Stem Cell Isolation

a) Sources for stem cell isolation
Stem cells can be isolated from different sources. They can be extracted from embryonic tissues with high efficiency because immature tissues contain many undifferentiated (stem) cells. Furthermore, in the embryo, there exist different populations of stem cells that are hierarchically arranged. This means that different stem cells can be isolated from a developing organism, and all of these, in a general sense, may be considered ot be embryonic stem cells. In fact stem cells have been isolated from the morula, blastocyst, placenta, amniotic fluid, organ primordia (e.g. embryonic brain), umbilical cord blood and so on. However, the term embryonic stem (ES) cell in a narrow sense has been reserved only for cells yielded from the inner cell mass of the blastocyst. Historically these were the first stem cells isolated from the embryo (murine ES in 1981, human ES in 1998). Since then the pluripotent ES cells have been cultured beyond the Hayflick limit without changing their phenotype and genotype. As a result ES cells can be distributed form one laboratory to another and can be used as a source for in vitro production of all cell types of our body or for the generation of chimaeric animals. The stem cells do not disappear from mature tissues postnatally. They remain there and become "diluted" among differentiated cells but they change their properties in response to the change of niche and new demands of the organism. Tissue-specific stem cells can be harvested from different tissues and at different time-points: from children tissues, adult tissues, tissues of elderly people and even post mortem. In fact adult stem cells are the most frequently discussed in terms of therapeutic applications. Stem cells also occur in tumours and these cells are responsible for the growth of cancers.

b) Methods of stem cell isolation
There are different approaches for the isolation of stem cells. Factors influencing the method of choice depend on the source and type of cells and the donor age. Many methods involve the enrichment of stem cells in the cell suspension by elimination of unwanted differentiated cells, rather than direct stem cell isolation. This may be achieved, e.g. by targeting cell specific markers with corresponding antibodies to label the differentiated cells with ferromagnetic nanoparticles (immunomagnetic separation) or to lyze them after incubation with complement proteins. Similar results may be reached in vitro by removal of factors that support survival of differentiated cells, e.g. by removal of pro-differentiation factors in serum and by using plastcic dishes that do not support cell adhesion. These methods are often combined with those that stimulate proliferation of stem cells while preserving their undifferentiated status, e.g. cell cultivation with the help of mitotic growth factors. Cell cultures permit propagation of stem cells in vitro and their expansion (under stimulation with growth factors, stem cells may undergo symmetric divisions and increase their numbers). The cells grow and divide, eventually covering the dish in which they are cultured. A long-term cultivation of cells is achieved by the cell passaging, i.e. replating them in a new culture dish after the cells reached a high density (e.g. 70% confluence). Since few stem cell-specific factors have also been identified, these may be recognized by antibodies used, e.g. for fluorescence-activated cell sorting (click here then 'other' then 'flow cytometry' to see an animation, click here to see an explanation from wikipedia). For stem cell isolation, one can also take advantage of their special physical properties (for example they may have a large nucleocytoplasmic ratio) and enrich these cells in separators after gradient centrifugation against different buoyant densities. An interesting approach is to enrich stem cells as side population (SP) cells due to their capacity to efflux Hoechst dye. A novel opportunity is provided by a method of transfection of target cells with stem cell-associated genes, which yields induced pluripotent stem (iPS) cells.