines, the growth factors should be added in the following concentrations: 20 ng/mL LIF, 60 ng/mL SCF, and 20 ng/mL bFGF. All cultures are maintained in a humidified incubator at 37°C with 5% CO2. Cultures are fed every day with fresh medium and growth factors, and monitored for possible yeast and/or bacterial contamination. It beyond the scope of this chapter to describe the effects of all tested growth factors and cytokines on the growth and differentiation of PGCs. An excellent review of these experiments is contained in ref. 21 and references therein.

If the generation of EG cell lines is desired, a secondary culture is generated from the primary culture after 10 d. It is a good idea to stain some portion of the primary cultures for alkaline phosphatase activity to be sure that the PGCs are present and growing before proceeding further. If there are no alkaline-positive cells in the primary culture, the experiment should be stopped at this time. To begin a secondary culture, primary cultures are washed in PBS and trypsinized for 5 min at 37°C. Add an equal volume of serum-containing culture medium to inactivate the trypsin, and pipet up and down 5-10 times to obtain a single-cell suspension. This suspension is then distributed onto fresh feeder plates at a 1:4-1:10 dilution. Secondary cultures need to be monitored daily for the appearance of EG colonies. These colonies can then be isolated and grown as cell lines, although it is not clear that they are clones and they will undergo changes in culture (16). Once EG cell lines are generated, they can be grown in the same manner as ES cell lines on feeder layers from either primary mouse embryo fibroblasts or STO fibroblasts, with only LIF added to the medium, since bFGF and SCF are no longer necessary. Single-cell suspensions of individual cell lines should be frozen at low passage numbers in a freezing medium of 10% DMSO, 20% fetal bovine serum in culture medium, and stored indefinitely under liquid nitrogen. It should be noted that despite many attempts in this laboratory and others (16), EG cell lines have not been generated derived with PGCs from late genital ridges (over 12.5 d pc).

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