15 Nov 2002

The replacement of dying neurons in Alzheimer’s disease with neural stem cells remains a pie in the sky, but at least researchers now appear to have gotten their hands on the filling. In the November 11 online Nature Neuroscience, investigators at the University of Texas Medical Branch in Galveston write that they have managed, for the first time, to generate large numbers of cholinergic neurons in a region-specific pattern by transplanting specially primed human neural stem cells into adult rat brains.

Cholinergic neurons are a key ingredient to realize the dream of a stem cell therapy because they are among the most affected in Alzheimer’s, where, for example, a projection of cholinergic neurons from the medial septum to the hippocampus degenerates early on. Prior stem cell studies, write Ping Wu, Richard Coggeshall, Yongjia Yu, and colleagues, have not been able to generate this type of neuron in significant numbers.

Wu et al. started out tackling the problem that human neural stem cells do not develop into neurons, but rather stay immature or turn into glia when transplanted into non-neurogenic (that is, most) areas of the adult brain. Like several labs in the field, they were looking for in-vitro culture conditions that would nudge those stem cells toward neuronal differentiation before transplanting them in vivo. They claim to have found that magic cocktail with a variation of culture conditions reported previously. Wu et al. played with combinations of different tropic factors or other chemicals known to be important in the differentiation of cholinergic neurons (from leukemia inhibitory factor to sonic hedgehog peptide to BDNF and others). They discovered the right conditions when culturing cells from two established human neural lines in a mix of human basic fibroblast growth factor (bFGF) and heparin (which assists bFGF signaling) for six days before injecting them. This combination has been used before; the trick here appears to lie in not culturing the stem cells floating as little blobs in solution, but to allow them to spread out on laminin-coated coverslips, the authors report.

First characterizing the differentiating cells in vitro, Wu et al. report that about 27 percent of them develop into large multipolar cells that bear neuronal markers as well as cholinergic markers, such as the synthetic enzyme choline acetyltransferase (ChAT) and Islet-1. Glutamatergic, GABAergic neurons, astrocytes, and nestin-positive undifferentiated cells were also in the mix, as well, but no other reagent cocktail produced cholinergic neurons, the authors write. These ChAT-positive cells generated action potential when stimulated, suggesting they are functional neurons.

The researchers next injected the primed stem cells into various brain areas of adult rats, including hippocampus (which is neurogenic in its dentate gyrus region), prefrontal cortex, medial septum, and spinal cord, (all of which are not). To be able to trace the stem cells, the scientists first transduced them with green fluorescent protein (GFP), which spreads throughout neurons including their distant processes. The findings, if repeated and extended, sound promising: One month after injection, 19 percent had survived in hippocampus, 16 percent in medial septum, and 13 percent in cortex, though only five percent in spinal cord. Integrated cells had migrated and, in the AD-relevant CA1 region of the hippocampus, looked morphologically like typical pyramidal neurons and sported dendritic spines on their extended processes. Neuron-specific antibodies such as NeuN, TuJ1 indicated that most injected cells had become neurons in all four brain areas, with only a few turning into astrocytes and none remaining undifferentiated. This is the paper’s key advance over prior work, the authors write.

Importantly, the differentiation into neuron types followed a region-specific pattern, the authors report. In medial septum and spinal cord (which are rich in cholinergic neurons), 61 and 55 percent, respectively, of the GFP-positive cells were cholinergic. In prefrontal cortex, 51 percent were glutamatergic, and in the CA1 region of hippocampus, 71 percent were GABAergic.

As yet, none of this has been done in a degenerating brain or in a damaged spinal cord to get a sense of its therapeutic utility. Nor is it clear whether these neurons project to their proper targets or can be functional, or whether adult human stem cells would react similarly to the priming cocktail described here.—Gabrielle Strobel

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