Induction of In Vitro Differentiation in Embryonic Stem Cells

By the suspension culture of cellular aggregates, ES cells can be induced to differentiate along pathways thought to be analogous to those of early embryonic development, leading to the formation of embryoid bodies. These may be simple structures comprising an outer layer of endodermal cells or may progress into fluid-filled, cystic embryoid bodies. These comprise an inner layer of ectodermal-like cells, with a Reichert's membrane separating a presumed outer layer of parietal endodermal cells. These cystic embryoid bodies contain alpha-fetoprotein and transferrin and are thus analogous to the visceral yolk sac of the postimplantation-stage mouse embryo (23).

1. Suspension culture is conducted in agarose-coated dishes. These are prepared by applying a base layer consisting of 2% (w/v) agarose (Type 1; Sigma) in PBS, which is dissolved and sterilized by autoclaving. Approximately 1.5 mL is added per 6-cm dish to give an even layer and is left to set at room temperature. A second, thin layer is then applied using 1% (w/v) agarose in PBS. Once this has set, 5 mL of DMEM10 + 0.1 mM P-mercaptoethanol is added to each dish and incubated to allow equilibration. Before use, the medium should be replaced.

2. ES cells are lightly trypsinized with TEG for 1-2 min. By gently rocking the flask, large clumps of cells detach and the trypsin should then be immediately neutralized with DMEM10 medium + 0.1 mM p-mercaptoethanol.

3. An approx 1/20 aliquot of this aggregate suspension is dispensed into a 6-cm agarose-coated dish. At higher seeding densities, the individual aggregates adhere to each other.

4. Cultures are fed regularly, aspirating the old medium by either transfer ring the suspension into a conical universal to allow the embryoid bodies to settle, or by simply tilting the dish, before fresh medium is added.

5. Utilizing these procedures, simple embryoid bodies form within 2-4 d and become cystic after 7-10 d.

6 If simple embryoid bodies are allowed to attach to a tissue-culture surface, the resulting differentiation is chaotic and a wide range of different cell types form, which may be identified utilizing standard histology or specific cell markers.

3.4.5. Karyotype Analysis of Embryonic Stem Cells Karyotype analysis is used to determine the sex and chromosome complement of a cell line. In order to obtain germ-line transmission of the ES cell genotype in chimeras, it is vital that a high proportion of cells within the cell line have an euploid chromosome complement and a modal number of 40 chromosomes. It is important to check the karyotype of an ES cell line routinely with increasing time in culture, since there is the risk of selecting aneuploid cells, which exist within most ES cell lines. To regenerate a karyotypically normal cell line, the ES cells may be single-cell cloned, and diploid cultures identified and reexpanded.

This section describes the methods to prepare cells in metaphase in order to perform chromosome counts and G-banding analysis, which involves the denaturation of the chromosomes with hot saline and trypsin in order to identify individual chromosomes on the basis of their unique banding pattern, and to determine whether any abnormalities are present. Preparation of Metaphase Spreads

Metaphase spreads of ES cells may be prepared from cultures exposed to colcemid or, preferably, by utilizing cultures in an exponential phase of cellular growth, in order to maximize the number of cells in mitosis. The quality of the mitotic spreads is generally superior when colcemid is not used.

1. ES cells are trypsinized and pelleted in a 20-mL conical universal as described previously (Section 3.4.2.).

2. The medium is aspirated and the cell pellet disrupted, before a Pasteur pipet and bulb are used to introduce dropwise, around 0.5 mL of a 0.56% (w/v) KC1 solution. Once mixed, excess hypotonic KC1 solution is added to make 10 mL and left at room temperature for 15 min, to allow the cells to swell.

3. Following centrifugation (1000 rpm for 5 rain) and aspiration of the supernatant, freshly prepared fixative (3 vol of absolute methanol to 1 vol of glacial acetic acid) is slowly added dropwise, while flicking the tube to prevent the cells from forming clumps.

4. Excess fixative is added to make 10 mL and the cells are left for 5 min, at room temperature, before pelleting (1000 rpm for 5 min) and aspiration of the supernatant. This cycle is repeated two further times, each time with freshly prepared fixative. The cells are finally suspended in around 0.5 mL of fixative and are then ready for producing mitotic spreads.

5 Metaphase spreads of the fixed, swollen cells are made on clean, wet, glass microscope slides that have been chilled on ice. A quantity of the fixed suspension is drawn into a hand-pulled Pasteur pipet (tip diameter ca 1 mm) with a bulb. Several drops of suspension are released onto the slide from a height of around 100 cm.

6. The undersurface of the slide is wiped dry, and evaporation of the fixative is aided by warming the slide briefly in the flame of a Bunsen burner, as well as blowing across the surface of the slide (taking care not to inhale the fixative vapor). The height from which the cells fall and the rate of fixative evaporation are both important variables in maximizing the rupture of the swollen cells and, hence, the spreading of the chromosomes.

7. For determining the modal chromosome number, the slides can be stained immediately in a 3% (v/v) solution of Gurr's Giemsa stain in PBS for 15 min. The slides are then rinsed in two changes of distilled water and allowed to air-dry before counting chromosomes. For the purposes of G-banding, the best staining results are obtained by first storing the slides in a dust-free place for 10-14 d after preparation of the mitotic spreads. G-Banding Analysis

1. Slides are incubated in 2X SSC at 60°C for 1 h. The slides are then rinsed four to five times in distilled water and stored temporarily in a rack under water.

2. Each slide is individually immersed in a 0.25% (w/v) trypsin solution in Gurr's phosphate buffer (pH 6.8) for between 7 and 15 s at room temperature.

3. The trypsin remaining on the slide after this digestion is neutralized in Gurr's buffer containing 5% (v/v) NCS.

4. The slides are rinsed further in two changes of buffer before being stained in freshly prepared 5% (v/v) Giemsa Gurr's R-66 stain in Gurr's buffer (pH 6.8) for 8-10 min.

5. The slides are finally rinsed in two changes of buffer, followed by two changes of distilled water, and allowed to air-dry. The incubation times of the slides in trypsin and stain should be determined empirically in order to optimize the clarity of the banding patterns.

6. G-banded metaphase spreads are examined with a standard format, bright-field microscope utilizing oil immersion, objective lenses (maximum magnification around 1000X). Several suitable mitotic spreads with no or minimal overlapping of chromosomes should be photographed. G-banded chromosomes are identified according to the Standardized Genetic Nomenclature for mice (38), and karyograms constructed to determine the sex of the ES cell line and whether populations exist within the cell line that possess chromosomal abnormalities.

4. Notes

1. In the mouse, ES cells have been isolated from the blastomeres of 16- to 20-cell morulae (33), from the inner cell mass (ICM) of blastocyst-stage embryos (1,2), from the primitive ectoderm of the d 5.5 pc egg-cylinder-stage embryo (19) and recently from d 8.5 pc primordial germ cells (39). ES cell lines derived from the ICM have been obtained from fertilized d 3.5 pc or implantationally delayed blastocysts and from parthenogeneti-cally (40,41) and androgenetically produced blastocyst embryos (42).

Historically, the majority of workers have isolated ES cell lines from the readily available d 3.5 pc blastocyst-stage embryo. ES cell cultures have been obtained from both intact blastocysts and immunosurgically isolated ICMs with equal success (ca. 10%; 28). From experience, blastocysts that have undergone a period of implantational delay are expected to generate, on average, a threefold increase m the efficiency of ES cell isolation compared to nondelayed, d 3 5 pc embryos (19,43). The increase in the potential of delayed embryos to yield ES cells may arise either as a consequence of the small, yet significant increase that occurs in the number of cells in the ICM (44), or may be a result of some epige-netic change in gene expression. Although primitive endoderm has formed in the implantationally delayed embryo, no further differentiation of the ICM occurs (45). Therefore, the normal pattern of expression of genes responsible for the differentiation of the ICM is halted, and this may be an essential feature in the establishment of ES cell lines in vitro.

Recent evidence indicates that there is a period of embryonic development, from d 2.5 to d 8 5 pc, from which pluripotent cells present within the murine embryo can be successfully isolated and maintained in culture as ES cells. Eistetter (33) has reportedly isolated ES cells from individual blastomeres of decompacted morulae with significantly greater effi ciency than from blastocysts cultured conventionally (37 vs 8%). Although possible, the establishment of ES cell lines from postimplantation-stage embryos is both technically more demanding and less successful than from preimplantantion stages (19,39). Since detailed chimeric analyses have not been reported, it is not known whether these blastomere-, primitive ectoderm-, and primordial germ cell-derived cell lines are of the same lineage as ICM-derived ES cells, or if their in vivo developmental potentials are "frozen" at the embryonic stage from which they are isolated.

2. For both ES cell isolation and routine culture, no antibiotics are used. If a culture becomes infected (especially if by fungus or yeast), it is generally wise to discard it from the laboratory. If, however, a particularly valuable culture becomes infected with bacteria, it may be extensively washed in Ca2+- and Mg2+-free PBS before medium containing 50 IU/mL penicillin (sodium salt) and 50 |ig/mL streptomycin is added.

3. Smith and Hooper (35) found that medium conditioned by Buffalo rat liver (BRL) cells contained a factor that was a potent inhibitor of stem cell differentiation. This factor was termed stem cell differentiation inhibitory activity (DIA). Structural and functional comparisons have shown that DIA is identical to the murine myeloid leukemia inhibitory factor (LIF; 15,16). Furthermore, a third growth factor, human interleukin for DA cells (HILDA), has been shown to be essentially identical to the human LIF protein (46,47).

4. It has been found that the osmolarity of DMEM, as supplied by Gibco, is approx 345 mosM/kgH20, and is still considered very high after all of the media supplements and sera have been added (ES2o: ca. 335 mosM/ kg H20 and ES10: ca. 340 mosM/kg H20; 48). Although an initial study found no significant differences in the efficiency of ES cell isolation with media of varying osmolarities (Wells, unpublished data), the plating efficiency of an established ES cell line was observed to have been optimal with medium of 290 mosM/kg H20 (J. McWhir, IAPGR, Edinburgh, Scotland, personal communication). Hence, it is recommended that the osmolarity of both ES10 and ES20 media be reduced to around 290 mosM/kg H20 by the addition of 13% (v/v) sterile water (see Section 2.3.).

5. The function of the feeder cells, in addition to providing a more suitable attachment surface for direct coculture (20), is in the active suppression of stem cell differentiation. It has recently been shown that a factor known as DIA/LIF (see Note 3), which inhibits the differentiation of stem cells, is produced by these feeder cells as both a diffusible protein and in an immobilized form, associated with the extracellular matrix (21). Furthermore, it has been shown that in the coculture sys tem, ES cells secrete a heparin-binding growth factor responsible for the stimulation of DIA/LIF expression in the feeder cells (49). It has also been recently shown that recombinant DIA/LIF, added in the culture medium, can replace feeder cells in the isolation of ES cell lines, which may retain their capacity for germ-line transmission (50,51). However, it is recommended that feeder cell layers be used for the isolation of new ES cells lines (but not necessarily for their maintenance once established; see Section 3.4.1.), since the coculture system may aid in producing cell lines with an euploid chromosome complement.

6. ES2o medium is used routinely for ES cell isolation. However, others have demonstrated the use of medium conditioned on PSA-1 embryonal carcinoma cells (2) or BRL cells (13) to facilitate the isolation of ES cells. However, Robertson and Bradley (43) have observed that although conditioned medium may enhance the growth of primary colonies, the majority tend to be of a "pretrophoblast" lineage and ultimately differentiate. Furthermore, a comparative study has shown no significant effect of cell-conditioned medium on the efficiency of ES cell isolation (28).

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