Download Lineage-restricted neural precursors survive, migrate, and

Experimental Neurology 194 (2005) 230 – 242
www.elsevier.com/locate/yexnr
Lineage-restricted neural precursors survive, migrate, and differentiate
following transplantation into the injured adult spinal cord
A.C. Lepore, I. FischerT
Department of Neurobiology and Anatomy, 2900 Queen Lane, Drexel University College of Medicine, Philadelphia, PA 19129, USA
Received 4 January 2005; revised 4 January 2005; accepted 15 February 2005
Available online 5 April 2005
Abstract
Fetal spinal cord from embryonic day 14 (E14/FSC) has been used for numerous transplantation studies of injured spinal cord. E14/FSC
consists primarily of neuronal (NRP)- and glial (GRP)-restricted precursors. Therefore, we reasoned that comparing the fate of E14/FSC with
defined populations of lineage-restricted precursors will test the in vivo properties of these precursors in CNS and allow us to define the
sequence of events following their grafting into the injured spinal cord. Using tissue derived from transgenic rats expressing the alkaline
phosphatase (AP) marker, we found that E14/FSC exhibited early cell loss at 4 days following acute transplantation into a partial hemisection
injury, but the surviving cells expanded to fill the entire injury cavity by 3 weeks. E14/FSC grafts integrated into host tissue, differentiated
into neurons, astrocytes, and oligodendrocytes, and demonstrated variability in process extension and migration out of the transplant site.
Under similar grafting conditions, defined NRP/GRP cells showed excellent survival, consistent migration out of the injury site and robust
differentiation into mature CNS phenotypes, including many neurons. Few immature cells remained at 3 weeks in either grafts. These results
suggest that by combining neuronal and glial restricted precursors, it is possible to generate a microenvironmental niche where emerging glial
cells, derived from GRPs, support survival and neuronal differentiation of NRPs within the non-neurogenic and non-permissive injured adult
spinal cord, even when grafted into acute injury. Furthermore, the NRP/GRP grafts have practical advantages over fetal transplants, making
them attractive candidates for neural cell replacement.
D 2005 Elsevier Inc. All rights reserved.
Keywords: Fetal graft; Neural progenitor; Neural stem cell; Rat; Spinal cord injury
Introduction
Numerous studies over the last two decades have shown
that grafting of embryonic day-14 fetal spinal cord tissue
(E14/FSC) into the injured spinal cord rescues axotomized
neurons from retrograde cell death and atrophy (Bregman
and Reier, 1986; Mori et al., 1997), reduces scar formation
(Houle, 1992), and promotes host axonal regeneration
(Diener and Bregman, 1998b; Tessler et al., 1988). In the
adult, host axons penetrate the graft, but do not transverse
Abbreviations: AP, human placental alkaline phosphatase; E14/FSC,
embryonic day-14 fetal spinal cord; GRP, glial-restricted precursor; NRP,
neuronal-restricted precursor; NSC, multipotent neural stem cell.
T Corresponding author. Fax: +1 215 843 9082.
E-mail address: [email protected] (I. Fischer).
0014-4886/$ - see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.expneurol.2005.02.020
transplants to reenter host neuropil. Graft-derived fibers
also project into host tissue (Jakeman and Reier, 1991;
Reier et al., 1986). Although FSC grafts have been
successful in improving functional recovery following
complete spinal transection of neonates (Diener and Bregman, 1998a; Howland et al., 1995; Miya et al., 1997), little
recovery occurs in adult models (Bregman et al., 1993;
Stokes and Reier, 1992). Despite potential benefits, the use
of fetal transplants has serious shortcoming including issues
of supply, storage, cellular heterogeneity, and quality
control.
E14/FSC transplants consist primarily of lineagerestricted neural precursors, both neuronal (NRP)- and
glial(GRP)-restricted precursors, as well as small populations of multipotent neural stem cells (NSCs) and differentiated cells (Kalyani and Rao, 1998). Previous studies
A.C. Lepore, I. Fischer / Experimental Neurology 194 (2005) 230–242
231
have shown that NSCs grafted into non-neurogenic regions
of intact and injured adult CNS survive poorly (Lepore et al.,
2004) and differentiate mostly into glial cells (Cao et al.,
2001). In contrast, lineage-restricted precursors survive for
long periods of time and differentiate into neurons and glia,
respectively (Han et al., 2002, 2004; Lepore et al., 2004).
However, previous studies have raised concerns about the
ability of grafted NSCs and NRPs to survive in the toxic
environment of the injury site and to differentiate into
mature neurons without the necessary instructive environment (Cao et al., 2002). We therefore reasoned that
comparing the fate of E14/FSC transplants and transplants
of a defined population of lineage-restricted precursors
(NRPs and GRPs) in the injured adult spinal cord, using
cells derived from alkaline phosphatase (AP) transgenic rats,
will allow for elucidating the sequence of events that
follows grafting of fetal cells in the injured adult CNS,
including survival, migration, and differentiation.
Using a lateral funiculus injury model, we found an early
and extensive cell loss following acute transplants of E14/
FSC. Surviving precursors expanded to fill the entire lesion
by 3 weeks post-transplantation. E14/FSC grafts integrated
and extended long processes into host spinal cord, differentiated into neurons, astrocytes, and oligodendrocytes and
showed variability in migration out of the transplant site.
Grafts of defined NRP/GRP cells filled the cavity without
significant early cell loss, differentiated into mature CNS
phenotypes and showed consistent cellular migration out of
the injury site, even when grafted into the acute injury. This
work suggests that mixed lineage-restricted precursor grafts
generate a microenvironment that protects the cells from the
detrimental effects of the injured CNS and provides them
with a permissive niche for survival, differentiation, and
migration. Furthermore, NRP/GRP grafts represent a
practical alternative to fetal tissue transplants because of
the ability to isolate, expand, store, and genetically
manipulate these cells.
medium [DMEM-F12, BSA (1 mg/mL; Sigma; St. Louis,
MO), B27 (Invitrogen), bFGF (10 Ag/mL; Peprotech; Rocky
Hill, NJ), Pen-Strep (100 IU/mL; Invitrogen), N2 (10 AL/
mL; Invitrogen), NT-3 (10 Ag/mL; Peprotech)] on poly-llysine (13.3 Ag/mL; Sigma)- and laminin (20 Ag/mL;
Invitrogen)-coated dishes.
Methods
Adult spinal cord injury and transplants
Cell isolation and culture
Lateral funiculus injuries were created at the cervical 4
spinal cord level. Adult female Sprague–Dawley rats
(approximately 250 g) received intraperitoneal injections
of anesthetic cocktail [acepromazine maleate (0.7 mg/kg;
Fermenta Animal Health, Kansas City, MO), ketamine (95
mg/kg; Fort Dodge Animal Health; Fort Dodge, IA), and
xylazine (10 mg/kg; Bayer, Shawnee Mission, KS)]. The
back musculature was excised, and a laminectomy was
performed at the cervical 3/4 level. The dura was incised
above the dorsal root entry zone. Microscissor cuts were
created at the rostral and caudal extents of the injury.
Aspiration was used to selectively ablate only the lateral
white matter tracts, as well as a minimal portion of the
dorsal and ventral gray matter. The dorsal columns and
central canal were unaffected. Once hemostasis was
NRPs and GRPs were isolated from embryonic day-13.5
transgenic Fischer 344 rats that express the marker gene,
human placental alkaline phosphatase (AP). This transgenic
animal has previously been characterized (Kisseberth et al.,
1999; Mujtaba et al., 2002). Briefly, embryos were isolated
in DMEM/F12 (Invitrogen; Carlsbad, CA). Trunk segments
were incubated in collagenase Type I (10 mg/mL; Worthington Biochemicals; Lakewood, NJ)/dispase II (20 ng/
mL; Roche Diagnostics; Indianapolis, IN)/HBSS (Cellgro;
Herndon, VA) solution for 8 min at room temperature to
remove meninges from the cords. Cords were dissociated
using a 0.05% trypsin/EDTA (Invitrogen) solution for
20 min at 378C. Cells were then plated in NRP complete
Preparation of cells for grafting
Following embryonic dissection, NRPs and GRPs were
co-cultured for 3–10 days prior to transplantation. A mixed
population of NRPs and GRPs was dissociated from
culture flasks using 0.05% trypsin/EDTA, washed and resuspended at a concentration of 200,000 cells/AL [in Type
1 collagen matrix-Vitrogen (Cohesion; Palo Alto, CA)/
basal media mixture] for transplantation. Cells were placed
on ice during surgery. After completion of surgery, cell
viability was assessed using the trypan blue assay.
Viability was always greater than 90%. The composition
of the NRP/GRP cultures, with respect to the presence of
undifferentiated neural precursors devoid of mature cells,
was verified before grafting by staining for the immature
neural marker nestin, for markers for NRPs (E-NCAM)
and GRPs (A2B5), as well as for markers of mature cell
types.
Preparation of fetal spinal cord tissue (E14/FSC) for
grafting
Whole FSC tissue was isolated from embryonic day-14
rats as previously described (Bregman and McAtee, 1993).
To obtain labeled tissue, we used transgenic Fischer 344 rats
that express the AP marker gene. Briefly, embryos were
isolated in DMEM/F12. Trunk segments were dissected, and
the surrounding meninges were removed. Square pieces of
tissue (1–3 mm) were kept in DMEM-F12 on ice until
placed directly into the spinal cord lesion site (approximately 10 min).
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achieved, NRP/GRP cells (mixed in Type I Collagen matrixVitrogen) or whole FSC tissue pieces were implanted into
the injury cavity using either a 10-AL Hamilton Gastight
syringe (Hamilton; Reno, NV) or forceps, respectively. The
cell/matrix mixture and the tissue grafts both completely
filled the cavities. Approximately 400,000 cells in the mixed
NRP/GRP transplants were grafted into the injury site. Dura
was closed with 9-0 suture; muscle was re-apposed; skin
was closed with wound clips. Animals received Bupranorphin and methylprednisolone (Pharmacia and Upjohn;
Kalamazoo, MI) postoperatively. Animals were immunosuppressed by subcutaneous administration of cyclosporine
A (10 mg/kg; Sandoz Pharmaceuticals, East Hanover, NJ)
daily beginning 3 days before grafting and continuously
until sacrifice.
The care and treatment of animals in all procedures was
conducted in strict accordance with the guidelines set by
the Drexel University IACUC and the Institute of
Laboratory Animal Resources, U.S. National Academy of
Sciences.
Tissue processing
Animals were sacrificed at various time points (4 days, 3
and 5 weeks) following transplantation by transcardial
perfusion with 0.9% saline, followed by ice-cold 4%
paraformaldehyde (Fisher Scientific; Pittsburgh, PA). Spinal
cords were removed from the animals, followed by
cryprotection in 30% sucrose (Fisher Scientific)/0.1 M
phosphate buffer at 48C for 3 days. The tissue was
embedded in OCT (Fisher Scientific), fast frozen with dry
ice, and stored at 808C until processed. Spinal cord tissue
blocks were cut in the sagittal or transverse plane at 20 Am
thickness. Sections were collected on gelatin- and poly-llysine-coated glass slides and stored at 808C until
analyzed.
Alkaline phosphatase histochemistry
Serial sections were analyzed by AP histochemistry to
assess the presence, location, migration, and morphology of
graft-derived cells in the spinal cord. Sections were washed
with PBS, heated at 608C for 1 h to inactivate endogenous
enzyme activity, washed briefly in AP buffer (100 mM Tris,
100 mM NaCl, 50 mM MgCl2, pH 9.5; all Fisher Scientific)
and finally incubated at room temperature in the dark with
AP staining solution [(NBT; 1.0 mg/mL; Sigma), (BCIP;
0.1 mg/mL; Sigma), (levamisole; 5 mM; Sigma) in AP
buffer] for 1.5–2.0 h. Slides were cover-slipped in hard-set
Vectashield (Vector; Burlingame, CA) and visualized with
light microscopy.
Immunohistochemistry/phenotypic analysis
Tissue sections and cultured cells were washed in PBS,
blocked in 10% goat serum (Invitrogen) for 1 h at room
temperature and incubated with primary antibody solution
at 48C overnight. Both monoclonal and polyclonal (1:200;
Accurate, Westbury, NY) antibodies against human placental alkaline phosphatase were used to identify graftderived cells. A number of primary antibodies were used
to assess the phenotype of cells. Nestin (1:1000; monoclonal; Pharmingen; San Diego, CA) was used to identify
undifferentiated neural precursor cells (NSCs, NRPs, and
GRPs). Sox-2 (1:200; polyclonal; Chemicon; Temecula,
CA) was used to identify NSCs. Dividing cells were
identified using Proliferating Cell Nuclear Antigen
(PCNA; 1:200; monoclonal; Chemicon). NRPs were
identified specifically using E-NCAM (1:200; monoclonal;
Chemicon), while GRPs were identified using A2B5
(1:500; monoclonal; Chemicon). Neurons were identified
using the antibodies: MAP2 (1:100; monoclonal; Chemicon); NeuN (1:100; monoclonal; Chemicon). Astrocytes
were identified using GFAP (1:100; monoclonal; Chemicon). Oligodendrocytes precursors were identified using
the antibodies: NG2 (1:250; polyclonal; Chemicon), Sox10 (1:200; polyclonal; Chemicon). Mature oligodendrocytes were identified with RIP (1:1000; monoclonal;
Chemicon). Samples were incubated for 2 h at room
temperature with goat anti-mouse and goat anti-rabbit
secondary antibodies (1:200; Jackson, West Grove, PA)
conjugated to rhodamine or FITC. Samples were counterstained with DAPI (1:1000; Sigma) to identify nuclei, and
cover-slipped with anti-fade mounting media (Fluorosave,
CN Biosciences; La Jolla, CA). Slides were subsequently
stored at 208C. Images were acquired on either a Leica
DMRBE fluorescence microscope (Leica Microsystems;
Bannockburn, IL) using a Photometric Sensys KAF-1400
CCD camera (Roper Scientific; Trenton, NJ) or on a Leica
TCS SP2 laser confocal microscope (Leica). Images were
analyzed using either IP Lab (Scanalytics; Fairfax, VA) or
Leica confocal software version 2.0 (Leica). Adobe
Photoshop CS (Adobe, San Jose, CA) was used to prepare
figures.
Results
Graft composition
E14 spinal cord grafts
The phenotypic composition of E14/FSC was assessed
to determine the specific cell types that make up the graft.
DAPI staining shows the cellular pattern of E14/FSC
(Fig. 1A). A large proportion of the cells expressed nestin
(Fig. 1B), an early neural marker expressed by both NSCs
and lineage-restricted precursors. Cells of the E14 FSC
also expressed either E-NCAM (Fig. 1C) or A2B5 (Fig.
1D), markers of NRPs and GRPs, respectively. Only small
numbers of cells expressed markers of NSCs (Sox-2) or
neurons (NeuN) (data not shown). These results indicate
that while E14/FSC grafts consisted mainly of lineage-
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233
Fig. 1. Graft characterization. Prior to transplantation, E14/FSC (A–D) and NRP/GRP cultures (E–F) were characterized to define their phenotypic profile.
DAPI staining shows the cellular pattern of the tissue (A). Cells within the E14/FSC express the early neural marker nestin (B), as well as the cell surface
antigens, E-NCAM (C) or A2B5 (D), markers of NRPs and GRPs, respectively. Cultures of NRPs and GRPs were harvested from E13.5 FSC. All cells in this
population display the morphological characteristics of small phase-bright bipolar lineage-restricted cells (E). Similar to E14/FSC, NRP/GRP cells express
nestin (F), as well as either E-NCAM (G) or A2B5 (H). Scale bars: 100 Am (A–D) or 50 Am (E–F).
restricted precursors (NRPs and GRPs), they also included
NSCs and differentiated neurons.
NRP/GRP grafts
NRPs and GRPs were isolated from the caudal neural
tube of E13.5 AP rats and co-cultured for 3–10 days. During
the culturing period, the cells showed the characteristic
morphology of small, phase-bright, lineage-restricted precursors (Fig. 1E). Before transplantation, the phenotypic
composition of the grafted cells was again verified. We
found that at the time of grafting, all cells in the NRP/GRP
cultures expressed the early neural marker, nestin (Fig. 1F),
showing that all cells were neural precursors. NRP/GRP
cells did not express mature markers of neurons (NeuN),
astrocytes (GFAP), or oligodendrocytes (RIP, NG2, and
Sox-10) (data not shown), showing that cultures did not
contain differentiated cell types. Cells in the NRP/GRP
cultures expressed either E-NCAM (Fig. 1G) or A2B5 (Fig.
1H). Together, these results suggest that the cultures were
composed almost exclusively of lineage-restricted precursors, both NRPs and GRPs. The mixed lineage-restricted
cells used for grafting consisted of various ratios of NRPs
and GRPs, dependent on the time spent in culture, and were
devoid of NSCs and mature cells types. Furthermore,
cultures of NRPs and GRPs maintained for multiple
passages (up to 10 days) continued to be nestin-positive,
without expressing mature markers. These results demonstrate that lineage-restricted precursors could be expanded in
an undifferentiated state for extended periods of time.
Mixed lineage-restricted cells used for grafting consisted
of NRPs and GRPs at ratios that were dependent on the
culturing time, but were always devoid of NSCs and mature
cells types.
Survival and migration
E14 spinal cord grafts
Small tissue segments of E14/FSC were grafted acutely
into a lateral funiculus injury of the cervical spinal cord.
Animals were sacrificed at 4 days, and 3 and 5 weeks postengraftment. Serial sections were assessed by AP histochemistry to detect the presence of AP-labeled cells. At the
time of transplantation, the entire injury cavity was
completely filled with graft tissue, but 4 days later only a
small portion of the lesion site was filled with grafted cells
(Figs. 2A–B). By 3 and 5 weeks post-transplantation, the
entire lesion cavity was completely refilled with APlabeled cells, forming an even interface with the host
(Figs. 2C–G; 3A, C). In addition, at the 3- and 5-week time
points, long graft-derived fibers could be seen projecting
from the transplant into the surrounding intact spinal cord
(Figs. 2C–G). Graft-derived cells also migrated out of the
transplant/injury site into the host parenchyma (Figs. 3A,
B, D). The extent of migration of the transplanted cells and
the extension of graft-derived fibers into the host spinal
cord were variable and not observed in all animals (Fig.
3C). These observations show that after an initial cell loss,
the grafted cells filled the injury site, most likely by
proliferation, and appeared to have integrated with the host
tissue.
NRP/GRP grafts
Mixed populations of NRPs and GRPs (400,000 cells/
transplant at various ratios) were grafted acutely into the
lesion cavity via direct injection. These grafts were analyzed
using the same protocols as with E14/FSC grafts. Despite
the small number of precursor cells grafted relative to the
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Fig. 2. Grafting of E14/FSC into the injured spinal cord: Survival and process extension. Undissociated E14/FSC was grafted acutely into a lateral funiculus
injury of the cervical spinal cord. AP histochemistry was used to detect the presence of transplanted cells. Grafts initially filled the entire lesion cavity (data not
shown), but suffered extensive cell loss by 4 days post-transplantation (A–B). By 3 weeks post-transplantation, E14/FSC grafts completely filled the lesion
cavity and integrated with the adjacent host tissue (C–G). Graft-derived fibers project out of the transplant site into the host spinal cord in both the sagittal
(C–E) and transverse (F–G) planes. These fibers project into both white (C–E) and gray (F–G) matter, and extend for distances greater than 5 mm in both the
rostral (C) and caudal (D) direction. Panels are sagittal (A, C–E) or transverse (B, F–G) sections, with the dorsal aspect facing upwards. Scale bars: 1 mm
(A–D, F) or 0.5 mm (E, G).
number of cells contained in fetal tissue transplants, NRP/
GRP cells showed excellent survival in the injury site at 4
days (Figs. 4A–B). Grafts completely filled the injury cavity
by 3 and 5 weeks, with no signs of a large cell mass
suggestive of tumor formation (Fig. 4C). By 3 to 5 weeks
post-engraftment, the NRP/GRP transplants robustly and
consistently migrated out of the injury cavity along white
matter tracts of the intact cord in both the rostral and caudal
direction (Figs. 4C–E) to distances of up to 15 mm. This
behavior has a general similarity to the fate of the E14/FSC
grafts, but also underscores several differences between the
NRP/GRP and E14/FSC transplants. These differences
include characteristics of graft survival, migration, and
process extension.
Fig. 3. Grafting of E14/FSC into the injured spinal cord: Variability in migration and process extension. Undissociated E14/FSC was grafted acutely into a
lateral funiculus injury of the cervical spinal cord. AP histochemistry was used to detect the presence of transplanted cells. By 3 weeks post-transplantation,
E14/FSC-derived cells migrate out of the graft site along white matter tracts of the intact host spinal cord (A–B, D). Cells migrate in both the rostral and caudal
directions (A–B) for distances greater than 10 mm. However, FSC grafts demonstrate variability in migration and process extension. All FSC grafts survive and
completely fill the lesion cavity, but some show only modest migration or process extension into the host spinal cord (C). All panels are sagittal sections, with
the dorsal aspect facing upwards. Scale bars: 1 mm (A, C), 0.5 mm (B), or 0.1 mm (D).
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235
Fig. 4. Grafting of NRPs/GRPs into the injured spinal cord. Mixed populations of NRPs and GRPs were grafted acutely into a lateral funiculus injury of the
cervical spinal cord. AP histochemistry was used to detect the presence of transplanted cells. NRP/GRP grafts filled the injury cavity only partially by 4 days
post-transplantation (A–B), but expanded to fill the cavity by 3 weeks post-transplantation (C). NRP/GRP grafts demonstrated robust and consistent migration
into the host spinal cord selectively along white matter tracts in both rostral and caudal directions (C–E). Cells were found at distances up to 15 mm by 3 weeks
post-transplantation. All panels are sagittal sections, with the dorsal aspect facing upwards. Scale bars: 1 mm (A–C), 0.5 mm (D), or 0.1 mm (E).
Phenotypic fate
Fetal spinal cord grafts
The presence of the transgenic AP marker allowed us
to follow the phenotypic fate of transplant-derived cells at
various time points after engraftment using double
staining and confocal microscopy. We used co-localization of the AP transgene with markers of neural
precursors (nestin), neurons (NeuN and MAP2), astrocytes (GFAP), and oligodendrocytes (RIP, Sox-10, and
NG2). We found that at 4 days post-engraftment, a large
proportion of graft-derived cells were still undifferentiated, expressing the early neural marker nestin (Figs. 5A–
C). In addition, graft-derived neurons (Figs. 6A–C) could
be found throughout the injury site, most likely originating from differentiated cells that were present in the E14/
FSC graft. In addition, astrocytes (Figs. 7A–C) were
found in the injury site at 4 days post-engraftment. In
contrast, no graft-derived oligodendrocytes could be
detected at this early time point (Figs. 7G–I), consistent
with the paucity of these cells in the E14 spinal cord. At
3 and 5 weeks post-engraftment, almost no graft-derived
nestin-positive cells remained (Fig. 5D); whereas, numerous graft-derived neurons (Figs. 6D–F), astrocytes (Figs.
7D–F), and oligodendrocytes (Figs. 7J–L) were present
throughout the transplant site. The grafted cells also
expressed synaptophysin, a marker of synaptic vesicles, at
4 days (Figs. 6G–I) and at 3 and 5 weeks (Figs. 6J–L).
Assessment of cell division of engrafted cells was
conducted using the proliferation marker, PCNA. A large
proportion of the grafted cells were proliferating at 4
days post-transplantation, while very few graft-derived
cells continued to divide by 5 weeks (data not shown).
Table 1 provides a summary of the fate of E14/FSC
grafts.
NRP/GRP grafts
The vast majority of NRP/GRP graft-derived cells
were nestin-positive at 4 days (Figs. 5E–G), confirming
that the transplanted cells were undifferentiated prior to
engraftment. No graft-derived cells had differentiated into
neurons (Figs. 8A–C) or oligodendrocytes (Figs. 9G–I) by
4 days, but some cells differentiated into astrocytes (Figs.
9A–C). In contrast, very few graft-derived cells expressed
nestin at 3 and 5 weeks post-transplantation (Fig. 5H),
while most of the cells differentiated into neurons (Figs.
8D–F), astrocytes (Figs. 9D–F), and oligodendrocytes
(Figs. 9J–L) throughout the injury site at these later time
points. Grafted cells also expressed synaptophysin by 3
weeks post-transplantation (Figs. 8J–L). In addition to the
injury/graft site, graft-derived cells that had migrated into
the host spinal cord expressed markers of neurons,
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Fig. 5. Persistence of grafted neural precursors. Both E14/FSC and NRP/GRP transplants were analyzed for nestin expression to determine if graft-derived
precursors were still present. The majority of cells in both the E14/FSC (A–C) and NRP/GRP (E–G) grafts were positive for nestin at 4 days. Double
immunofluorescence staining for AP (B and F, green) and nestin (C and G, red) shows the presence of graft-derived precursors (A and E, overlay). However,
almost no graft-derived precursors were found at 5 weeks in both the E14/FSC (D) and NRP/GRP (H) grafts. Arrowheads denote double-labeled cells. All
panels are sagittal sections. Scale bars: 30 Am.
Fig. 6. E14/FSC grafts differentiate into neurons and express synaptic markers in the injured spinal cord. Graft-derived neurons are found at both 4 days (A–C)
and 5 weeks (D–F) post-transplantation. Double immunofluorescence staining for AP (B and E, green) with NeuN (C and F, red) and MAP2 (data not shown)
shows the presence of graft-derived neurons (A and D, overlay). E14/FSC grafts express synaptic markers at both 4 days (G–I) and 5 weeks (J–L) posttransplantation. Double immunofluorescence staining for AP (H and K, green) and synaptophysin (I and L, red) suggests the possible association of grafted
cells with synapse formation (G and J, overlay). Arrowheads denote double-labeled cells. All panels are sagittal sections. Scale bars: 30 Am.
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237
Fig. 7. E14/FSC grafts differentiate into astrocytes and oligodendrocytes in the injured spinal cord. Graft-derived astrocytes are found at both 4 days (A–C) and
5 weeks (D–F) post-transplantation. Double immunofluorescence staining for AP (B and E, green) and GFAP (C and F, red) shows the presence of graftderived astrocytes (A and D, overlay). Graft-derived oligodendrocytes are found at 5 weeks (J–L) post-transplantation, but not at 4 days (G–I). Double
immunofluorescence staining for AP (K, green) with RIP (L, red), Sox-10 (data not shown) and NG2 (data not shown) shows the presence of graft-derived
oligodendrocytes (J, overlay). Arrowheads denote double-labeled cells. All panels are sagittal sections. Scale bars: 30 Am.
astrocytes, and oligodendrocytes (data not shown). However, the neurons were found predominantly close to the
injury site, while their glial counterparts were also found
at greater distances. This finding is in agreement with
previous reports that demonstrated the limited migratory
capacity of NRPs compared to GRPs (Han et al., 2002,
2004). Similar differentiation results were obtained from
cells maintained in culture for 3–10 days and were in
agreement with the grafting results into the intact spinal
cord (Lepore et al., 2004). Similar to E14/FSC transplants, it appears that a significant proportion of the
grafted cells expressed PCNA at 4 days post-transplantation, while very few cells continued to divide by
5 weeks (data not shown). Table 1 provides a summary
of the fate of NRP/GRP grafts.
Discussion
Fetal spinal cord tissue has been used as a transplant
source for two decades, but the events that follow grafting
into the injured spinal cord were not well understood
because of difficulties in cell detection and the limited
knowledge of neural precursor biology at the time. Our
studies were designed to elucidate the sequence of events
that follow FSC transplantation into the injured spinal cord
and at the same time address important issues concerning
the fate of neural precursor cells in the injured CNS. The
close relationship between these topics stems from the
finding that E14/FSC, a transplant source for many of the
grafting studies, consists primarily of neuronal- and glialrestricted precursors (Rao, 1999). In this study, we
Table 1
Summary of graft fate
Neural precursors
Cell division
Neurons
Astro’s
Oligo’s
4 days
Synaptic proteins
4 days
5 weeks
4 days
5 weeks
4 days
5 weeks
4 days
5 weeks
E14/FSC
NRP/GRP
++
++
+
+
++
++
+
+
+
++
++
+
+
++
++
5 weeks
4 days
5 weeks
+
+
+
++
++
Long-term survival
Initial loss
Fill cavity (5 w)
Migration
Tumors
Process extension
E14/FSC
NRP/GRP
Yes
Yes
Large
Small
Yes
Yes
Variable
Consistent
None
None
Variable
Minimal
This table provides a summary of the fate of both E14/FSC and NRP/GRP grafts at 4 days and 5 weeks following transplantation into the injured spinal cord.
(+) Denotes few cells; (++) denotes many cells; ( ) denotes no cells.
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Fig. 8. NRP/GRP grafts differentiate into neurons and express synaptic markers in the injured spinal cord. Graft-derived neurons are found at 5 weeks (D–F)
post-transplantation, but not at 4 days (A–C). Double immunofluorescence staining for AP (E, green) with NeuN (F, red) and MAP2 (data not shown) shows
the presence of graft-derived neurons (D, overlay). NRP/GRP grafts express synaptic markers at 5 weeks (J–L) post-transplantation, but not at 4 days (G–I).
Double immunofluorescence staining for AP (K, green) and synaptophysin (L, red) suggests the possible association of grafted cells with synapse formation
(J, overlay). Arrowheads denote double-labeled cells. All panels are sagittal sections. Scale bars: 30 Am.
Fig. 9. NRP/GRP grafts differentiate into astrocytes and oligodendrocytes in the injured spinal cord. Graft-derived astrocytes are found at both 4 days (A–C)
and 5 weeks (D–F) post-transplantation. Double immunofluorescence staining for AP (B and E, green) and GFAP (C and F, red) shows the presence of graftderived astrocytes (A and D, overlay). Graft-derived oligodendrocytes are found at 5 weeks (J–L) post-transplantation, but not at 4 days (G–I). Double
immunofluorescence staining for AP (K, green) with RIP (L, red), Sox-10 (data not shown) and NG2 (data not shown) shows the presence of graft-derived
oligodendrocytes (J, overlay). Arrowheads denote double-labeled cells. All panels are sagittal sections. Scale bars: 30 Am.
A.C. Lepore, I. Fischer / Experimental Neurology 194 (2005) 230–242
employed a well-defined population of lineage-restricted
neural precursors, cells whose properties have been previously characterized in vitro (Mayer-Proschel et al., 1997;
Rao and Mayer-Proschel, 1997) and in the normal uninjured
CNS (Han et al., 2002, 2004; Lepore et al., 2004).
Furthermore, we were able to clearly interpret the grafting
experiments because the tissue and the cells were obtained
from alkaline phosphatase transgenic rats, allowing us
unambiguously to track the grafts (Kisseberth et al., 1999;
Mujtaba et al., 2002).
Previous studies have shown that E14/FSC, grafted into
adult models of traumatic spinal cord injury, rescue
axotomized neurons (Mori et al., 1997), induce regeneration
into but not through the transplant (Tessler et al., 1988), and
promote limited recovery of function (Bregman et al., 1993;
Stokes and Reier, 1992). The efficacy of these grafts can be
enhanced with delayed transplantation, together with the
administration of exogenous growth factors (Bregman et al.,
2002; Coumans et al., 2001). Despite their limited success,
the analysis of fetal grafts derived from various developmental ages has yielded important information with respect
to the survival and fate of grafted cells. For example, studies
examining FSC derived from different embryonic ages have
shown excellent survival and differentiation at the E12–15
range, while the survival rate decreases dramatically in
tissue taken from older fetuses (Bernstein et al., 1985; Reier
et al., 1983). We now recognize that at the optimal
embryonic age (e.g., E13–14), FSC contains primarily
lineage-restricted precursor cells (Rao, 1999). At later times
(e.g., NE15), there are increasing numbers of mature cell
types such as post-mitotic neurons, which survive poorly
following transplantation into the adult CNS (Sieradzan and
Vrbova, 1991). Furthermore, our previous grafting experiments into the adult CNS demonstrated excellent survival
and robust differentiation of lineage-restricted precursors
derived from E13.5, compared with poor survival of NSCs
derived from E10.5 (Lepore et al., 2004). We have therefore
argued that lineage-restricted cells, rather than NSCs,
represent the cell population critical for successful grafting
into the spinal cord (Han et al., 2002, 2004; Lepore et al.,
2004). Nevertheless, it is important to note that NSCs can
produce neurons when grafted into a neurogenic environment such as the SVZ-rostral migratory stream (Fricker
et al., 1999), hippocampus (Shihabuddin et al., 2000), or the
developing CNS (Campbell et al., 1995).
While these grafting studies indicate that lineagerestricted precursors derived from the developing spinal
cord are potentially an excellent source for transplantation
into the adult CNS, it has remained unclear whether these
properties will stand the challenges of the injured spinal
cord. The main concern raised by previous work has been
the ability of the grafted cells to survive the toxic site of the
injury and subsequently differentiate into mature neurons in
a non-neurogenic environment. For example, various
classes of isolated glial-restricted precursors robustly
survive and differentiate in vivo (Han et al., 2004; Roy et
239
al., 1999; Windrem et al., 2004; Zhang et al., 1999), but
neuronal-restricted precursors survive poorly and fail to
differentiate into neurons in the injured adult spinal cord
(Cao et al., 2002). The failure to obtain neurons in the injury
site suggests that the microenvironment of the graft needs to
be modified to improve survival and to include instructive
cues for neuronal differentiation that are missing. Examples
of such strategies include in vitro manipulation of grafted
cells (Le Belle et al., 2004; Wu et al., 2002), delayed
engraftment (Ogawa et al., 2002), co-grafting with other
cells (Schumm et al., 2004; Wilby et al., 1999), immune
modulation (Zhang et al., 2003), genetic modification of
transplanted cells (Castellanos et al., 2002; Ostenfeld et al.,
2002), and exogenous administration of mitogenic (Martens
et al., 2002), neuroprotective (Cicchetti et al., 2002; Helt
et al., 2001), or differentiation factors (Chmielnicki et al.,
2004).
The present results represent an effective example of
overcoming the non-neurogenic and non-permissive environment of injured spinal cord and the lack of instructive
cues in the adult CNS. We show that mixed grafts of
neuronal- and glial-restricted precursors support survival,
neuronal, and glial differentiation, and migration by creating
a microenvironmental niche that is both protective and
instructive, resembling a neurogenic CNS environment.
Potential explanations for the survival and neuronal differentiation abilities of mixed NRP/GRP grafts include the use
of lineage-restricted precursors committed to a neuronal fate
and the presence of glial cells in the form of noninflammatory astrocytes, via differentiation of transplanted
GRPs. The fetal-derived astrocytes provide factors supporting precursor survival and neuronal maturation. Astrocytes
are known to secrete a wide variety of trophic molecules
that can enhance survival (Banker, 1980; Lindsay, 1979;
Muller et al., 1995) and regeneration (Kliot et al., 1990)
such as GDNF (Schaar et al., 1993), CNTF (Dallner et al.,
2002; Sendtner et al., 1994), NT-3 (Dreyfus et al., 1999;
Rudge et al., 1995), and NGF (Houlgatte et al., 1989; Rudge
et al., 1992). Interestingly, hippocampal but not spinal cord
astrocytes have been shown to play an important role in
neurogenesis by instructing NSCs to adopt a neuronal fate
(Song et al., 2002; Ueki et al., 2003). Our studies suggest
that while astrocytes derived from spinal cord may not be
able to instruct NSCs toward a neuronal lineage choice, they
can promote or at least support neurogenesis of lineagerestricted neuronal precursors. It is also possible that
astrocytes derived from GRPs of embryonic origin are more
permissive than adult spinal cord astrocytes with respect to
supporting neurogenesis. The latter interpretation is less
likely given that neurons are generated before astrocytes
during development (Lee et al., 2000).
Interestingly, E14/FSC and NRP/GRP grafts both show
selective migration out of the injury site into white matter of
the intact host CNS. Previous work has demonstrated that
both endogenous (Arvidsson et al., 2002; Nakatomi et al.,
2002; Parent and Lowenstein, 2002; Parent et al., 1997) and
240
A.C. Lepore, I. Fischer / Experimental Neurology 194 (2005) 230–242
grafted precursors (Aboody et al., 2000; Snyder et al., 1997;
Svendsen et al., 1997) can migrate to sites of CNS
pathology. Presumably, factors produced by dying/dysfunctional cells, invading immune cells, and other potential
players stimulate this precursor response. In vitro studies
have demonstrated that microglia (Aarum et al., 2003),
chemokines, and chemokine receptors such as SDF-1a and
CXCR4, respectively (Ehtesham et al., 2004; Ji et al., 2004;
Peng et al., 2004; Tran et al., 2004), and inflammatory
cytokines such as TNFa and IFNg (Ben-Hur et al., 2003)
and PDGF (Armstrong et al., 1990; Forsberg-Nilsson et al.,
1998) can stimulate migration of neural precursor. Many of
these pro-migratory elements are found at spinal cord injury
sites (Bartholdi and Schwab, 1997; Wang et al., 1996), and
thus may be playing a role in instructing grafted NRPs and
GRPs to migrate, possibly along degenerating white matter
tracts. However, our data indicate that while the E14/FSC
grafts show variable migration out of the transplant site, the
NRP/GRP grafts show consistent and robust migration. We
propose that these differences may be the result of the
extensive cell loss and the presence of adherent cellular
matrix in the E14/FSC graft, as well as the variable graft–
host interface associated with E14/FSC grafts, altogether
generating conditions that compete and interfere with
signals directing migration out of the grafting site. The
migration of NRPs and GRPs in the injured spinal cord is
particularly attractive for CNS repair. NRPs, which by and
large remain at the grafting site, have the potential of
becoming relays for reconstructing circuits and creating a
permissive environment at the injury site. The migration
stream, consisting mostly of grafted GRPs and subsequently
glial cells, can potentially guide and myelinate growing
axons.
Despite the relatively small number of NRP/GRP cells
injected, the mixed precursor grafts show excellent survival
that is equivalent, or greater, than E14/FSC transplants. In
this case, we propose that the increased cell death observed
with fetal tissue transplants may be a product of poor
survival of multipotent and/or mature cell types present in
the heterogeneous fetal tissue, as well as the poor access of
nutrients to the grafted tissue. In contrast, the survival of
NRP/GRP grafts reflects the stable microenvironment
generated by the mixed population of neuronal and glial
precursors and good nutrient supply accessible to cultured
cells. In both cases, we found that the grafted cells
continued to divide for at least 4 days after grafting, a
process that can explain how these grafts were able to fill
the lesion cavity. In both graft types, we found neither
significant proliferation at 5 weeks post-transplantation nor
any signs of a large cell mass suggestive of tumor formation.
Regardless of their potential efficacy, FSC grafts face
daunting ethical and practical problems with respect to their
clinical use in CNS repair, including tissue availability,
cellular heterogeneity and logistics of harvesting and storing
tissue. We therefore argue that because mixed NRP/GRP
grafts are able to recapitulate the utility of the FSC grafts,
along with added benefits of increased survival and
migration, they should be considered as preferred candidates
for therapy. In addition, lineage-restricted precursor cell
transplantation offers practical benefits such as the capacity
to appropriately select specific classes of precursors
according to the specific pathological needs and the ability
to derive these cells from alternative sources such as
embryonic stem cells.
Acknowledgments
We acknowledge all the members of our group for their
assistance, particularly Carla Tyler-Poltz for excellent tissue
culture, Dr. Birgit Neuhuber and Dr. Steve Han for critical
discussions, Dr. Timothy Himes and Theresa Connors for
their technical expertise, and Andrea Ketschek for help in
early experiments. A.C.L. thanks the Neuroscience graduate program for support and Stephen, Leopold, and Molly
for a much needed diversion. I.F. is supported by NIH
grants NS24707 and NS 37515. This work has been
inspired and encouraged by the continued collaboration
with Dr. Mahendra Rao.
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