January 15, 2006 (Vol. 26, No. 2)
Culture and Maintenance
The beginning of embryonic stem cell (ESC) research can be pegged to 1981 when the first reports of the derivation of mouse embryonic stem cells (mESC) were made.1,2
Scientists subsequently showed that mESC could be genetically manipulated in vitro. In addition, it was demonstrated that the manipulated cells retained their ability to integrate into the inner cell mass (ICM) of the mouse blastocyst and contributed to tissues of the developing embryo, including the germ cells through the generation of chimeric animals derived from the engineered mESC line and the host blastocyst.3
In the years following these initial studies methods have been developed and refined that allow for relatively straightforward manipulation of the mouse genome via mESC intermediary.4
However, the quality of the mESC line used for the in vitro manipulation is often the major stumbling block to developing the mouse model containing the modified gene locus.
The Figure depicts an overview of the applications of in vitro and in vivo work resulting from murine ESC development. This article will describe the major factors that affect the quality of the ESC and its ability to generate germline chimeras.
To efficiently integrate into the ICM and produce germline chimeras, mESC cultures must be robust, fast growing, and maintained under conditions that allow for proliferation, but limit differentiation. This is determined by the overall laboratory conditions and operations, the quality of the culture medium, the quality of the feeder layer, and the quality of the mESC line when experiments are initiated.
Undertaking an ESC project is expensive and time consuming. ESCs require more care and attention than most cell types cultured in the laboratory.
The best results are achieved by dedicating appropriate space, equipment, and personnel to the project. This is true whether the experiments are in vitro differentiation or in vivo mouse model development. Upfront investment of the proper resources will lead to more predictable and successful experiments and will save time and money in the long run by limiting the amount of down time and re-work.
The culture lab should (at a minimum) be located in a quiet, low traffic area and be equipped with a 4-foot tissue culture Class II biosafety cabinet; ESC-only CO2 incubator; a phase contrast microscope with 4x, 10x objectives; access to: 4C, -20C, -80C, -140C refrigerator and freezer space for storage of media, reagents, and cell stocks.
The quality of the mESC line at the time of initiating the experiment is one of the most important factors determining the success of an experiment, whether the experiment is in vitro differentiation or in vivo production of chimeric mice. In general, the passage number has the biggest overall impact on the pluripotency of ESC lines.
The most widely used mESC lines have been derived from the 129 strain of mouse. This strain has several substrains and each has a different genotype and phenotype.5
When using 129-derived ESC lines it is a good idea to learn from what substrain the line was derived and to use this substrain for developing inbred lines. Differences in substrains can also affect the targeting efficiency depending on the level of conservation of the locus between substrains of the cell line and the targeting vector.
In general, it is a good idea to know the exact strain of mouse from which the ESC line has been derived.
Assuming that the ESC line has been properly cultured in high quality medium and by trained personnel, the greatest impact on the lines pluripotency is the passage number. The longer a cell line is maintained in culture the more genetic and epigenetic changes it acquires.6,7 Eventually these small mutations tip the scales and the line is no longer pluripotent.
Therefore, frequently freezing back vials as the line is expanded then starting experiments with vials of cells from validated batches is crucial to success when working with ESC and should be part of standard operating procedures.
ESCs require daily attention, which is best provided by dedicated personnel who are able to monitor day-to-day changes in the culture and develop the skills required to determine when and how to passage the cells, the proper time to initiate experiments, freeze stocks, or terminate the culture and start over.
While a few mESC lines have been converted from conventional culture conditions to growth on gelatin without a mouse embryonic fibroblast (MEF) feeder layer or in other minimal media these lines are the exception.8 mESC growth on a MEF feeder layer in high quality medium that contains fetal bovine serum (FBS) and mouse leukemia inhibitory factor (LIF) is the traditional, most common, and most successful culture method to date.
The incubator should be dedicated to ESC culture and access in and out of the incubator should be limited. It is best to limit the time that the culture is out of the incubator and the incubator doors are open to the air. The cells are sensitive to changes in pH.
While most laboratories culture their ESC at 5% CO2, it is important to know the pH of the basal medium at that concentration of CO2. DMEM was originally formulated with bicarbonate levels that work optimally to maintain mammalian physiological pH in a 10% CO2 environment.
When using the traditional formulation of DMEM basal media, Primogenix (www.primogenix.com) cultures at 7.5% CO2 with a temperature of 37C.
High-quality basal media and supplements are crucial to successful and predictable experiments. Today there are many good and reliable sources of basal media and supplements.
However, there is a certain amount of variability in culture media, which comes primarily from the addition of serum or serum substitutes, which are either wholly or partially, animal derived.
Serum is a biological product; therefore variation exists from lot to lot. Finished FBS is typically produced in large batches of up to 3,000 liters originating from several thousand individual animals. This pooling method minimizes the variability of cell culture performance.
However, raw serum samples can vary in the age of animal at collection, animal diet, country of origin, and various other factors. With large lots, the likelihood of consistent cell culture performance increases. Additionally, many vendors now test lots for compatibility with ESC culture.
Serum substitutes and replacements, which are more defined than serum, may improve ESC experimentation by providing a more predictable culture medium and eliminating some of the variability found in serum products. But it is important to remember that currently available serum substitutes are biological materials derived from serum.
With this potential for variability in mind, thorough testing should be performed to determine long-term stability and pluripotency of an ESC line both when changing media or culture conditions.
Monitoring and Analysis
Morphology in Culture. Mouse ESC grow rapidly, dividing every 1020 hours depending on the line. Often the faster growing lines, doubling in the 1012 hour range, produce the best germline chimeras.
The morphology of ESC culture is dependent on the substrata. When grown on MEFs, undifferentiated mESC cultures have a distinctive morphology. The cells have a high nuclear to cytoplasmic ratio; they grow in small, tightly clustered colonies with tight phase bright borders. It is difficult to identify individual cells within a colony of undifferentiated mESC.
Karyotype. Monitoring the karyotype of the ESC line is critical. While a normal karyotype is no guarantee that a line is lacking genetic abnormalities, detection of an abnormal karyotype will allow one to eliminate clones, subclones, stocks, and even ESC lines from further experimentation.
Since human ESC lines are often carried continuously in culture for six months or longer, it is recommended that they be karyotyped every 1015 passages9.
Markers of Pluripotency. The list of pluripotency markers keeps growing and it is clear that when ESC stop expressing some of these markers, the cells are no longer pluripotent and are unlikely to produce chimeras or predictably differentiate in vitro.
Germline transmission remains the the gold standard for mouse-derived ESC lines. Despite all the new markers of pluripotency identified, the best test for pluripotency of mouse-derived ESC lines is to inject them into a blastocyst to determine how efficiently they produce germline chimeras.
Although scientists have been using mESC for nearly 25 years, ESC science is in its early stages. The use of mESC to generate mouse knockouts has led to a better understanding of gene function in vivo.
Thomsons 1998 report in Science of the derivation of human ESC lines10 sparked a renewed effort to understand the basic biology of ESC self renewal and in vitro differentiation capacity of ESCs, both mouse and human.
Many labs are developing culture media and paradigms that will allow for directed and predictable differentiation into specific cell types. Others are working to define conditions that will allow for more routine culture in an undifferentiated state.
While new culture media are being developed, the media, supplements, and sera substitutes should be carefully tested so that one is aware of and understands the effect these new media have on the determinants used to describe the characteristics of ESCs such as morphology, expression of markers, and, ultimately, their function.
As methods to measure and define pluripotency and direct differentiation improve, confidence in these new media will increase, and fully defined culture media that either promotes proliferation or directs differentiation are likely to be developed. This will be a boon to the science as well as the potential therapeutic application of stem cells.