Guidelines for Human Embryonic Stem Cell Research

Inner Cell Mass

Unfertilized Egg Fertilized Egg Oocyte Zygote

Morula (3-4 days)

Blastocyst (5 days)

FIGURE 2.1 Preimplantation development. The oocyte (unfertilized egg) combines with sperm to form a zygote (fertilized egg). Each gamete (oocyte or sperm) is haploid (has a single set of chromosomes); the zygote and all later cells are diploid (have two sets of chromosomes). The zygote then divides approximately once a day. Since there is no growth during this period of cell division (cleavage), the cells become progressively smaller. By 3-4 days, a ball of cells (morula) has formed. By 5 days, it has become hollowed out to form a blastocyst, which consists of a sphere 0.1-0.2 mm in diameter comprising two cell types—an outer shell of trophectoderm cells and an inner collection of 30-34 cells called the inner cell mass. By day 6, the blastocyst would normally implant into the uterine wall, the trophecto-derm would begin to form the placenta, and the inner cell mass would begin to form the cells and tissues of the fetus. At the blastocyst stage, cells of the inner cell mass are undifferentiat-ed and pluripotent; that is, they have the potential to differentiate into all cells of the fetus except the placenta. If separated from the blastocyst and cultured, the cells of the inner cell mass can be converted into embryonic stem cells that are also pluripotent and can be propagated extensively while maintaining that potential. Blastocyst picture from http:// stemcells.nih.gov/info/scireport/chapter3.asp.

organisms. The latter subject raises possibilities of cell-based therapies to treat disease, often referred to as regenerative medicine.

Scientists discovered how to obtain or derive embryonic stem cells from mouse blastocysts in the early 1980s (Evans and Kaufman, 1981; Martin, 1981) by cultur-ing inner cell masses on feeder layers of mouse fibroblasts. It was later discovered that feeder cells could be replaced with culture medium containing the growth factor leukemia inhibitory factor (LIF)(Smith et al., 1988; Williams et al., 1988). Mouse ES cells (mES cells) have been studied in the laboratory, and a great deal has been learned about their essential properties and what makes them different from specialized cell types.

mES cells are shown to be pluripotent using three kinds of tests. The first and most rigorous test is to inject mES cells into the blastocoel cavity of a blastocyst (Stewart, 1993). The blastocyst is then transferred to the uterus of a pseudopreg-nant female (a female primed to accept implanted blastocysts). If the mES cells are pluripotent, the resulting progeny will be a chimera because it consists of a mixture of tissues and organs derived from both the donor mES cells and the recipient blastocyst. In some cases, a fetus can be derived entirely from mES cells by providing trophectoderm cells from another source (Nagy et al., 1990, 1993). However, mES cells cannot themselves form a functional placenta and therefore are not equivalent to an intact blastocyst. The ability of mES cells to generate a complete embryo tends to decline with the number of times the cells have divided (or been "passaged") in culture.

A second approach for testing pluripotency of mES cells is to inject them into the testis or under the skin or kidney capsule of an immunodeficient mouse. If pluripotent, the injected cells form benign tumors known as teratomas. The terato-mas contain differentiated tissues from all three germ layers (ectoderm, mesoderm, and endoderm). Such structures as gut, muscle (smooth, skeletal, and cardiac), neural tissue, cartilage, bone, and hair are found, but they are arranged in a disorganized manner (Martin, 1981).

A third approach for testing pluripotency of mES cells is by in vitro differentiation (Wiles, 1993). Spontaneous differentiation can occur if the mES cells are grown in suspension without feeders or LIF. The cells will form fluid-filled clumps called embryoid bodies, which will differentiate along the ectoderm, mesoderm, and endo-derm pathways. If the embryoid bodies are allowed to attach to the tissue culture dish, they will differentiate into multiple tissue types much like teratomas.

Developmentally relevant signaling factors can also be used to induce mES cells to differentiate into specific cell types in vitro, including hematopoietic stem cells, beating cardiac muscle cells, neuronal progenitors, endothelial cells, and bone cells. In some cases, those differentiated cell types can be transplanted into animals to form functional tissues (Lanza et al., 2004). Such work engenders excitement about regenerative medicine using hES cells. One of the milestones of mES cell research was the development of methods to modify the cells genetically (Doetschman et al., 1987; Thomas and Capecchi, 1987). The evolution of those methods has revolutionized animal models for biomedical research by allowing one to modify endogenous genes or to tag the cells so that they can be easily visualized in the animal.

Bongso et al. (1994) first described isolation and culture of cells of the inner cell mass of human blastocysts in 1994, and techniques for deriving and culturing stable hES cell lines were first reported in 1998 (Thomson et al., 1998). The trophectoderm was removed from day-5 blastocysts, and the inner cell mass, consisting of only 30-34 cells, was placed into tissue culture. Cell lines similar to mES cells were derived after fairly extensive culture and passaging of the cells. Cells with similar properties were reported at about the same time from culturing cells isolated from fetal genital ridges—so-called human embryonic germ (hEG) cells (Shamblott et al., 1998). It had previously been shown that the germ cells in fetal mouse gonads can give rise to permanent pluripotent stem cell lines in culture, mEG cells (Matsui et al., 1992; Resnick et al., 1992). Under appropriate culture conditions, hES cells were shown to be pluripotent by differentiating into multiple tissue types (Itskovitz-Eldor et al., 2000; Reubinoff et al., 2000). Since 1998, research teams have refined the

techniques for growing hES cells in vitro (Amit et al., 2000; Itskovitz-Eldor et al., 2000; Klimanskaya and McMahon, 2004; Reubinoff et al., 2000). Collectively, the studies indicate that it is now possible to grow karyotypically normal hES cells (that is, with correct chromosome number) for more than a year in serum-free medium on mouse fibroblast feeder layers. Both XX (female) and XY (male) hES cell lines have been established. The cells express markers characteristic of pluripotent and proliferating cells. Work with hEG cells has also shown pluripotency and extended self-renewal, but more extensive work has been done with hES than with hEG cells.

There are differences between mouse and human ES cells (Pera and Trounson, 2004). For example, mES cells grow as rounded colonies with indistinct cell borders, while hES cell colonies are flatter and display more distinct cell borders. The two cell types also demonstrate differences in growth regulation. In general, both mES and hES cells require fibroblast feeder cell support. Current attempts to substitute for that support have required different approaches for the two species. The soluble growth factor, LIF, can substitute for a feeder cell layer in maintaining mES cells, but hES cells require a solid extracellular matrix (Matrigel) in place of the fibroblasts (Xu et al., 2005). Those examples of interspecies differences indicate that if one is to identify signals that cause stem cells to differentiate into specialized cells, work needs to continue with both hES and mES cells.

Embryonic stem cells have three important characteristics that distinguish them from other types of cells. First, hES cells express factors—such as Oct4, Sox2, Tert, Utfl and Rex—that are associated with pluripotent cells (Carpenter and Bhatia, 2004). Second, they are unspecialized cells that renew themselves through many cell divisions. A starting population of stem cells that proliferates for many months in the laboratory can yield millions of cells. An important research challenge is to understand the signals that cause a stem cell population to remain unspecialized and to continue to proliferate until they are needed for repair of a specific tissue.

A third characteristic of hES cells is that under some physiological or experimental conditions in tissue culture they can be induced to become cells with special functions, such as cardiomyocytes (the beating cells of the heart), liver cells, nerve cell precursors, endothelial cells, hematopoietic cells, and insulin-secreting cells (Assady et al., 2001; Chadwick et al., 2003; Kaufman et al., 2001; Kehat et al., 2001; Levenberg et al., 2002; Mummery et al., 2002; Reubinoff et al., 2001; Reubinoff et al., 2000; Xu et al., 2002; Zhang et al., 2001). However, because hES cells have not yet been used in blastocyst chimera studies, researchers have been able to assess in vivo differentiation only after injection of hES cells into immunodefi-cient mice. There, the cells create teratomas in which tissues of the three embryonic germ layers are found (Thomson et al., 1998). Examples are bone and cartilage tissue, striated muscle, gut-like structures, neural rosettes, and glomerulus-like structures. More organized structures—such as hair follicles, salivary glands, and tooth buds—also form. hES cells will also create embryoid bodies and differentiate in vitro (Itskovitz-Eldor et al., 2000). However, those types of differentiation assays do not provide conclusive evidence that the resulting cell types are functioning normally, nor whether hES cells have the capacity to participate in normal development in the context of the three-dimensional embryo in the reproductive tract. Such conclusive evidence requires testing in blastocyst chimeras as is routinely done with mES cells.

Understanding why ES cells are able to proliferate essentially indefinitely and retain the ability to be induced to differentiate and stop proliferating will provide important information about the regulation of normal embryonic development and the uncontrolled cell division that can lead to cancer. It is known that external signals for cell differentiation include chemicals secreted by other cells, physical contact with neighboring cells, and molecules in the microenvironment. Identifying such factors would allow scientists to find methods for controlling stem cell differentiation in the laboratory and thereby allow growth of cells or tissues that can be used for specific purposes, such as cell-based therapies.

Several methods have been shown to be effective for delivering exogenous genes into hES cells, including transfection by chemical reagents, electroporation, and viral infection (Eiges et al., 2001; Gropp et al., 2003; Ma et al., 2003; Pfeifer et al., 2002; Zwaka and Thomson, 2003). Those are all critical methodological objectives that must be met if hES cells are to be used as the basis of therapeutic transplantation.

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