A scientific rationale for studying cloned stem cells

[Wise Young]

We have heard a lot of opinions expressed about the pros and cons of cloned and adult stem cells but, to date, I have not heard a clear explanation for the scientific value of having some embryonic and cloned stem cells available for human studies. Before doing so, let define the different kinds of stem cells.

Embryonic stem cells are stem cells that are obtained from a cloned or fertilized egg about two weeks after fertilization. The term "embryonic" is a misnomer in that the cells are obtained from a blastocyst which is before the egg becomes an embyro. Fetal stem cells are obtained from aborted fetuses. Umbilical cord blood stem cells are obtained from umbilical or placental blood of newborn babies and represent neonatal blood; note that most of the stem cells in such blood are hematopoeitic stem cells or cells that produce blood cells and only about 1% of the cells are pluripotent stem cells. Adult stem cells are obtained from adult tissues, generally from two sources: the brain and bone marrow. Much less than 1% of the cells in the bone marrow are mesenchymal stem cells that have been shown to be pluripotent. Peripheral blood has some mesenchymal stem cells and are very rare. While stem cells have been reported in other tissues, such as fat, they are very very rare and may actually be mesenchymal stem cells from blood.

Scientists want to study embryonic stem cells (ESCs) and fetal stem cells (FSCs) for several reasons. First, they are the cells that produce all the other cells in the body. We don't know how they do this. Second, they are likely to have greater ability for producing all different kinds of cells and much greater capacity to divide and produce many cells. For example, an adult stem cell probably will not divide more than 30-70 times while embryonic stem cells can divided hundreds of time. Third, stem cells from embryonic, fetal, and neonatal sources are able to reproduce themselves, i.e. produce more stem cells.

Cloned stem cells are obtained from a blastocyst that results from transferring a nucleus to a blastocyst (somatic nuclear transfer) or simply tricking an egg to develop into a blastocyst (parthenogenesis). No fertilization is involved. The cells are obtained from the blastocyst in the same way as other embryonic stem cells. A cloned stem cell would have the same genetic makeup as the transferred nucleus in the case of somatic nuclear transfer or the mother in the case of parthenogenesis.

Why do we need to study cloned stem cells? If embryonic stem cells from another individual is transplanted to somebody, the recipient's immune system may recognize the cells as being foreign and reject the cells. If a cloned stem cell is transplanted into the person from which the stem cells are cloned, the likelihood of rejection should be less.

However, foreign antigens is not the only reason that transplanted cells are rejected. Our immune systems are exquisitely tuned to detect growing cells that may be cancerous. Cloned stem cells may be rejected for that reason. Note that cells have some genes outside of the nuclei, specifically the mitochondria. There is some suggestion that mitochondrial genes may induce immune rejection although this is only one explanation for the rejection of cloned stem cells. Cloned stem cells may be rejected because they are growing and the recipient's immune system may mistaken them for cancer cells.

In the coming years, there will probably be clinical trials of embryonic stem cells and even cloned embryonic stem cells in humans. These will undoubtedly be compared against adult stem cells. If adult stem cells work, that would of course be great and I doubt that anybody would go to the extent of using embryonic stem cells or cloned embronic stem cells. However, if adult stem cells do not work and embryonic stem cells are beneficial, there will be a need for a source of embryonic stem cells that are less likely to be rejected by the immune system. Therefore, cloned embryonic stem cells may be necessary.

In the end, however, I believe that we will know what genes are expressed by embryonic stem cell and how to make any cell into a stem cell. When this happens, there will be no shortage of cells for therapy. But, a lot of work and study of embryonic stem cells will be needed to reach this point. Without access to human embryonic stem cells for study, it will be many years before we discover how to make stem cells.

If implanted embryonic stem cells fail to engraft (i.e. survive and grow), this may be because they were recognized as foreign cells by the immune system and rejected. It may also be because the embryonic stem cells express antigens that make the immune system think that they are cancerous cells and the immune system will reject them. To differentiate between these possibilities, it will be useful to compare cloned and non-cloned embryonic stem cells.

At the present, many laboratories are developing techniques to differentiate stem cells into neuronal, astrocytic, and oligodendroglial precursor cells. These studies are being done on mice and the approaches developed for mouse may not be the same as for humans. There is a need to develop these methods for human stem cells as well. The future of transplantation will be in the direction of transplanting mixtures of defined progenitor cells rather than the stem cells themselves. At the present, the only method that we have for producing progenitor cells is from stem cells. If stem cell therapies are to become a reality, we need to have facilities and procedures for manufacturing stem cells or progenotpr cells in large quantities for clinical application.

Without NIH funding, development of stem cell faciities will be very slow because most pharmaceutical companies are not investing in such resarch or facilities. Whether the cells come from embryonic, fetal, umbilical, or adult sources, such facilities are still necessary for production of stem cells for therapies. Adult stem cells are more rare, more difficult to culture, possibly less pluripotent, and less able to divide rapidly to produce millions of cells for therapeutic purposes.

In summary, embryonic stem cells differ from adult stem cells in their pluripotency, ability to divide many times, and possibly ability to reproduce themselves. The immune system may reject an implanted stem cell because it thinks that it is a foreign cell or a cancerous cell. Even cloned stem cells may be rejected if the latter is the case. It is therefore important to have access to heterologous embryonic stem cells, cloned embryonic stem cells, and homologous adult stem cells.

Wise.

[This message was edited by Wise Young on 03-04-03 at 18:17.]

[davecarl]

Wise, do you have an update to the article. In particular, what is your current view on Embryonic Stem Cells and the current Research.


Thank you,

Dave Menaker

[Wise Young]

Wise, do you have an update to the article. In particular, what is your current view on Embryonic Stem Cells and the current Research.


Thank you,

Dave Menaker

Dave,

We currently do not have a practical source of stem cells to treat millions of people. This statement actually applies to any kind of cell, including blood. There is no question in my mind that cellular therapy will be a very significant part of the cure for many diseases and conditions. This is not just my opinion but that of most scientists and doctors. Given this belief, it is astounding that our government has been obstructing research that could be developing sources of cells that can be used to treat millions of people. Let me give just two examples to illustrate the problem that we face.

• Many millions of people suffer from hemapoietic and autoimmune diseases, such as thalessemia, sickle cell anemia, immune-deficiency syndromes, lupus erythematosus, multiple sclerosis, multiple myeloma, leukemia, and a multitude of conditions that could be cured by replacement of hematopoietic and immune stem cells. At the present, we only have bone marrow and umbilical cord blood stem cells to treat these conditions and much data suggest that these cells can *cure* the condition. For example, recent studies suggest that 80% of thalessemia can be cured by umbilical cord blood transfusions. This is a horrendous disease that probably affect over 35 million people around the world and that is currently treated by repeated blood transfusions. There are probably at most 200,000 units of umbilical cord blood available around the world for transplantation, sufficient to provide matched units for 20,000 people per year. In other words, for this one condition alone, we currently don't have cells to treat even 1% of the people with this affliction. If a nuclear disaster were to occur (such as Chernobyl or a "dirty" bomb) that expose even a relatively small city to high levels of radioactivity, hundreds of thousands of people will die from radiation-induced destruction of their bone marrows. At the present, we have no way of treating so many people with available bone marrow or umbilical cord blood stem cells. Millions of people will die because we do not have the cells to treat them.

• Stem cell transplants are an important approach to treating neurological disorders such as spinal cord injury, traumatic brain injury, stroke, motoneuronal disease, and degenerative diseases such as Alzheimer's and Parkinson's disease. Probably over 100 million people in the world suffer from these conditions. Can you imagine what would happen if a series of clinical trials were to show that stem cells can prevent or even reverse neurological deficits in even one of these conditions? Not only will millions of people die if we don't have the cells to treat them but what price can be placed on the cells? What will we do? Have a lottery for the cells or have them go to the highest bidder? This would be a moral catastrophe that is worse that anything that we have faced. To a certain extent, we are already facing this problem with organ transplantation. Thousands of people are dying while awaiting cadaver kidneys and hearts for transplantation. This will be multiplied a hundredfold if and when the first stem cell therapy has been shown to be effective for any neurological condition.

Embryonic stem cells are imbued with two qualities that make them attractive as sources of cells. First, they can make many kinds of cells. Second, they can produce many cells. We do not yet fully understand the biology of these cells and how they do what they do. While studies of animal stem cells have given us significant insights into stem cells, the research also suggests that human stem cells may be quite different. The methods that we have for growing the cells are still very primitive and empirically based. Much basic research is needed to determine what allows these cells to make many kinds of cells and to be virtually immortal. This knowledge would allow us to make and grow any kind of cells from any cell of the body.

There are two additional great unsolved problems. One is immune rejection of the cells. While everybody has been aiming at cloning as a means of producing patient-specific cells, this is not necessarily the only or even practical solution to the problem. There is relatively little research being done to find out why and how the immune system recognize transplanted cells, particularly in the central nervous system. One solution to the problem of immune rejection is to teach the immune system to tolerate certain transplanted cells. If so, we should be able to create a "universal" stem cell that could be transplanted into all or most people.

The second major unsolved problem is teaching the stem cells to do the "right thing". Many of us assume that stem cells "know" what to do when they are transplanted. How does a stem cell "know", when it has been transplanted into the spinal cord, that it should be creating type I astrocytes to bridge the injury site, to spew out the right combination of growth factors to stimulate regeneration, to migrate to the right places, to myelinate axons, and to replace neurons? The cells may also do the "wrong thing" when transplanted. A stem cell that produces the wrong type or the wrong number of cells is, by definition, a tumor. We should also not be assuming that adult stem cells "know" any better than embryonic stem cells.

These problems lie at the core of biology. Solutions to these problems will not only provide sources of cells for therapy but are likely to represent cures for cancer and a huge variety of other diseases for which we currently have little or no effective therapies. The discovery of adult pluripotent stem cells in our body, including the brain, occurred only recently, less than a decade ago. Stem cells clearly play a major role in our bodies, not only for repair of tissues but also in aging of tissues. It is astonishing, given the importance of stem cells to biology and medicine, that the National Institutes of Health (NIH) currently is investing less than $250 million into all of human stem cell research, both embryonic and adult. This is less than 1% of the total NIH budget of over $28 billion per year. By comparison, for example, our government is spending over $8 billion per year on bioterrorism research and vaccines for anthrax and smallpox.

How is it possible that the politics of stem cells have resulted in such gross misallocation of our medical research priorities? Some people blame this solely on religious politics and the misguided policies of George Bush. But, the real problem, in my opinion, is the failure of Congress and people to understand what stem cells do, why the research is so important, and what needs to be done to bring stem cell therapies to practical clinical applications. Both opponents and proponents of stem cell research mistakenly believe that stem cells are not only pluripotent but omnipotent (all-knowing). There is a general public perception that all we have to do is plop the cells into somebody and they will be cured. Many in our own community are willing to go anywhere in the world to get "stem cell" therapies. Most of the time, the cells that are being transplanted not only may not be stem cells but the doctors who are transplanting them have no idea what they are transplanting.

It is so important to give scientists the opportunity and the time to understand the biology of stem cells. We currently don't know what signals stem cells respond to in tissues that tell them to produce the right kind and number of cells. We don't understand why certain stem cells divide a certain number of times and others will keep dividing for as long as they are observed. We have precious few tools to persuade stem cells to produce certain kinds of cells. We are only beginning to understand what turns on stem cells and what controls them. Without such understanding, stem cell therapies will be mostly misses and hopefully a few hits.

Curiuously, the failure of the federal government to invest adequately into stem cell research has led to the unprecedented emergence of state funding of biomedical research. Since World War II, the federal government has been the major source of biomedical research funding. This funding is what allowed the United States to become the dominant source of biomedical technology for the world. Over the last six years, the federal government has ceded their leadership in stem cell research to other countries and to the state governments. Starting this coming year, California alone will be spending more money on stem cell research than the National Institutes of Health. Other states such as New Jersey are following suit.

The economic impact of the Bush Administration's stem cell policy will be much greater than we think. As I have pointed out above, when the first stem cell therapies are shown to be effective for treating diseases, there will be an catastrophic shortage of cells for therapy. Stem-cell producing countries and states will dominate world economy, much like oil-producing countries who are currently dominating the world economy. The only difference is that there is no price too high for a cure.

Wise.

[mimij5]

Wise-
This post is a wonderfully comprehensive and pointful overview of the stem cell situation in this country, considering both (lack of) consiousness and funding. Our pals in the Russel Building should read it, imho.
Mimi

[Agios]

Dr. Young,

1) Which researchers do you consider to be the leaders in adult stem cells and hesc research in regards to motor neuron regeneration either here in the USA or elsewhere?

2) Is credible motor neuron regeneration research taking place outside the USA? If so, where?

3) Is there much collaberation and sharing of information within the field?

[Wise Young]

Dr. Young,

1) Which researchers do you consider to be the leaders in adult stem cells and hesc research in regards to motor neuron regeneration either here in the USA or elsewhere?

• There are many but the ones that come to mind include John Gearhart, Doug Kerr, and others at Johns Hopkins. There are also good groups at Harvard, Cornell, and other places. I list some abstracts below.

2) Is credible motor neuron regeneration research taking place outside the USA? If so, where?

• Canada, Japan, China

3) Is there much collaberation and sharing of information within the field?

• Yes, I think so.

1. Liu Z and Martin LJ (2006). The adult neural stem and progenitor cell niche is altered in amyotrophic lateral sclerosis mouse brain. J Comp Neurol 497: 468-88. Amyotrophic lateral sclerosis (ALS) is a fatal adult human disease caused by motor neuron degeneration. Stem cell therapy might be a treatment for ALS. The adult mammalian forebrain has neural stem cells (NSCs) and neural progenitor cells (NPCs) in the anterior subventricular zone (SVZa), rostral migratory stream (RMS), olfactory bulb (OB) core, and dentate gyrus (DG). These cells could be used to rescue or replace degenerating upper and lower motor neurons through endogenous recruitment or autologous/allogenic transplantation. We evaluated the competency of forebrain NSCs and NPCs in transgenic (tg) mice harboring human mutant superoxide dismutase-1 (mSOD1), a model of ALS. Tg human wild-type SOD1 (wtSOD1) mice and non-tg mice were controls. Bromodeoxyuridine (BrdU) labeling of cells, a marker for cell proliferation and other events, was reduced in a niche-specific pattern in presymptomatic and symptomatic mice, with the SVZa having greater reductions than the RMS, OB, and DG. Different NSC and NPC complements were evaluated by localizing nestin, neural cell adhesion molecule, distalless-2 transcription factor, vimentin, and glial fibrillary acidic protein. In symptomatic mice, NSC markers were reduced, whereas NPC markers were unchanged or elevated. Neurogenesis was preserved in symptomatic mSOD1 mice. NSC/NPC competence assessment in vitro revealed that mSOD1 SVZa cells had the ability to proliferate and form neurospheres but had an impaired response to mitogen stimulation. We conclude that adult mSOD1 ALS mice have abnormalities in forebrain NSCs, but the essential features of NSC/NPCs remained in presymptomatic and symptomatic mice. J. Comp. Neurol. 497:468-488, 2006. (c) 2006 Wiley-Liss, Inc. Department of Pathology, Division of Neuropathology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2196. http://www.ncbi.nlm.nih.gov/entrez/q..._uids=16736475
   
2. Soundararajan P, Miles GB, Rubin LL, Brownstone RM and Rafuse VF (2006). Motoneurons derived from embryonic stem cells express transcription factors and develop phenotypes characteristic of medial motor column neurons. J Neurosci 26: 3256-68. Embryonic stem (ES) cells differentiate into functional motoneurons when treated with a sonic hedgehog (Shh) agonist and retinoic acid (RA). Whether ES cells can be directed to differentiate into specific subtypes of motoneurons is unknown. We treated embryoid bodies generated from HBG3 ES cells with a Shh agonist and RA for 5 d in culture to induce motoneuron differentiation. Enhanced green fluorescent protein (eGFP) expression was used to identify putative motoneurons, because eGFP is expressed under the control of the Hb9 promoter in HBG3 cells. We found that 96 +/- 0.7% of the differentiated eGFP+ motoneurons expressed Lhx3, a homeobox gene expressed by postmitotic motoneurons in the medial motor column (MMCm), when the treated cells were plated on a neurite-promoting substrate for 5 d. When the treated embryoid bodies were transplanted into stage 17 chick neural tubes, the eGFP+ motoneurons migrated to the MMCm, expressed Lhx3, projected axons to the appropriate target for MMCm motoneurons (i.e., epaxial muscles), and contained synaptic vesicles within intramuscular axonal branches. In ovo and in vitro studies indicated that chemotropic factors emanating from the epaxial muscle and/or surrounding mesenchyme likely guide Lhx3+ motoneurons to their correct target. Finally, whole-cell patch-clamp recordings of transplanted ES cell-derived motoneurons demonstrated that they received synaptic input, elicited repetitive trains of action potentials, and developed passive membrane properties that were similar to host MMCm motoneurons. These results indicate that ES cells can be directed to form subtypes of neurons with specific phenotypic properties. Department of Anatomy and Neurobiology, Dalhousie University, Halifax, Nova Scotia, B3H 1X5, Canada. http://www.ncbi.nlm.nih.gov/entrez/q..._uids=16554476
   
3. Hamada M, Yoshikawa H, Ueda Y, Kurokawa MS, Watanabe K, Sakakibara M, Tadokoro M, Akashi K, Aoki H and Suzuki N (2006). Introduction of the MASH1 gene into mouse embryonic stem cells leads to differentiation of motoneuron precursors lacking Nogo receptor expression that can be applicable for transplantation to spinal cord injury. Neurobiol Dis 22: 509-522. ES cells transfected with the MASH1 gene yielded purified spinal motoneuron precursors expressing HB9 and Islet1. The cells lacked the expression of Nogo receptor that was of great advantage for axon growth after transplantation to an injured spinal cord. After transplantation, mice with the complete transection of spinal cord exhibited excellent improvement of the motor functions. Electrophysiological assessment confirmed the quantitative recovery of motor-evoked potential in the transplanted spinal cord. In the grafted spinal cord, gliosis was inhibited and Nogo receptor expression was scarcely detected. The transplanted cells labeled with GFP showed extensive outgrowth of axons positive for neurofilament middle chain, connected to each other and expressed Synaptophysin, Lim1/2 and Islet1. Thus, the in vivo differentiation into mature spinal motoneurons and the reconstitution of neuronal pathways were suggested. The grafted cell population was purified for neurons and was free from teratoma development. These therapeutic strategies may contribute to a potent treatment for spinal cord injury in future. Department of Immunology and Medicine, St. Marianna University School of Medicine, 2-16-1, Sugao, Miyamae-ku, Kawasaki, Kanagawa 216-8511, Japan; Department of Orthopedic Surgery, St. Marianna University School of Medicine, 2-16-1, Sugao, Miyamae-ku, Kawasaki, Kanagawa 216-8511, Japan; Department of Emergency Critical Care Medicine, St. Marianna University School of Medicine, 2-16-1, Sugao, Miyamae-ku, Kawasaki, Kanagawa 216-8511, Japan. http://www.ncbi.nlm.nih.gov/entrez/q..._uids=16497507
   
4. Corti S, Locatelli F, Papadimitriou D, Donadoni C, Del Bo R, Crimi M, Bordoni A, Fortunato F, Strazzer S, Menozzi G, Salani S, Bresolin N and Comi GP (2006). Transplanted ALDHhiSSClo neural stem cells generate motor neurons and delay disease progression of nmd mice, an animal model of SMARD1. Hum Mol Genet 15: 167-87. Spinal muscular atrophy with respiratory distress type 1 (SMARD1) is an infantile autosomal-recessive motor neuron disease caused by mutations in the immunoglobulin micro-binding protein 2. We investigated the potential of a spinal cord neural stem cell population isolated on the basis of aldehyde dehydrogenase (ALDH) activity to modify disease progression of nmd mice, an animal model of SMARD1. ALDH(hi)SSC(lo) stem cells are self-renewing and multipotent and when intrathecally transplanted in nmd mice generate motor neurons properly localized in the spinal cord ventral horns. Transplanted nmd animals presented delayed disease progression, sparing of motor neurons and ventral root axons and increased lifespan. To further investigate the molecular events responsible for these differences, microarray and real-time reverse transcription-polymerase chain reaction analyses of wild-type, mutated and transplanted nmd spinal cord were undertaken. We demonstrated a down-regulation of genes involved in excitatory amino acid toxicity and oxidative stress handling, as well as an up-regulation of genes related to the chromatin organization in nmd compared with wild-type mice, suggesting that they may play a role in SMARD1 pathogenesis. Spinal cord of nmd-transplanted mice expressed high transcript levels for genes related to neurogenesis such as doublecortin (DCX), LIS1 and drebrin. The presence of DCX-expressing cells in adult nmd spinal cord suggests that both exogenous and endogenous neurogeneses may contribute to the observed nmd phenotype amelioration. Dino Ferrari Centre, Department of Neurological Sciences, University of Milan, IRCCS Foundation Ospedale Maggiore Policlinico, Mangiagalli and Regina Elena, Milan, Italy. http://www.ncbi.nlm.nih.gov/entrez/q..._uids=16339214
   
5. Singh Roy N, Nakano T, Xuing L, Kang J, Nedergaard M and Goldman SA (2005). Enhancer-specified GFP-based FACS purification of human spinal motor neurons from embryonic stem cells. Exp Neurol 196: 224-34. Human embryonic stem (hES) cells may generate all major somatic cell types, yet no neuronal subtype has yet been specifically generated in useful purity from hES culture. We report here the selective induction and isolation of functional spinal motor neurons (MNs) from human ES cells. hES cells of the H1 line were transfected with plasmids encoding GFP placed under the control of an MN-specifying enhancer within the 5'-regulatory region of the gene encoding the transcription factor Hb9 and treated with sonic hedgehog (Shh) and retinoic acid (RA). As MNs were induced under the influence of Shh and RA, they activated Hb9-driven GFP expression, permitting their isolation by fluorescence-activated cell sorting (FACS). The MNs thereby generated and isolated became cholinergic and achieved functional maturation in vitro, as evidenced by their fast sodium currents and action potentials on whole-cell patch-clamp and alpha-bungarotoxin-identified clustering of AChR receptors on co-cultured skeletal myoblasts. The serial combination of these two approaches, motor neuron phenotypic induction followed by Hb9 enhancer-based FACS, permitted the high-efficiency induction and isolation of functional motor neurons from hES cells. These results suggest the utility of promoter/enhancer-based FACS for the isolation of specific phenotypes from hES cell populations as a means of purifying clinically appropriate vectors for cell therapy. Department of Neurology, Cornell University Medical College, NYC, NY 10021, USA. ner2004@med.cornell.edu http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&li st_uids=16198339''
   
6. Klein SM, Behrstock S, McHugh J, Hoffmann K, Wallace K, Suzuki M, Aebischer P and Svendsen CN (2005). GDNF delivery using human neural progenitor cells in a rat model of ALS. Hum Gene Ther 16: 509-21. Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive loss of spinal cord, brainstem, and cortical motor neurons. In a minority of patients, the disease is caused by mutations in the copper (2+)/zinc (2+) superoxide dismutase 1 (SOD1) gene. Recent evidence suggests that astrocytes are dysfunctional in ALS and may be a critical link in the support of motor neuron health. Furthermore, growth factors, such as glial cell line-derived neurotrophic factor (GDNF), have a high affinity for motor neurons and can prevent their death following various insults, but due to the protein's large size are difficult to directly administer to brain. In this study, human neural progenitor cells (hNPC) isolated from the cortex were expanded in culture and modified using lentivirus to secrete GDNF (hNPC(GDNF)). These cells survived up to 11 weeks following transplantation into the lumbar spinal cord of rats overexpressing the G93A SOD1 mutation (SOD1 (G93A)). Cellular integration into both gray and white matter was observed without adverse behavioral effects. All transplants secreted GDNF within the region of cell survival, but not outside this area. Fibers were seen to upregulate cholinergic markers in response to GDNF, indicating it was physiologically active. We conclude that genetically modified hNPC can survive, integrate, and release GDNF in the spinal cord of SOD1 (G93A) rats. As such, they provide an interesting source of cells for both glial replacement and trophic factor delivery in future human clinical studies. Waisman Center and Department of Anatomy, University of Wisconsin, Madison, WI 53703, USA. http://www.ncbi.nlm.nih.gov/entrez/q..._uids=15871682
   
7. Chen J, Magavi SS and Macklis JD (2004). Neurogenesis of corticospinal motor neurons extending spinal projections in adult mice. Proc Natl Acad Sci U S A 101: 16357-62. The adult mammalian CNS shows a very limited capacity to regenerate after injury. However, endogenous precursors, or stem cells, provide a potential source of new neurons in the adult brain. Here, we induce the birth of new corticospinal motor neurons (CSMN), the CNS neurons that die in motor neuron degenerative diseases, including amyotrophic lateral sclerosis, and that cause loss of motor function in spinal cord injury. We induced synchronous apoptotic degeneration of CSMN and examined the fates of newborn cells arising from endogenous precursors, using markers for DNA replication, neuroblast migration, and progressive neuronal differentiation, combined with retrograde labeling from the spinal cord. We observed neuroblasts entering the neocortex and progressively differentiating into mature pyramidal neurons in cortical layer V. We found 20-30 new neurons per mm(3) in experimental mice vs. 0 in controls. A subset of these newborn neurons projected axons into the spinal cord and survived >56 weeks. These results demonstrate that endogenous precursors can differentiate into even highly complex long-projection CSMN in the adult mammalian brain and send new projections to spinal cord targets, suggesting that molecular manipulation of endogenous neural precursors in situ may offer future therapeutic possibilities for motor neuron degenerative disease and spinal cord injury. Department of Neurosurgery and Program in Neuroscience, Massachusetts General Hospital-Harvard Medical School Center for Nervous System Repair, Harvard Medical School, Massachusetts General Hospital, Boston, MA 02114, USA. http://www.ncbi.nlm.nih.gov/entrez/q..._uids=15534207
   
8. Miles GB, Yohn DC, Wichterle H, Jessell TM, Rafuse VF and Brownstone RM (2004). Functional properties of motoneurons derived from mouse embryonic stem cells. J Neurosci 24: 7848-58. The capacity of embryonic stem (ES) cells to form functional motoneurons (MNs) and appropriate connections with muscle was investigated in vitro. ES cells were obtained from a transgenic mouse line in which the gene for enhanced green fluorescent protein (eGFP) is expressed under the control of the promotor of the MN specific homeobox gene Hb9. ES cells were exposed to retinoic acid (RA) and sonic hedgehog agonist (Hh-Ag1.3) to stimulate differentiation into MNs marked by expression of eGFP and the cholinergic transmitter synthetic enzyme choline acetyltransferase. Whole-cell patch-clamp recordings were made from eGFP-labeled cells to investigate the development of functional characteristics of MNs. In voltage-clamp mode, currents, including EPSCs, were recorded in response to exogenous applications of GABA, glycine, and glutamate. EGFP-labeled neurons also express voltage-activated ion channels including fast-inactivating Na(+) channels, delayed rectifier and I(A)-type K(+) channels, and Ca(2+) channels. Current-clamp recordings demonstrated that eGFP-positive neurons generate repetitive trains of action potentials and that l-type Ca(2+) channels mediate sustained depolarizations. When cocultured with a muscle cell line, clustering of acetylcholine receptors on muscle fibers adjacent to developing axons was seen. Intracellular recordings of muscle fibers adjacent to eGFP-positive axons revealed endplate potentials that increased in amplitude and frequency after glutamate application and were sensitive to TTX and curare. In summary, our findings demonstrate that MNs derived from ES cells develop appropriate transmitter receptors, intrinsic properties necessary for appropriate patterns of action potential firing and functional synapses with muscle fibers. Department of Anatomy and Neurobiology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7. http://www.ncbi.nlm.nih.gov/entrez/q..._uids=15356197
   
9. Liu Y and Rao MS (2004). Glial progenitors in the CNS and possible lineage relationships among them. Biol Cell 96: 279-90. Glial cells are derived from stem cells that mature through specific stages of development to generate fully differentiated astrocytes and oligodendrocytes. Several types of intermediate precursors have been described and in some cases lineage relationships identified although this remains a subject of controversy. We review recent findings and discuss some possibilities. Motoneuron-oligodendrocyte precursors (MNOPs), white matter progenitor cells (WMPCs), polydendrocytes, glial restricted precursors (GRPs), astrocyte precursor cells (APCs), and oligodendroblasts are likely all derived from earlier appearing stem cells but segregate at different stages in development. Some of these precursors persist in the adult, and it is these glial progenitors rather than stem cells that respond after injury and participate in the repair process. Although which specific glial progenitor responds remains unclear, the availability of new markers will likely resolve this issue. We believe that the development of consensus sets of markers and an improvement in our ability to define stages of glial maturation will lead to a clearer appreciation of the importance of glia in the etiopathology of disease. Laboratory of Neurosciences, National Institute on Aging, Baltimore, MD 21224, USA. http://www.ncbi.nlm.nih.gov/entrez/q..._uids=15145532
   
10. Harper JM, Krishnan C, Darman JS, Deshpande DM, Peck S, Shats I, Backovic S, Rothstein JD and Kerr DA (2004). Axonal growth of embryonic stem cell-derived motoneurons in vitro and in motoneuron-injured adult rats. Proc Natl Acad Sci U S A 101: 7123-8. We generated spinal motoneurons from embryonic stem (ES) cells to determine the developmental potential of these cells in vitro and their capacity to replace motoneurons in the adult mammalian spinal cord. ES cell-derived motoneurons extended long axons, formed neuromuscular junctions, and induced muscle contraction when cocultured with myoblasts. We transplanted motoneuron-committed ES cells into the spinal cords of adult rats with motoneuron injury and found that approximately 3,000 ES cell-derived motoneurons (25% of input) survived for >1 month in the spinal cord of each animal. ES cell-derived axonal growth was inhibited by myelin, and this inhibition was overcome by administration of dibutyryl cAMP (dbcAMP) or a Rho kinase inhibitor in vitro and in vivo. In transplanted rats infused with dbcAMP, approximately 80 ES cell-derived motor axons were observed within the ventral roots of each animal, whereas none were observed in transplanted rats not treated with dbcAMP. Because these cells replicate many of the developmental and mature features of true motoneurons, they are an important biological tool to understand formation of motor units in vitro and a potential therapeutic tool to reconstitute neural circuits in vivo. Department of Neurology, Johns Hopkins University School of Medicine, Pathology 627C, 600 North Wolfe Street, Baltimore, MD 21287, USA. http://www.ncbi.nlm.nih.gov/entrez/q..._uids=15118094
    
11. MacDonald SC, Fleetwood IG, Hochman S, Dodd JG, Cheng GK, Jordan LM and Brownstone RM (2003). Functional motor neurons differentiating from mouse multipotent spinal cord precursor cells in culture and after transplantation into transected sciatic nerve. J Neurosurg 98: 1094-103. OBJECT: One of the current challenges in neurobiology is to ensure that neural precursor cells differentiate into specific neuron types, so that they can be used for transplantation purposes in patients with neuron loss. The goal of this study was to determine if spinal cord precursor cells could differentiate into motor neurons both in culture and following transplantation into a transected sciatic nerve. METHODS: In cultures with trophic factors, neurons differentiate from embryonic precursor cells and express motor neuronal markers such as choline acetyltransferase (ChAT), Islet-1, and REG2. Reverse transcription-polymerase chain reaction analysis has also demonstrated the expression of Islet-1 in differentiated cultures. A coculture preparation of neurospheres and skeletal myocytes was used to show the formation of neuromuscular connections between precursor cell-derived neurons and myocytes both immunohistochemically and electrophysiologically. Following various survival intervals, precursor cells transplanted distal to a transection of the sciatic nerve differentiated into neurons expressing the motor neuron markers ChAT and the alpha1 1.2 (class C, L-type) voltage-sensitive Ca++ channel subunit. These cells extended axons into the muscle, where they formed cholinergic terminals. CONCLUSIONS: These results demonstrate that motor neurons can differentiate from spinal cord neural precursor cells grown in culture as well as following transplantation into a transected peripheral nerve. Department of Physiology, University of Manitoba, Winnipeg, Manitoba, Canada. http://www.ncbi.nlm.nih.gov/entrez/q..._uids=12744371

[JimmyMack]

Wise,
Both of your posts #1 and #3 are elegant, I hope every CC member reads them. You have opened my eyes to a whole new set of dynamics concerning stem cells, availability,immunability, coaxing stem cells to get the right job done in the right place with reliabilty etc.

I do have a question, where are the philanthropic donations, certainly there must be a person or foundation willing to plunk a billion down, I'm surprised Soros or Gates hasnt step up yet, they certainly have a more liberal stance and I would imagine that they would wholeheartedly support ESC research, they could hide behind foundations if they are afraid of bad press.

I think I might understand why venture capitalists don't cough up the cash the ROI has to be almost a gimme and immediate, do you think once the ball gets going and we see progress the VC's will jump on board?


JimmyMack

[lilsister]

Thank you Dr Wise, just happened that I wanted just that concise info tonight for more media blitz here in Wisconsin. I have been getting questions from Republicans after speaking for re-election for Doyle here. I strongly feel that the explanations have to be simple and understandable to John Q Public or their interest wanes, or the information gets distorted. The cloning seems to be the biggest issue here, many are fearful of " people being cloned". Do you feel that the research being done here in Wisconsin is important? Deb

[Leif]

I do have a question, where are the philanthropic donations, certainly there must be a person or foundation willing to plunk a billion down, I'm surprised Soros or Gates hasnt step up yet, they certainly have a more liberal stance and I would imagine that they would wholeheartedly support ESC research, they could hide behind foundations if they are afraid of bad press.

Jimmy. Good questions. Lately there was this news that Mike Bloomberg donated a lot of money, some of it to ESCR as well. I think donations like this of course is excellent and very good but have problems to see if this alone would be enough to speed up things. For speeding up things State funding as we see a lot of these days is picking up but to really have boosted it Federal funding is mandatory I think. This is why it is so critical in EU as well that the FP7 will become a good budget, above that we can see heavy funding from some countries, especially from United Kingdom which has funded in the £100 million level.

Here is a snip regarding Bloomberg's donations;

The billionaire mayor has donated hundreds of millions of dollars to the school, where the public health division bears his name.

Most recently, he gave $100 million, part of which goes to support research at the medical school's Institute for Cell Engineering. The school does some of its research with embryonic stem cells...
Source.

[Wise Young]

Wise,
Both of your posts #1 and #3 are elegant, I hope every CC member reads them. You have opened my eyes to a whole new set of dynamics concerning stem cells, availability,immunability, coaxing stem cells to get the right job done in the right place with reliabilty etc.

I do have a question, where are the philanthropic donations, certainly there must be a person or foundation willing to plunk a billion down, I'm surprised Soros or Gates hasnt step up yet, they certainly have a more liberal stance and I would imagine that they would wholeheartedly support ESC research, they could hide behind foundations if they are afraid of bad press.

I think I might understand why venture capitalists don't cough up the cash the ROI has to be almost a gimme and immediate, do you think once the ball gets going and we see progress the VC's will jump on board?


JimmyMack

Jimmy, thanks.

Regarding philanthropic donations, much has gone into fighting the political battle for and against stem cell research. For five years, many millions of dollars but untold numbers of volunteer hours have been spent on convincing Congress to come to a decision concerning stem cell research, convincing the state governments to fund the research, and educating the public concerning stem cells. The public relations battle for Proposition 71, for example, alone probably took well over $30 million to accomplish. The effort in New Jersey spent much less money but took place over a three year effort through the work of hundreds of volunteers from 90 advocacy groups. We should also remember the amount of money that is being spent to oppose stem cell research.

I estimate that probably over a billion per year of private stem cell research funding is being spent on stem cell research. Organizations like the Howard Hughes Foundation have probably spent over a hundred million per year to fund basic stem cell research in many institutions around the country. Almost every state has one or more stem cell institute that has been started at a university or private organization. The stem cell efforts at Harvard and Johns Hopkins, for example, were both largely privately funded. Many of these have not received much public notice. I estimate that as much as a billion dollars per year has been spent on stem cell research outside of the federal government in the past year. Without these expenditures, the United States would not be a player in the field.

Research takes time to organize, start, and move. It takes months to develop the plans to spend money, to establish the infrastructure for research, to set up the laboeratories, and transform money into research. Buildings and laboratories take time to build, scientific teams take time to recruit, move, build, and start up, and research project get up to speed slowly. The courtship process for scientists to move to institutes alone can take a year or more. It takes over a million dollar to move a scientist and his/her operation.

Wise.