SCIENTIFIC CHALLNGES FACING CELL TRANSPLANT THERAPY
Wise Young, Ph.D., M.D.
last updated: 26 June 2006

Many people talk about cell transplantation to the spinal cord as if all that we have to do is plop the cells in the spinal cord and they will not only survive but know what to do to repair the spinal cord. That is expecting a lot of stem cells. Many factors determine whether transplants will "engraft" into the recipient, i.e. survive, grow, and participate in the function of an organ. I would like to discuss the three great challenges that must be solved before cell transplantation becomes standard therapy that can be used in people: immune compatability, differentiation, and tumor prevention.

Immune Compatability

Blood Transfusion. For probably over 100 years, we knew that blood can be transferred from person to person, so long as one matched the A, B, and Rh antigens that are expressed on blood cells. A simple nomenclature was developed to express these antigens, AB±. For example, people who express A and Rh antigens are called A+, people who express B but not Rh are B-, people who express none are called O-. Blood that is O- is called universal donor blood because it does not express any of the antigens and therefore would not be rejected by the recipient. On the other hand, people who are O- cannot get any blood that contain A, B, or Rh.

Organ Transplants. It was not until the 1960's that scientists realized that another set of cell antigens determined immune rejection of other tissues. Called human leukocyte antigen (HLA), these antigens are much more complex than AB±. The three most important families of these antigens, named by letters of the alphabet but preceded by HLA, e.g. HLA-A, HLA-B, HLA-DR. There are many more HLA families but these three turn out to be the most important for immune rejection of organs. Each family has dozens of members. For example, HLA-A and HLA-B have several dozen members, about 15 of which are common. HLA-B has many dozens members. Each of us have two sets of HLA genes, one from our mother and the other from our father. Because HLA-A, -B, and -DR are the most important antigens, people usually refer to HLA matching as the number of genes that match out of 6. So, 6 out of 6 HLA matching is a "perfect" match and most tissues can be transplanted, including bone marrow. Anything less has a high probability of being rejected. For reasons that are not well understood, most umbilical cord blood will engraft with 4 out of 6 HLA-match.

CNS Transplants. For many years scientists thought that the central nervous system is "immunologically privileged" because the blood brain barrier limits the movement of cells and antibodies into the brain. While this is true, this usually means that the immune system takes several weeks or even months to recognize the cells as being "foreign". Eventually, the cells are rejected. However, again, it seems that rejection depends on the cells being transplanted. For example, neurons seem to be less likely to be rejected. For that reason, fetal neuronal transplants to humans with Parkinson's disease appear to survive and are usually not rejected even though no attempt is made to match HLA antigens. It is sometimes hard to tell the difference between immune rejection and simple death of the cells. However, if one uses the immunosuppressants cyclosporin (CyA) or tacrolimas (FK506) that prevent activation of lymphocytes in response to new antigens, immune rejection does not occur. Note that both CyA and FK506 does not prevent immune response to something that the immune system is already sensitive to and it simply prevents lymphocytic activation to new antigens. I want to remind people that the first successful transplants of any cell to the spinal cord was only reported in the 1970's and not consistently until the 1980's.

Differentiation

Stem cells, by definition, are "pluripotent" cells, i.e. that can produce many kinds of cells. Contrary to what everybody thinks, pluripotency is not necessarily a desirable feature of cells for transplantation. When one transplants stem cells, it is often not clear what the cells will produce. For example, one would definitely not want skin or hair to grow in the spinal cord. In fact, embryonic stem cell tumors are called "dermoid cysts" because embryonic stem cells often produce skin and hair when transplanted. It is therefore essential that the cells be pre-differentiated before being transplanted.

Retinoic Acid. When embryonic stem cells are treated with a chemical called retinoic acid which pushes the cells down its path of differentiation. It is interesting that one of the first stage of development of embryonic stem cells is into neural cells, i.e. cells that form the central nervous system. Embryonic stem cells have receptors to retinoic acid. It is a chemical that is often used in cosmetics to produce smoother skin.

[b]Serum[/u]. Another approach is to grow the cells in serum-containing media (which contains many factors since serum is the "soup" that have been through all tissues and contain factors from all tissues). Serum is what is left over when one collects blood, allows it to clot, and then centrifuge the clot out. The serum that seems to enables cells to survive and to grow usually comes from fetuses. The most common source of fetal serum comes from cows, e.g. fetal bovine serum (FBS).

SHH and BMP. Once the cells have differentiated into neural precursor cells, further treatment of the cells with a factor called sonic hedgehog (SHH) will push the cells to produce more neurons. Treatment of the cells with a factor called bone morphogenic protein (BMP) tends to push them to produce more astrocytes. Many other factors are being used to differentiate cells into hemotopoietic (blood-producing) cells, chondroblasts (cartilage forming cells), myoblasts (muscle-forming cells), etc.

De-differentiation. Although vehemently denied by some scientists, de-differentiation refers to cells becoming less differentiated and possibly becoming pluripotent, i.e. capable to producing many kinds of cells. It is a misnomer because the assumption is that a stem cell is less differentiated than other cells. I believe that a stem cell is actually a very specialized cell, a cell that has the ability to make many different kinds of cells. Several genes have been associated with embryonic stem cells and many studies have shown that expression of these genes in cells of the body (somatic cells) can cause the cells to become pluripotent. Many laboratories are working feverishly on this problem because the first to succeed in reliably producing stem cells from any kind of somatic cell will be able to produce stem cells at will.

Tumor Formation.

Definition of a tumor. A tumor, by definition, is a growth of the wrong number or type of cells. Thus, for example, a wart is a kind of tumor. A scar that does not resolve is also a kind of tumor. Clearly, a stem cell must respond to the tissue in which it has been transplanted. Much recent research suggest that adult stem cells require the presence of several other cells, called a niche, before it can behave like a stem cells to produce cells that are required for the particular tissue. This is true for skin, bone marrow, and the central nervous system. It is also likely to be true for liver, heart, lung, and pancreas (where the insulin-producing cells reside).

Niches. Because tumors are undesirable, stem cells have evolved to require very specific signals in the body before they begin behaving like stem cells. If they did not, since we have many mesenchymal stem cells coursing through out our body, we would have tumors growing all over our bodies. In fact, there is currently a theory that most of the solid tumors (such as prostate, colon, lung, and breast) provide niches of cells that stimulate incoming stem cells to produce tumor cells. That may be one reason why almost all the chemotherapies that are effective against cancer are also very effective in killing stem cells.

Creating niches. Much progress is being made in identifying "niches" in bone marrow, central nervous system, liver, muscle, and pancreas, as well as some of the factors that seem to signal mesenchymal stem cells to produce the correct kind of cells. Unfortunately, the brain does not seem to contain many "niches" that promote the production of neurons. For this reason, many stem cells implanted into the brain or spinal cord seldom behave like stem cells. Umbilical cord blood cells, for exampl, just sit in the tissue and produce more of themselvs. One way of getting around the problem of niches is to transplant mixture of cells that not only provide the stem cells but also the niches that produce the right type of cells.

Immune rejection of tumors. One important function of the immune system is elimination of tumors. Our immune systems can recognize many cells that become cancerous. In fact, this is probably the reason why people with acquired immunodeficiency syndromes (AIDS) often develop multiple tumors. Likewise, immune-suppression can also lead to increased risk of cancer and tumors. It is probably not a good idea to implant cells that cannot be detected by the immune system because, if such cells became tumors, the immune system cannot detect them and get rid of them. On the other hand, it may be possible to genetically modify stem cells so that they can be induced to produce tumor-like antigens that would stimulate the immune system to get rid of them, when they are no longer needed.

Basic Research

Much basic research is now going on, defining not only the biology of stem cells but also the response of the immune system and different tissues to stem cells. This research is very important because they will provide the technology that can circumvent our current bottleneck of having to derive stem cells from only certain sources, such as blastocyts, fetuses, umbilical cord blood, and bone marrow. A clear and deep understanding of stem cell biology and the immune system will be very important to future therapies involving stem cells. Some of the most exciting possibilities include tolerizing the immune system to certain cells, putting inducible genes into the cells, and reprogramming somatic cells to become stem cells.

Tolerizing the immune system. While a great deal of attention has been paid to HLA-matching of cells or "cloning" of stem cells that would produce cells that are genetically identical to the person, it is important to realize that several more practical methods are likely to be available earlier. It is well known that early during development, the immune system of the fetus "learns" what is its own cells. This is done by exposing certain lymphocytes of the immune system (called b-lymphocytes) to cells and then raising the level of glucocorticoid hormone that causes all activate b-lymphocytes to die. This way, all the cells that respond to one's own cells are killed off and cannot produce auto-immune disease. We know that it is possible to "tolerize" the immune system. It is frequently done to eliminate allergies. Also, many scientists have shown that when cells are transplanted early during fetal development, the cells are accepted and the immune system does not attack the cells.

Inducible genes. It is now possible to genes into the cells and turn them on with specific drugs. Bacteria have evolved genes that detect antibiotics. For example, the antibiotic tetracycline turns on certain bacterial genes. Therefore, if one uses a tetracycline-sensitive promoter of a stem cell gene, one may be able to construct cells would transform into a stem cell or some other kind of cell, by giving the person. It is also possible to induce apoptosis genes, i.e. genes that kill the cell, allowing us to eliminate the cells when we no longer need them. Finally, it may be possible to introduc differentiation genes, i.e. genes that push the cells into particular directions of development. Such genetically modified cells will be future of stem cell therapy because they would be safer and more controllable.

Reprogramming. The discovery that somatic cell nuclei can be transferred into an egg and then fooled to develop into an organism was the first definitive demonstration that nuclei can be reprogrammed, not only to become other cells but to become the ultimate cell that develops into all the other cells of the body. It essentially eliminated the dogma that "de-differentiation" cannot occur. Cells must have many highly evolved mechanisms to prevent de-differentiation. These mechanisms must be very powerful and effective because we would otherwise have many more stem. It is also apparent that the "programming factors" are present in the cytoplasm. This is why somatic cell nuclear transfer (SCNT) can work. The nucleus placed inside an egg realizes that it can behave like an egg nucleus.

In summary, much essential and important stem cell research is going on. This research is very likely to revolutionize all our current assumptions about stem cells, the need for embryonic stem cells, somatic cell nuclear transfer, and differentiation of cells.