Retinal degenerative diseases result from the death of nerve cells in the retina, the part of the eye which converts light into electrical signals that are then processed as images by the brain. Like the brain, the retina is primarily made of cells called neurons and glia. One strategy for treating these diseases is to make new retinal cells from neural precursor cells grown in petri dishes. These new cells could then be transplanted into retinas where they would grow and replace dying cells that cause blindness. However, initial studies demonstrated many problems with making neural precursor cells. The cells could produce only a few types of neurons, many cells grew uncontrollably and became cancerous, and they could not be regrown in petri dishes. A recent NEI-funded study demonstrates a new method for making neural precursor cells that may have solved many of these problems.
The study by Li et al demonstrated a faster and more robust way to convert human embryonic stem cells (hESCs) into stable neural precursor cells (NPCs) than currently available. Until recently this process was long and complicated. Conversion recipes often involved growing stem cells in the presence of other cells types for 30 days followed by mechanical extraction or chemical selection of the NPCs from the rest of the cells.
In 2009, Chambers et al demonstrated a faster and simpler process. They showed that over 80% of hESCs grown in a petri dish could be converted to NPCs in thirteen days by treating them with a couple of ‘small molecule’ drugs called Noggin and SB431542. These drugs inhibit chemical reactions induced by transforming growth factor beta proteins (TGF-ß), which regulate cell growth and fate throughout the body.
Li et al tried a slightly different ‘cocktail’ of small molecule drugs. They treated hESCs with SB431542 and two other drugs called CHIR99021 and Compound E. CHIR99021 blocks the activity of the metabolic enzyme glycogen synthase kinase 3 (GSK3) and Compound E blocks the activity of gamma secretase enzymes which help regulate neuronal development by controlling a series of chemical reactions called the Notch signaling pathway. This new combination worked faster and more robustly than the Chambers cocktail, converting almost all (~96%) of the hESCs grown in a petri dish into NPCs in just seven days.
Treating the new NPCs with neuron-inducing growth factors for two weeks converted most (~ 75%) of the cells into mature neurons. Like many neurons throughout the nervous system these cells were stained well by antibodies raised against the cytoskeletal protein microtubule associated protein 2 (MAP2). Electrical recordings indicated that the new neurons conducted many of the currents seen in mature neurons and formed the types of fast chemical synapses found throughout the nervous system.
NPC are found throughout the nervous system and each one makes slightly different proteins. Antibodies, raised against some of these different proteins, such as Nestin, Orthodenticle Homolog 2 (Otx2), and Nuclear Receptor Related 1 protein (Nurr1), stained the newly converted NPCs suggesting they could potentially replace neurons throughout the nervous system. When the NPCs were implanted into various brain regions in mice the cells often turned into the expected neurons, further suggesting that they could be used to replace neurons throughout the nervous system including the retina. Surprisingly, implantation of NPCs did not produce tumors as seen with NPCs produced by other means.
One of the most important findings is that the NPCs could be converted into a variety of neurons even after they had been divided and regrown about 30 times. These results suggested that NPCs made with the Li cocktail may be a very convenient source of new nerve cells for regenerative therapies.
Public Impact Statement/Significance:
Retinal degenerative diseases are the leading cause of blindness. This study may greatly advance replacement cell therapy for treating eye diseases and other nervous system disorders.
“Using neural precursor cells is appealing for their potential to cure blindness caused by such diseases as macular degeneration and retinitis pigmentosa,” said Kang Zhang, M.D., Ph.D., an ophthalmologist at the Shiley Eye Center, at the University of California San Diego and a co-author on the papers.
These studies were partially supported by grant (NEI EY021374) designed to fund exceptionally innovative and ground-breaking research. The results demonstrate that the grant may be very effective at advancing medicine. Dr. Zhang shares the grant with Sheng Ding, Ph.D., a senior investigator at The Gladstone Institute of Cardiovascular Disease at the University of California San Francisco and with Thomas Reh, Ph.D., Director of the Department of Neurobiology and Behavior at the University of Washington School of Medicine in Seattle.
By Christopher G. Thomas, Ph.D.
- Chambers SM et al. “Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling.” Nat Biotechnol, March 1, 2009, vol. 27(3): 275-280. PubMed
- Li W et al. “Rapid induction and long-term self-renewal of primitive neural precursors from human embryonic stem cells by small molecule inhibitors.” PNAS, May 17, 2011, vol. 108(20), pp. 8299-8304. PubMed
Last Reviewed: December 2011