Embryonic Stem Cells
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Embryonic Stem Cells
Embryonic stem cells (ESCs), by definition, are primary cell cultures that have the ability to proliferate indefinitely with unlimited differentiation potential, both in vitro and vivo. Each ESC has the property of pluripotency, or rather the ability to differentiate into any cell type found in somatic tissues, including germ cells. All ESCs maintain the property of self-renewal, whereas each ESC can divide to form two clonal daughter cells, each with the exact same properties as the original ESC. An ESC may divide asymmetrically as well, meaning one clonal ESC is produced as well as another daughter cell destined for differentiation. With these combined properties, the current and potential applications of ESCs for use in biomedical and biotechnological applications is advancing rapidly.
Beginning in the mid-twentieth century, several investigators began working with an specific type of tumor. This tumor, termed a teratocarcinoma, contained within it many fully-developed tissues normally associated with embryonic and fetal development (including teeth, hair, gut-epithelial tissue, etc.). The disorganized tumor containing fully differentiated tissues suggested that certain primordial cell types were directing its aberrant growth. Eventually, it was discovered that these tumors were related to germ-cell tumors, specifically those arising from the testis. These germ-cell carcinomas led directly to modern embryonic stem cell basic research as a first-cause investigation into the origin of the programming that led to the tumors.
By the 1980's research into mouse embryogenesis was advanced enough to allow for the first isolation and characterization of mouse embryonic stem cells (mESCs) by Evans and Kaufman in the 1980's. These cells were isolation on a monolayer of mitotically-inactivated mouse primary fibroblast feeder cells, aka "feeders" or "MEFs". It was found that the addition of these support cells was necessary for the maintenance of the undifferentiated state of the mESCs in fetal bovine serum-containing media. The replicative and proliferative properties were noticed immediately by researchers, and so more thorough investigations into the mechanisms that allowed these cells to grow like cancer cell lines, but have the properties of primary cell cultures.
Shortly thereafter it was discovered by Dr. Austin Smith that the primary contribution of the feeders is Leukemia Inhibitory Factor (LIF), an IL-6 cytokine family member, that is secreted into the growth media and activates the LIF-JAK-STAT3 pathway to promote mESC self-renewal. These cells were further analyzed to express the canonical markers of pluripotency: Oct3/4, the master transcription factor responsible for the "stem-ness" of the cell; Sox-2, another transcription factor suggested to help re-model the epigenome; Nanog, like Oct3/4, a master pluripotency regulator, and Rex-1, a gene helps regulate the pluripotency network established by the previous three genes. LIF-supplemented culture medium has been sufficient thus far in maintaining most mouse ESC lines for lengthy passages (>P50). However, many "feeder-free" cell lines have adapted to the differing culture conditions by becoming karyotypically abnormal. This again, reflects the need to understand the fundamental self-renewal mechanism to better control these cells in vitro.
Recently, however it has been demonstrated that for feeder-dependent cells lines, LIF is not enough to maintain the pluripotent undifferentiated state once the feeders are removed from the culture system. This so-called mESC "crisis" was first described by Sir Martin Evans. Investigations have tried to identify another soluble factor that may promote self-renewal or actively block differentiation cues, however no known factor has been implicated as yet. Several studies have suggested that the cell-dependent contact is the missing factor, and so many studies have implicated the extra-cellular matrix as a likely candidate. So far, though, the elusive self-renewal factor in mESCs remains un-identified. For most basic research applications however, the current culture system, although hetereogenous, is satisfactory for conducting rigorous testing.
The first human embryonic stem cell (hESC) line was derived in 1998 by Dr. James Thomson at the University of Wisconsin. These cells, like their murine counterparts, require a feeder cell layer to remain undifferentiated. Unlike mouse cells however, is their insensitivity to LIF for self-renewal. hESCs require bFGF, and in some cases, Activin A. A consistent media formulation is still being being debated by different researchers, so there is considerable variation between laboratories in the manner in which the hESC lines are maintained. This debate has fueled the dialogue necessary to develop a standard media for both human and mouse ESCs. Scientists in the field are trying to rid the culture system of the feeders, in hopes of homogenizing culture conditions. But a limited understanding of which signal pathway(s) are involved in both mouse and human ESCs are hindering this movement. Another obvious problem is the lack of homology between human and mouse ESC lines. Other than a few pluripotency markers, such as Oct3/4, Sox-2, Nanog, and Rex-1, differentiation and self-renewal protocols used for each species is disparate at best. Yet, the potential applications for each are still better in comparison to the current tools available to researchers and clinicians now.