Biomedical Engineering Reference
In-Depth Information
fully determined for any stem cell family, though there have been considerable improve-
ments toward this goal.
Isolating Stem Cells for Scientific and Clinical Purposes
Methods have been developed to isolate stem cells or enrich the stem cell content of a
cell population. At present no single marker has been identified for use as a definitive stem
cell marker. Therefore, most effective isolation protocols use multiparametric isolation
strategies. These strategies may comprise immunoselection for cells with specific antigenic
profiles that are used in combination with cell selection methods such as diameter, cell den-
sity, and levels of “granularity” (the extent of cytoplasmic particles such as mitochondria).
The published protocols include
flow cytometry
and/or immunoselection with magnetic
columns, affinity columns, and counterflow elutriation. Once purified, validation of the
identity of stem cells can be achieved either ex vivo or in vivo. Ex vivo assays typically
involve clonogenic expansion assays, in which a single cell is expanded in culture under
precise conditions, and the ability to give rise to daughter cells of more than one fate iden-
tifies the original cell as a stem cell. In vivo assays involve transplanting the putative stem
cell into an animal to observe whether multiple tissue types can be regenerated.
Types of Stem Cells
Embryonic stem cells are the most pluripotent cell type, since they can give rise to essen-
tially any tissue in the body. These cells are typically derived from very early stages of
embryonic development (usually the blastocyst stage). They have high potential to create
large populations of specialized cell types, but their use is controversial because of ethical
issues. In addition, their use for therapeutic cell transplantation is in question because they
have the immune profile of the original embryo and therefore are necessarily an allogeneic
therapy. For these reasons, scientists have very actively searched for alternatives to embry-
onic stem cells. The technique of somatic nuclear transfer involves replacing the nuclear
material of an egg cell with that from a specialized somatic cell. The resulting cell is thereby
“reprogrammed” to form a blastocyst with the genetic identity of the original somatic cell.
This technique is the basis for cloning in animals and is being investigated for “therapeutic
cloning,” which involves creating cell banks that are genetically identical to the somatic cell
donor for potential therapeutic use. It also is controversial because of the potential to be
used for “reproductive cloning,” which involves recreation of an entire organism based
on the original reprogrammed cell.
A recent exciting discovery is the ability to reprogram cells by transfecting a few key
regulatory genes (as opposed to the entire nuclear material). The resulting cells are termed
inducible pluripotent stem (iPS) cells
to reflect the fact that they have been induced to revert
to a pluripotent state. In this technique, a specific set of two to six genes is introduced into
the nucleus of a somatic cell. The gene set is chosen because of its ability to reprogram the
target cell to a near-embryonic state, and currently there are several different gene sets that
can achieve this goal. The resulting cell line then can theoretically be propagated and then
differentiated into the desired specialized cell type. This technique has energized the stem cell
research community, and the applications to tissue engineering are being investigated.
In the last decade there has been a large amount of research devoted to finding, charac-
terizing, and using adult stem cells for tissue engineering and regenerative medicine. It is
now recognized that there are a number of cell types in various tissues of the adult that
