Human stem cells require a feeder cell medium in which to grow, and it is best to use feeder cells from a human not an animal. However, there is a risk that if a patient is growing their stem cell lines on growth medium derived from another human, that it could transmit viruses, infecting the patient with conditions such as HIV, hepatitis B, or another as-yet-unidentified disease.
One novel approach to this problem is to use feeder cells from the actual patient. A typical scenario of this approach would be to for a patient wishing to undergo autograft of bone marrow stem cells. As well as the bone marrow collection, the patient undergoes a small skin biopsy. The skin cells acquired from the skin biopsy are then allowed to transform into fibroblasts, and these fibroblasts are used to grow the patient's bone marrow stem cells into differentiated cells needed for the therapy (Bongso, 2004).
An allograft from one individual to another often results in tissue rejection of the grafted cells, if immunosuppressive drugs aren't used.
It is already known that in kidney transplants, if the recipient first gets bone marrow from the donor before the actual transplanted kidney, then chimeric bone barrow develops in the recipient and the kidney is less likely to be rejected (Mummery, 2004).
Applying this knowledge to stem cells, it now appears that if one first transplants early stem cells from the donor, before later transplanting the actual desired more differentiated stem cells, that it will induce a long term graft acceptance, thereby reducing the risk of rejection (Faendrich, 2002).
Consider the cases of stem cell therapy that are replacing a missing gene product (often an enzyme, protein, or a secreted chemical). The therapy thus would involve inserting stem cell lineages into the patient that can produce large enough amounts of the missing genetic product One option is to just splice the desired gene directly into the embryonic cell's chromosome. However, the drawback is that often this has negative effects on the gene expression. Similar to what happens to many retroviruses that integrate into chromosomes, the gene is progressively silenced with each generation. This would result in insufficient replacement product for the therapy.
The alluring alternative is so-called “extrachromosomal replication”, which uses plasmids instead. A plasmid is a small circular piece of DNA that can self-replicate inside the cell, without needing to insert itself into the host chromosomes.
One can use a particular plasmid called polyoma virus (Gassman, 1995), which has been genetically modified. The virus is modified so that it cannot leave and infect new cells or cause disease, but can still self-replicate. The plasmid is further modified by inserting two new genes: a gene for the desired gene product for the therapy and an antibiotic resistance gene. The antibiotic-resistance selects out the viruses that have successfully taken up the two new genes, then the modified plasmids are inserted into the stem cells. The plasmid progressively self-replicates and makes the desired gene product, without interfering with the stem cell division and differentiation process (Aubert, 2002).
Consider a case of a patient whose cells are secreting too much of a gene product. Therefore the patient's stem cell therapy requires cells that don't secrete the product. One option would be to try to genetically modify the stem cell by either removing that gene from the chromosome, or splicing some junk DNA into that gene on the chromosome to stop proper expression. However, there is still the problem that any transformation of the DNA can have negative effects. This is the where the alternative strategy of RNA interference comes in.
RNA interference is a type of the so-called “knock-down gene” phenomenon whereby double stranded RNA induces the destruction of RNA molecules that have a homologous sequence.
The strategy begins by creating a double stranded RNA that is homologous to the messenger RNA of the unwanted gene product (Caplen, 2003). Then this formulated double-stranded RNA sequence is inserted into the embryonic stem cell. When the stem cell chromosome then reads the unwanted gene and makes a messenger RNA, the inserted double-stranded RNA binds to the unwanted messenger RNA and causes the degradation of the messenger RNA. Without the messenger RNA, there is no unwanted gene product, giving the desired therapeutic effect for the cell (Yang, 2001). The biggest challenge in this strategy has been the way to get sustained amounts of the double-stranded RNA into the stem cells.
Some organs are injured or diseased beyond repair, and no amount of stem cell infusions into the damaged organ will restore it back to adequate function. In cases such as these, the entire organ may need replacement, but organ transplantation currently suffers from a lack of qualified donors. Waiting lists are long, and some patients die while awaiting a suitable match. One attractive theory is to use stem cell techniques to generate a full replacement organ in vitro, and then transfer it into the patient. This would address not only the issue of organ availability, but also of transplant rejection.
Organogenesis is the term given to the spontaneous appearance and development of individual organs during embryonic development. By using the process of organogenesis on in vitro stem cells, it is hoped that replacement organs could be cultured. This technique is currently still very far from the clinical trial stages.
While the full mechanisms behind organogenesis are still not yet unidentified, some key factors have been uncovered which may lead to viable in vitro organogenesis. For example, SDF-1 chemokine molecules are expressed during organogenesis (McGrath, 1999). It is also becoming clear that the stem cells polarize themselves during embryonic development (Gardner, 1999). The molecular mechanisms of this polarization, such as laminin, are being uncovered (Li, 2003). It is also increasingly clear that the organogenesis process is very cell-specific (Edgar, 2004).
As well as unlocking the cellular mechanisms of organogenesis, the issue of growth of the three-dimensional structure needs to be addressed. However, this is also under study, and is described in the following section.
Some graft replacements would require not an insertion of not just individual stem cells, but a complex three-dimensional structure such as a replacement joint composed of cartilage and bone (Stock, 2001).
This requires a scaffold onto which the stem cells can replicate. A novel approach is to make the scaffold from a biodegradable scaffold, so that once the stem cells have sufficiently constructed the structure, the underlying scaffold can be absorbed into the body (Fodor, 2003; Holmes, 2002).
Mesenchymal stem cells are attractive choices for growing on the scaffolds because of their relative ease of culturing, proliferation and differentiations (Bianco, 2001).
Several different scaffolds are currently available. Some are polymers of biological materials such as collagen type I or fibronectin, isolated from the extracellular matrix or from plants. Others are synthetic, composed of compounds such as hydroxyapatite and tri-calcium phosphate ceramic (Kassem, 2004).
Initial experiments in animal models have shown success. For example, the technique has been used for treatment of defective long bones in sheep (Kon, 2000), as well as individual human case reports.
As described earlier, implanted embryonic stem cells have a tendency to create teratoma tumors because stem cells have many cancer-like properties.
Apoptosis is the process in which cells commit suicide, thereby preventing cancerous growth. The apoptosis process is increasingly well studied, and various genetic and cellular mechanisms have been uncovered. Thus, one novel approach to the stem-cell tumorigenesis problem is to genetically modify apoptosis regulatory genes such as bcl-2 in the stem cell graft, prior to transplantation.
Currently one of the best-studied niches is the hematopoietic stem cell niche, which has yielded several exciting advances. Bone marrow contains endosteum, which is the layer of cells lining the inner surface of the bone marrow, and contains osteoblast cells that form osseous matrix.
It was initially found that if someone transplants hematopoietic stem cells into a recipient, the inserted stem cells home in towards the endosteum (Nilsson, 2001; Askensay, 2002). Then came the breakthrough finding that through manipulating the osteoblast cells within the endosteum, one could regulate the number of stem cells (Calvi, 2003; Reya, 2003).
The niche findings also shed light on the cellular mechanisms involved, termed the Notch and Wingless (Wnt) signaling pathways (Calvi, 2003; Reya, 2003). It has been found that the resulting proteins, Notch1 and Wnt3a, are proteins that work through cell-to-cell contact, instead of through the usual soluble transmitter-receptor interactions (Bonde, 2004).
Finally, it has been found that the marrow's hematopoietic primitive stem cells stay in the areas of the bone marrow with the least amount of oxygen. It appears that this hypoxic area enhances both the survival and replication of primitive stem cells. The differentiated cells then follow an oxygen gradient: the original stem cells prefer the part of the marrow with decreased oxygen, but as the offspring cells differentiate and mature, they migrate towards the oxygen-rich parts of the marrow (Danet, 2003; Ceradini, 2004).
Many questions still remain (Taichman, 2005), but a full understanding of the hematopoietic stem cell niche will contribute greatly to not only marrow-based therapies, but also stem cell treatments in general.
One issue that arises in stem cell therapy is evaluation of its continued success. Once the stem cell graft is place, there needs to be a way to ensure the transplanted cells are continuing to thrive and regenerate as desired. This is a problem currently being resolved with eloquent tracking technologies.
In one technique concept, the stem cells are labeled with biologically-compatible magnetic nanoparticles before implantation. These iron-oxide nanoparticles stay inside the stem cell and are harmless. Then the patient would visit the hospital for a magnetic resonance imaging (MRI) scan. An MRI is a non-invasive way to visualize a three-dimensional image of the patient. The labeled nanoparticles of the stem cells would then light up in the MRI images, thus showing the viable stem-cell graft cells. Moreover, during each stem cell division, approximately half of the nanoparticles go to each daughter cell, so a medical team could assess if the stem cells were merely living, or were actively dividing (Perez, 2004).
Another approach would follow the same idea, but using semiconductor quantum dots instead of magnetic nanoparticles, as the way to label the stem cells. This would be then tracked in the patient not with an MRI scan, but with a illumination system based on laser beams (Gao, 2004).
As described earlier, many of the most useful stem cells require an appropriate niche to develop into a clinically useful final product. The niche secretes appropriate chemicals to direct the differentiation and development. A suboptimal method in the laboratory is to coarsely dump the required chemicals onto the cells.
A much better solution is currently under investigation: growing the stem cells on a so-called “lab-on-a-chip”. This is a silicon chip with nano reservoirs. The chip surface contains about a thousand reservoir cavities, with each reservoir only about 500 nanometers across. A reservoir holds a small amount of liquid chemicals similar to what the stem cells would be exposed to in the niche. Each reservoir is sealed with a lipid bilayer equivalent to a cell membrane. These reservoir bilayers also contain the same voltage-gated channels found in cells. A small charge of electricity can then be applied to any individual reservoir to open the channels allow the chemicals to spill out, delivering them to any particular stem cell at any specified time of development. The concern of the electrical voltage negatively affecting the stem cells is addressed by recessing the reservoirs, increasing the distance between the stem cells and the electrical field.
The nano reservoir chip technology also allows the possibility of growing cells layer by layer, making compound tissues, which are otherwise difficult to produce. For example, a graft tissue could be created that is bone on one side and cartilage on the other (Choi, 2005).