Znop asked if I would start a discussion concerning embryonic and adult stem cells. I said that I would start with a summary and hope that others will contribute to the discussion. ]\
Before we begin the discussion, it is important that we define the terms because there is so much misunderstanding and misuse of the terms:
- Stem cells. These are cells that can make many different kinds of cells, as well as themselves. There are many types of stem cells
- Embryonic stem cells. These are cells that are derived from blastocysts, a very early stage of development that occurs during the first two weeks after fertilization. A blastocyst is a small ball that contains about 200-300 stem cells. The blastocyst is pre-embryonic. A developing fertilized egg becomes an "embyro" only after a midline appears. This usually occurs shortly after implantation of the egg into the uterus and a "primitive streak" develops. So, the term "embryonic stem cells" is a misnomer. They should be called "pre-embryonic stem cells".
- Fetal stem cells. These are cells that are derived from fetuses. A fetus refers to the stage of development when body parts are evident, including a head, arms, and legs, usually starting 6 weeks after conception and continuing until birth. Many types of stem cells are present in fetuses, associated with the various organs of the body that are developing. These include:
- Fetal neural stem cells. These are obtained from the developing brain.
- Fetal bone marrow stem cells. These are obtained from developing bone.
- Fetal stem cells isolated from various tissues, including skin, blood, bone, etc.
- Neonatal stem cells. These are cells that are derived from cells collected from newborns. These include:
- Umbilical cord blood stem cells. These are obtained from umbilical cord blood collected from the umbilical cord shortly after birth.
- Umbilical cord and placental stem cells. These are obtained from the umbilical cord and placenta shortly after birth.
- Adult stem cells. These are cells that are derived from cells collected from any person older than newborn, from several days to elderly. They include:
- Bone marrow stem cells. These are cells obtained from bone marrow, usually autologous or from the same person that will receive the transplant. Although the markers for bone marrow stem cells are well defined in humans, many investigators use CD34+ which is a maker for hematopoietic stem cells (i.e. stem cells that make blood cells). Please note that we did not know that there were pluripotent stem cells in bone marrow until 1999.
- Mesenchymal stem cells. These are cells collected from a variety of tissues, including peripheral blood. There are some markers, such as CD144+ but the markers are controversial. Tuli, et al. (2003) for example reports that mesenchymal derived from human trabecular bone are CD73(+), STRO-1(+), CD105(+), CD34(-), CD45(-), CD144(-) while Mayer (2004)]Mayer (2004)[/url] suggests that bone cells are CD13+, CD44+, CD90+, CD147+, CD14-, CD34-, CD45- and CD144- in elderly women, Mitchell, et al. (2006) suggests that such cells obtained from adipose (fat) tissues are CD13, CD29, CD44, CD63, CD73, CD90, CD166 are initially low but increase progressively with passage. Kuwana, et al. (2006) report that human monocyte derived multipotential cells are expressed CD31, CD144, vascular endothelial growth factor (VEGF) type 1 and 2 receptors, Tie-2, von Willebrand factor (vWF), endothelial nitric oxide synthase, and CD146, but CD14/CD45 expression was markedly down-regulated.
- Neural stem cells. We have only known since the mid-1990's that there are neural stem cells in adult brain that continue to make neurons throughout adult life.
- Enteric glia. These are stem cells from the gut. Although known for a long time to be able to produce neurons, these cells are now believed to be a type of stem cell or a neuroprogenitor cell.
- Progenitor cells. These are cells that make several different kinds of cells but are usually more limited than stem cells and may not be able to make themselves. The difference between progenitor and stem cells is not clear and may become moot as if becomes clear that it is possible to "dedifferentiate" cells to become stem cells.
- Precursor cells. These are even more limited than progenitor cells and make only a few types of cells and cannot make themselves. These are further down the differentiation path from progenitor cells.
There are several possible goals for using stem cells for treating spinal cord injury.
- Bridging the gap. The injury site is frequently a "bombed out" tissue that has lost many cells and is filled with macrophages and other inflammatory cells in the weeks that follow injury. In the months and years after injury, it may be taken over by reactive glial cells that may express substances that repel axonal growth. One of the goals of spinal cord injury repair is to fill this site with cells that are conducive to axonal growth.
- Growth factors. Many stem cells and progenitor cells are believed to secrete growth factors that stimulate growth of cells. Many of these factors are also produced by other cells. These include neurotrophins (that stimulate neurons), proliferative factors such as fiberblast growth factor (FGF) and epidermal growth factor (EGF), survival and protective factors such as glial-derived neurotrophic factor, insulin-like growth factor (IGF), and others.
- Remyelinating axons. Injury damages oligodendroglial cells (the cells that provide myelin for axons). Oligodendroglia come from oligodendroglial precursor cells called O2A. These cells may be in short supply or cannot migrate well into the injury to to remyelinate axons. Note that regenerated axons are "naked" and need to be remyelinated in order to conduct efficiently.
- Replacing neurons. Neurons may be lost, particularly when the injury is close to the lumbar enlargement where the neurons from the legs are present or in the cervical enlargement where the neurons for the arms are located. The sacral tip of the spinal cord or conus contain neurons that innervate the bladder, anal sphincter, and other important functions.
Tissue niches for stem cells. As investigators gain more experience with stem cell transplants, it is becoming clear that many tissues don't have the factors that tell a stem cell what to do, what kind of cells to produce. In adult tissues, stem cells must interact with other cells, sometimes called a "niche" in order to produce other kinds of cells. This may be a very important regulator of stem cells so that they do not produce the wrong type or wrong number of cells. For example, it would not be good if bone marrow stem cells produced blood cells in the spinal cord. In general, many investigators attempt to differentiate stem cells in culture before transplantation so that they will produce the right type of cells. For example, one can differentiate embryonic stem cells by treating them with a factor called retinoic acid which pushes them to differentiate towards neural stem cells and then using factors such as sonic hedghog (SHH) to differentiate the cells further to become neurons. These approaches have worked to produce neurons in the brain and motoneurons in the spinal cord.
Neural stem cells. During development, the stem cells of the central nervous system are radial glial cells. These are very specialized looking cells that have[ur long processes that go all the way from the ventricles to the surface of the brain. These cells disappear with maturation and are absent from adult brain. However, certain populations of cells that look like glial cells (astrocytes) remain in certain areas of the brain (such as the subventricular zone or SVC) and these cells migrate to various parts of the brain and can make new neurons in the hippoaompus and the olfactory bulb. While stem cells are believed to be present in the spinal cord and several investigators have successfully isolated cells from the spinal cord that behave like stem cells, it is not clear what form these cells are in the spinal cord itself. One possibility is that they are microglia (the small non-descript cells that are activated by injury to produce large numbers of macrophages. Astrocytes also can be transformed by injury and can produce additional astrocytes. The oligodendroglial precursor O2A cells make oligodendroglial cells.
Stem cells versus differentiated cell transplants. Most of cells transplanted into the spinal cord in animal studies and human clinical trials are not stem cells and, even if they are, it is not clear that they are performing as stem cells. For example, olfactory ensheathing glia are not stem cells. Likewise, while nasal mucosa may contain some stem cells, it is not clear transplanted nasal mucosa are producing neurons in the spinal cord after transplantation. The only exception has been embryonic stem cells. These cells appear to behave like stem ells when transplanted into the spinal cord, producing some neurons, astrocytes, and oligodendroglia. To date, neither adult bone marrow nor umbilical cord blood cells have been reported to produce neurons or glia in the spinal cord after transplantation. In fact, most umbilical cord blood and bone marrow cells simply remain undifferentiated when they are transplanted into the spinal cord. While they may proliferate (i.e. produce additional cells), it seems that they produce more of themselves and not necessarily neurons, astrocytes, or oligodendroglia. However, they may stimulate endogenous cells to remyelinate the spinal cord axons.
Drug stimulation of stem cells. Some drugs seem to stimulate certain cells to produce more growth factors. For example, lithium seems to do this and this may be one of the reasons for the beneficial effects of lithium when used to treat depression. Lithium has been reported to stimulate bone marrow cells to grow. Erythropoeitin is known to stimulate bone marrow stem cells. Several bone marrow stimulation factors are known to stimulate bone marrow cell prolieration and differentiation. This approach may enhance the neuroregenerative and remyelinative effects of bone marrow and umbilical cord blood cells. This is one of the reasons why we are interested in assessing the effects of lithium on spinal cords that have been transplanted with umbilical cord blood mononuclear cells. Mononuclear cells presumably include mesenchymal stem cells. There is also substantial interest in the effects of lithium on neural stem cells because this is one of the theories as to why lithium is beneficial as a treatment of manic depression. It would be of interest to see if lithium stimulates transplanted bone marrow cells as well.
Many therapies regenerate the spinal cord without stem cells. Marie Filbin and her colleague have reported that increased cAMP levels inside neurons will allow axons to grow despite the presence of growth inhibitors. Thus, there was a great deal of excitement when Mary Bunge and colleagues at the Miami Project showed that treatment with rolipram and dibutyryl cAMP and Schwann cells (a source of growth factors and a cell that supports axonal growth) allowed large numbers of axons to grow across the contusion site of injured spinal cords, associated with improved functional recovery. Schwann cells are not stem cells. Likewise, olfactory ensheathing glial cells are not stem cells.
Embryonic stem cells need to be combined with other therapies. Douglas Kerr and colleagues has shown that human embryonic stem cells transplanted to the spinal cord will not only survive and produce motoneurons but, when stimulated with rolipram and dibutyryl cAMP, send axons out of the spinal cord to re-innervate muscle. So far, only embryonic stem cells have been shown to do this. But, it is important to note that embryonic stem cells alone cannot do this. They must be pre-differentiated and combined with other therapies in order to achieve the objective of replacing neurons for these neurons to regenerate and reconnect with muscle.
Stem cells must be differentiated before they are transplanted. Much evidence suggest that it is important to differentiate the stem cells before they are transplanted. In the case of embryonic stem cells, it is necessary to differentiate them or else they may produce inappropriate types and numbers of cells in the spinal cord. Note that it is not necessary for embryonic stem cells to become cancers (i.e. cells that have lost their growth control). All they have to do is produce the wrong number and types of cells. For example, it would not be good if embryonic stem cells produced fibroblasts (skin cells) or hair cells in the spinal cord. To avoid this, most investigators pre-differentiate embryonic stem cells before they transplant them into the spinal cord. Thus, what is being transplanted are not stem cells but rather progenitor or even precursor cells that produce astrocytes, oligodendroglia, or neurons in the spinal cord. In fact, Steven Davies and his colleagues recently reported that it is helpful to differentiate fetal neural stem cells into a particular kind of astrocyte before transplanting them into the spinal cord, to encourage regeneration.
Embryonic stem cells are important. Embryonic stem cells are potentially a source of all the cells in the body. They provide the possibility of producing cells rather than having to harvest the specific cells from different organs for transplantation. While this is controversial, I personally believe that we (not our group but scientists in general) someday should be able make many kinds of cell of the body behave like stem cells. However, in order to reach this goal, it is critical that scientists be allowed to study human embryonic stem cells, find out what makes them pluripotent, identify factors that cause them to produce or differentiate into different kinds of cells, and how to regulate them. Why can't this be done in animal cells? There are important differences between animal and human cells. In fact, for reasons that we still don't understand, scientists have had very little success growing rat embryonic stem cells even though we can grow mouse, human, and primate embryonic stem cells. The fact that we cannot grow rat embryonic stem cells should give people an idea how little we understand of the factors that control and sustain embryonic stem cells and that there are species differences that we don't understand.
Embryonic stem cells alone are not a cure. It is important that people don't expect a cure from just by transplanting human embryonic stem cells into the spinal cord for several reasons. First, there is no reason why embryonic stem cells should or would know what to do when they are plugged into an injured spinal cord. Second, the studies with embryonic stem cell transplants in animal spinal cord injury models have been done shortly after injury and in chronically injured spinal cords. Third, there is the problem of immune rejection of transplanted cells. Although several laboratories have hypothesized that embryonic stem cells are not rejected when transplanted into the spinal cord, this is not the experience of most investigators. That is the reason why there is strong interest in cloning of embryonic stem cells. Fourth, the mechanisms of immune rejection in the central nervous system may be different from immune rejection in other parts of the body.
Adult stem cells alone are not a cure. Likewise, it is important that people don't expect a cure from just infusing umbilical cord blood cells into people. Many clinics are advertising umbilical cord blood treatments as if umbilical cord blood stem cells injected into the bloodstream will go directly to the spinal cord and start to produce the right types of cells and the right types of growth factors to repair the spinal cord. I am very skeptical of claims that are saying that umbilical cord blood cells are doing this. For the past two years, at the Rutgers Keck Center, we have been transplanting umbilical cord blood cells into the spinal cord and finding that the cells do not produce neurons, astrocytes, or oligodendroglial cells. Yes, when they are transplanted directly into the spinal cord, umbilical cord blood cells do produce growth factors that may be beneficial for the spinal cord. However, when we have injected human or rat neonatal blood cells intravenously into rats after spinal cord injury, we find that few or none of the cells go into the spinal cord, even when we suppress the immune system.
Stem cells are one of several tools that we should use to repair the spinal cord and stimulate regeneration. Scientists must be allowed to study a diversity of human embryonic stem cells to understand what and how they do what they do. By understanding stem cell biology, we should be able to make any cell behave like a stem cell. After all, a stem cell is just a cell that is expressing certain genes. Also, it is important to understand how the central nervous system recognize "foreign" cells and reject them. If we know the mechanisms, we may be able to trick the spinal cord to accept transplanted cells are "native" to the spinal cord. Finally, we need to know the factors needed to get the cells to do what we would like them to do, to stimulate regeneration, to provide a substrate that is conducive to axonal growth, to replace neurons, and to remyelinate the spinal cord.
No reason to be discouraged. After reading the above, many may conclude that it will take a long time before treatments become available to restore function to people with spinal cord injury. I don't think so for the following reasons. First, stem cells do provide an important substrate of regeneration in the spinal cord. For regeneration, it may not be necessary for the transplanted cells to stay there forever. For example, once the cells have created a bridge and the axons have grown across, it may not be necessary to keep the bridge. In fact, it might even be a good idea for the bridge to go away. In fact, this may be one reason why stem cell transplants have been relatively safe. Second, stem cells are a very efficient way of delivering growth factors to the spinal cord. I am quite excited by the discovery that umbilical cord blood cells secrete most of the growth factors that are known to stimulate regeneration and remyelination in the spinal cord. Third, although we may not understand the mechanisms, many studies have reported beneficial effects of stem cell transplants to the spinal cord.
Nogo receptor blockers. Many studies indicating that blockade of axonal growth inhibitors will allow regeneration to occur in the spinal cord. For example, there are phase 1 trials of the Nogo antibody by Novartis and Cethrin (the blocker of Nogo receptor intracellular messenger rho) by Bioaxone. Dozens of studies have shown the beneficial effects of chondroitinase in animal studies. Likewise, Biogen has identify other blockers of the Nogo receptor. These need to be taken to clinical trial. When these are combined with cell transplants and sustained growth factor support, I hope that we will see substantial regeneration in human spinal cords. It is not trivial to get such complicated combination therapy trials tested. We have to develop the clinical trial infrastructure now so that we are ready when the therapies are available to be tested in humans.