The characteristics of stem cells are self-renewal (ability to divide indefinitely without differentiating), maintaining a full diploid karyotype, generating any tissue when introduced into an embryo, and colonizing the germ lines of recipient embryos. It is difficult to grow stem cells in culture because of the spontaneity of differentiation. Stem cells must be cultured on a medium that provides signals for maintaining the undifferentiated state or they will proliferate and differentiate without control. The culture, manipulation, and characterization of embryonic stem cells, embryonic germ cells, and adult stem cells in vitro require a mix of nutrients, hormones, growth factors, and blood serum.
Human embryonic stem cells derived from an inner cell mass of blastocysts have the pluripotency to form all three embryonic germ layers; some scientists prefer the term totipotency, because it means the cell can produce any cell in the body, though the suggestion of totality could also be misleading because an individual stem cell has not been shown to be capable of producing an embryo. Traditional culture techniques using mouse fibroblast cells are fine for research but are unsuitable when the stem cell line is intended for human clinical use because of the potential to introduce animal pathogens into the treatment.
Established protocols using a wide range of materials (some available that have been specifically created for embryonic stem cell culturing) and a variety of chemical/biological substrates allow researchers to grow stem cells in vitro. These culture techniques usually include mimicking the in vivo environment in a laboratory by adding molecular components similar to those found naturally within the stem cell niches of the body.
feeder culture
The first step in using a feeder culture is growing the feeder cells or using a prepared formula available from a variety of suppliers. Feeder cells—often mouse (mitotically inactive primary mouse embryonic fibroblasts) or human fibroblasts—are used in culture protocols to keep the stem cells from differentiating. The feeder cells provide secreted factors (many of which have not been identified), extracellular matrix, and cellular contact to keep the stem cells from differentiating and to maintain the normal karyotype. Researchers plate embryonic stem cells onto feeder layers. A limitation of working with feeder cells is cell overcrowding between the feeder cells and the embryonic stem cell colonies. An additional key factor in using feeder cells is to ensure that the density of the feeder cell is sufficient for the delivery of the right amount of factors to maintain the cells in an undifferentiated state without depleting nutrients in the coculture environment, and therefore diminishing the capacity of growth of stem cell colonies.
A Japanese patent has been filed for the use of an immobilized notch ligand protein as a feeder for culturing stem cells. The Notch pathway plays an important role in in vivo stem cell niches of the hematopoietic system, gut, mammary gland, and muscles. The patent calls for using a notch ligand protein on a human cell membrane to maintain the stem cells in an undifferentiated state. The hope is that using this type of feeder will allow the stem cells to be used for cell transplantation and genetic therapy. Other possible feeders to be used for stem cell culturing include human fetal muscle and skin, adult fallopian tube epithelium, and human fibro-blasts from foreskin, skin, endometrial, embryo and placenta, and breast parenchyma cells.
feeder-free culture
In order to culture cells in the absence of a feeder cell layer, coating the culture plate with a permissive substrate is necessary. The substrate provides important adhesion and contact dependent factors necessary to maintaining undifferentiated cell cultures. Mouse embryonic stem cells grow without feeder cells in a supplement of leukemia-inhibiting factor (LIF) to maintain symmetric division and inhibit differentiation. Human cells do not respond to LIF in the same way. To use human embryonic stem cells for clinical therapy, the culture medium for growing and differentiating stem cells needs to be able to provide the nutrients and necessary factors without the possibility of transferring animal or human viruses to the stem cells. A feeder-free culture system is designed to keep the stem cells from differentiating while protecting the stem cells from direct contact with the feeder in an effort to prevent cross-contamination or passing nonhuman pathogens into the stem cells.
Research is progressing with the development of a variety of protocols. For example, increasing the dose of basic fibroblast growth feeder in a serum-free culture, altering the concentration of serum replacement supplements, changing the concentration of growth factors and other nutrients, or using different permissive substrates (e.g., fibronectin) will allow human embryonic stem cells to grow without differentiation, for prolonged periods of culturing and to maintain the pluripotency and normal karyotype of the stem cells. Furthermore, a feeder-free culture system allows for reducing the exposure of growing cells to animal viruses.
A possible substrate for feeder-free culturing is Matrigel (a trade-name protein mixture from BD Biosciences derived from mouse tumor cells; contains laminin and collagen as well), which can be used in a thin layer or as a three-dimensional gel. The gel is appropriate for maintaining undifferen-tiated embryonic stem cells. Laminins are derived from the basal lamina and are glycoproteins creating the structural scaffold in tissues.
