Researchers reported that embryonic stem cells can be induced to differentiate into neural subtypes, that the resulting dopaminergic neurons survive long-term after transplantation into the striatum, and that such cells can be used to treat Parkinson’s disease in animals. The study by Tiziano Barberi, MD, Lorenz Studer, MD, and colleagues was presented in the October Nature Biotechnology.

The investigators reported that their new protocol permitted quick and efficient production of most central nervous system phenotypes, including neural stem cells, astrocytes, oligodendrocytes, and neurons. The cells were tweaked into becoming gamma-aminobutyric acid (GABA), dopamine, serotonin, or motor neurons by culture conditions that copied the series of signals directing central nervous system development in vivo, they explained. Transplantation of the resulting dopaminergic neurons into the striatum corrected chronic circling behavior in mice with 6-hydroxy-dopamine-induced brain lesions, a common model for Parkinson’s disease.

THE CLONE CURES

“I think there are two main points,” Dr. Studer told NEUROLOGY REVIEWS. “The first is that we reported an improved protocol for selective generation of dopaminergic, serotonergic, cholinergic, and GABAergic neurons as well as astroglia and oligodendroglia from cultured embryonic stem cells. The second is that we reported the first proof-of-principle demonstration of therapeutic cloning in a model of neural disease.” Dr. Studer is an Assistant Professor of Cell Biology and Genetics at Cornell University’s Weill Medical College and member of the Laboratory of Stem Cell and Tumor Biology at Memorial Sloan-Kettering Cancer Center, both in New York City.

The treated animals were studied for up to two months, and the correction of parkinsonism appears to be permanent. “At the therapeutic level, I was surprised by the small variability between animals, suggesting that the therapeutic effect occurs in nearly every single animal,” Dr. Studer said.

This work is an advance over previous approaches because the therapeutic effects do not require overexpression of a transgene and appear to be safer. According to Dr. Studer, previous work had required overexpression of transcription factor Nurr1. This led to “overcompensation” in some animals, apparently due to too much of the transcription factor. No such problems were seen in Dr. Studer’s study, which the investigators admitted “was surprising, because some mice had more than 40,000 surviving tyrosine hydroxylase-expressing cells in the striatum, a number that exceeds the roughly 10,000 midbrain dopamine neurons present in the adult mouse midbrain.”

To induce neural differentiation, undifferentiated embryonic stem cells were cocultured in serum replacement medium with mouse bone marrow–derived stromal feeder cell lines or with primary stromal feeder cells obtained from the aorta-gonad-mesonephros region. Within six days all embryonic stem cell–derived colonies showed neural precursor markers such as nestin and neural cell adhesion molecules. These cells were then cultured in N2 medium plus basic fibroblast growth factor (bFGF), which produced neural precursor cells. Differentiation was induced by withdrawing bFGF and adding ascorbic acid. Dr. Studer noted that nearly all clonally derived colonies that proliferated in the presence of bFGF “showed multilineage central nervous system potential.” Cells were steered toward a final identity by sequential exposure to molecular signals that mimicked in vivo neural development.

CONTROLLING FATE

“[O]ur study demonstrates that regional fate specification can be controlled by manipulation of external medium conditions and by sequential patterning cues that seem to recapitulate in vivo development,” the investigators wrote. In contrast to previous systems, this one also allows rapid differentiation into astroglia and oligodendroglia as well as neurons.

“I was surprised by the extremely close temporal correlation between in vitro and in vivo development,” Dr. Studer commented. “This suggests that our in vitro system will be a very powerful tool for the study of normal brain development,” he noted.

A POTENTIALLY POWERFUL TOOL

The fact that dopaminergic cells could be developed from all of the embryonic stem cell lines tested is encouraging, since therapeutic usefulness would be unlikely if culture conditions had to be adapted to each individual cell line. The yield is encouraging also: about 1,000 dopaminergic neurons for each embryonic stem cell initially plated, compared to two or three dopaminergic cells per embryonic stem cell using the older embryoid body-based protocols.

In addition to Parkinson’s disease, Dr. Studer expects this approach to be useful in developing treatment for demyelination (including multiple sclerosis), Huntington’s disease, amyotrophic lateral sclerosis, and other motor neuron diseases. “Protocols have to be tested with human embryonic stem cells. This work is progressing quickly in our lab and in other labs, and we should have these answers very soon.”

There are fundamental questions about cell transplantation therapy in Parkinson’s disease that also need to be addressed to proceed, he noted. “Particularly, the occurrence of side effects observed in fetal tissue grafts will be a factor that needs to be considered before clinical trials. In my opinion it is likely that stem cell technology will allow us to overcome most of the problems observed with fetal tissue grafts, but this will require significant additional research,” Dr. Studer said.

NR

—Janis Kelly

Suggested Reading
Barberi T, Klivenyi P, Calingasan NY, et al. Neural subtype specification of fertilization and nuclear transfer embryonic stem cells and application in parkinsonian mice. Nat Biotechnol. 2003;21:1200-1207.
Kim JH, Auerbach JM, Rodriguez-Gomez JA, et al. Dopamine neurons derived from embryonic stem cells function in an animal model of Parkinson’s disease. Nature. 2002;418: 50-56.

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