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Jan05
STEM CELL TRANPLANT IN SPINAL CORD INJURY
INTRODUCTION

Stem cell research is advancing knowledge about how an organism develops from a single cell and how healthy cells replace damaged cells in adult organisms. This promising area of science is also leading scientists to investigate the possibility of cell-based therapies to treat disease, which is often referred to as regenerative or reparative medicine.


WHAT ARE STEM CELLS?

Stem cells are cells found in most multi-cellular organisms. They have two important characteristics that distinguish them from other types of cells. First, they are unspecialized cells that renew themselves for long periods through mitotic cell division and differentiation. The second is that under certain physiologic or experimental conditions, they can be induced to become cells with special functions such as the beating cells of the heart muscle or the insulin-producing cells of the pancreas.

The two broad types of mammalian stem cells are: embryonic stem cells that are isolated from the inner cell mass of blastocysts, and adult stem cells that are found in adult tissues.

Stem cells are important for living organisms for many reasons. In the 3- to 5-day-old embryo, called a blastocyst, stem cells in developing tissues give rise to the multiple specialized cell types that make up the heart, lung, skin, and other tissues. In some adult tissues, such as bone marrow, muscle, and brain, discrete populations of adult stem cells and progenitor cells act as repair system for the body, replenishing specialized cells, but also maintaining the normal turnover of regenerative organs, such as blood, skin, or intestinal tissues.
Stem cells can now be grown and transformed into specialized cells with characteristics consistent with cells of various tissues such as muscles or nerves through cell culture.
Highly plastic adult stem cells from a variety of sources, including umbilical cord blood and bone marrow, are routinely used in medical therapies. Embryonic cell lines and autologous embryonic stem cells generated through therapeutic cloning have also been proposed as promising candidates for future therapies.[3




UNIQUE PROPERTIES OF STEM CELLS
Stem cells differ from other kinds of cells in the body and regardless of their origin have three general properties. They are capable of dividing and renewing themselves for long periods; they are unspecialized; and can give rise to specialized cell types.
Stem cells do not have any tissue-specific structures that allow it to perform specialized functions. A stem cell cannot work with its neighbours to pump blood through the body (like a heart muscle cell); it cannot carry molecules of oxygen through the bloodstream (like a red blood cell), nor can it fire electrochemical signals to other cells that allow the body to move or speak (like a nerve cell), but, more importantly, it can give rise to the development of these specialized cells by a process known as differentiation.
The internal signals are controlled by a cell's genes, which are interspersed across long strands of DNA, and carry coded instructions for all the structures and functions of a cell. The external signals for cell differentiation include chemicals secreted by other cells, physical contact with neighbouring cells, and certain molecules in the microenvironment.
Unlike the above mentioned specialized cells, stem cells are capable of proliferating for long periods. A starting population of stem cells that proliferates for many months in the laboratory can yield millions of cells. If the resulting cells continue to be unspecialized, like the parent stem cells, the cells are said to be capable of long-term self-renewal.
Adult stem cells typically generate the cell types of the tissue in which they reside. A blood-forming adult stem cell in the bone marrow, for example, normally gives rise to the many types of blood cells such as red blood cells, white blood cells and platelets. Until recently, it had been thought that a haematopoietic stem cell in the bone marrow could not give rise to the cells of a very different tissue, such as nerve cells in the brain. However, a number of experiments over the last several years have raised the possibility that stem cells from one tissue may be able to give rise to cell types of a completely different tissue, a phenomenon known as plasticity. Examples of such plasticity include blood cells becoming neurons, liver cells that can be made to produce insulin and haematopoietic stem cells that can develop into heart muscle.









HISTORY OF STEM CELL RESEARCH
• 1908 - The term "stem cell" was proposed for scientific use by the Russian histologist Alexander Maksimov (1874–1928) at congress of hematologic society in Berlin. It postulated existence of haematopoietic stem cells.
• 1960s - Joseph Altman and Gopal Das present scientific evidence of adult neurogenesis, ongoing stem cell activity in the brain; like André Gernez, their reports contradict Cajal's "no new neurons" dogma and are largely ignored.
• 1963 - McCulloch and Till illustrate the presence of self-renewing cells in mouse bone marrow.
• 1968 - Bone marrow transplant between two siblings successfully treats SCID.
• 1978 - Haematopoietic stem cells are discovered in human cord blood.
• 1981 - Mouse embryonic stem cells are derived from the inner cell mass by scientists Martin Evans, Matthew Kaufman, and Gail R. Martin. Gail Martin is attributed for coining the term "Embryonic Stem Cell".
• 1992 - Neural stem cells are cultured in vitro as neurospheres.
• 1997 - Leukemia is shown to originate from a haematopoietic stem cell, the first direct evidence for cancer stem cells.
• 1998 - James Thomson and coworkers derive the first human embryonic stem cell line at the University of Wisconsin–Madison.[60]
• 2000s - Several reports of adult stem cell plasticity are published.
• 2001 - Scientists at Advanced Cell Technology clone first early (four- to six-cell stage) human embryos for the purpose of generating embryonic stem cells.[61]
• 2003 - Dr. Songtao Shi of NIH discovers new source of adult stem cells in children's primary teeth.[62]
• 2004–2005 - Korean researcher Hwang Woo-Suk claims to have created several human embryonic stem cell lines from unfertilised human oocytes. The lines were later shown to be fabricated.
• 2005 - Researchers at Kingston University in England claim to have discovered a third category of stem cell, dubbed cord-blood-derived embryonic-like stem cells (CBEs), derived from umbilical cord blood. The group claims these cells are able to differentiate into more types of tissue than adult stem cells.
• 2005 - Researchers at UC Irvine's Reeve-Irvine Research Center are able to partially restore the ability of mice with paralyzed spines to walk through the injection of human neural stem cells.
• August 2006 - Rat Induced pluripotent stem cells: the journal Cell publishes Kazutoshi Takahashi and Shinya Yamanaka.[63]

• October 2006 - Scientists at Newcastle University in England create the first ever artificial liver cells using umbilical cord blood stem cells.[64][65]
• January 2007 - Scientists at Wake Forest University led by Dr. Anthony Atala and Harvard University report discovery of a new type of stem cell in amniotic fluid.[66] This may potentially provide an alternative to embryonic stem cells for use in research and therapy.[67]
• June 2007 - Research reported by three different groups shows that normal skin cells can be reprogrammed to an embryonic state in mice.[68] In the same month, scientist Shoukhrat Mitalipov reports the first successful creation of a primate stem cell line through somatic cell nuclear transfer[69]
• October 2007 - Mario Capecchi, Martin Evans, and Oliver Smithies win the 2007 Nobel Prize for Physiology or Medicine for their work on embryonic stem cells from mice using gene targeting strategies producing genetically engineered mice (known as knockout mice) for gene research.[70]
• November 2007 - Human induced pluripotent stem cells: Two similar papers released by their respective journals prior to formal publication: in Cell by Kazutoshi Takahashi and Shinya Yamanaka, "Induction of pluripotent stem cells from adult human fibroblasts by defined factors",[71] and in Science by Junying Yu, et al., from the research group of James Thomson, "Induced pluripotent stem cell lines derived from human somatic cells":[72] pluripotent stem cells generated from mature human fibroblasts. It is possible now to produce a stem cell from almost any other human cell instead of using embryos as needed previously, albeit the risk of tumorigenesis due to c-myc and retroviral gene transfer remains to be determined.
• January 2008 - Robert Lanza and colleagues at Advanced Cell Technology and UCSF create the first human embryonic stem cells without destruction of the embryo[73]
• January 2008 - Development of human cloned blastocysts following somatic cell nuclear transfer with adult fibroblasts[74]
• February 2008 - Generation of pluripotent stem cells from adult mouse liver and stomach: these iPS cells seem to be more similar to embryonic stem cells than the previous developed iPS cells and not tumorigenic, moreover genes that are required for iPS cells do not need to be inserted into specific sites, which encourages the development of non-viral reprogramming techniques.[75]
• March 2008-The first published study of successful cartilage regeneration in the human knee using autologous adult mesenchymal stem cells is published by clinicians from Regenerative Sciences[76]
• October 2008 - Sabine Conrad and colleagues at Tübingen, Germany generate pluripotent stem cells from spermatogonial cells of adult human testis by culturing the cells in vitro under leukemia inhibitory factor (LIF) supplementation.[77]
• 30 October 2008 - Embryonic-like stem cells from a single human hair.[78]
• 1 March 2009 - Andras Nagy, Keisuke Kaji, et al. discover a way to produce embryonic-like stem cells from normal adult cells by using a novel "wrapping" procedure to deliver specific genes to adult cells to reprogram them into stem cells without the risks of using a virus to make the change.[79][80][81] The use of electroporation is said to allow for the temporary insertion of genes into the cell.[82][83][84][85]
• 28 May 2009 Kim et al. announced that they had devised a way to manipulate skin cells to create patient specific "induced pluripotent stem cells" (iPS), claiming it to be the 'ultimate stem cell solution'.[86]
DEVELOPMENT OF STEM CELL RESEARCH

The spinal cord needs more protection then any other organ or system because unlike other organs, the spinal cord once damaged, cannot regenerate. While the majority of cells found in the central nervous system are born during the embryonic and early postnatal period, scientists recently discovered that new neurons are continuously added to two specific regions of the adult mammalian brain (Reynolds and Weiss 1992). Neural stem cells were isolated from the dentate gyrus of the hippocampus and the walls of the ventricular system called the ependymal layer. The progeny of these stem cells differentiate in the granule cell layer, meaning neurogenesis continues late into adult rodent life. These stem cells also migrate along the rostral migratory stream to the olfactory bulb, where they differentiate into neurons and glial cells (Luskin, 1993). Nerve cell differentiation has been witnessed in vivo, as well as in vitro when stimulated with an epidermal growth factor (Gage, 1995).
Along with pluripotent stem cells progenitor cells, a more restricted type of stem cells, are found in the hippocampus and ependymal layer. These cells are immature cells that are predetermined to differentiate into neurons, oligodendrocytes, and astrocytes. In 1995 Frissen observed that the presence of nestin increases in response to spinal cord injury. Nestin is a protein expressed by stem cells: presence of it indicates neural stem cells are much more active then previously believed. Our brain naturally increases the production of stem cells to aid an injured CNS. In 1999, Johansson and Momma observed that the only active progenitor cells were differentiating into astrocytes. They labeled ependymal cells with a Dil injection so migration could be followed. After making lesions in the spinal cord they waited four weeks and then observed the progress of the ependymal cells. They tested the cells found in the scar tissue around the site of injury and found that all DIL marked cells were astrocytes. This indicates that the progeny from ependymal cells had only differentiated to astrocytes. Stem cells do respond to spinal cord injury, just not for the purpose of reestablishing connection between neurons.
This realization sparked scientist’s interest in understanding what triggers these progenitor cells to proliferate. Scientists began to focus on neurotrophic factors that triggered this differentiation, specifically the presence of brain derived neurotrophic factors (BDNF) and neurotrophin 3 and 4 (NT-3 and NT-4). In the early 90’s these trophic factors were targeted as what triggered axon growth during early development. NT-3 also is expressed in greater amounts in response to spinal cord injury. In 1994 Schwab reported dramatic increase in function, and regrowth of a partially severed cord of rats after treatment with NT-3. In 1997 Grill, Gage, and colleagues published a paper examining the effects of transplanted NT-3 on motor skills and morphology after induced spinal injury in mice. They focused on the corticospinal tract, the pathway in charge of making voluntary movements. NT-3 has been previously observed to promote regrowth of corticospinal axons, and preserves degenerating motor neurons.
Grill and colleagues induced lesions in the dorsal hemisection of adult rat’s spinal cord, resulting in severely limited motor ability. Next grafts of syngenic fibroblasts, genetically altered to produce NT-3, were transplanted into the lesion cavity of the experimental group. These rats were kept alive for three months and put though a series of tests to monitor motor improvement. These tests examined coordination, ability to walk on inclined surfaces and precision of foot placement. After three months these rats were killed for the purpose of a quantitative cell count.
Recipients of the NT-3 secreting grafts showed significant improvement in motor skills over the control group, although they did not recover to the full ability they had before injury. After three months recipients of the NT-3 grafts demonstrated growth of corticospinal axons up to 8 mm from where the stem cells were transplanted. Only the injured axons at the lesion site showed any sign of regrowth. Uninjured axons showed no effort to reestablish connections across the site of injury. This suggests that NT-3 only responds when corticospinal axons are injured. If scientists could pinpoint signals triggering this response there is potential to manipulate the process in a manner causing neural cells to differentiate.
Triggering neurotrophic factors in hopes of inducing progenitors to proliferate is one of two major areas of study in spinal cord regeneration. Scientists also can derive undifferentiated embryonic stem cells (ES cells) from foetal spinal cord tissue and then mature them into cells that are suitable to implant into the damaged spinal cord. When using ES cells, researchers have two options: they can treat ES cells, allowing them to mature into CNS cells in vitro before transplantation, or they can directly implant differentiated cells and depend on signals from the brain mature the cells. This technique became possible when Reynolds and Weiss found that stem cells taken from the brain could be propagated in vitro. This allowed labs to duplicate what occurs naturally in the brain, and attempt to use the product to re-grow the damaged cells.
In December of 1999 McDonald and colleagues from Washington University School of medicine successfully implanted ES cells in laboratory rats. McDonald induced thoracic spinal cord injury in rats using a metal rod 2.5 mm in diameter resulting in paralysis. Nine days after the injury McDonald and colleagues transplanted roughly 1 million ES embryoid bodies pre-treated with retinoic acid into the syrinx that had formed around the contusion. During the nine days that passed between injury and transplantation, all the standard events following a spinal cord injury occurred. At the time of injury some cells died immediately, followed by a second wave of apoptosis within the first 24 hours. The centre of the bruised spine filled with fluid becoming a cyst referred to as syrinx. McDonald injected the ES cells into this cavity.
Two weeks after the transplantation ES stem cells filled the area normally occupied by glial scarring. After five weeks the stem cells had migrated further away from the implantation site. Although a number of them had died, there was still enough for the rats to have a growing supply of neurons and glial cells. Most of the surviving cells were oligodendrocytes and astrocytes, but some neurons were found in the middle of the cord. The rats regained limited use of their legs. Paralysis had been cured!!
McDonalds work in 1999 represented new successes in stem cell technology but this technology is yet to be tested in humans. A major obstacle remains: although scientists are achieving results, they don’t understand the factors responsible for what occurs. In McDonalds study, the regaining of functions could result from the few differentiated neurons. Another possibility could be that the high differentiation of oligodendrocytes re-myelinated enough axons to reestablish communication. Or perhaps functions regained due to ES cells producing growth factors—more research will have to be done before these options are narrowed down. Additional to unclear understanding of the process, other complications exist. Any introduction of foreign cells into the body triggers the immune system. ES cells would not simply be accepted into the host CNS. McDonald used cyclosporine to prevent rejection in the rats, but things get more complicated when testing begins on humans. The brain and spinal cord is complex, mysterious realms of the body—until science can predict the exact affect of evolving technologies, no testing on humans can occur.
A major motivation behind spinal cord research has been Christopher Reeve. Injured in a horseback riding incident, Christopher Reeve suffered a cervical spinal cord injury that left him quadriplegic. Christopher Reeve began the Christopher Reeve Paralysis Foundation (CRPF). CPRF funds research to treat or cure paralysis resulting from spinal cord injury or other CNS disorders. CPRF supports a Research Consortium, which collaborates the work of nine laboratories, as well as funds an international individual grants program. Several of the labs involved in the Research consortium focus on stem cells, making a lot of progress. The Salk Institute, run by Dr. Fred Gage examines the progenitor cells differentiating into glial cells. Someday they hope to manipulate these progenitor cells, inducing differentiation into neural cells.
STEM CELL TRANSPLANT VS ETHICS
There are a lot of people who find stem cell research extremely unethical. Scientists have found the most success with ES cells taken from embryoid spinal cords: although the ES cells are taken from embryos consisting at most of 64 cells, they still have potential to develop into a human being. People who believe life begins at conception remain morally against stem cell research. Justification is that the stem cells are derived from embryos discarded from fertility clinics. These embryos would be wasted if not used for stem cell research. Christopher Reeve published a position paper in response to the moral concerns and President Bush's decisions on stem cell researching. CPRF supports responsible stem cell research, recognizing the fine ethical boundaries existing in this technology.
TRANSPLANTATION OF STEM CELLS INTO SPINAL CORD
The original cell transplantation technology has been developed in the Centre for treating SCI patients. After surgically disrupting an intramedullary cyst (see Figure 1), the spinal cord defect is entirely filled up with the special gel containing foetal-derived, immature stem cells (see Figure 2). Moreover, during several months after the surgery each patient is subarachnoidally grafted with foetal-derived cells one or more times. The donor cell combination that is highly effective in generating regenerative processes in an adult nervous tissue has been previously determined by special experimental studies.

Figure 1. Dissection of the connective tissue cyst and opening access to the cord defect.

Figure 2. Infill of the spinal cord defect with the cell- containing gel implant.

FUTURE OF STEM CELLS

It has been hypothesized by scientists that stem cells may, at some point in the future, become the basis for treating diseases such as Parkinson's disease, diabetes, and heart disease.
As scientists learn more about stem cells, it may become possible to use the cells not just in cell-based therapies, but also for screening new drugs and toxins and understanding birth defects.
N.B.: This article was an e learning exercise by medical student Ms Kausalyaa Krishnabalan of Melaka Manipal Medical College , Malaysia


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