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Embryonic Stem Cells

physiological differences genetic clinical human gene mouse mice

Mammalian embryonic stem (ES) cells have the special property of being able to differentiate into virtually every cell type. Because ES cells can be genetically manipulated in vitro and can be transplanted into embryos and adults, they are a powerful tool in biological experiments and hold promise for future medical therapies.

The ability to differentiate into all cell types, a property known as pluripotency, arises from the fact that ES cells are isolated from in vitro outgrowths of early stage embryos (in the mouse, at three and one-half days, at the blastocyst stage). These outgrowths are cultured in specialized conditions—often in the presence of support cells, called feeder cells, which do not proliferate, and specific growth factors. The ES cells proliferate rapidly in culture, and clonal (identical) populations can readily be initiated from single cells.

Until 1998 the only mammalian embryonic stem cells isolated were those from the mouse. The first human embryonic stem cells were isolated in 1998 from embryos created through in vitro fertilization. In the United States, government funding for research on human ES cells was restricted in 2002 to a small set of existing human ES cell lines. Only privately funded research remained able to use other sources of cells.

In the following years, most publicized research on human ES cells will concentrate on manipulating cells in culture, attempting to understand the signals that cells require to proliferate and differentiate into various adult cell types, such as nerve or muscle cells.

In 2001 there was a highly publicized report of human cloning using human ES cells, but the resulting embryos did not progress past the twelve-cell stage, and there was no reliable evidence that the ES cells ever contributed to the development of the embryos. Nonetheless, most researchers thought obstacles would likely be overcome, and that the use of ES cells for therapy or cloning would become technically possible.

Mouse ES cell research is much further advanced. Research in the mouse has included injection of ES cells into blastocysts, an early stage in embryo development. Once injected, ES cells fully participate in embryonic development and form part of every tissue in the embryo. The resulting mouse is called a chimera, since it usually contains a mixture of cells derived from Figure 1. Mouse embryonic stem cells in culture. both the ES cells and the host blastocyst. An important feature of a chimera is that its ES cells will contribute to forming the germ cells, which eventually form the gametes of the animals. Consequently, mating of the chimera with recipient females allows the (ES cell derived) sperm to deliver their genetic material into an egg, and subsequent offspring will carry genes from the ES cell. Thus genetic manipulations made in ES cells in culture can be transmitted through chimeras to intact animals.

As with other cell lines grown in vitro, it is possible to add new genes, termed "transgenes," or to modify existing genes in ES cells using a technique known as gene targeting. Gene targeting involves using specialized DNA vectors that share substantial DNA sequence similarity with the gene that needs to be modified. When introduced into cultured ES cells, the genetargeting vector undergoes "homologous recombination," a process in which the vector is integrated into the existing chromosomal gene and leaves the ES cells genetically modified.

Removing a gene product is often referred to as creating a "knockout," since it prevents a protein from being expressed. Modifying a specific gene Figure 2. A typical chimera. ES cell-derived tissue is identified as light patches in the mouse coat. in the genome to include a single DNA nucleotide change (mutation) or incorporating a genetic marker (such as a gene that produces a colored protein that can be seen with a microscope) is accomplished by a similar method and is often referred to as creating a "knock-in"—since a piece of DNA is inserted into a specific part of the genome.

The genetically modified ES cells can be used to create mouse chimeras, whose offspring will carry the genetic modifications into the ES cells. These mice can be identified by analysis of DNA made from a small tissue sample, usually taken from the mouse's tail. The genomic DNA can be analyzed by polymerase chain reaction (PCR) or Southern blot to identify the mice carrying the genetic change. These offspring will be heterozygous for the introduced mutation and can be interbred to generate a second generation, some of which will be homozygous for the introduced mutation. The homozygous mice will lack both copies of the normal gene—any differences to normal development displayed by the mice as a result of losing the gene of interest can be analyzed. This, in turn, can provide valuable clues to the function of the gene and its normal role in mouse physiology.

This experimental approach to genetic manipulation of mice, which was pioneered in the 1980s, has been used to modify thousands of mouse genes and has played a significant role in understanding many physiological and pathological processes. For example, mutations in the p53 gene show its involvement in cancer, and mutations in the psd-95 gene show involvement in learning and memory. Variations on these methods are also used to model human disease mutations in mice and to test drug therapies.

ES cells also have potential for treating human diseases through "cell-based" therapy. In this context, the ES cells (or their differentiated derivative cells) are transplanted into patients whose own cells are defective or degenerated. This strategy could have applications for diseases such as Parkinson's disease (which affects brain cells), diabetes (pancreatic cells) and heart disease (which affects heart muscle cells). An important clinical issue at this point will be whether ES cells not derived from the patient will be rejected by the patient's immune system. If so, one strategy for dealing with this problem would be to use a patient's own cells to create an embryo by nuclear transfer, from which ES cells compatible with that patient could then be derived.

The use of human blastocysts in both research and therapy remains controversial because it is necessary to destroy the blastocyst to generate an ES cell line from it. As a result, work with human embryos is governed by strict regulations in many countries.

Seth G. N. Grant

and Douglas J.C. Strathdee

Bibliography

Donehower, L. A., et al. "Mice Deficient for p53 Are Developmentally Normal but Susceptible to Spontaneous Tumors." Nature 356 (1992): 215-221.

Holland, Suzanne, Karen Lebacqz, and Laurie Zoloth, eds. The Human Embryonic Stem Cell Debate: Science, Ethics, and Public Policy. New York: MIT Press, 2001.

Juengst, Eric, and Michael Fossel. "The Ethics of Embryonic Stem Cells—Now and Forever, Cells without End." Journal of the American Medical Association 284 (2000): 3180-3184.

Migaud, M., "Enhanced Long-Term Potentiation and Impaired Learning in Mice with Mutant Postsynaptic Density-95 Protein." Nature 396 (1998): 433-439.

Thomson, James, et al. "Embryonic Stem Cell Lines Derived from Human Blastocysts." Science 282 (1998): 1145-1147.

Turksen, Kursad, ed. Embryonic Stem Cells: Methods and Protocols. Totowa, NJ: HumanaPress, 2002.

Internet Resources

Embryonic Stem Cells. University of Wisconsin-Madison. <http://www.news.wisc.edu/packages/stemcells/>.

Stem Cell Primer. National Institutes of Health. <http://www.nih.gov/news/stemcell/primer.htm>.

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