Innovative Solutions for Stem Cell Biology

By: Soverin Karmiol, BioFiles 2008, 3.11, 3.

BioFiles 2008, 3.11, 3.
Soverin Karmiol
Manager, Research and Development, Regenerative Medicine
soverin.karmiol@sial.com

Multicellular organisms arise from a single cell. The process by which this occurs is an immense challenge in biology and has significant implications in medicine. A fundamental notion in this process is that the information required to define the entire organism is imbedded in this single cell; in other words this single cell is able to differentiate into the many mature cell types that constitute the adult state. This concept is captured by the term, totipotency. Embryonic stem cells are characterized by their ability to renew themselves by cell division while maintaining the undifferentiated state and differentiate into the various specialized cell types.

Embryonic stem cells can be generated from blastocysts which are an early stage of embryonic development morphologically characterized as a hollow sphere containing a group of cells known as the inner cell mass. The cells comprising the outer layer of the sphere will eventually become the placenta while the cells of the inner cell mass will give rise to later structures of the adult organism; it is for this reason that embryonic stem cells are pluripotent and not totipotent.

Embryonic stem cells provide a unique opportunity to study the developmental process and provide clues as to the inter-relationships that exist between cell types that make up the various specialized tissues of the body. Presently, in tissue culture stem cells can be differentiated into a few specialized cell types, cardiomyocytes, neuronal cells, hepatocytes and pancreatic beta-cells, that have characteristics similar to cells found in normal tissue. These developments have applications in cell therapy, tissue engineering, regenerative medicine, and tissue models for drug discovery and toxicity testing.




Another type of stem cell is found in adult tissues and has the capacity to differentiate into various specialized cells types; however, they do not have the pluripotency potential of embryonic stem cells. The adult stem cells are further along their differentiation pathway than embryonic stem cells but still have the capacity to differentiate into more terminally differentiated cells, so that adult stem cells are multipotent rather than pluripotent. The function of these cells is to repair damage due to injury or disease and to support the normal cell turnover that occurs throughout the body. Clinical applications of adult stems cells have been in use for a few decades; most, notably, the adult blood forming stem cells, CD 34+, from bone marrow have been used for transplant being able to reconstitute the entire hematopoietic system, treating leukemia and other related blood cancers. Adult stem cells having neuronal, astroglial, oligodendroglial and hepatocyte characteristics have been isolated from cord blood and a rich source of mesenchymal stem cells has been found in the jelly-like connective tissue surrounding the blood vessels of the human umbilical cord. The adult tissues that contain stem cells include brain, bone marrow, peripheral blood, blood vessels, skeletal muscle, skin, liver and adipose tissue.

Bone marrow has been a source of not only hematopoietic stem cells but mesenchymal stem cells that can differentiate into bone, cartillage and adipopse tissue and have been used to enhance the success of bone marrow transplantations. Recently, a very small embryonic-like (VSEL) stem cell has been observed in the bone marrow of adults. These cells are characteristic of the stem cells isolated from a blastocyst. It is believed that during early embryonic development stem-like cells are deposited in various primitive organs and remain in the stem-like state into adulthood. The VSELs are believed to be the source of the mesenchymal stem cells. This is an interesting prospect and may provide a new source of pluripotent stem cells.

There are two strategies used to re-program adult cells into pluripotent stem cells. The first is called somatic cell nuclear transfer and requires the removal of the nucleus from a donor oocyte followed by the introduction of an adult nucleus. The oocyte environment is capable, through the actions of unknown factors, to reprogram the adult nucleus from a highly specialized state to a pluripotent state; this process is also referred to as cloning since the cells of the inner cell mass are clones of the donor. This process has been used to clone many animals, such as sheep, mice, cows, and goats.

More recently, reprogramming has been achieved without the use of a donor oocyte. Transcription factors associated with the pluripotent state were transfected by viral vectors into human adult differentiated cells. Two protocols were employed by two separate labs; one used OCT4, SOX2, Klf4 and c-myc genes while the second lab used OCT4, SOX2, NANOG and LIN28 genes. The action of these pluripotency associated genes on an adult cell is to silence genes associated with differentiation and allow for the expression of a pluripotent state. These cells are called induced pluripotent stem cells (iPS) and resemble human embryonic stem cells with respect to their cell surface markers, morphology, karyotype and the expression of multilineage differentiation in embroid bodies (a cell culture structure used to test pluripotency). This type of reprogramming does not require the use of an oocyte and is therefore attracting a considerable amount of attention.

The prospects of iPS cells are far reaching. These cells have the same genetic make-up as the donor and provide the potential of supplying the desired tissue for transplantation; in cell therapy and regenerative medicine the ability to provide differentiated tissue customized for an individual not needing immunosuppressants is a great boon. Also, it is possible to generate human disease specific models by generating iPS cells from individuals with these diseases; these models would allow research into the disease process and drug development not possible at the present moment. Since viral vectors are presently being used to generate iPS cells and c-myc and OCT4 have been associated with tumor tissues there is a concern in using them for transplant purposes. However, their existence has spurred the investigation in developing methods that are more clinically friendly.

The fruition of stem cells for tissue models and transplantation will require their assembly into highly organized structures that will allow the expression of the structure and function observed within the human body to be duplicated in vitro. To this end the coordination of various disciplines ranging from cell and molecular biology, biomedical engineering, immunology and surgery will be required. To a large extent these disciplines are engaged and it is an exciting time to be on the threshold of a series of events that will have a profound effect on basic science and medicine.

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