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Stemness et. al.

Ever since Leroy Stevens observed testicular teratocarcinomas in 1% of male mice (Stevens et al., 1954), the discovery, isolation and culturing of human embryonic stem cells has been described as one of the most significant breakthroughs in biomedicine of the century. Cells, that formed the typical colonies, which can differentiate and proliferate, were distinguished as the stem cells of the tumours and so became known as Embryonal Carcinoma Cells (ECCs) (Stevens, 1964). When cultured in bacteriological dishes, and therefore unable to attach to plastic, the cells formed clumps (Martin, 1975) that developed into simple embryoid bodies containing a core of stem cells surrounded by epithelial cells (Martin et al., 1975). They were hard to develop and had unstable chromosomes from the formation of the teratocarcinomas in the Intra Cellular Masses (Stevens 1964). These tumors were hypothesized to contain a pool of malignant stem cells and benign non cancerous cells (Dixon, 1993) and conclusively proved for the derivation of testicular tumors from multi potential germ cells and the ability of normal cells, particularly embryonic normal cells, to reduce or eliminate the malignant potential of neighboring cancer cells, presumably by inducing differentiation (Kleinsmith et al., 1964).

Few years later the embryonic nature of the stem cells in teratocarcinomas were confirmed when retransplantable teratocarcinomas from early mouse embryos grafted to extra uterine sites (Stevens, 1970) and thus led to pluripotency (Solter et al., 1970). Similar differentiated and undifferentiated cells in squamous cell carcinomas were observed, thereby not limiting these cells only to the testicles. (Pierce et al., 1971). A xenotransplantion was attempted into hamster cheek pouch as well (Pierce et al., 1957). Stevens came up with another strain of mice, the 129/terSv, which had and elevated level of the incidence of teratocarcinomas from 1% to 30% ( Stevens, 1973). From then on, until 30 years later, that gene which when mutated, caused such high teratoma incidence was cloned and identified as Dnd1 (dead-end-homologue) (Youngren et al., 2005). Attempts were also made to culture teratocarcinoma fragments and to dissect the process of differentiation from pluripotent stem cells to adult cell types (Pierce et al., 1961). A refinement of culture techniques, most notably the introduction of the cell feeder layer, allowed reliably to subclone mass cultures of pluripotent teratocarcinoma cell lines (Martin, 1975). The first mouse ES cell lines were derived independently by two groups from mouse blastocysts grown on a feeder layer of division-incompetent mouse fibroblasts (Evans et al., 1981 and Martin, 1981). Irrespective of the different methods used, the presence of the feeder layer was crucial even for the derivation of the first human ES cell lines (Thomson et al., 1981) and human embryonic germ-cell lines (Shamblott et al., 1998).

The derivation and characterization of mouse and human ECC lines depended on the existence of suitable markers, which would be able to identify the live cell and had to be absolutely unique to the embryonal carcinoma cell as well as it also had to be lost on differentiation. The first marker was expression of the enzyme alkaline phosphatase, which was highly expressed in mouse and human embryonal carcinoma cells, in the cells within the inner cell mass of the mouse blastocyst, and in ectoderm and primordial germ cells (Benham et al., 1981). With the development of monoclonal antibodies, anti-stage-specific embryonic antigen 1 (SSEA1) proved to be especially versatile and effective in monitoring embryonal carcinoma cell differentiation and isolation of primordial germ cells and is widely used today (Stern et al., 1978). Two further monoclonal antibodies, one raised against mouse embryos (Shevinsky et al., 1982) (SSEA3) and another against human embryonal carcinoma cell lines (Kannagi et al., 1983) (SSEA4), proved to be highly specific for human embryonal carcinoma cells. The transcription factors Oct4, Sox2, and Nanog have essential roles in early development transcriptional circuitry and are required for the propagation of undifferentiated embryonic stem (ES) cells in culture and contribute to their pluripotency and self-renewal ( Rodda et al., 2005).

The first report of a specific chemical compound, retinoic acid, either alone or in combination with dibutyryl cAMP, (also hexamethylbisacetamid (Jakob et al., 1978)) can induce the nullipotent embryonal carcinoma cell line F9 to differentiate into cells that resemble the parietal endoderm led to news frontiers in differentiation (Strickland et al., 1978).

The only true ES cell lines today are only mouse ES cells. As the ability to form germline chimeras are the mandatory criteria but not in case of human ES cells (where the ability to differentiate and the expression of suitable markers) (Gardner et al., 2004). Once it was realized that differentiated derivatives of human ES cells could be used in the therapy of many degenerative diseases and injuries, it also became apparent that it would be of clear advantage if the cells transplanted into patients were genetically identical to the recipients, therefore obviating the need for life-long immunosuppression (Solter et al., 1999). The most obvious way to achieve this is to produce ES cells from the patient by a procedure now known as somatic cell nuclear transfer (SCNT), which is also called therapeutic cloning. Using not the stem cells directly, but the media in which they grow have been shown to have therapeutic potentials which no immunological constraint (Timmers et al., 2007).

However, two groups (Takahashi et al., 2006 and Ludwig et al., 2006) made headlines when they described methods to reprogramme adult human cells to a pluripotent state. These cells, called induced pluripotent stem (iPS) cells, are genetically modified by the integration of up to four DNA-transcription factors into the adult cell genome and not necessarily from embryonic cell fate.

Not a day goes by without news of another advance in steering stem cells, and the cells derived from them towards therapies for treatment (Parson, 2006). Clearly, stem cells and the cells derived from them have great potential to serve medicine – from therapies, to drug testing, to teaching us more about the body’s biology. The technological constraints lie in cell selection, lineage restriction (cell fate determination), stepping up the production level – from petri plates to bio-reactors and therapeutic applications from bench to bedside. Viral vectors were not recommended for therapeutic processes involving human subjects, but of late adenoviral vectors have been used to generate Induced Pluripotent Stem Cells Generated without Viral Integration (Stadtfeld et al., 2008) and that can contribute to cell based therapy greatly.

To sum up, stem cell research has proved something of a political, ethical, social and legal minefield, creating challenges for regulatory bodies, policy makers and scientists as they traverse their way through a tangled web of regulations and moral proselytizing, which only proper dissemination of knowledge can help decode.



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