Neuronal stem cells
Stem cells are self regenerating multipotential cells, found in the human brain which have the potential to differentiate into neurons, astrocytes and oligodendrocytes, and to self renew sufficiently to provide adequate number of cells in the brain. Neural stem cell grafts have been studied in a variety of animal models for various diseases like metabolic disorders, muscular dystrophies, neurodegenerative disorders, spinal cord repair, brain tumors and demyelinating disease. Stem cells may be derived from autologus, allogeneic or xenogenic sources. Histocompatibility is prerequisite for transplantation of allogeneic stem cells. Fetal tissue is the best current tissue source for human neural stem cells, however ethical issues are a major concern. Thus the prospect that stem cells could potentially be used to promote neurogenesis following injury and disease may seem attractive, yet the inherent problems associated with isolation and rejection in case of stem cells from another source, the potential to form tumors and ethical issues are the major challenges.
In 1913 the great Spanish neuroscientist Santiago Ramón y Cajal pronounced “that in adult centers the nerve paths are something fixed, ended, immutable. Everything may die, nothing may be regenerated”. For many years neuroscientists believed not just that brain damage was irreparable, but also that no process to replace lost neurons existed in our brain. Both beliefs turned out to be false, though the extent of reparability and the classes of neurons, which might be replaced, remain unclear. Lately, stem cells, which are self-renewing and can give rise to a diverse progeny have been found in the adult human brain. These self-regenerating cells have attracted a great amount of interest, as a potential therapeutic application for a variety of neurological disorders. For the present review we scrutinized all journals on Medline and Pubmed using the following key words from 1990-2003 including all cross references: stem cells and neurogenesis; fetal ventral mesencephalic (FVM), xenotransplant; human retinal pigment epithelial cells and Parkinson's disease; stem cells and leucodystrophy, multiple sclerosis, spinal cord injury, stroke, epilepsy, Alzheimer's disease; hemotopoeitic stem cells transplantation and Krabbe's disease.
Stem cells are multipotential cells that have the capacity to proliferate in an undifferentiated state, to self-renew, and to give rise to all the cell types of a particular tissue. To be considered a stem cell in the CNS, a cell must have the potential to differentiate into neurons, astrocytes, and oligodendrocytes, and to self renew sufficiently to provide the number of cells in the brain. There are three types of stem cells, categorized by their ability to form other cells and correlated with the body's development process:
v Totipotent: Totipotent stem cells are those that can form an entire organism. A fertilized egg is totipotent.
v Pluripotent: Pluripotent stem cells are those that can form any cell in the body but can't form an entire organism. After several cycles of cell division, a developing embryo's totipotent cells become pluripotent.
v Multipotent: Multipotent stem cells are those that can only form specific cells in the body, such as blood cells. By childhood, most pluripotent stem cells have become multipotent. There are no totipotent stem cells and only a few hard-to-isolate pluripotent stem cells.
Neural stem cells have been discovered in the adult liver, pancreas, and brain. Neural stem cells reside within the periventricular subependymal zone and hippocampus, are multipotent and can give rise to all three types of cells of the central nervous system. This plasticity has made stem cells a target for medical researchers, who hope to use them to cure as well as to repair tissue.
Stem cells from embryos offer the most treatment potential because they are pluripotent. In transplantation, a distinction is made between use of the patient's own stem cells (autologous) and stem cells from another donor (allogeneic).
Sources of the patient's own stem cells (autologous) are either the cells from patient's own body or his or her cord blood. For autologous transplants physicians now usually collect stem cells from the peripheral blood rather than the marrow. This procedure is easier, unlike a bone marrow harvest, it can take place outside of an operating room and the patient does not have to be under general anesthesia. Patients also tend to recover more quickly when transplanted with stem cells from the peripheral blood, require fewer red blood cell and platelet transfusions, have shorter hospital stays and is less costly. A few days before the procedure, donors are generally given a medication (G-CSF [filgrastim], GM-CSF [sargramostim] or a combination of the two to mobilize or force stem cells from the marrow into the circulating blood. These agents can cause flu-like symptoms in the days preceding and following stem cell harvest. To circumvent these two problems, researchers have attempted to isolate adult stem cells from mammalian skin, a highly accessible tissue source. The possibility that skin contains a precursor capable of generating neural cell types was indicated by the finding that Merkel cells, the neural sensory receptors found in the dermis, can be generated in adult skin in rodents.
Sources of stem cells from another donor (allogeneic) are primarily relatives (familial-allogeneic) or completely unrelated donors (unrelated-allogeneic). The stem cells in this situation are extracted from either the donor's body or cord blood. Histocompatibility is prerequisite for transplantation of allogeneic stem cells. Fetal tissue is the current tissue source for human neural stem cells, raising important ethical issues. Moreover, the use of human fetal tissue, even if acceptable by society, involves heterologous transplantation. Other sources of allogenic adult stem cells include the central nervous system,, bone marrow,,, retina and skeletal muscle.,
In this stem cells from different species are transplanted, e.g. striatal porcine fetal ventral mesencephalic (FVM) xenotransplants for Parkinson's disease. This has no major ethical concerns and a large amount of tissue is available, however life long immunosupression and risk of rejection are the major limitations.
All of these stem cells preferentially generate differentiated cells of the same lineage as their tissue of origin However, several recent transplant studies indicate that at least a fraction of stem cells in these populations can generate cells of a different embryonic lineage in vivo. For example, neural stem cells can generate blood and skeletal muscle cells, and can contribute to many embryonic tissues when transplanted into blastocysts. Similarly, transplanted bone marrow stem cells can contribute to skeletal muscle, and liver, and can generate cells producing neuronal markers in the brain.,
The standard method of isolating neural stem cells in vitro is to dissect out a region of the fetal or adult brain that has been demonstrated to contain dividing cells in vivo, for example the subventricular zone (SVZ) or the hippocampus. The tissue is disaggregated and then the dissociated cells are exposed to a high concentration of mitogens such as fibroblast growth factor-2 (FGF-2) or epidermal growth factor (EGF) either a defined or supplemented medium as a matrix and as a substrate for binding. After some proliferation, the cells are either induced to differentiate by withdrawing the mitogens, or by exposing the cells to another factor that induces some of the cells to develop into different lineages. The isolation of these cells is complicated because their culturing inevitably leads to a mixed population of progenitor and stem cells, best described as expanded neural precursors (ENP). In addition, proliferation of ENP's in cultures is not indefinite.
Neural stem cells grafts have been studied in a variety of animal models for various diseases.
In an animal model of Parkinson disease, human precursor cells grafted into the striatum can replace degenerated dopamine-producing neurons in the nigrostriatal pathway and promote limited functional recovery. However, ethical issues apply to the use of mesencephalic progenitors derived from human fetuses. Alternative attractive sources of multipotent stem cells are the human retinal pigment cells (hRPE), which can be derived from a single donor, with successful results seen in rhesus monkeys. In this study hRPE cells attached to gelatin microcarriers (GM) were transplanted intrastriatally in 3 MPTP-treated hemiparkinsonism monkeys. All 3 animals were implanted with 10,000 hRPE-GM per site into 5 targets in the left striatum, and the third monkey was implanted at six additional sites 5mm apart in the left parietal white matter using MRI guided stereotactic technique. Improvement in the motor scores of unified Parkinson's disease rating scores (UPDRS) was seen in the first and second monkeys, while 18 F-dopa PET imaging 6 weeks after transplantation revealed an area of high uptake in parietal white matter at the site of transplantation in the third monkey. Histological examination of the brains of the first two monkeys revealed cells that morphologically resembled hRPE cells, with minimal inflammatory response. Further, in a blinded placebo controlled trial, 16 MPTP-treated monkeys showed significant improvement in motor part of UPDRS at 3 months. In an innovative procedure, retinal cells were implanted in the brains of patients with advanced Parkinson's disease (PD). In this phase I trial, retinal pigment epithelial (RPE) cells attached to gelatin micro- carriers were implanted into the brains of six patients with advanced PD. RPE cells, normally found in the back of the eye, were cultured in the laboratory to produce cells for this treatment. These cells provide a source of increased dopamine production. After 12 months follow up improvement was seen in their tremor, stiffness, slowness of movements and balance, common motor functions affected in Parkinson's disease. Half of the participants also showed improvement of dyskinesia, a complication seen in advanced PD.
Lysosomal storage disease
Hematopoetic stem cell transplantation (HSCT) was performed in 15 infants with symptoms and a family history of infantile onset Krabbe's disease, 3 with marrow from matched donors and 12 with unrelated cord blood. Outcome assessments included engraftment studies, MRI, CSF proteins measurement, nerve conduction studies and neurodevelopmental evaluation. It was seen that in 14 of the 15 patients, engraftment occurred. A total of 10 patients were followed up for 12-59 months (median 30 months), rest dying before 12 months. CSF proteins decreased in all, MRI improved/stabilized in 6 of 8 patients and serial nerve conduction studies improved in 2 neonatal patients. Four neonatatally diagnosed patients with no/minimal symptoms at HSCT developed age appropriate language function and maintained motor skills usually lost by first year, while those who were transplanted after developing substantial symptoms did not show any improvement. Thus age and neurological status at HSCT were found to be primary determinants of outcome.
Grafts of neural stem cells have also shown to be effective in cases of widespread neural degeneration. For example, in a genetic model of demyelination, both the pathology and symptoms were reversed by transplantation of neural stem cells into the cerebral ventricles at birth. Cultured neural stem cells were injected into the brain ventricles of newborn mice from a mutant strain that develops severe tremors by 2 to 3 weeks of age. The tremor develops because the mice lack a key protein needed to make myelin. The lack of normal myelin in these mice mimics the defect seen in many human demyelinating disorders, such as multiple sclerosis and a group of childhood disorders such as leukodystrophies. The researchers found that most of the transplanted cells migrated throughout the brain and matured into normal-looking oligodendrocytes. These oligodendrocytes produced a significant amount of the missing protein and began to cover nearby nerve fibers with myelin just as normal oligodendrocytes would. Moreover, tremors disappeared almost completely in 60% of the tested mice that received the transplant. The grafted stem cells migrated extensively throughout the brain, integrated into the host cytoarchitecture, and corrected the myelination process during subsequent developmental stages.
In another study, role of high dose immunosuppressive therapy (HDIT) rescued with autologous stem cell transplantation in the management of severe, non responsive multiple sclerosis (MS) was performed in 26 patients, including primary progressive MS (n=7), secondary progressive MS (n=18) and relapsing remitting MS (n=1). Eligibility requirements included an EDSS from 5-8 and deterioration of 1 or more point over previous year. Twenty one patients had been treated earlier with interferon-beta with poor results, and 15 had failed multiple therapies including copaxone, prednisone and methotrexate, with a median follow up of 12 (3-36) months. With a minimum follow up of 12 months it was seen that majority patients remained neurologically stable with unchanged EDSS.
Spinal cord repair
Grafted neural stem cells could potentially replace cells lost to injury, reconstitute the neuronal circuitry, and provide a relay station between the injured pathways above and below the lesion. Furthermore, intraspinal stem cells transplants can be genetically modified to provide therapeutic factors that prevent cell death and promote regeneration. Cells to be transplanted into the injured spinal cord need to be readily obtained, easily expanded and stored, and amenable to genetic modification. They should also be able to survive for extended periods within the injury site, to integrate with host tissue, to rescue injured neurons from cell death and atrophy, to promote axonal regeneration, and, ultimately, to restore function. Neural stem cells and neural precursors theoretically fit many of the above requirements; the challenge is to demonstrate their efficacy and safety for clinical applications. Among the most promising sources of cells for spinal cord repair are neuronal-restricted precursors (NRPs) derived from the developing spinal cord. These cells can be expanded in vitro and have the potential to differentiate into numerous neuronal types including motoneurons. In the ex vivo modality of gene therapy, therapeutic genes are introduced into cultured cells that are subsequently transplanted into the CNS. Genetically modified stem cells have not yet been grafted into the injured spinal cord; however, transplantation of brain-derived neurotropic factor-producing fibroblasts has been carried out in the laboratory using a rat spinal cord injury model of partial cervical hemisection. These grafts resulted in long distance regeneration of axons from brainstem neurons and partial recovery of motor function. Ongoing experiments with genetically modified fibroblasts are examining the effects of other growth factors, as well as adhesion molecules and growth-associated genes.
Stem cell therapy has also been suggested as a possible strategy for replacing damaged circuitry and restoring learning and memory and abilities in Alzheimer's disease. Grigorian et al lesioned these areas of the brain of rats in order to simulate the effects of Alzheimer's disease. Then they grafted neural stem cells to the sites of the lesions. They found that the rats with the stem cells grafts improved on memory tasks. Furthermore they found that some of the stem cells moved to areas that they were not implanted in, but were also lesioned.
Despite a paucity of direct evidence for roles of neurogenesis in human CNS regeneration, there in evidence that adult rodent brain attempts self repair in various disease states like stroke and epilepsy. Because of ethical dilemmas of embryonic stem cell research and the limitations of xenotransplantation, immortalized cell lines are being utilized as alternative graft sources. Most studies of stem cells transplants in stroke models using rodents have shown improved motor behaviour and task learning, better results with intravenous delivery as compared to striatal implant, reduction in infarct volume, migration of transplanted cells selectively to the site of injury when the cells were transplanted intracisternally and into non-lesioned side. Cells transplanted have been derived from various sources such as filgratism (granulocyte colony stimulating factor) mobilized peripheral blood progenitor cells, human umblical cord blood, subventricular zone (SVZ) cells, bone marrow stem cells. Only a single phase I clinical trial done so far has assessed the safety of intrastriatal transplantation of NT2N (graft derived from human testicular germ cell tumor) in patients with basal ganglia infarcts and stable motor deficits 6 months to 6 years before transplantation. Preliminary findings in 12 patients have shown no evidence of malignant transformation. PET scanning at 6 months showed more than 15% relative uptake of Fluorodeoxyglucose at transplant site in 6 patients. Though this is not a case of true stem cell transplant, it provides evidence of usefulness of another source of cells in stroke patients. A second study of 18 patients with controls is underway.
Researchers need to learn how to create large batches and how to direct stem cells to become what they want. Stem cell therapy can alleviate or even cure some of the mentioned diseases- but what about the ethics, if the stem cells come from embryos? which are currently considered as the best source of stem cells. Though the use of fetal tissue is regulated by guidelines, elective termination of pregnancy is considered ethically unacceptable by many as a foetus is unable to give consent. Also their number is small and therefore will not be able to provide a realistic approach to cell therapy. Human stem cell (HSC) lines thought to be pluripotent have been derived both from primordial germ cells developing in fetal tissue [embryonic germ (EG) cell line], from early pre-implantation embryos no longer required for infertility treatment [embryonic stem (ES)] cell lines and human ES cell lines derived from donated oocytes fertilized for the purpose of stem cell derivation and from transfer of somatic cell nucleus (SCNT). For those who believe the human embryo from the one-cell stage onwards has absolute moral value, equal to that of a newborn baby or adult, any embryo research is ethically unacceptable, as it is tentamounts to murder. Also would the use of SCNT be ethical? The principle objection put forward is that it might facilitate reproductive cloning in humans.
The problem of immune rejection also needs to be addressed if stem cells from a source other than the person requiring treatment is used. It is known that embryonic stem cells have the potential to form tumors, as they grow vigorously. These problems could be circumvented by modifying culture conditions, use of transducing vectors encoding an immortalizing oncogene, and using immunosuppressive drugs. Gene transduction of pluripotent human hematopoietic stem cells (HSCs) is used for successful gene therapy of genetic disorders involving hematolymphoid cells. Evidence for transduction of pluripotent hematopoetic stem cells (HSCs) can be deduced from the demonstration of a retroviral vector integrated into the same cellular chromosomal DNA site in myeloid and lymphoid cells descended from a common HSC precursor. In a long methods which requires inverse PCR and sequencing to confirm identity, these procedures are capable of transducing human HSC. However, in clinical practice long term use of immuno-suppressive therapy is used. The potential benefit of using SCNT stem cells is that they can be made from the patient's own somatic cell nuclei, custom built for immunological compatibility and so transplant rejections are not an issue. The downside is the ethical dilemma presented by the resource tissue. Such research would require a large supply of unfertilized eggs, and SCNT being an inefficient process, many eggs would be required to produce a single ES cell line.
Till recently, brain cells were not supposed to possess capability of replicating and recovery from injury, infarction and neurodegeneration. In late 70's and 80's it was believed that there is the phenomenon of neuro-plasticity whereby if the injury occurs early on in the life, there is possibility of recovery, albeit partial. The swing is now turning to the other end, and research in stem cells has opened up new horizons in the area of treatment of disorders such as stroke, epilepsy, neuro-degeneration and trauma. Current research is aimed at finding the appropriate source of stem cells for a given indication, ways of expanding and perpetuating these cells in culture, best route of administration of these cells and methods to overcome rejection. Brain being sensitive to volume changes access through intracisternal, intraventicular, intrastriatal and intravenous or intraarterial routes are studied. The advantage of endovascular route is potential for widespread distribution, ability to deliver large volumes, limited perturbation of brain tissue and the feasibility of repeated administration. Restricted diffusion and presence of blood brain barrier are some of the limiting factors being looked into.
Among the tissues which can be transplanted, marrow derived cells (stimulated by granulocyte colony stimulating factor), mobilized (by granulocyte colony stimulating factor) peripheral blood, human umbilical cord blood, amniotic fluid cells, subventricular zone (SVZ) cells, human retinal pigment epithelial (RPE) cells, fetal ventral mesencephatic (FVM) cells are being investigated.
Ethical issues are of paramount importance especially when foetal allogenic grafts and therapeutic cloning are considered. Producing a foetus/clone to harvest neuronal stem cells for adult will certainly amount to murder, which no society can allow. Considering these issues the best option would be to obtain peripheral blood derived cells, those derived from human umbilical cord blood or amniotic fluid. Such sources are free from ethical issues and host rejection. We are still far from our goal but certainly moving towards it.