Stem cell research holds a promise for therapeutic intervention for a range of diseases. While there are certainly some extra disease-specific requirements, there is a common set of steps needed to treat most of conditions for which stem cell therapy is being considered, each with its own specific challenges.
Firstly, there must be a supply of stem cells of some kind. For many diseases, the most desirable therapeutic choice is human embryonic stem cells, as they have the much-desired characteristics of pluripotency, clonality and stable karyotype.
Autografts of the person's own stem cells would often be the most desirable to reduce rejection. A possible source is a female's own egg collected during in vitro fertilization.
Embryonic stem cells may not be required; it may be an application in which multipotent adult progenitor cells may be equally suitable.
If the person's own stem cells are unavailable for use, there are still other possible sources. Allografted stem cells might be harvested from a relative having a close match. A xenograft from animal stem cells may be applicable in some uncommon cases such as organogenesis. Finally, one could use one of the few existing stem cell lines that are available. All non-autograft sources do have a pitfall of graft rejection by the patient's immunity.
Some further pitfalls plague the use of one of the few existing stem cell lines as the source of the donor material. Stems cells can continue to be indefinitely replenished through contant division, and there is still the correct number of chromosomes in the cultivated stem cell lines (Buzzard, 2004). However, there is no guarantee that errors are not accumulating over time. It is estimated that for every cell division, one mutation occurs (Kunkel, 2000). Therefore, after the thousands of cell divisions of the cultured stem cell lines, there are likely thousands of defects. Many of these would be of no consequence, but mutations in the differentiation capacity or mutations of proto-oncogenes are a profound negative for their use in stem cell therapeutics. Furthermore, many of the existing stem cell lines have been exposed to animal culture mediums at some point, raising a specter of passing disease from animals to humans.
A novel, yet controversial, strategy of acquiring the stem cells is through the process of so-called “therapeutic cloning”. This is the process that was used to famously create the cloned sheep named Dolly (Wilmut, 1997). Applying the technique to stem cells, the patient would first donate a somatic cell from his body. The nuclear material from the patient's cell would be then inserted into a human egg that had all its own nuclear material removed. The stem cell would then be allowed to grow in vitro and differentiate into the desired tissue type. Major ethical and legal concerns are obviously involved in this strategy. Nonetheless, the proof of concept of this approach has already been performed and reported by groups in South Korea (Hwang, 2004). There are still unanswered questions, however, such as the possible negative impact of having no spermatic imprinting on the stem cell when it was created (Mann, 2001).
Based on the origin of the donor stem cell, the stem cell genetic properties may need to be modified to add therapeutic characteristics. This may include replacing a missing gene, correcting a point mutation, suppressing an overactive gene. The usual genetic technologies are described in the Standard stem cell genetic manipulation techniques section.
As mentioned earlier, upon transplantation into a site of the body other than the uterus, undifferentiated early embryonic cells have a tendency to turn into either teratomas or teratocarcinomas (Thomson, 1998).
This finding is expected, considering how closely stem cells resemble cancer cells in many respects: unlimited dividing, no contact inhibition, and so on. Furthermore, if a stem cell undergoes cell fusion with an existing cell, the nuclei of the stem cells could also fuse with the two sets of chromosomes already in the cell, creating a tetraploid nucleus, which is more tumorigenic (Mummery, 2004).
An effective stem-cell therapy must somehow reduce this tendency towards tumors. This is a difficult item to change, by all measures. Fortunately continued research one helpful finding: some teratomas are the result of not just the transplanted embryonic stem cells, but the transplantation of differentiated cell cells that were derived from those embryonic stem cells (Kim, 2002). Therefore, purification of cell lines, as described in the next section, may assist in decreasing the tumor potential.
Since there is a possibility of tumors from both the undifferentiated embryonic stem cells and the undesired lineages of differentiated cells, one needs to successfully purify out the desired lineages needed for the particular therapy.
The cell lineages chosen are often lineages that are tissue specific progenitors that have a capped upper limit of replication, thereby reducing cancer risk.
The purification strategy requires some method of specifying the desired lineages from the contaminating ones. There are several methods that have studied: cell surface markers, fluorescent sorting, culture conditions, magnetic sorting and drug sensitivity. Some of these techniques will be discussed in a further detail below.
The drug sensitivity strategy involved placing a gene for drug resistance under a promoter that was specific to a certain lineage (Li, 1998). The desired lineage, neuroepithelial cells, had resistance to the drug neomycin. Therefore, addition of neomycin selected out a seemingly pure neuroepithelial population that went to differentiate into valuable neural cells (Li, 1998).
Fluorescent sorting involves adding a gene to the desired lineage that expresses a fluorescent protein, and then using a cell sorter that can select based on the ability of a cell to fluoresce. A similar strategy is used in magnetic sorting, except instead of a fluroscent protein, magnetic microbead-tagged antibodies are used for the selection.
Because no purification strategy is fully selective, combinations of multiple selection methods could be used. One experiment simultaneously used drug resistance and fluorescent markers to try to get a pure sample of endothelial cells from the source embryonic stem cells (Marchetti, 2003).
After the selected lineage is purified, the lineage or tissue must be placed into the patient. Delivery can be complex, if targeting deep tissues such as the heart or brain.
There are molecules on the surfaces of most cells called Major Histocompatibility Complex (MHC), which mark a person's tissue from someone else's foreign tissue invading the body. Since donor stem cells might not be from the patient, but from someone else, the MHC markers can be different. The invading tissue's MHC molecules signal the immune system to mount an attack against the invading tissue, leading to graft rejection. There are several strategies to try to avoid this:
The first strategy is classical immunosuppression, as is normally used for organ transplants (Gage, 1998). The patient would take some medications to prevent his body from mounting an immune response against the donor stem cell tissue. There is a suite of accompanying complications with immunosuppressive therapy including drug toxicity, increased infections, skin cancer and lymphoproliferative diseases.
Another option would be a stem cell donor bank. People who are interested in either donating or receiving stem cells would visit the stem cell bank. Recipients would be matched to the available donated stem cells on file, checking for close MHC matches. The costs of isolating and maintaining the cell lines would be very high (Wobus, 2005). However, stem cell banks have already been created in Sweden in the United Kingdom (Mummery, 2004).
In the third strategy, the genes that are responsible for immune rejection could be excised from the stem cell chromosome, or otherwise knocked out by a method such as RNA interference. A strong benefit of this approach is that one could create stem cells that don't express any MHC at all on their surfaces, and thus that one line of self-renewing stem cells could be a supply suitable for many future donors (Boheler, 1999; Drukker, 2002). Mouse stem cells have been successfully created with their MHC molecules knocked out (Grusby, 1995). However, MHC isn't the only thing that can trigger an immune response. For example, wood particles have no MHC on them, but a foreign piece of wood in the flesh triggers a response, so simply knocking out MHC won't fully solve the rejection problem.
The final strategy would be “therapeutic cloning”, described in the section entitled Acquire supply of stem cells. In the technique, a somatic cell's DNA from a donor is inserted into a chromosome-free egg. The resulting tissue would then be very desirable for transplant since it would have a perfectly matching MHC complex (Lanza, 1999).