Stem Cell Cultures: Maintenance, Characterization and Application

Read this article to learn about the maintenance, characterization and applications of stem cell cultures.

The cells that retain their proliferative capacity throughout life are regarded as stem cells. When the stem cells divide, they can generate differentiated cells and/or some more stem cells. These stem cells are capable of regenerating tissue after injury.

The lack of tissue-specific differentiation markers is a characteristic feature of stem cells.

Embryonic Stem (ES) Cells:

As the embryonic development occurs, cells of the inner mass of embryo (i.e. those contributing to future fetus) represent embryonic stem (ES) cells. They continue to divide and remain in an un­differentiated to totipotent state. It has been possible to establish and maintain cell lines for ES cells. The ES cells isolated from mouse blastocyst are the most commonly used in the laboratory. The most widely used embryonic stem lines are the various 3T3 lines, WI-38, MRC-5 and other human fetal lung fibroblasts.

Advantages of ES cells:

In general, the cultures from embryonic tissues survive, and proliferate better than those from the adult. This is due to the fact that ES cells are less specialized with higher proliferative potential.

Limitations of ES cells:

In some cases, the ES cells will be different from the adult cells, and thus there is no guarantee that they will mature to adult-type cells. Therefore, it is necessary to characterize the cells by appropriate methods.

Epithelial Stem Cells:

The epithelial cells (e.g. epidermis lining of gut) are constantly being shed from their outer surface. This cellular loss is compensated by a continuous replacement process. The replacement occurs in a highly organized and a regulated fashion.

It is estimated that in humans the entire outer layer of skin is shed daily. The entire epithelial lining of the mouse gut is replaced once in 3-4 days. This process of shedding and replacement continues throughout life.

The epidermis of the skin has a proliferative compartment containing stem cells and post mitotic cells, besides some transit amplifying population of cells. The transit amplifying cells, produced from stem cells with limited life span are shed from the epidermis.

Maintenance of Stem Cells in Culture:

The basic criteria to maintain stem cell in vitro is to ensure that they possess the same characteristics and differentiating abilities when they are present in the tissue in vivo. The maintenance of epidermal and non-epidermal epithelial cells in the in vitro cultures is briefly described.

Epidermal stem cells in culture:

The epidermal stem (or keratinocyte stem) cells can be successfully maintained by co-culturing with 3T3 feeder layer. By this technique, it is possible to achieve long term maintenance of cells, besides retaining their capacity for both proliferative and differentiating characteristics.

It has been demonstrated that the so maintained stem cells when placed into nude mice could form stratified and differentiated epithelium. Serum-free media with added growth factors were found to be more efficient in maintaining the epidermal stem cells in culture.

Epithelial cells in culture:

Several types of non-epidermal epithelial cells can be grown and maintained in cultures. As in the case with epidermal stem cells, use of feeder layer is advantageous for epithelial cell culture. Epithelial cells of prostate gland have been successfully grown in suspension cultures in the presence of 3T3 feeder layer. The same method is also used for culturing human breast epithelial cells and colorectal carcinoma cells.

Characterization of Stem Cells:

Immunological techniques are widely used for the characterization of different populations of stem cells. These techniques are mostly based on immunocytochemistry using fluorescent microscopy or staining technique involving colour reactions. The cells of the tissues produce specific cell surface and cytoplasmic proteins. The cell surface proteins such as integrin’s and the members of CD (cluster of differentiation) antigens (e.g. CD₁₀, CD₃₁, CD₄₄) can be used as markers of epithelial cell types. Further, the cytoplasmic proteins of epithelial cells (of cytokeratin family) are also useful for their identification.

Applications of Cultured Stem Cells:

Embryonic stem cells in tissue repair:

The culture stem cells can be used for the repair of tissues with functional impairment that may occur due to damage or ageing. The cultured embryonic stem cells can be manipulated to produce cultures characteristic of a particular tissue.

Thus, there exists a possibility of treating the following diseases.

i. Diabetes with pancreatic insulin producing cells.

ii. Parkinson’s disease with cultured dopamine- producing neurons.

Embryonic stem cells are useful for the production of defined transgenic animals. It is also possible to modify ES cell genome by gene targeting using in vitro transformation and selection.

Applications of tissue specific stem cells:

Stem cells, isolated from different tissues of humans and animals, and cultured in vitro are less toitpotent than ES cells. They usually differentiate into a single cell type and are referred to as unipotent. However, stem cells from bone marrow and brain are capable of forming different cell types though to a lesser extent when compared to ES cells. In mouse lacking bone marrow, when the cultured neuronal cells are placed, they develop into blood cells.

Tissue specific culture stem cells are used for the following purposes:

i. In surgical repair and tissue grafting.

ii. In gene therapy.