Region-specific generation of cholinergic neurons from fetal hu #4

[Guest guest]

>

> >> Discussion

>

> Here we report a simple and efficient priming procedure to treat fetal

> hNSCs in vitro before further differentiation and transplantation in vivo.

> This procedure allowed us to obtain cholinergic neurons in vitro, as well

> as a nearly pure population of neurons in vivo, from long-term

> mitogen-expanded fetal hNSCs.

>

> The priming cocktail contains bFGF, heparin and laminin. Our initial

> screening indicated that both bFGF and heparin were necessary to obtain

> large cholinergic neurons from fetal hNSCs in vitro. An adherent laminin

> substrate included in the priming media was also required for an optimal

> spreading of neurospheres, which presumably function by allowing cells

> inside the spheres to be exposed evenly to the bFGF/heparin treatment.

> Using this priming method, many large, multipolar cholinergic neurons

> differentiated from fetal hNSCs following further incubation in the B27

> medium without bFGF. In contrast, direct plating of mitogen-expanded fetal

> hNSCs onto laminin-coated culture dishes generated only small bipolar

> GABAergic and glutamatergic neurons, even when cells were treated with

> various neurotrophic factors<A

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

nt/full/#B23 " >23</A>. Similar effects were observed on fetal hNSC

> differentiation in vitro when cells were treated with RA, EGF, LIF, ShhN

> and heparin, alone or in combination.

> Our in vivo transplantation data also showed that both bFGF/heparin and

> adhesive culture were involved in initiating neuronal differentiation of

> fetal hNSCs. Under certain conditions, bFGF favors neuronal differentiation<A

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

nt/full/#B30 " >

> 30</A>, <A

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

nt/full/#B31 " >31</A>. Furthermore, heparin may potentiate the biological

activity of bFGF

> through its helper effect on the binding of bFGF to its tyrosine kinase

> receptor<A

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

nt/full/#B32 " >32</A>, <A

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

nt/full/#B33 " >33</A>. Lack of heparin, therefore, might contribute in part to

the

> absence of neuronal differentiation from rat neural stem cells when they

> are transplanted into non-neurogenic spinal cord<A

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

nt/full/#B16 " >16</A>, <A

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

nt/full/#B20 " >20</A>. Two other groups

> were able to obtain neuronal differentiation in non-neurogenic striatum

> from grafted hNSCs, which were pre-cultured in medium containing

> bFGF/heparin. However, neuronal differentiation in these studies was

> limited. For example, one study<A

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

nt/full/#B17 " >17</A> reports that a small number of immature

> neurons differentiated from fetal hNSCs 6 weeks after grafting into

> striatum, while another<A

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

nt/full/#B27 " >27</A> shows that numbers of neurites were gradually

> increased over a 5-month period. In contrast, we were able to obtain a

> nearly pure population of mature neurons with many neurites from fetal

> hNSCs in a much shorter time after transplantation. Although the hNSCs in

> most other studies were cultured with bFGF/heparin prior to

> transplantation, we used adhesive culture instead of clustered cells in

> suspension as used by the other groups. In more detail, fetal hNSCs were

> cultured adhesively over a flat surface with bFGF/heparin for 6–7 days,

> which allowed cells to spread out from the aggregates and thus be evenly

> exposed to the same concentrations of bFGF/heparin. Thus, our present

> findings, combined with previous reports, suggest that both bFGF/heparin

> and adhesive culture of hNSCs in the priming procedure is responsible for

> the generation of a large number of mature neurons from hNSCs in a

> relatively short period of time (1 month) after grafting into

> non-neurogenic areas of normal adult CNS. Although underlying mechanisms

> remain to be defined, the bFGF/heparin treatment of adhesively cultured

> hNSCs for 6–7 days may prime stem cells evenly toward a plastic

> intermediate stage, in which over 90% of cells remain nestin-positive. They

> then differentiate into neurons (about 45%) and astroglial cells (about

> 35%) under in vitro differentiation conditions within 10–14 days. Among the

> neurons, three phenotypes were detected: cholinergic, glutamatergic and

> GABAergic. This indicates that fetal hNSCs that originated from cortex have

> an intrinsic capability to differentiate into at least these three neuronal

> subtypes.

> In contrast to the relatively low percentage of neuronal differentiation in

> vitro from primed fetal hNSCs, a nearly pure population of neurons (about

> 95%) with region-specific subtypes was generated in vivo from the primed

> fetal hNSCs when grafted into either neurogenic or non-neurogenic areas in

> intact rat CNS. In particular, significant numbers of hNSCs-derived

> cholinergic neurons were detectable in medial septum (61%) and spinal cord

> (55%), but not in prefrontal cortex and hippocampus. Both medial septum and

> spinal cord are areas in the CNS that contain cholinergic neurons, whereas

> the other two regions do not have significant numbers of such neurons. Thus

> this showed a regional specificity, and as none of the primed hNSCs before

> transplantation had a cholinergic differentiation, additional cues such as

> astrocytes<A

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

nt/full/#B34 " >34</A> and/or a three-dimensional configuration in the adult host

> environment may be necessary to ensure more complete neuronal

> differentiation and subtype specification<A

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

nt/full/#B22 " >22</A>, <A

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

nt/full/#B35 " >35</A>. It remains to be

> determined whether this region-specific cholinergic neuronal

> differentiation is due to an instructive effect (that is, inducing primed

> cells to become cholinergic neurons), a selective effect (preferential

> survival of cholinergic neurons in medial septum or spinal cord) or both.

> Further studies are also needed to determine whether this region-specific

> differentiation pattern is retained in other areas of the brain, as well as

> in degenerated or damaged CNS.

> The two cell lines, K048 and K054, derived from human fetuses, were used

> for in vitro studies. They behaved identically in terms of differentiation

> patterns when exposed to various priming conditions. Thus, the FHL priming

> cocktail induced many large cholinergic neurons in both lines, indicating

> that FHL may have a universal effect on fetal human neural stem cells in

> vitro. Although it is likely that the two lines will behave similarly in

> vivo, further studies are needed to confirm this. Furthermore, it is not

> known whether our priming procedure has the same effect on ES cells and

> adult neural stem cells. Other critical issues, such as whether these fetal

> hNSC-derived neurons could project to correct targets or functionally

> replace dead neurons, need to be addressed before using the stem cell

> technology to treat the various neurological disorders that arise from loss

> of neurons. >> Methods

>

> Cell culture and rAAV vector. Fetal human neural stem cells, K048 and K054,

> originally derived from the cortices of 8-week and 10-week human fetuses,

> respectively, were provided by C.N. Svendsen, University of Wisconsin<A

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

nt/full/#B5 " >5</A>, <A

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

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

> Isolation and propagation of these hNSCs have been extensively described<A

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

nt/full/#B5 " >5</A>.

> Briefly, cells were cultured initially in medium containing EGF plus bFGF

> and then EGF alone. After passage 20, cells were cultured in a basic medium

> that consisted of DMEM:F12 (3:1, Invitrogen, Carlsbad, California), 15 mM

> HEPES (Sigma, St. Louis, Missouri), 1.5% glucose (Sigma), 2 mM L-glutamine

> (Sigma) and 1 penicillin/streptomycin (Sigma), which was then supplemented

> with N2<A

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

nt/full/#B36 " >36</A>, 20 ng/ml EGF (R & D Systems, Minneapolis, Minnesota), 10

ng/ml

> bFGF (R & D Systems), 2.5 g/ml heparin (Sigma) and 10 ng/ml LIF (Chemicon,

> Temecula, California). Once every 10 days, expanded neurospheres were

> passaged by dissociation into single cells with 0.025% Trypsin (Sigma) and

> trituration using a fire-polished Pasteur pipette, and re-plated in a mix

> of equal volumes of fresh and conditioned media. For in vitro priming,

> neurospheres were adhesively cultured in basic medium plus N2, 20 ng/ml

> bFGF, 5 g/ml heparin and 1 g/ml laminin (Invitrogen) (FHL) for 5–7 days. In

> some experiments, Shh-N (R & D Systems) was also added at concentrations

> ranging from 0.002 to 1 g/ml (SFHL cocktail). A half-volume of medium was

> replaced with fresh medium once every 1–2 days. For differentiation studies

> in vitro, small spheres (3–4 days post-passage) were seeded at 6–7 104

> cells/cm2 on glass coverslips pre-coated with 0.01% poly-D-lysine (PDL)

> (Sigma) and 0.5–1 g/cm2 laminin (Invitrogen). After 5–7 days of priming,

> cells were switched to basic medium plus B27 (1:50, Invitrogen) alone or

> with other neurotrophic factors for an additional 7–14 days. For

> transplantation, neurospheres from passages 19–55 were plated in T25

> culture flasks pre-coated with 0.01% PDL. Cells were primed with FHL or

> SFHL for a total of 6–7 days and treated with the CAG-egfp rAAV vector at a

> multiplicity of infection (MOI) of 2–5 (transducing particles/cell) for 3–

> 4 days before grafting. The CAG-egfp viral stock was prepared and titrated

> as previously described<A

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

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

>

> Electrophysiological recording. Resting and action potentials of cells were

> recorded at room temperature (20–23°C) using an Axopatch-200A patch clamp

> amplifier (Axon Instruments, City, California). Cells were kept on

> glass coverslips in the basic differentiation medium for 7–14 days after

> FHL or SFHL priming, and then transferred to a recording chamber with the

> medium containing 140 mM NaCl, 4 mM KCl, 10 mM HEPES, 10 mM glucose, 2 mM

> CaCl2 and 1 mM MgCl2 (pH 7.4). The pipette solution contained 120 mM

> KMeSO3, 20 mM KCl, 1 mM CaCl2, 1 mM BAPTA, 10 mM HEPES and 2 mM Mg-ATP (pH

> 7.2). The recorded signals were filtered at 2 kHz, sampled at 200 s per

> point and analyzed with the IGOR programs (WaveMetrics, Lake Oswego,

> Oregon).

> Transplantation. All surgical protocols were established according to the

> National Institutes of Health (NIH) guidelines for the care and use of

> laboratory animals and approved by the University of Texas Medical Branch

> IACUC. Male Sprague-Dawley rats (Harlan, Indianapolis, Indiana), 240–270 g,

> were immunosuppressed with Neoral cyclosporine (Novartis Pharmaceuticals,

> East Hanover, New Jersey) at 100 g/ml in drinking water 3 days before

> surgery and thereafter. For each individual experiment, the same batch of

> AAV-labeled hNSCs, primed or unprimed, was grafted into either brains or

> spinal cords in a given day. Dissociated cells (2–5 104 in 2 l) were

> stereotaxically injected into prefrontal cortex (in mm from skull: AP,

> +2.7; ML, -0.8; DV, -3.0), medial septum (AP, +0.7; ML, +0.2; DV, -7.0) or

> hippocampus (AP, -4.3; ML, +2.5 mm; DV, -3.0). Those cells were shown to

> have similar differentiation patterns and survival rates when grafted into

> brain or spinal cord. Transplantation of hNSCs in spinal cord (in mm from

> dura: ML, +1; DV, -1.5) was done as previously described<A

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

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

> anteroposterior axis; ML, mediolateral axis; DV, dorsoventral axis.

> Immunocytochemistry. Cells for in vitro studies were fixed with 4%

> paraformaldehyde (PFA). Animals were perfused with 4% PFA 1 week to 1 month

> after grafting, cryosectioned (coronally for brain and longitudinally for

> spinal cord) at 48 m. Cells or sections were subjected to immunofluorescent

> staining<A

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

nt/full/#B26 " >26</A> using mouse anti-Class III -tubulin (TuJ1) (1:4,000,

Covance,

> Richmond, California), mouse anti-Islet I (1:50, Developmental Studies

> Hybridoma Bank, Iowa City, Iowa), goat anti-ChAT (1:100, Chemicon), rabbit

> anti-synapsin I (1:500, Chemicon), mouse anti-NeuN (1:100, Chemicon), mouse

> anti-human nuclei (1:20, Chemicon), rabbit anti-rat carboxyl terminal of

> cytochrome P450scc (1:400, Chemicon), rabbit anti-GABA (1:1,000, Sigma),

> rabbit anti-glutamate (1:5,000, Sigma), rabbit anti-GFAP (1:1,000,

> Chemicon), rabbit anti-TH (1:500, Chemicon), mouse anti-human Nestin

> (1:200, C.A. Messam, NIH)<A

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

nt/full/#B37 " >37</A> or mouse anti-GalC (1:100, Chemicon). The a

> Fluo 594-conjugated secondary antibodies, goat anti-mouse, goat anti-rabbit

> or donkey anti-goat (all from Molecular Probes, Eugene, Oregon) were used

> at 1:200. Cell nuclei were counterstained with 1 g/ml DAPI (Sigma).

> Quantification. For quantitative analyses of cell phenotypes of hNSCs

> primed and/or differentiated in vitro, ten monolayer fields (more than 200

> cells) were randomly chosen for each sample. The percentage of any given

> phenotype in a sample was obtained by averaging proportions of a specific

> cell type in each of the 10 fields. At least four samples were counted for

> each treatment group.

> To determine survival rates of grafted hNSCs in brain and spinal cord of

> adult rats, total numbers of surviving GFP+ cells in each region were

> counted stereologically based on our previous description<A

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

nt/full/#B38 " >38</A>. Briefly, nine

> sections (48 m) per region in each animal were taken in a uniform random

> pattern. Upper and lower boundaries of optical dissectors were set at

> appropriate confocal planes using an Olympus Fluoview confocal microscope

> (Leeds Precision Instruments, Irving, Texas) with a 20 objective, with

> attention to 3-dimensional exclusion and inclusion lines, and green cell

> numbers were estimated by a fractionator analysis<A

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

nt/full/#B38 " >38</A>. Survival rates of

> grafted hNSCs were then calculated by dividing the number of green cells in

> each region by the total number of GFP+ cells originally injected. These

> values were then averaged for ten rats grafted with primed cells and for

> four rats with unprimed cells.

> To determine the percentages of double-labeled hNSCs, cell profiles were

> counted using an Olympus Fluoview confocal microscope with a 20 objective.

> Nine or three semi-serial sections 150–240 m apart were immunostained with

> ChAT or other antibodies (Nestin, TuJ1, GFAP, NeuN, GABA, glutamate),

> respectively. The number of GFP-labeled cells (representing grafted hNSCs)

> and the number of double-labeled cells (for each phenotype) were counted in

> three randomly chosen confocal sections (1 m thickness) and averaged for

> each cryostat section (48 m). Moreover, averaged percentages of

> double-labeled cell profiles over nine or three cryostat sections per rat

> were further averaged from ten animals for each cell phenotype in each

> grafted areas of the CNS. Repeated-measures analysis of variance (ANOVA)

> was used for statistical analyses using the InStat program (GraphPad

> Software, San Diego, California).

> Received 4 October 2002; Accepted 22 October 2002; Published online 11

> November 2002.

>

[Guest guest]

>

> >> Discussion

>

> Here we report a simple and efficient priming procedure to treat fetal

> hNSCs in vitro before further differentiation and transplantation in vivo.

> This procedure allowed us to obtain cholinergic neurons in vitro, as well

> as a nearly pure population of neurons in vivo, from long-term

> mitogen-expanded fetal hNSCs.

>

> The priming cocktail contains bFGF, heparin and laminin. Our initial

> screening indicated that both bFGF and heparin were necessary to obtain

> large cholinergic neurons from fetal hNSCs in vitro. An adherent laminin

> substrate included in the priming media was also required for an optimal

> spreading of neurospheres, which presumably function by allowing cells

> inside the spheres to be exposed evenly to the bFGF/heparin treatment.

> Using this priming method, many large, multipolar cholinergic neurons

> differentiated from fetal hNSCs following further incubation in the B27

> medium without bFGF. In contrast, direct plating of mitogen-expanded fetal

> hNSCs onto laminin-coated culture dishes generated only small bipolar

> GABAergic and glutamatergic neurons, even when cells were treated with

> various neurotrophic factors<A

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

nt/full/#B23 " >23</A>. Similar effects were observed on fetal hNSC

> differentiation in vitro when cells were treated with RA, EGF, LIF, ShhN

> and heparin, alone or in combination.

> Our in vivo transplantation data also showed that both bFGF/heparin and

> adhesive culture were involved in initiating neuronal differentiation of

> fetal hNSCs. Under certain conditions, bFGF favors neuronal differentiation<A

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

nt/full/#B30 " >

> 30</A>, <A

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

nt/full/#B31 " >31</A>. Furthermore, heparin may potentiate the biological

activity of bFGF

> through its helper effect on the binding of bFGF to its tyrosine kinase

> receptor<A

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

nt/full/#B32 " >32</A>, <A

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

nt/full/#B33 " >33</A>. Lack of heparin, therefore, might contribute in part to

the

> absence of neuronal differentiation from rat neural stem cells when they

> are transplanted into non-neurogenic spinal cord<A

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

nt/full/#B16 " >16</A>, <A

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

nt/full/#B20 " >20</A>. Two other groups

> were able to obtain neuronal differentiation in non-neurogenic striatum

> from grafted hNSCs, which were pre-cultured in medium containing

> bFGF/heparin. However, neuronal differentiation in these studies was

> limited. For example, one study<A

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

nt/full/#B17 " >17</A> reports that a small number of immature

> neurons differentiated from fetal hNSCs 6 weeks after grafting into

> striatum, while another<A

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

nt/full/#B27 " >27</A> shows that numbers of neurites were gradually

> increased over a 5-month period. In contrast, we were able to obtain a

> nearly pure population of mature neurons with many neurites from fetal

> hNSCs in a much shorter time after transplantation. Although the hNSCs in

> most other studies were cultured with bFGF/heparin prior to

> transplantation, we used adhesive culture instead of clustered cells in

> suspension as used by the other groups. In more detail, fetal hNSCs were

> cultured adhesively over a flat surface with bFGF/heparin for 6–7 days,

> which allowed cells to spread out from the aggregates and thus be evenly

> exposed to the same concentrations of bFGF/heparin. Thus, our present

> findings, combined with previous reports, suggest that both bFGF/heparin

> and adhesive culture of hNSCs in the priming procedure is responsible for

> the generation of a large number of mature neurons from hNSCs in a

> relatively short period of time (1 month) after grafting into

> non-neurogenic areas of normal adult CNS. Although underlying mechanisms

> remain to be defined, the bFGF/heparin treatment of adhesively cultured

> hNSCs for 6–7 days may prime stem cells evenly toward a plastic

> intermediate stage, in which over 90% of cells remain nestin-positive. They

> then differentiate into neurons (about 45%) and astroglial cells (about

> 35%) under in vitro differentiation conditions within 10–14 days. Among the

> neurons, three phenotypes were detected: cholinergic, glutamatergic and

> GABAergic. This indicates that fetal hNSCs that originated from cortex have

> an intrinsic capability to differentiate into at least these three neuronal

> subtypes.

> In contrast to the relatively low percentage of neuronal differentiation in

> vitro from primed fetal hNSCs, a nearly pure population of neurons (about

> 95%) with region-specific subtypes was generated in vivo from the primed

> fetal hNSCs when grafted into either neurogenic or non-neurogenic areas in

> intact rat CNS. In particular, significant numbers of hNSCs-derived

> cholinergic neurons were detectable in medial septum (61%) and spinal cord

> (55%), but not in prefrontal cortex and hippocampus. Both medial septum and

> spinal cord are areas in the CNS that contain cholinergic neurons, whereas

> the other two regions do not have significant numbers of such neurons. Thus

> this showed a regional specificity, and as none of the primed hNSCs before

> transplantation had a cholinergic differentiation, additional cues such as

> astrocytes<A

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

nt/full/#B34 " >34</A> and/or a three-dimensional configuration in the adult host

> environment may be necessary to ensure more complete neuronal

> differentiation and subtype specification<A

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

nt/full/#B22 " >22</A>, <A

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

nt/full/#B35 " >35</A>. It remains to be

> determined whether this region-specific cholinergic neuronal

> differentiation is due to an instructive effect (that is, inducing primed

> cells to become cholinergic neurons), a selective effect (preferential

> survival of cholinergic neurons in medial septum or spinal cord) or both.

> Further studies are also needed to determine whether this region-specific

> differentiation pattern is retained in other areas of the brain, as well as

> in degenerated or damaged CNS.

> The two cell lines, K048 and K054, derived from human fetuses, were used

> for in vitro studies. They behaved identically in terms of differentiation

> patterns when exposed to various priming conditions. Thus, the FHL priming

> cocktail induced many large cholinergic neurons in both lines, indicating

> that FHL may have a universal effect on fetal human neural stem cells in

> vitro. Although it is likely that the two lines will behave similarly in

> vivo, further studies are needed to confirm this. Furthermore, it is not

> known whether our priming procedure has the same effect on ES cells and

> adult neural stem cells. Other critical issues, such as whether these fetal

> hNSC-derived neurons could project to correct targets or functionally

> replace dead neurons, need to be addressed before using the stem cell

> technology to treat the various neurological disorders that arise from loss

> of neurons. >> Methods

>

> Cell culture and rAAV vector. Fetal human neural stem cells, K048 and K054,

> originally derived from the cortices of 8-week and 10-week human fetuses,

> respectively, were provided by C.N. Svendsen, University of Wisconsin<A

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

nt/full/#B5 " >5</A>, <A

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

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

> Isolation and propagation of these hNSCs have been extensively described<A

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

nt/full/#B5 " >5</A>.

> Briefly, cells were cultured initially in medium containing EGF plus bFGF

> and then EGF alone. After passage 20, cells were cultured in a basic medium

> that consisted of DMEM:F12 (3:1, Invitrogen, Carlsbad, California), 15 mM

> HEPES (Sigma, St. Louis, Missouri), 1.5% glucose (Sigma), 2 mM L-glutamine

> (Sigma) and 1 penicillin/streptomycin (Sigma), which was then supplemented

> with N2<A

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

nt/full/#B36 " >36</A>, 20 ng/ml EGF (R & D Systems, Minneapolis, Minnesota), 10

ng/ml

> bFGF (R & D Systems), 2.5 g/ml heparin (Sigma) and 10 ng/ml LIF (Chemicon,

> Temecula, California). Once every 10 days, expanded neurospheres were

> passaged by dissociation into single cells with 0.025% Trypsin (Sigma) and

> trituration using a fire-polished Pasteur pipette, and re-plated in a mix

> of equal volumes of fresh and conditioned media. For in vitro priming,

> neurospheres were adhesively cultured in basic medium plus N2, 20 ng/ml

> bFGF, 5 g/ml heparin and 1 g/ml laminin (Invitrogen) (FHL) for 5–7 days. In

> some experiments, Shh-N (R & D Systems) was also added at concentrations

> ranging from 0.002 to 1 g/ml (SFHL cocktail). A half-volume of medium was

> replaced with fresh medium once every 1–2 days. For differentiation studies

> in vitro, small spheres (3–4 days post-passage) were seeded at 6–7 104

> cells/cm2 on glass coverslips pre-coated with 0.01% poly-D-lysine (PDL)

> (Sigma) and 0.5–1 g/cm2 laminin (Invitrogen). After 5–7 days of priming,

> cells were switched to basic medium plus B27 (1:50, Invitrogen) alone or

> with other neurotrophic factors for an additional 7–14 days. For

> transplantation, neurospheres from passages 19–55 were plated in T25

> culture flasks pre-coated with 0.01% PDL. Cells were primed with FHL or

> SFHL for a total of 6–7 days and treated with the CAG-egfp rAAV vector at a

> multiplicity of infection (MOI) of 2–5 (transducing particles/cell) for 3–

> 4 days before grafting. The CAG-egfp viral stock was prepared and titrated

> as previously described<A

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

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

>

> Electrophysiological recording. Resting and action potentials of cells were

> recorded at room temperature (20–23°C) using an Axopatch-200A patch clamp

> amplifier (Axon Instruments, City, California). Cells were kept on

> glass coverslips in the basic differentiation medium for 7–14 days after

> FHL or SFHL priming, and then transferred to a recording chamber with the

> medium containing 140 mM NaCl, 4 mM KCl, 10 mM HEPES, 10 mM glucose, 2 mM

> CaCl2 and 1 mM MgCl2 (pH 7.4). The pipette solution contained 120 mM

> KMeSO3, 20 mM KCl, 1 mM CaCl2, 1 mM BAPTA, 10 mM HEPES and 2 mM Mg-ATP (pH

> 7.2). The recorded signals were filtered at 2 kHz, sampled at 200 s per

> point and analyzed with the IGOR programs (WaveMetrics, Lake Oswego,

> Oregon).

> Transplantation. All surgical protocols were established according to the

> National Institutes of Health (NIH) guidelines for the care and use of

> laboratory animals and approved by the University of Texas Medical Branch

> IACUC. Male Sprague-Dawley rats (Harlan, Indianapolis, Indiana), 240–270 g,

> were immunosuppressed with Neoral cyclosporine (Novartis Pharmaceuticals,

> East Hanover, New Jersey) at 100 g/ml in drinking water 3 days before

> surgery and thereafter. For each individual experiment, the same batch of

> AAV-labeled hNSCs, primed or unprimed, was grafted into either brains or

> spinal cords in a given day. Dissociated cells (2–5 104 in 2 l) were

> stereotaxically injected into prefrontal cortex (in mm from skull: AP,

> +2.7; ML, -0.8; DV, -3.0), medial septum (AP, +0.7; ML, +0.2; DV, -7.0) or

> hippocampus (AP, -4.3; ML, +2.5 mm; DV, -3.0). Those cells were shown to

> have similar differentiation patterns and survival rates when grafted into

> brain or spinal cord. Transplantation of hNSCs in spinal cord (in mm from

> dura: ML, +1; DV, -1.5) was done as previously described<A

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

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

> anteroposterior axis; ML, mediolateral axis; DV, dorsoventral axis.

> Immunocytochemistry. Cells for in vitro studies were fixed with 4%

> paraformaldehyde (PFA). Animals were perfused with 4% PFA 1 week to 1 month

> after grafting, cryosectioned (coronally for brain and longitudinally for

> spinal cord) at 48 m. Cells or sections were subjected to immunofluorescent

> staining<A

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

nt/full/#B26 " >26</A> using mouse anti-Class III -tubulin (TuJ1) (1:4,000,

Covance,

> Richmond, California), mouse anti-Islet I (1:50, Developmental Studies

> Hybridoma Bank, Iowa City, Iowa), goat anti-ChAT (1:100, Chemicon), rabbit

> anti-synapsin I (1:500, Chemicon), mouse anti-NeuN (1:100, Chemicon), mouse

> anti-human nuclei (1:20, Chemicon), rabbit anti-rat carboxyl terminal of

> cytochrome P450scc (1:400, Chemicon), rabbit anti-GABA (1:1,000, Sigma),

> rabbit anti-glutamate (1:5,000, Sigma), rabbit anti-GFAP (1:1,000,

> Chemicon), rabbit anti-TH (1:500, Chemicon), mouse anti-human Nestin

> (1:200, C.A. Messam, NIH)<A

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

nt/full/#B37 " >37</A> or mouse anti-GalC (1:100, Chemicon). The a

> Fluo 594-conjugated secondary antibodies, goat anti-mouse, goat anti-rabbit

> or donkey anti-goat (all from Molecular Probes, Eugene, Oregon) were used

> at 1:200. Cell nuclei were counterstained with 1 g/ml DAPI (Sigma).

> Quantification. For quantitative analyses of cell phenotypes of hNSCs

> primed and/or differentiated in vitro, ten monolayer fields (more than 200

> cells) were randomly chosen for each sample. The percentage of any given

> phenotype in a sample was obtained by averaging proportions of a specific

> cell type in each of the 10 fields. At least four samples were counted for

> each treatment group.

> To determine survival rates of grafted hNSCs in brain and spinal cord of

> adult rats, total numbers of surviving GFP+ cells in each region were

> counted stereologically based on our previous description<A

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

nt/full/#B38 " >38</A>. Briefly, nine

> sections (48 m) per region in each animal were taken in a uniform random

> pattern. Upper and lower boundaries of optical dissectors were set at

> appropriate confocal planes using an Olympus Fluoview confocal microscope

> (Leeds Precision Instruments, Irving, Texas) with a 20 objective, with

> attention to 3-dimensional exclusion and inclusion lines, and green cell

> numbers were estimated by a fractionator analysis<A

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

nt/full/#B38 " >38</A>. Survival rates of

> grafted hNSCs were then calculated by dividing the number of green cells in

> each region by the total number of GFP+ cells originally injected. These

> values were then averaged for ten rats grafted with primed cells and for

> four rats with unprimed cells.

> To determine the percentages of double-labeled hNSCs, cell profiles were

> counted using an Olympus Fluoview confocal microscope with a 20 objective.

> Nine or three semi-serial sections 150–240 m apart were immunostained with

> ChAT or other antibodies (Nestin, TuJ1, GFAP, NeuN, GABA, glutamate),

> respectively. The number of GFP-labeled cells (representing grafted hNSCs)

> and the number of double-labeled cells (for each phenotype) were counted in

> three randomly chosen confocal sections (1 m thickness) and averaged for

> each cryostat section (48 m). Moreover, averaged percentages of

> double-labeled cell profiles over nine or three cryostat sections per rat

> were further averaged from ten animals for each cell phenotype in each

> grafted areas of the CNS. Repeated-measures analysis of variance (ANOVA)

> was used for statistical analyses using the InStat program (GraphPad

> Software, San Diego, California).

> Received 4 October 2002; Accepted 22 October 2002; Published online 11

> November 2002.

>