Fwd: Region-specific generation of cholinergic neurons from fetal hu #2

November 17, 2002

> Priming alone does not generate ChAT+ neurons To determine whether

> FHL-priming alone could direct fetal hNSCs to become cholinergic neurons in

> vitro, K048 cells from passage 33 were plated onto PDL-laminin-coated glass

> coverslips and treated for 6 days with FHL. Cells were fixed immediately

> for immunofluorescent staining with various antibodies. The number of cells

> stained with each antibody was counted in ten randomly chosen monolayer

> fields per sample, performed in triplicate. The majority of cells (91.1

> 0.7%) remained nestin+ (<A

HREF= " http://www.nature.com/neuro/journal/vaop/ncurrent/fig_tab/nn974_F3.html " >F\

ig. 3a</A>), indicating their stem/progenitor cell

> phenotypes. Only 0.8 0.4% of the total cell population became GFAP+

> astrocytes (<A

HREF= " http://www.nature.com/neuro/journal/vaop/ncurrent/fig_tab/nn974_F3.html " >F\

ig. 3b</A>), whereas 9.9% 1.1% differentiated into TuJ1+ neuronal

> cells (<A

HREF= " http://www.nature.com/neuro/journal/vaop/ncurrent/fig_tab/nn974_F3.html " >F\

ig. 3c</A>). However, negative staining with a mature neuronal marker,

> NeuN, suggested that these neurons are not fully developed. Further

> phenotypic studies revealed that 2.1 0.7%, which is one fifth of the TuJ1+

> neurons, were stained with a GABA-specific antibody in cell bodies (<A

HREF= " http://www.nature.com/neuro/journal/vaop/ncurrent/fig_tab/nn974_F3.html " >F\

ig. 3d</A>

> ). The rest of the TuJ1+ neurons did not acquire any of the phenotypes that

> we tested using specific ChAT, glutamate and tyrosine hydroxylase (TH)

> antibodies. Furthermore, cells transduced with a recombinant

> adeno-associated viral (rAAV) vector containing an enhanced green

> fluorescent protein (eGFP) did not alter this phenotypic pattern

> (transducing vector abbreviated CAG-egfp). These results suggest that

> additional conditions after priming are required for generating mature

> neurons and distinct neuronal subtypes from FHL-primed fetal hNSCs.

> Primed hNSCs become neurons in adult rat CNS To determine whether hNSCs

> primed by FHL or SFHL for 6â€“7 days in vitro could become cholinergic

> neurons in vivo, we injected primed cells into a neurogenic region

> (hippocampus)<A

HREF= " http://www.nature.com/cgi-taf/DynaPage.taf?file=/neuro/journal/vaop/ncurre\

nt/full/#B24 " >24</A> and several non-neurogenic regions, including prefrontal

> cortex, medial septum and spinal cord in adult rats. To trace the grafted

> cells, primed hNSCs were transduced with a CAG-egfp vector. This method was

> chosen because GFP labels both cell bodies and processes without leakage<A

HREF= " http://www.nature.com/cgi-taf/DynaPage.taf?file=/neuro/journal/vaop/ncurre\

nt/full/#B25 " >25</A>,

> and can be directly visualized without further staining procedures. About

> 80% of cells become GFP+ without apparent cytotoxicity. Furthermore, GFP+

> hNSCs maintained neuronal and glial differentiation profiles that were

> similar to those of untransduced hNSCs<A

HREF= " http://www.nature.com/cgi-taf/DynaPage.taf?file=/neuro/journal/vaop/ncurre\

nt/full/#B26 " >26</A>.

>

> One to two weeks after grafting 5 104 primed and labeled fetal hNSCs into

> either rat brain or spinal cord, GFP+ cells were found mainly near the

> injection sites (data not shown). Migration and integration could be seen

> one month after transplantation (<A

HREF= " http://www.nature.com/neuro/journal/vaop/ncurrent/fig_tab/nn974_F4.html " >F\

ig. 4aâ€“d</A>). The extent of migration was

> variable depending on the regions injected. In prefrontal cortex, for

> example, GFP+ cells were usually located within 0.4â€“2 mm of the injection

> site. However, cells migrate much farther, up to 1â€“2 cm, when grafted into

> medial septum, hippocampus or spinal cord. More interestingly, the

> distribution of transplanted cell bodies and processes followed endogenous

> patterns in the highly organized CNS regions we examined, including frontal

> cortex (<A

HREF= " http://www.nature.com/neuro/journal/vaop/ncurrent/fig_tab/nn974_F4.html " >F\

ig. 4a</A>), medial septum (<A

HREF= " http://www.nature.com/neuro/journal/vaop/ncurrent/fig_tab/nn974_F4.html " >F\

ig. 4b</A>), hippocampus (<A

HREF= " http://www.nature.com/neuro/journal/vaop/ncurrent/fig_tab/nn974_F4.html " >F\

ig. 4c</A>) and spinal

> cord (<A

HREF= " http://www.nature.com/neuro/journal/vaop/ncurrent/fig_tab/nn974_F4.html " >F\

ig. 4d</A>). GFP-labeled neuronal fibers were detectable as early as 1

> week after transplantation, and much more abundant in 1-month grafts. This

> is in contrast to a previous study using undifferentiated cells, which

> observed neuronal fibers mainly in 20-week grafts<A

HREF= " http://www.nature.com/cgi-taf/DynaPage.taf?file=/neuro/journal/vaop/ncurre\

nt/full/#B27 " >27</A>. Stereological analyses

> (Methods) revealed higher survival rates of grafted primed hNSCs in various

> brain regions (13.8 2.1% in cortex, 16.4 3.8% in medial septum, 19.8

> 5.0% in hippocampus) than in spinal cord (5.1 0.5%). The latter most

> likely resulted from the trauma of the spinal cord grafting procedure.

>

November 17, 2002