A team of researchers from Harvard and Columbia University Medical Center have reprogrammed skin cells from an 82-year-old woman suffering from amyotrophic lateral sclerosis to generate first stem cells and then motor neurons. This is a significant advance which could aid in the development of drug treatments and cell replacement therapies for the condition and related neurodegenerative disorders.
The study, due to be published in the journal Science, demonstrates that skin cells from a chronically diseased elderly patient can be induced to de-differentiate into stem cells and then re-differentiate into motor neurons, which is an important technical achievement. More importantly though, because the technique used resulted in cell lines that are genetically identical to the patient, it will provide researchers with a better model for investigating how the condition develops. It also bypasses the need to obtain stem cells from human embryos.
Amyotrophic lateral sclerosis (ALS) is a form of motor neuron disease characterized by loss of motor neurons in the brain and spinal cord. These cells are involved in movements, which are generated by the sequential activity of cells in three regions of the central nervous system: they are planned by the activity of neurons in the premotor cortex (whose activity is monitored by brain-computer interfaces), and executed when this activity is relayed to motor neurons in the ventral horn of the spinal cord, via the cells in the primary motor cortex.
Consequently, the degeneration of motor neurons in the motor cortex and spinal cortex leads to progressive paralysis. This normally starts subtlely, with cramping or stiffness of the muscles, slurred speech or difficulty with swallowing. As the disease progresses, the muscle weakness spreads, and patients have greater difficulty walking and swallowing. Eventually, they cannot breath without the aid of a ventilator. The sequence in which these symtpoms emerge, and the rate of progreesion, varies from one patient to the next. In the vast majority of cases, however, there is no deficit in cognitive function (one of the best known people afflicted with ALS is the physicist Stephen Hawking).
The new study, which was led by Kevin Eggan from the Harvard Stem Cell Institute, involved the recruitment of ALS patients and healthy controls. Among those in the experimental group were two sisters, aged 82 and 89, who are among the oldest known patients with a rare familial (or hereditary) form ALS. Both have one copy of a rare form the SOD1 gene, and so suffer from a slowly progressing form of the disease. The younger sibling presented with ALS symptoms such as difficulty in swallowing and weakness in the arms and legs, whereas the older one exhibited no clinical symptoms, but was found upon close physical examination to have signs of primary motor cortical neuron degeneration, such as exaggerated reflexes.
The researchers first carried out skin biopsies on the participants, then isolated from the tissue samples cells called fibroblasts, which are the main constituent of connnective tissue and play an importnat role in wound healing. Four genes known to be expressed by pluripotent embryonic stem (ES) cells were then introduced into approximately 30,000 of the fibroblasts. They therefore de-differentiated into ES cells, and regained the ability to re-differentiate into any type of cell in the body. Two weeks after the biopsies, the researchers had several small colonies of these cells, and they used DNA fingerprinting to confirm that they were genetically identical to the donors.
During development, ES cells are exposed to chemical signals which are generated at certain times and places within the embryo. These signals are under tight genetic control and cause the ES cells in a given region of the embryo to differentiate along a particular pathway, such that nerve cells are generated in a specific place, skin cells in another, and so on. Because many of these signals have now been elucidated, Eggan and his colleagues were able to introduce two other genes into the ES cells, causing them to re-differentiate into both neurons and glia.
The identity of these cells was confirmed by antibody staining. 20% of the cells expressed the motor neuron-specific gene HB9; 90% of these also expressed Islet 1 and Islet 2, which are known to be required for motor neuron development, and more than half expressed the genes encoding enzymes which synthesize the neurotransmitter acetylcholine. They also expressed the microtubule protein Tuj1 (labelled green in the image at the top), and had a structure that is characteristic to spinal motor neurons. The cultures also contained cells expressing glial fibrillary acidic protein (GFAP, labelled orange) which, as its name suggests, is specific for glial cells.
Thus, the ES cells obtained from the patients' fibroblasts were competent to respond to the signals which cause differentiation and patterning of the embryo. It is significant that both motor neurons and glia were generated, because, although it is the neurons that degenerate, glia are now known to be involved in the disease process - they synthesize and secrete various factors which are toxic to motor neurons.
This study shows that large numbers of motor neurons can be obtained from ALS patients. It provides a method for obtaining patient-specific cell lines that can be used to investigate the intrinsic defects of neurons and glial cells in ALS, as well as the cellular processes of pathogenesis. Secondly, the method used in the study may overcome one of the major problems of regenerative medicine - the rejection of donor cells by the immune system. Because they are genetically identical to the patient, they would not produce an immune response if they are re-introduced into the patient after having their genetic defects repaired.
However, such cell replacement therapies have not yet been developed, and could still be a long way off. A number of genes have been implicated in ALS, and the mutations need to be properly characterized before they can be rectified. Furthermore, the method developed in this study for obtaining cells used oncogenes (cancer-causing genes) and retroviruses to shuttle the genes into the fibroblasts and ES cells. It will therefore remain unfeasible for clinical use until a safer method of reprogramming cells is developed. Finally, the study involved patients with a rare familial form of ALS; more than 90% of ALS cases are sporadic, and are therefore likely to have other combinations of mutations associated with them.
Dimos, J.T., et al (2008). Induced Pluripotent Stem Cells Generated from Patients with ALS Can Be Differentiated into Motor Neurons. Science DOI: 10.1126/science.1158799
Technically, the fibroblasts were turned into induced pluripotent stem cells (iPS cells or iPSCs), which are ES-like cells. They are not ES cells in their own right.