Download Region-specific generation of cholinergic neurons from fetal human

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Region-specific generation of
cholinergic neurons from fetal human
neural stem cells grafted in adult rat
Ping Wu1,2, Yevgeniya I. Tarasenko1, Yanping Gu1, Li-Yen M. Huang2,3, Richard E. Coggeshall1,2
and Yongjia Yu4
Departments of 1Anatomy & Neurosciences, 3Physiology & Biophysics and 4Radiation Oncology, 2Marine Biomedical Institute, University of Texas
Medical Branch, Galveston, Texas 77555, USA
Correspondence should be addressed to P.W. ([email protected])
Published online 11 November 2002; doi:10.1038/nn974
Pluripotent or multipotent stem cells isolated from human embryos or adult central nervous system
(CNS) may provide new neurons to ameliorate neural disorders. A major obstacle, however, is that the
majority of such cells do not differentiate into neurons when grafted into non-neurogenic areas of the
adult CNS. Here we report a new in vitro priming procedure that generates a nearly pure population of
neurons from fetal human neural stem cells (hNSCs) transplanted into adult rat CNS. Furthermore, the
grafted cells differentiated by acquiring a cholinergic phenotype in a region-specific manner. This
technology may advance stem cell–based therapy to replace lost neurons in neural injury or
neurodegenerative disorders.
Advances in stem cell research have enabled the isolation and
propagation of human embryonic stem (ES) cells1 and germ
(EG) cells2. These cells are pluripotent—they can become any
cell type in the human body, including neurons. Multipotent
neural stem cells—another source for neural cells—have also
been isolated successfully from either fetal3–7or adult8–10 human
central nervous system (CNS). Their properties of self-renewal
and multipotential differentiation make stem cells an attractive
and presumably unlimited donor source for cell replacement
therapy to treat neurological disorders.
Human and rodent stem cells are able to differentiate into
specific neuronal types when grafted into either developing
CNS11–14 or neurogenic areas of the adult CNS15–17. However,
these cells remain undifferentiated or become mainly glial cells
when transplanted into non-neurogenic regions of the adult
CNS16–20, indicating that in vitro priming or some differentiation prior to grafting is necessary for these cells to develop specific neuronal subtypes. In particular, there have been no reports
as yet of the generation of a significant number of cholinergic
neurons from long-term mitogen-expanded human stem cells.
As these neurons are centrally involved in motor function, learning and memory, they are highly relevant to clinical applications.
For example, human stem cell–derived cholinergic neurons may
be used to replace motoneurons lost in amyotrophic lateral sclerosis (ALS or Lou Gehrig’s disease) or spinal cord injury. Here
we report a simple yet efficient priming procedure that caused
mitogen-expanded primary fetal human neural stem cells
(hNSCs) to become almost pure neurons when grafted into both
non-neurogenic and neurogenic areas of adult rat CNS. Most
importantly, a large number of these transplanted cells developed into cholinergic neurons when grafted in medium septum
and spinal cord.
nature neuroscience • volume 5 no 12 • december 2002
RESULTS
Primed hNSCs become cholinergic neurons in vitro
To obtain cholinergic neurons, we treated K048 hNSCs with
tropic factors or other chemicals that are important in the development of cholinergic neurons21,22, including recombinant
human basic fibroblast growth factor (bFGF), epidermal growth
factor (EGF), leukemia inhibitory factor (LIF), mouse sonic
hedgehog amino-terminal peptide (Shh-N), all-trans retinoic
acid (RA), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3),
neurotrophin-4 (NT-4), natural mouse laminin and heparin.
The K048 cell line was originally derived from the cortex of an
8-week legally aborted human fetus5 and mitogen-expanded
in vitro without viral or chemical treatments for immortalization. In this study, K048 hNSCs have been passaged continuously in vitro for over two years (85 passages) without changes
in their proliferation and differentiation patterns, or in their
normal diploid karyotype. These long-term epigenetically
expanded hNSC spheres (19–55 passages or 38–52 weeks) were
plated onto poly-D-lysine (PDL) and laminin-coated dishes and
treated with the above agents in vitro at various concentrations
either alone or in combination, concurrently or sequentially.
The combination consisting of bFGF, heparin and laminin
(abbreviated as FHL) had unique effects on fetal hNSCs. Thus,
a one-day exposure to FHL, with or without Shh-N (combination of Shh-N and FHL abbreviated as SFHL), resulted in a rapid
spreading of large planar cells in culture (Fig. 1a). All other oneday treatments (such as bFGF plus laminin, abbreviated FL), in
contrast, gave rise to a limited radial spread of spindle-shaped
cells, with the cells remaining close to the cores of the spheres
(Fig. 1b), similar to a previous description5. After 6 days of priming and a 10-day further differentiation in medium containing
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Fig. 1. Morphological, immunocytochemical and electrophysiological
characterization of primed and differentiated K048 fetal hNSCs in vitro.
(a–d) Phase contrast images of FHL-primed (a, c) or bFGF/laminintreated cells (b, d) for 1 day (a, b) or for 6 days followed by a 10-day differentiation in B27 basic medium (c, d). (e–h) Immunofluorescent
staining on FHL-primed and differentiated hNSCs with specific antibodies, viewed under a regular Nikon epifluorescent microscope. Note that
differentiated neurons are stained with TuJ1, Islet 1 (Isl), ChAT or
synapsin I (Syn). (i) Phase contrast image of typical cells used for a electrophysiological recording in (j–k). Note that the action potential
evoked by a supra-threshold stimulus (j) was blocked by tetrodotoxin
(TTX) treatment (k). Scale bars, 40 µm.
B27, large multipolar neuron-like cells were found near the edge
of the FHL-primed spheres (Fig. 1c), while most of the neuronlike cells were small and bipolar or unipolar in other treatment
groups (Fig. 1d).
Immunocytochemical analyses indicated that the small bipolar or unipolar cells (<20 µm) were either GABAergic or glutamatergic (data not shown), as found previously23. On the other
hand, many of the large multipolar cells near the edge of FHLprimed spheres, whose neuronal phenotype was shown by
immunocytochemical staining using a monoclonal antibody
against the neuron-specific type III β-tubulin (TuJ1; Fig. 1e),
were cholinergic because they showed immunoreactivity to markers specific for cholinergic neurons such as Islet-1 (Fig. 1f) and
choline acetyltransferase (ChAT; Fig. 1g). Some of these neurons
also expressed synapsin I (Fig. 1h). From five independent experiments, we estimated that of the total cell population in the
monolayer regions, 45.5 ± 4.7% differentiated into TuJ1+ neurons, whereas cholinergic (ChAT+) neurons accounted for 27.8
± 4.2% of the same population. However, multiple layers in and
around the core of spheres prevented absolute quantification of
any given phenotype in the total population. Furthermore,
ChAT+ neurons became undetectable if differentiated cells were
subjected to further dissociation and re-plating, a procedure
described previously23. In addition to the cholinergic neurons,
significant numbers of small neurons (glutamatergic, 6.3 ± 0.5%;
GABAergic, 11.3 ± 1.4%), astrocytes (35.2 ± 2.8%) and nestin+
cells (18.9 ± 2.0%) were also detected in FHL-primed neu-
rospheres even after the additional 10 days of differentiation
in vitro. On the other hand, no ChAT or Islet-1 positive cells were
detected in hNSCs untreated or treated with other reagents, in
which they either became astrocytes or small glutamatergic or
GABAergic neurons. Astrocytes and small neurons ranged from
40–70% and 10–60% of the total cells, respectively.
To determine whether the large cells have the electrical characteristics of neurons, resting potentials and action potentials
were monitored using the whole-cell patch clamp recording technique. Seven days after FHL-priming, most of the large multipolar cells (Fig. 1i) had resting potentials (−29.0 ± 2.0 mV,
n = 6), but no action potentials could be evoked. These resting
potentials were much more negative (−63.6 ± 3.0 mV, n = 5)
14 days after FHL treatment, and action potentials were seen
when depolarizing currents were injected (Fig. 1j). The action
potentials were blocked by 1 µM tetrodotoxin (TTX, Fig. 1k).
Thus, our data suggested that the FHL-priming procedure directed some of fetal hNSCs in vitro to become functional neurons.
To determine whether FHL priming has the same effect on
other fetal hNSCs apart from K048, we tested the K054 cell line,
which is derived from the cortex of a 10-week human fetus5. K054
Fig. 2. Morphological and immunocytochemical characterization of
primed and differentiated K054 fetal hNSCs in vitro. (a–d) Phase contrast images of FHL-primed (a, c) or Shh-N/laminin-treated cells (b, d)
for 1 day (a, b) or for 6 days followed by a 10-day differentiation in B27
basic medium (c, d). (e–h) Immunofluorescent staining on FHL-primed
(e, g–h) or SL-treated (f) K054 cells with specific antibodies TuJ1(e, f)
and ChAT (g, h). (h) is a higher magnification of the inset in (g). Note
that numerous TuJ1- or ChAT-stained cells are differentiated from FHLprimed hNSCs. Scale bars, 40 µm.
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Fig. 3. Immunocytochemical characterization of FHL-primed K048
fetal hNSCs without further in vitro differentiation. Note that the
majority of cells are undifferentiated, as they are stained with a human
specific nestin antibody (a). Only a few cells start differentiation:
arrows show cells specifically stained with antibodies against GFAP (b),
TuJ1 (c) and GABA (d). Scale bars, 40 µm.
cells (20 passages for 42 weeks) behaved very similarly to K048
cells in terms of their differentiation patterns and responses to
different priming treatments (Fig. 2a–f). Thus, FHL-priming for
6 days followed by a 10-day further differentiation resulted in the
appearance of cholinergic neurons (30.9 ± 3.1%) in vitro
(Fig. 2g–h). Other cell phenotypes included nestin + cells
(17.7 ± 1.8%), GFAP + astrocytes (35.8 ± 4.5%) and TuJ1 +
(45.3 ± 4.1%), glutamatergic (5.6 ± 0.7%) and GABAergic
(8.7 ± 1.1%) neurons. Exclusion of FH produced small GABA or
glutamate neurons, but not ChAT+ neurons.
with a GABA-specific antibody in cell bodies (Fig. 3d). 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.
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+
(Fig. 3a), indicating their stem/progenitor cell phenotypes. Only
0.8 ± 0.4% of the total cell population became GFAP+ astrocytes
(Fig. 3b), whereas 9.9% ± 1.1% differentiated into TuJ1+ neuronal cells (Fig. 3c). 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
Primed hNSCs become neurons in adult rat CNS
To determine whether hNSCs primed by FHL or SFHL for 6–7
days in vitro could become cholinergic neurons in vivo, we injected primed cells into a neurogenic region (hippocampus)24 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 vec-
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Fig. 4. Neuronal differentiation and
integration of primed fetal hNSCs
one month post-grafting in various
regions of brain and spinal cord of
adult rats. 5 × 104 cells were injected
into each region. (a–d) Confocal
images (merged from 30 1.2-µm confocal sections) of GFP-labeled hNSCs
grafted in prefrontal cortex (a),
medial septum (b), hippocampus (c)
and spinal cord (d). (e) GFP-labeled
cells were double-labeled with a
human specific nucleic marker, hN
(red). (f) GFP-labeled cells were not
labeled with a polyclonal antibody
specifically against rat cytochrome
P450scc. (g) hNSCs acquired a typical morphology of pyramidal neurons
in the CA1 region of the hippocampus. (h) The majority of hNSCs were
double-labeled with a neuron-specific
marker, NeuN (red). 1-µm confocal
sections from prefrontal cortex
(CX, i–k), dentate gyrus of hippocampus (DG, l–n), medial septum
(MS, o–q) and spinal cord (SC, r–t)
were labeled with another neuronspecific marker, TuJ1 (red). Scale bars,
100 µm. Midline indicated by (∗).
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tor. This method was chosen because GFP labels both cell bodies
and processes without leakage25, 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 hNSCs26.
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 (Fig. 4a–d). The extent of migration was variable
depending on the regions injected. In prefrontal cortex, for
example, GFP+ cells were usually located within 0.4–2 mm of
the injection site. However, cells migrate much farther, up to
1–2 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 (Fig. 4a), medial septum (Fig. 4b), hippocampus
(Fig. 4c) and spinal cord (Fig. 4d). 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 grafts27. 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.
To exclude the possibility of host cells picking up gfp DNA or
GFP proteins leaked from damaged fetal hNSCs, we performed
control transplantations using freeze-thawed hNSCs, which were
pre-treated with the same priming procedure and rAAVtransduction as the experimental groups. We did not detect any
green fluorescent (GFP+) cells in the host tissue in these control
rats for at least ten days after surgery (data not shown). In animals transplanted with live fetal hNSCs, all nucleated GFP+ cells
originated from the grafted hNSCs as verified by positive labeling
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Fig. 5. Confocal images of CAG-egfp-transduced fetal hNSCs without
priming one month post-grafting in various regions of brain and spinal
cord of adult rats. 2 × 104 cells were injected into each region. GFPlabeled fetal hNSCs grafted in (a) prefrontal cortex (CX), (b) medial
septum (MS), (c) CA1 region of hippocampus and (d) spinal cord (SC)
were double-labeled with a human specific nestin monoclonal antibody
(yellow cells), indicating that most of grafted cells remain undifferentiated. (e) Some of the GFP-labeled cells in the dentate gyrus (DG) of hippocampus were double-labeled with a neuron specific TuJ1 marker
(arrow). (f) Some GFP-labeled hNSCs in spinal cord (SC) were doublelabeled with a glial marker, GFAP (yellow cells indicated by arrows).
Scale bars, 100 µm.
with a monoclonal antibody specifically against human nuclei
(Fig. 4e), and negative labeling with a specific antibody against
rat cytochrome P450 side chain cleavage enzyme (P450scc,
Fig. 4f). Therefore, our data indicate that the GFP+ cells were
indeed of human origin, and that host cells were not labeled by
GFP leakage from damaged hNSCs or by spontaneous fusion
between human and rat cells28,29.
Morphological examination of fetal hNSC grafts showed that
GFP+ cells in the pyramidal cell layer of the CA1 region of the
hippocampus acquired typical pyramidal cell characteristics
(Fig. 4g). Numerous spines on their processes indicated a functional maturation of these grafted hNSCs. Immunocytochemical analyses using the neuron-specific markers NeuN (Fig. 4h)
and TuJ1 (Fig. 4i–t) revealed that most GFP+ cells acquire neuronal labeling 1 month after transplantation into cortex
(Fig. 4i–k), hippocampus (Fig. 4l–n), medial septum (Fig. 4o–q)
or spinal cord (Fig. 4r–t). Only a few scattered GFP+ cells were
double-labeled with an astrocyte-specific marker—glial fibrillary acidic protein (GFAP) (data not shown). No GFP+ cells were
immunoreactive to a monoclonal antibody against galactocerebroside (GalC, data not shown), indicating the absence of oligodendrocyte differentiation from grafted hNSCs. In addition,
negative staining using an undifferentiated neural stem cell marker, nestin (data not shown), suggested that all grafted cells had
differentiated by one month after transplantation.
Unprimed hNSCs become astrocytes in adult rat CNS
Although CAG-egfp transduction did not alter the in vitro differentiation patterns of hNSCs with or without priming, as previously reported26, it is necessary to determine whether this is
true for these cells grafted in vivo. To exclude the possibility that
GFP expression in fetal hNSCs could be responsible for the neuronal differentiation observed in vivo, we grafted CAG-egfptreated but not FHL-primed hNSCs into spinal cord, cortex,
medial septum and hippocampus (2 × 104 cells and n = 4 for
each region).
One month after transplantation, stereological analyses
showed that percentages of surviving hNSCs were 11.8 ± 2.0%
in cortex, 13.8 ± 3.5% in medial septum, 16.8 ± 3.2% in hippocampus and 4.8 ± 0.6% in spinal cord. These survival rates
were similar to those of their primed counterparts. Many of
the surviving cells, 61 ± 6.2% in cortex (Fig. 5a), 60.1 ± 4.7% in
medial septum (Fig. 5b), 58.7 ± 3.8 in CA1 of hippocampus
(Fig. 5c), 48.9 ± 2.3 in dentate gyrus (DG) of hippocampus
and 56.9 ± 5.6% in spinal cord (Fig. 5d), still expressed nestin
one month after transplantation. Neither neurons nor their
GFP + fibers were detected in the non-neurogenic areas we
examined. However, some grafted hNSCs became TuJ1+ in the
neurogenic dentate gyrus of the hippocampus (Fig. 5e, 11.2 ±
4.8%), which is consistent with previous observations17. On
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the other hand, 38.6 ± 8.7%, 39.9 ± 6.0%, 40.7 ±3.7%,
39.6 ± 2.9% and 42.7 ± 5.1% of grafted cells became GFAP+
when they were grafted into cortex, medial septum, CA1, DG
and spinal cord (Fig. 5f), respectively, which is similar to previous reports20. In summary, CAG-egfp-modified hNSCs without FHL priming remained either undifferentiated or acquired
an astroglial phenotype when grafted into non-neurogenic
areas of the adult rats. Therefore, both in vitro and in vivo data
support the conclusion that the FHL priming procedure, but
not CAG-egfp transduction, is responsible in part for the neuronal differentiation of fetal hNSCs.
Generation of ChAT+ neurons is region-specific
Subtypes of fetal hNSC-derived neurons in rat CNS were identified by single-channel or merged confocal xy images with a
z-thickness of either 1 µm or 0.3 µm after immunofluorescence
analyses with various specific antibodies. Many of the GFP+
neurons were double-labeled with the ChAT-specific antibody
in medial septum (Fig. 6a–c) and spinal cord (Fig. 6d–i) of the
host brain, which are regions containing many endogenous
ChAT+ neurons. A few GFP+/ChAT+ double-labeled neurons
Fig. 6. Region-specific differentiation of neuron subtypes from primed
fetal hNSCs after grafting in brain or spinal cord of adult rats. Primed
hNSCs differentiated into cholinergic neurons, double-labeled with a
ChAT antibody, in medial septum (MS) (a–c) and spinal cord (SC) (d–i).
In contrast, the majority of grafted cells become glutamatergic neurons
in cortex (CX) (j–I) or GABAergic neurons (m–o) in the CA1 region of
hippocampus. Green, GFP-labeled fetal hNSCs (a, d, g, j, m); red,
immunostaining with specific antibodies against ChAT (b, e, h), glutamate (k) or GABA (n). (a–f) and (j–o), 1-µm confocal images; (g–i), 0.3µm confocal images. Scale bars, 100 µm.
were found in prefrontal cortex, an area with a limited number
of endogenous ChAT neurons. We did not find any
GFP+/ChAT+ neurons in the hippocampus, where there are no
endogenous ChAT neurons. In the spinal cord, some transplanted ChAT+ neurons had a size and morphology that was
indistinguishable from endogenous α motoneurons (Fig. 6g–i).
Immunohistochemical analyses with antibodies that specifically recognize other neuronal subtypes also revealed region-specific patterns. For example, glutamate immunoreactivity was
found in the majority of GFP + cells grafted into cortex
(Fig. 6j–l), some cells in spinal cord and the dentate gyrus of
hippocampus, a few in medial septum, and none in the CA1
region of hippocampus (data not shown). In contrast, the
majority of grafted GFP+ cells in the CA1 region (Fig. 6m–o)
as well as some cells in all other regions tested (data not shown)
were double-labeled with a GABA antibody. No GFP+ cells were
immunoreactive to TH.
To quantify neuronal differentiation of grafted fetal hNSCs,
we counted the number of GFP+ cell profiles and the number
of each phenotype. The vast majority of surviving GFP+ cells
differentiated into neurons, as determined by TuJ1 staining,
including 94.9 ± 1.8% in cortex, 95.4 ± 0.9% in medial septum,
96.3 ± 1.0% in CA1, 95.1 ± 1.1% in DG and 94.8 ± 1.9% in
spinal cord. In contrast, only small percentages of surviving
cells became GFAP+ astrocytes: 4.4 ± 0.9% in cortex, 3.9 ± 1.1%
in medial septum, 3.4 ± 0.7% in CA1, 4.8 ± 0.7% in DG and
4.4 ± 0.8% in spinal cord. As no significant differences were
observed between FHL-primed and SFHL-primed hNSCs
(n = 5 for each region in each group), ten animals for each
region were pooled to obtain quantitative analyses of neuronal
subtypes (ChAT, glutamate or GABA) with the means (± s.e.m.)
(Fig. 7). Specifically, 61.3 ± 5.4% and 55.5 ± 3.2% of the GFP+
cells become cholinergic neurons when grafted in medial septum and spinal cord, respectively. Fetal hNSC-derived glutamatergic neurons were mainly detected in prefrontal cortex
(51.1 ± 1.5%) and in a much lower percentage (13.9 ± 1.7%) in
spinal cord. Whereas 71.3 ± 4.9% of GFP+ cells turned into
GABAergic neurons in the CA1 region of hippocampus, smaller fractions (20–30%) of such neurons were observed in all the
other areas that we transplanted.
Fig. 7. Quantitative analyses of region-specific differentiation of neuronal subtypes from grafted fetal hNSCs in adult rat CNS from four separate experiments. The y-axis represents the percentage of each
neuronal phenotype over the total number of GFP-labeled cells.
Cholinergic, glutamatergic and GABAergic neurons were identified by
immunostaining with antibodies against ChAT, glutamate and GABA,
respectively. CX, cortex; MS, medial septum; HIP, CA1 of hippocampus;
SC, spinal cord. Error bars, ± s.e.m. (n = 10).
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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 factors23. 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 differentiation30,31. Furthermore, heparin
may potentiate the biological activity of bFGF through its helper
effect on the binding of bFGF to its tyrosine kinase receptor32,33.
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 cord16,20. Two other
groups were able to obtain neuronal differentiation in nonneurogenic striatum from grafted hNSCs, which were pre-cultured
in medium containing bFGF/heparin. However, neuronal differentiation in these studies was limited. For example, one study17
reports that a small number of immature neurons differentiated
from fetal hNSCs 6 weeks after grafting into striatum, while another27 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 nonneurogenic 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.
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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 astrocytes34 and/or a three-dimensional configuration in the
adult host environment may be necessary to ensure more complete neuronal differentiation and subtype specification22,35. 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 Wisconsin5,26. Isolation and propagation of these hNSCs have been extensively described 5 . 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
N236, 20 ng/ml EGF (R&D Systems, Minneapolis, Minnesota), 10 ng/ml
bFGF (R&D Systems), 2.5 µg/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 µg/ml heparin and 1 µg/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 µg/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 µg/cm2 laminin (Invitrogen). After 5–7 days of priming, cells were
nature neuroscience • volume 5 no 12 • december 2002
articles
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 precoated 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 described26.
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, Foster 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 µs 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 µg/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 µl) 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 described26. 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 µm. Cells or sections were subjected to
immunofluorescent staining26 using mouse anti-Class III β-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 antiNeuN (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)37 or
mouse anti-GalC (1:100, Chemicon). The Alexa 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 µg/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 description38. Briefly, nine
sections (48 µm) 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
nature neuroscience • volume 5 no 12 • december 2002
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 analysis38. 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 µm 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 µm thickness) and averaged for each cryostat section (48 µm).
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. Repeatedmeasures analysis of variance (ANOVA) was used for statistical analyses
using the InStat program (GraphPad Software, San Diego, California).
Acknowledgments
The authors thank C.N. Svendsen and W.D. Willis for critical reading. We are
also grateful to Y. Ye and Z. Chen for technical assistance, as well as to
B.M. Walters for manuscript preparation. This work was supported by the John
Sealy Memorial Endowment Fund (P.W.), Mission Connect of the Institute for
Rehabilitation and Research Foundation (P.W.) and National Institute on Drug
Abuse (L.M.H.).
Competing interests statement
The authors declare competing financial interests; see the Nature Neuroscience
website (http://www.nature.com/natureneuroscience) for details.
RECEIVED 4 OCTOBER; ACCEPTED 22 OCTOBER 2002
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