Potential for Cell-Transplant Therapy with Human Neuronal Precursors to Treat Neuropathic Pain in Models of PNS and CNS Injury: Comparison of hNT2.17 and hNT2.19 Cell Lines

ArticleinPain Research and Treatment 2012(2090-1542):356412 · April 2012with42 Reads
DOI: 10.1155/2012/356412 · Source: PubMed
Abstract

Effective treatment of sensory neuropathies in peripheral neuropathies and spinal cord injury (SCI) is one of the most difficult problems in modern clinical practice. Cell therapy to release antinociceptive agents near the injured spinal cord is a logical next step in the development of treatment modalities. But few clinical trials, especially for chronic pain, have tested the potential of transplant of cells to treat chronic pain. Cell lines derived from the human neuronal NT2 cell line parentage, the hNT2.17 and hNT2.19 lines, which synthesize and release the neurotransmitters gamma-aminobutyric acid (GABA) and serotonin (5HT), respectively, have been used to evaluate the potential of cell-based release of antinociceptive agents near the lumbar dorsal (horn) spinal sensory cell centers to relieve neuropathic pain after PNS (partial nerve and diabetes-related injury) and CNS (spinal cord injury) damage in rat models. Both cell lines transplants potently and permanently reverse behavioral hypersensitivity without inducing tumors or other complications after grafting. Functioning as cellular minipumps for antinociception, human neuronal precursors, like these NT2-derived cell lines, would likely provide a useful adjuvant or replacement for current pharmacological treatments for neuropathic pain.

Full-text

Available from: Stacey Quintero Wolfe
Hindawi Publishing Corporation
Pain Research and Treatment
Volume 2012, Ar ticle ID 356412, 31 pages
doi:10.1155/2012/356412
Research Ar ticle
Potential for Cell-Transplant Therapy with
Human Neuronal Precursors to Treat Neuropathic Pain in
Models of PNS and CNS Injury: Comparison of
hNT2.17 and hNT2.19 Cell Lines
Mary J . Eaton,
1
Yerko Berrocal,
2
and Stacey Q. Wolfe
3
1
Miami VA Health System Center, D806C, 1201 NW 16th Street, Miami, FL 33199, USA
2
Department of Cellular Biology and Pharmacology, Herbert We rtheim College of Medicine,
Florida International University, Miami, FL, USA
3
Department of Neurosurgery, Tripler Army Medical Center, 1 Jarrett White Road, Honolulu, HI 96859-5000, USA
Correspondence should be addressed to Mary J. Eaton, meatonscience@gmail.com
Received 14 November 2011; Accepted 15 January 2012
Academic Editor: Nanna Brix Finnerup
Copyright © 2012 Mary J. Eaton et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Eective treatment of sensory neuropathies in peripheral neuropathies and spinal cord injury (SCI) is one of the most dicult
problems in modern clinical practice. Cell therapy to release antinociceptive agents near the injured spinal cord is a logical next step
in the development of treatment modalities. But few clinical trials, especially for chronic pain, have tested the potential of transplant
of cells to treat chronic pain. Cell lines derived from the human neuronal NT2 cell line parentage, the hNT2.17 and hNT2.19 lines,
which synthesize and release the neurotransmitters gamma-aminobutyric acid (GABA) and serotonin (5HT), respectively, have
been used to evaluate the potential of cell-based release of antinociceptive agents near the lumbar dorsal (horn) spinal sensory
cell centers to relieve neuropathic pain after PNS (partial nerve and diabetes-related injury) and CNS (spinal cord injury) damage
in rat m odels. Both cell lines transplants potently and permanently reverse behavioral hypersensitivity without inducing tumors
or other complications after grafting. Functioning as cellular minipumps for antinociception, human neuronal precursors, like
these NT2-derived cell lines, would likely provide a useful adjuvant or replacement for current pharmacological treatments for
neuropathic pain.
1. Introduction
Despite improvements [1] in surgical management, physical
therapy, and the availability of pharmacological agents,
with a variet y of delivery systems, many patients following
peripheral and central neural injuries continue to suer from
intractable chronic pain [2]. Although opioids are the most
commonly used agent for the control of pain, only about
32% of patients receive any significant relief with long-term
use [3], but this often leads to untoward eects associated
with drug dependence, tolerance, tolerability, drug diver-
sion, and other side eects [4], including opioid-induced
neurotoxicity. Non-opioid medications can attenuate some
types of neuropathic pain, but seldom remove completely
the painful sensation [5]. Recent attempts at classification of
neuropathic, nociceptive, and other pain, aided by an IASP
Taskforce [6], have been of help to understand mechanisms
and to improve and devise better treatments for chronic
pain. But with the frequency of inadequate or failed clinical
trials to advance treatment options for these problems,
especially for chronic neuropathic pain [5], the development
of translational cell therapies [7, 8], from stem cell [9, 10]or
cell line sources [11], and use of newer animal models [12],
is driving new interest in more sophisticated techniques for
these problems [1317].
Spinal cord injury (SCI) is a devastating clinical problem
with injury severity directly related to not only motor
paralysis, but also a large host of secondary complications
[18] that challenge the injured person and their support
network. The clinical presentation of chronic neuropathic
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2 Pain Research and Treatment
pain following SCI is common but under-reported and has
proven dicult to treat [19]. Neuropathic pain results from
the abnormal processing of sensory input due to damage to
the nervous system and onset of SCI pain is usually weeks to
months after injury [20]. Few research studies have examined
SCI pain [21], given the paucity of animal models for SCI
pain, and so far none has shown any drug to be eective
for a significant number of people. Some treatments, like
implanted morphine pumps, work well but only temporarily.
Pharmacological and surgical treatments are rarely success-
ful, given the lack of understanding of mechanisms and
focused, reliable interventions.
Painful peripheral neuropathies [13, 22] and diabetic
peripheral neuropathy (DPN) are important clinical pain
syndromes [23] with an estimated prevalence of about 5+
million in the USA. Presently, since there is little clear
understanding of underlying mechanisms [24, 25], there are
few eective long term treatments [26] for these conditions
and therefore eorts are needed to develop and test novel
therapeutic interventions.
Transplants of primary cultured cells near the dorsal
horn of the spinal cord that release peptides and neuro-
transmitters have oered a new direction in the treatment of
chronic pain. But, primary cells are dicult to obtain, non-
homogeneous, and would require that each batch be tested
before clinical use. Transplantation of immortalized cell lines
genetically modified to release neuroactive antinociceptive
peptides [27, 28], inhibitory neurotransmitters [29, 30]
and neurotrophins [31] in chronic pain, and to upregulate
inhibitory neurotransmitter synthesis oers a renewable
source of cells [10, 11, 32, 33] that c an act as cellular mini-
pumps, are able to respond to the microenvironment of the
spinal cord, and should reduce or eliminate side eects asso-
ciated with the large doses of pharmacologic agents required
for centrally-acting pain-reducing agents.
A human embryonal carcinoma cell line, NTera2cl.D/l
(NT2), when treated with retinoic acid (RA), dierentiates
irreversibly into several morphologically and phenotypically
distinct cell types, which include terminally dierentiated
postmitotic CNS neurons [34]. Successive replating of RA-
treated hNT2 cells, in the presence of growth inhibitors,
results in the isolation of purified human neurons [35] that
have been extensively characterized and tested in vivo in a
number of animal models of traumatic injury and neurode-
generative disease [36]. The potential application of hNT2
neurons in cell transplantation therapy for CNS disorders,
without tumor formation in humans after transplants of dif-
ferentiated cells [37], and their use as vehicles for delivering
exogenous proteins into the human brain for gene therapy
has been demonstrated [38, 39]. Such NT2 neurons have
been used in Phase II clinical trials for the treatment of stroke
[40
] and have been approved by FDA for such trials [41].
Two major phenotypes present within the NT2 popula-
tion are those which synthesize the inhibitory neurotrans-
mitters gamma-aminobutyric acid (GABA) and serotonin
(5HT) [42]. Taking advantage of rapid methods of prolifer-
ation and dierentiation of the NT2 parent cell line in vitro,
two subcloned novel human cell lines which are exclusively
neuronal and of a specific neurotransmitter phenotype, the
dierentiated GABA hNT2.17 and 5HT hNT2.19 cell lines,
have been studied over the past few years in a variety of
animal models of neuropathic pain [10, 11, 4347] for their
ability to attenuate the loss of sensory and motor function
after PNS and CNS injuries. Here, we summarize the conclu-
sions derived from those studies, with additional data results
from the transplant of these human cell lines in models
of diabetic peripheral neuropathy (DPN) pain, unilateral
chronic constriction injury (CCI) to the sciatic nerve, and
excitotoxic SCI, with an interest towards describing the pros
and cons involved in their further development as clinical
tools to induce recovery-of-function and, especially, to treat
neuropathic pain.
2. Materials and Methods
2.1. Development of the Human hNT2.17 and hNT2.19 Cell
Lines. Human neuronal cell lines were subcloned from the
parental NTera2cl.D/l (NT2; hNT2) [34] cell line by serial
dilution and analysis of multiple cell lines using a variety of
immunohistochemical markers, including GABA and 5HT,
to determine the dierentiated neurotransmitter phenotype
of the various cell lines. We took advantage of a rapid
aggregation method [48] for retinoic acid (RA) treatment
and dierentiation into the human NT2-derived neuronal
phenotype to select various cell lines, as reported previously
[43, 47]. Although we derived a number of human hNT2
neurotransmitter cell lines by these methods, we have used
the specific hNT2.17 and hNT2.19 cell lines for transplant in
models of neuropathic pain in the peripheral (PNS) and cen-
tral nervous system (CNS). The rapid aggregation method
[48] for RA treatment and dierentiation was also used
for the preparation of cultures of dierentiated hNT2.17
and hNT2.19 cells in vitro for characterization and trans-
plant. Briefly, proliferating cultures of hNT2.17 or hNT2.19
cells were grown to near confluence at 37
Cinprolifer-
ation medium: Dulbeccos Modified Eagle Medium/Hams
F12 (DMEM/F12, Gibco)/10% fetal bovine serum (FBS,
HyClone, Logan, Utah)/2 mM L-glutamine (Gibco) f reshly
added/1% Pen-Strep (P.S.; Gibco) with an every 3rd day
media change. When cells were near 100% confluent, the y
were replated to 100 mm Petri dish (VWR) in DMEM/high-
glucose (HG)/10% FBS/10 μM all-trans retinoic acid (RA)
(Sigma)/15 mM HEPES, pH 8.0/2 mM L-glutamine/1%Pen-
Strep, and continued for two weeks, with fresh media
changed every 2 days. After removal with 0.5 mM EDTA, cen-
trifugation and resuspension, cells were re-plated to 100 mm
tissue culture dishes (Falcon) which had been coated with
mouse laminin ((Biomedical Technologies, Stoughton, MA,
USA; 20 μg/mL in DPBS)/polyL-l-ysine (Sigma; 20 μg/mL
inPBS)).Thecellcultureswerethencontinuedin
DMEM/high-glucose (HG)/5% FBS/1% Pen-Strep (P.S.)/L-
glutamine, 2 mM, at a pH of 7.4, for 9–24 hrs, before the
addition of cytosine- D-arabinofuranoside (araC) (Sigma;
1 μM), plus uridine (Sigma; 10 μM), for non-neuronal
growth inhibition. After seven days, cells were briefly exposed
to warmed trypsin/0.5 mM EDTA, and adherent surface cells
(hNT2 neurons) removed with DMEM/HG/5% FBS/P.S./L-
glutamine, 2 mM, at a pH of 7.4. The cells were centrifuged,
Page 2
Pain Research and Treatment 3
re-suspended, and re-plated on 60 mm tissue culture dishes
(Falcon), which had been coated with mouse laminin
((Biomedical Technologies, Inc; 20 Φg/mL in DPBS)/poly-L-
lysine (Sigma; 20 μg/mL)) and continued in DMEMHG/5%
FBS/P.S./L-glutamine, 2 mM at a pH of 7.4 at 37
C for two
weeks before transplant, with media change every 2-3 days.
Three cell lines, the hNT2.17, hNT2.19, and negative control
hNT2.6, were isolated and developed for use in transplant
studies.
2.2. Immunohistochemistry of the hNT2.17 and hNT2.19 Cell
Lines In Vitro. Monoclonal antibody anti-bromodeoxyuri-
dine (BrdU; #347580; dilution 1 : 10) was purchased from
Becton-Dickson, San Jose, CA, USA. The polyclonal anti-
body anti-5HT (ab10385-50; dilution 1/100 (in vitro)) was
purchased from Abcam Inc, Cambridge, MA, USA. Mon-
oclonal antibody anti-beta-tubulin III (TuJ1, MO15013;
dilution 1 : 100) was purchased from Neuromics, Edina, MN,
USA. The polyclonal antibody anti-GABA (dilution 1 : 100)
was purchased from Protos Biotech Corporation, New York,
NY. Monoclonal antibody anti-NuMA (dilution 1 : 20 (in
vivo)) was purchased from Calbiochem, San Diego, CA,
USA. The hNT2.17 and Hnt-19 cells, after two weeks of
RA treatment and mitotic inhibitors, were re-plated to dif-
ferentiate in 8-well laminin/poly-L-lysine coated Permanox
slides, and dierentiation continued for 1-2 weeks before
immunostaining. The cells were then fixed for 10 min at
4
C with 4% paraformaldehyde and 0.1% glutaraldehyde in
0.1 M phosphate buer, pH 7.4. All immunohistochemistry
experiments included the use of a negative control, sub-
stitution of specific primary antibody with species IgG, to
insure that positive signal was specific for the antigen. For
the anti-BrdU immunostaining: after fixation and rinsing
in PBS, pH 7.4 at room temperature, hNT2.17 or hNT2.19
cells were incubated with 2 N HCl for 20 min at room
temperature, rinsed x3 with PBS, incubated with borate
buer (pH 8.5)/0.01 M boric acid/0.5 M Na borate (1 : 1)
for 1 5 min at room temperature, rinsed for three times with
PBS, and then per meabilized for 30 min at room temperature
with blocking buer before incubation with the primary
anti-BrdU antibody. For all other in vitro immunostaining
experiments: after fixation and rinsing in PBS, pH 7.4 at
room temperature, fixed hNT2.17 or hNT2-19 cells were
permeabilized for 30 min at room temperature with 0.5%
Triton X-100 in PBS in the presence of 5% normal goat
serum (the blocking buer), before the addition of the
individual primary antibody, usually overnight at 4
C. The
staining was completed by incubation with the specific anti-
species IgG secondary conjugated to Alexa Fluor 488 Green
(dilution 1 : 100), purchased from Molecular Probe, Eugene,
OR, USA, for two hours at room temperature. After stain-
ing, slides were cover-slipped using Vectashield mounting
medium with DAPI (Vector Laboratories, Burlingame, CA,
USA). Photo images were taken with a Zeiss microscope
(Axioplan II Metamorphosis program). All staining experi-
ments were independently repeated at least x3, to insure that
micrographs are representative.
2.3. Immunohistochemistry of the hNT2.17
and hNT2.19 Cell Lines In Vivo
2.3.1. Fixation. Spinal cords were fixed for examination
of cell graft survival, GABA, and nuclear matrix antigen
(NuMA) staining (for hNT2.17 grafts) or survival, 5HT, and
TuJ1 antigen stain (for hNT2.19) eight weeks after DPN,
QUIS, or contusive SCI. Transcardial perfusion with Lanas
fixative (4% paraformaldehyde and 0.1% glutaraldehyde in
PBS) was performed. Rats were euthanized for tissue fixation
by a combination of pentobarbital overdose (i.p. injection,
12 mg/100 g) and exsanguination. Once the appropriate le vel
of anesthesia was reached (i.e., no corneal or withdrawal
reflexes), r ats were transcardially per fused with aldehydes.
After perfusion, the spinal cords, including transplant, were
removed and histologically processed. After removal from
thevertebralcolumn,cordswerestoredinfixfor12hat
4
C. These cords were cryoprotected by equilibration in 30%
sucrose and PBS overnight at 4
C and then frozen and stored
at
80
C. Cords were embedded in Shandon-1 Embedding
Matrix (Thermo Electron Corp; Waltham, Ma, USA) and
sagittally cut in sequential 20 μmsectionswithaCryostat
(Leica CM3050 S Cryostat, Micro Optics of Florida Inc;
Davie, Fl, USA). They were collected on non-coated slides
(micro Slides, Snowcoat X-tra, Surgipath; Richmond, Il,
USA). The slides were stored in a
20
Cfreezerandremoved
for defrosting before the immunostaining procedures. Every
second section was stained for the anti-human marker
NuMA or GABA (for hNT2.17 grafts) or anti-human TuJ1
and 5HT (for hNT2.19 grafts) and dehydrated, cleared, and
mounted in Cytoseal 60 (Richard-Allan Scientific (Thermo
Electron Corp)) after antibody staining. Processed slides
were observed and photographed with a Zeiss Axioplan2
research microscope.
2.3.2. GABA Staining in hNT2.17 Grafts. Methods for stain-
ing lumbar spinal cord sections for GABA have been adapted
from methods descr ibed elsewhere [49]. Sections were
incubated with the primary antibody anti-GABA (1 : 500;
Protide Phar maceuticals, Inc.) with 0.4% Triton-X-100 in
0.1 M PBS and 10% NGS overnight at 4
C, followed a one
hour incubation at room temperature with the secondary
antibody solution, biotinylated guinea pig raised in goat
(Vector) in 0.4% Triton-X-100 in 0.1 M PBS and 10% normal
goat serum (NGS), a Peroxidase ABC reporter in 0.1 M PBS
(Vector) and “VIP” substrate ( Vector). Some sections were
stained in the absence of primar y antibody, and ser ved as the
negative controls.
2.3.3. 5HT Staining in hNT2.19 Grafts. Methods for staining
lumbar spinal cord sections for 5HT and grafted hNT2-
derived cell lines have been adapted from methods described
elsewhere [46]. Sections were incubated with the primar y
antibody anti-5HT (1 : 100) with 0.4% Triton-X-100 in 0.1 M
PBS and 10% NGS overnight at 4
C, followed a one hour
incubation at room temperature with the secondary anti-
body solution, biotinylated anti-rabbit IgG (H+L), made in
goat (Vec tor; 1/200) in 0.4% Triton-X-100 in 0.1 M PBS and
10% normal goat serum (NGS), a Peroxidase ABC reporter
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4 Pain Research and Treatment
in 0.1 M PBS (Vector) and “VIP” substrate (Vector). Some
sections were stained in the absence of primary antibody and
served as the neg ative controls.
2.3.4. NuMA Staining in hNT2.17 Grafts. Methods for stain-
ing spinal cord sections for the human nuclear matrix anti-
gen (NuMA) to identify hNT2 neurons after grafting have
previously been described [50].Thesectionswerewashed
with 0.1 M PBS pH 7.4 and permeabilized with 0.4% Triton-
X-100 in 0.1 M PBS, 10% normal goat serum (NGS) and
3% poly-D-lysine (Sigma) for one hour. The sections were
then incubated overnight at 4EC in the primary anti-NuMA
antibody (EMD Bioscience; 10 mg/mL DPBS), and the
permeabilizing solution, followed by a one-hour incubation
at room temperature with the secondary antibody solution,
biotinylated mouse raised in goat (Vector), a Peroxidase
ABC reporter in 0.1 M PBS (Vector) and “VIP” substrate
(Vector). Some sections were stained in the absence of pri-
mary antibody and served as the negative controls.
2.3.5. TuJ1 Staining in hNT2.19 Grafts. Modified methods
for staining spinal cord sections for the human neuron-spe-
cific class III beta-tubulin (TuJ1) to identify g rafted hNT2.19
neurons after grafting have previously been described [51].
The sections were washed with 0.1 M PBS pH 7.4 and
permeabilized with 0.4% Triton-X-100 in 0.1 M PBS, 10%
normal goat serum (NGS) for one hour. The sections were
then incubated overnight at 4
C in the primary anti-TuJ1
antibody (1 : 100 DPBS), and the permeabilizing solution,
followed by a one-hour incubation at room temperature with
the secondary antibody solution, biotinylated mouse IgG
raised in goat (Vector; 1 : 200), a Peroxidase ABC reporter
in 0.1 M PBS (Vector) and “VIP” substrate (Vector). Some
sections were stained in the absence of primary antibody and
served as the neg ative controls.
2.4. HPLC of Neurotransmitters in hNT2 Cell Lines In Vitro
2.4.1. GABA in hNT2.17 Cells In Vitro. In order to examine
the GABA content and release in dierentiated hNT2.17 cells,
cells were dierentiated for 2 wks at 37
C after plating in
35 mm laminin/poly-L-lysinecoated 6well plates. Cell num-
bers were determined in sister wells by trypan blue exclusion
and counting. Either GABA content (in cells) or GABA secre-
tion or release (into the media) was examined by HPLC to
determine the content or basal or stimulated level of GABA
secretion or release, respectively, into the media. For GABA
content: cells were collected into 1.5 mL centrifuge tube (in
distilled water), cells broken by two 10 sec bursts of ultra-
sound, tube contents centrifuged at 4
C, and the supernatant
collected for HPLC. Similar cell culture samples were also
incubated with either normal K+ (2.95 mM) KrebsRinger
buer or high K+ (100 mM) buer for 15 min at 37
C,
and the media collected to determine the levels of GABA
released into the media by membrane depolarization. The
media samples were kept on ice and immediately analyzed
by HPLC. An o-phthaladehyde (OPA) pre-column deriva-
tization and reverse-phase isocratic liquid chromatography
with electrochemical detection, as described previously
[52] were used. The HPLC system consisted of a solvent-
delivery pump (Waters 510 Pump); an autosampler (Waters
717 plus Autosampler) and an elec trochemical detector (ESA
Coulochem II; Electrode: ESA Model 5011 Analytic Cell;
Guard Cell: Model 5020). Elution was carried out at room
temperature with a reversed-phase column (3 μm, C18, 80
×
4.6, HR80, ESA) and a mobile phase of 0.1 M sodium acetate
(pH.5)-acetonitrile (73 : 27, v/v) at a flow rate of 0.6 mL/min.
To an OPA solution (2 mg of o-phthaldialdehyde (OPA)
in 0.2 mL methanol), first 0.8 mL of 0.1 M borax buer
(pH 10.4) and 5 μL of 2-mercaptoethanol were added. Four
minutes before the injection on the column, 1 : 4 volumes of
the OPA reagent and sample were mixed and incubated at
room temperature by autosampler. After injection, the GABA
peak appearance time was about 5 min in 27% Ace mobile
phase.
2.4.2. 5HT in hNT2.19 Cells In Vitro. In order to examine
the 5HT content, secretion, and release in dierentiated
hNT2.19, cells were dierentiated for 2 wks at 37
Cafter
plating in 35 mm laminin/poly-L-lysine-coated 6-well plates.
Cell numbers were determined in sister wells by trypan blue
exclusion and counting. Either 5HT content (in cells) or 5HT
secretion or release (into the media) was examined by HPLC
to determine the content or basal or stimulated level of 5HT
release into the media. For 5HT content, cells were collected
into 1.5 mL centrifuge tube (in distilled water), cells broken
by lysis w ith 0.05 N PCA (perchloric acid), tube contents cen-
trifuged at 4
C, and supernatant collected for HPLC. Similar
cell culture samples were also incubated with either normal
K+ (2.95 mM) Krebs-Ringer buer or high K+ (100 mM)
buer for 15 min at 37
C and the media collected to deter-
mine the levels of 5HT released into the media by membrane
depolarization. The media samples were kept on ice and
immediately analyzed by HPLC. The HPLC system consisted
of a solvent-delivery pump (Waters 510 Pump), an auto-
sampler (Waters 717 plus Autosampler), and an electro-
chemical detector (ESA Coulochem II); Electrode: ESA Mi-
crodialysis Cell 5014A (DC CH1: 150 mV, DC CH2: 300 mV,
500 mA); Guard Cell Model 5020 (GC 350 mV). Elution was
carried out at room temperature with a reversed-phase col-
umn (C18, 5 M, 150-3, BetaBasic-18, Thermo) and MDTM
mobile phase (ESA Inc. 70–1332); it consisted of 75 mM of
NaH2PO4, 1.7 mM of C
8
H
17
O
3
SNa, 100 μL/L of TEA, 25 M
of EDTA, 10% acetonitrile, pH 3.0 adjusted by H
3
PO
4
at
a flow rate of 0.6 mL/min. Ordinarily the 5HT appeared at
about 7.5 min.
2.5. Surgeries and Cell Transplant
2.5.1. Unilateral Chronic Constriction Injury (CCI) of the
Sciatic Nerve and hNT2 or hNT2.17 Cell Transplant. The sur-
gery to produce CCI was first described by Bennett and Xie
[53]. This model of injury has been used by our laboratories
and many others to test the eects of cell transplants to
relieve pain-related behaviors [54]. Under ketamine/xylazine
anesthesia, the right common sciatic nerve was exposed at
the level of the middle thigh by blunt dissection through
Page 4
Pain Research and Treatment 5
the biceps femoris. Proximal to the nerves trifurcation, a 5–
7 mm of nerve was freed of adhering tissue and 4 ligatures
(4.0 chromic gut) were tied loosely around it with 1 mm
spacing. Care was taken to tie the ligatures so that the dia-
meter of the nerve was barely constricted, so that vigorous
tactile allodynia ( TA) and ther mal hyperalgesia (TH) behav-
iors lasted at least 10 wks after CCI. The incision was closed
in layers and the entire surgery was repeated, minus the
ligatures, on the left side to create a sham-operated nerve.
At two weeks following the CCI, and following a partial
laminectomy with a small puncture of the dura, either viable
or nonviable parental hNT2 or hNT2.17 cells (predier-
entiated 2 wks; 10
6
cells/injection) were injected into the
subarachnoid space of the lumbar dorsal spinal cord, by a
dorsal/caudal entry into the dural puncture a few millimeters
with a small length of polyethylene (PE-10) tubing contain-
ing the cells, at spinal segment L1. For both CCI and trans-
plantation, animals were anesthetized with a mixture of ket-
amine, xylazine, and acepromazine, 0.65 mL/Kg . Animals
were allowed to recover at 37
C for 12 hrs, after which time
they were returned to the animal care facility, and housed
2/cage with rat chow and water ad lib on a 12/12 hr light/dark
cycle.
All surgical interventions, pre- and postsurgical a nimal
care, and euthanasia were performed in accordance with
the Laboratory Animal Welfare Act, Guide for the Care and
Use of Laboratory Animals (National Institutes of Health;
Department of Health, Education and Welfare, Pub. No. 78-
23, Revised 1978) and the guidelines provided by the Animal
Care and Use Committees of the Department of Veterans
Aairs Medical Center and the University of Miami, both in
Miami, Fl, USA. All behavioral testing was performed under
blinded conditions to eliminate experimental bias; the data
were analyzed and un-blinded by the statistician at the end
of the experiment.
2.5.2. Streptozotocin-Induced Diabetic Peripheral Neuropathy
and hNT2.17 Cell Transplant. Animals were administered an
IV injection (in the tail vein) of STZ (50 mg/Kg) dissolved
in 0.9% (w/v) physiological saline, m ade to approximately
10 mg/25 μL. Immediately before, about 3 days and peri-
odically post-STZ injection all rats had their blood glucose
measured with a glucometer, utilizing <1 μL of blood
removed by tail prick. Rats with blood glucose >250 mg/dL
were considered diabetic and were assigned to one of the
control groups or to receive cell transplants. In all animals,
except those not to receive cell grafts (at 5 days after STZ) and
the development and measure of sensory behaviors, animals
received lumbar subarachnoid grafts of human (nonviable
or viable hNT2.17) cells. A partial T13L1 laminectomy was
performed and 10
6
cells/injection in 5–10 μLsterileHanks
buered saline was injected into the subarachnoid space of
the lumbar dorsal spinal cord, by a dorsal/caudal entry into
the dural puncture a few millimeters with a small length of
small length of polyethylene (PE-10) tubing containing the
cells, at spinal segment L1. Following transplantation, the
exposed surface of the spinal cord was covered with dura-
film, the overlying musculature was sutured and the wound
closed with wound clips. Animals were allowed to recover on
warming blankets (under the cage corner) for 12 hrs, after
which time they were returned to the animal care facilit y and
housed 2/cage with rat chow and water ad lib on a 12/12 hr
light/dark cycle.
All surgical interventions, pre- and postsurgical animal
care, and euthanasia were performed in accordance with
the Laboratory Animal Welfare Act, Guide for the Care and
Use of Laboratory Animals (National Institutes of Health;
Department of Health, Education and Welfare, Pub. No. 78-
23, Revised 1978) and the guidelines provided by the Animal
Care and Use Committees of the Department of Veterans
Aairs Medical Center and the University of Miami, both in
Miami, Fl, USA. All behavioral testing was performed under
blinded conditions to eliminate experimental bias; the data
were analyzed and unblinded by the statistician at the end of
the experiment.
2.5.3. Excitotoxic Spinal Cord Injury (QUIS) and hNT2.17
or hNT2.19 Cell Transplant. The spinal QUIS injur y pro-
cedure has been previously described [55]. To produce this
excitotoxic injury, quisqualic acid (QUIS; non-synthetic,
Sigma), a glutamate receptor agonist, was administered
in sucient concentrations (125 mM) to cause neuronal
cell loss and demyelination. The animals were anesthetized
with a mixture of ketamine, xylazine, and acepromazine
(0.65 mL/kg). A laminectomy was performed between T12-
L1. The rat was then placed in a stereotaxic frame and the
dura and arachnoid incised. Using a micropipette attached to
a Hamilton syringe, the QUIS was unilaterally injected into
the dorsal horn, 1000 μm below the surface of the cord, in
three separate injections 500 μm apart. Each injection was
0.4 μL in volume for a total of 1.2 μL. Anatomically, the
injection was located midway between the central vein and
dorsal root entry zone, just lateral to the posterior columns.
On pathologic examination, these unilateral injections were
centered in the gray matter between the spinal laminae IV-VI.
A small piece of sterile dura-film was placed over the dura (to
protect the spinal cord and facilitate reopening the dura for
transplantation) and the fascia and skin were closed. Other
than the anesthesia, no additional perioperative analgesics
were given. The animal was recovered at 37
C for 12 hours
and then returned to the animal care facility. Two weeks later,
an aliquot of one million cells was prepared immediately
prior to each transplant to assure near 100% viability
at the beginning of the experiment; grafting was within
30 min of cell preparation. Nonviable hNT2.17 cells were
prepared by initially re-suspending one mil lion cells in sterile
water, centrifugation, checking viability, then resuspension
in CMF-HBSS for transplant.
The animals to be transplanted, one day after showing
a vigorous response to behavioral testing, were anesthetized
with a mixture of ketamine, xylazine, and acepromazine
(0.65 mL/kg). The previous laminectomy site (T12-L1) was
exposed. A small dural and arachnoidal incision was made
and a 2-3 mm segment of polyethylene (PE-10) tubing,
connected to a micropipette, inserted through the durotomy
in a caudal direction. The one million cells (either hNT2.17
or hNT2.19) were injected into the intrathecal space at spinal
segment L1-L3 and the fascia and skin closed. Again, no
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6 Pain Research and Treatment
additional analgesia was used. The animals were allowed
to recover at 37
C for 12 hrs, after which time they were
returned to the animal care. All rats, including those not
provided cell transplants, received immunosuppressive ther-
apy with cyclosporine A (CsA; 10 mg/Kg), injected i.p., which
began one day before cell transplant, or 13 days after QUIS
or saline injection, and continued daily for 14 days.
All surgical interventions, pre- and postsurgical a nimal
care, and euthanasia were performed in accordance with
the Laboratory Animal Welfare Act, Guide for the Care and
Use of Laboratory Animals (National Institutes of Health;
Department of Health, Education and Welfare, Pub. No. 78-
23, Revised 1978) and the guidelines provided by the Animal
Care and Use Committees of the Department of Veterans
Aairs Medical Center and the University of Miami, both in
Miami, Fl, USA. All behavioral testing was performed under
blinded conditions to eliminate experimental bias; the data
were analyzed and un-blinded by the statistician at the end
of the experiment.
2.5.4. Severe Contusive Spinal Cord Injury and hNT2.17 and
hNT2.19 Cell Transplant. Contusion injury was induced by
the weight-drop device developed at New York University
[56]. Animals were anesthetized using an i.p. injection of a
mixture of ketamine (35 mg/Kg) and xylazine (5 mg/Kg), al l
0.65 mL/Kg, and then placed on a surgical table on a heating
pad (37
C) with pedal and eye blink reflexes assessed for deep
anesthesia before beginning procedures. The back region
was shaved and aseptically prepared with betadine. Lacrilube
ophthalmic ointment (Allerg an Pharmaceuticals, Irvine, CA,
USA) was applied to the eyes to prevent drying and
Bicillin (0.02 mL/100 mg body weight, 300 U/mL; J. Buck,
Inc., Owings Mills, MO, USA) administered intramuscularly.
Following anesthesia, a vertical incision was made along
the thoracic vertebra and the superficial muscle and skin
retracted. A laminectomy performed at thoracic vertebra T7
exposed the dorsal surface of the spinal cord underneath
(T8) without disrupting the dura mater. Stabilization clamps
were placed around the vertebrae at T6 and T12 to support
the column during impact. The exposed spinal cord was
severely injured by dropping a 10.0 g rod from a heig ht of
25.0 mm. The contusion impact velocity and compression
were monitored to guarantee consistency-of-injury between
animals. After injury, the muscles were sutured in layers
and the skin closed with absorbable sutures (Ethicon Inc).
Theratswereallowedtorecoverinawarmedcagewith
water and food easily accessible. Bicillin (0.02 mL/100 mg
body weight, 300 U/mL, i.m.) was administered 2, 4, and
6 d after the contusion injury. The rats were maintained
for 8 wks after injury, including gentle twice daily manual
bladder expression to prevent the development of cystitis.
For cell transplant 2 weeks after QUIS SCI, viability and
cell counts were assessed by trypan blue exclusion, and the
cells were suspended in 10–20 μLofCa
2+
-Mg
2+
free Hank’s
buered saline solution (CMF-HBSS). An aliquot of one
million cells (10
6
cells/injection) was prepared immediately
prior to each transplant to assure near 100% viability at
the beginning of the experiment; grafting was within 30 min
of cell preparation. The a nimals to be transplanted, one
day after showing a vigorous response to behavioral testing,
were anesthetized with a mixture of ketamine, xylazine, and
acepromazine (0.65 mL/kg). For subarachnoid grafts, the
previous laminectomy site (T7) was exposed and a small
dural and arachnoidal incision was made and a 2-3 mm
segment of polyethylene (PE-10) tubing, connected to a
micropipette, inserted through the durotomy in a c audal
direction. The one million cells (hNT2.17 or hNT2.19 cells)
were injec ted into the intrathecal space at spinal segment
L1–L3 and the fascia and skin closed. Again, no additional
analgesia was used. The animals were allowed to recover at
37
C for 12 hrs, after which time they were returned to the
animal care facility. All rats, including those not provided cell
transplants, received immunosuppressive therapy with CsA,
injected i.p., which began one day before cell transplant and
continued daily for 13 days, unless otherwise noted.
All surgical interventions, pre- and postsurgical animal
care, and euthanasia were performed in accordance with
the Laboratory Animal Welfare Act, Guide for the Care and
Use of Laboratory Animals (National Institutes of Health;
Department of Health, Education and Welfare, Pub. No. 78-
23, Revised 1978) and the guidelines provided by the Animal
Care and Use Committees of the Department of Veterans
Aairs Medical Center and the University of Miami, both in
Miami, Fl, USA. All behavioral testing was performed under
blinded conditions to eliminate experimental bias; the data
were analyzed and un-blinded by the statistician at the end
of the experiment.
2.6. Sensory Behavioral Testing
2.6.1. Tactile Allodynia. Mechanical allodynia, the occur-
rence of foot withdrawal in response to normally innocuous
mechanical stimuli, was tested using an automated, elec-
tronic von Frey anesthesiometer (IITC, Inc) [57]. Animals
were placed in a plexiglass box with an elevated mesh floor.
After the animal was acclimated for 5 min, the device tip
was applied perpendicular to the midplantar area of each
hindpaw and depressed slowly until the animal withdrew the
paw from pressure. The value, in grams, was recorded for
each of the 3 trials. A single trial of stimuli consisted of three
to four applications of the von Frey tip within a 10-second
period, to ensure a consistent response. The values obtained
for each hindpaw were averaged and the SEM calculated. The
animals were tested 3 times, one week apart, for 2-3 wks prior
to the injury (baseline), and then weekly for the duration of
the experiment. In order to provide a robust baseline value
for comparison purposes, all baseline data was averaged to a
mean baseline based on the three baseline tests.
2.6.2. Thermal Hyperalgesia. Methods for testing thermal
hyperalgesia with a Hargreaves device have been described
elsewhere [58]. Animals were placed in a clear plexiglass
boxonanelevatedplexiglassfloor.Animalswereallowed
to acclimate for approximately 5 min. A constant intensity,
radiant heat source was aimed at the midplantar area of the
hind paws. The time, in seconds, from initial heat source
activation until paw withdrawal, was recorded. Five minutes
were allowed between assessments. Three to four latency
Page 6
Pain Research and Treatment 7
measurements for each paw were recorded and the mean
and standard error of the mean (SEM) calculated for each
hindpaw. Animals were tested 3 times, one week apart, for
2 wks prior to the injury (baseline) and then weekly for the
duration of the experiment. In order to provide a robust
baseline value for comparison purposes, baseline data was
averaged to a mean baseline based on the three baseline tests.
2.7. Motor Behavior Testing
2.7.1. Open-Field Motor Behaviors (BBB). Two weeks prior
to the injury, open-field locomotor functions of all animals
were assessed using the Basso, B eattie, and Bresnahan (BBB)
locomotor rating scale [59]. Behavioral assessments were
then performed on days 1 and 7 following the injury and
weekly thereafter. The BBB score was used to study the func-
tional recovery stages following the injury, by categorizing
the rat hindlimb movements, trunk position and stability,
coordination, stepping, and paw placement and tail position.
Rats were placed in a small, shallow, empty childrens swim-
ming pool and allowed to move freely for 60 mins of exercise,
during which their motor behaviors were observed and
scored according to the BBB scale. All observations were
made by at least two independent observers, who were una-
ware of the extent or nature of the injury. The animals were
ratedonascaleof0to21.
2.7.2. Fine Motor Behaviors (BBB Subscores). The BBB scale
alone may not reflect changes in the finer details of locomo-
tion (e.g., paw positioning, toe clearance) [60]. Therefore,
since paw position and toe clearance are routinely docu-
mented once animals are able to step consistently, BBB anal-
ysis was supplemented by subscoring the fine details of
locomotion at the plateau of the recovery phase (at weekly
time points before and before injury). A subscore, 0–5, was
given to each hindlimb based on paw rotation and toe clear-
ance of the hindlimb. Subscores were assigned as follows:
paw position, 0.5 rotation at initial contact and lifto,1.5
rotation at initial contact or liftoand parallel at initial con-
tact/lifto, 2.5 parallel at initial contact and lifto;andtoe
clearance, 0.5 no clearance, 1.5 occasional clearance (50% of
the time), 2.5, frequent clearance (51–95%), 3.5, consistent
(95%) clearance. T he cumulative scores of each hindlimb
were summed to yield a single score (maximum score of
10/rat). All observations were made by at least two observers,
who were unaware of the nature or extent of injury.
2.8. Statistical Analysis. Statistical analyses were perfor m ed
with PASW 17.0 for Windows. To determine dierences be-
tween the groups and between time points, one-way analysis
of variances (ANOVAs) and paired Student’ t-tests were used.
All t-tests were two-tailed, and Bonferroni correction to
adjust for multiple comparisons were used. A P value of .05
or less was considered statistically significant.
3. Results
3.1. Morphology and Phenotyp e of hNT2.17 and hNT2.19 Cells
In Vitro. It is essential that dierentiated cells useful for
clinical transplant be homogeneous and with a well-defined
phenotype, since the functionality of any clinical transplant
source depends on easily identifiable cell-synthesized agents.
Both the hNT2.17 [43, 61]andhNT2.19[47] cell lines have
been characterized previously. As seen in Figure 1,dier-
entiated hNT2.17 and hNT2.19 cells are distinctly dierent
in morphology and phenotype. The GABAergic hNT2.17
have small nuclei, with long neurites (Figure 1(a)), and stain
intensely for GABA (Figure 1(c)), while the 5HT NT2.19 cells
have very large nuclei and are generally multi- or bipolar,
with short neurites (Figure 1(b)), and stain brightly for 5HT
(Figure 1(d))afterdierentiation in vitro. Both cell lines are
exclusively neuronal following dierentiation [43, 47, 61],
expressing a variety of human and neuronal markers, includ-
ing neuron-specific enolase and neurofilament proteins, with
the hNT2.17 cell line able to synthesize high molecular-
weight neurofilament protein (NFH) slightly later than the
hNT2.19 cell line in culture, but both appear to express a
mature neuronal phenotype, including neurite extensions
and bouton-like structures, that co-localize other impor-
tant neurotransmitters, such as glycine, esp ecially in the
hNT2.17 cell line. Also, in the hNT2.17 cells both the GAT3
plasmolemma GABA re-uptake and vesicular inhibitory
amino acid transporter (VIAAT) are abundant at 2 weeks
of dierentiation [43], another marker of a mature neuron.
Important to any clinical use for transplant, both cell lines
can be kept for long periods (>30 days), and both form
increasingly dense neurite mats, with aggregate balls of cell
bodies, over long periods in culture.
3.2. Both Cell Lines Cease Proliferation with D ierentiation In
Vitro. The parental tumorigenic NT2 cell line is known to
change its phenotype to nontumorigenic after dierentiation
with RA in vitro [35], downregulating key tumor genes
after RA [62, 63], allowing it to be tr ansplanted into the
central nervous system (CNS) [38, 64] and used safely in
human studies [41, 6567]. Here, the hNT2.17 and hNT2.19
cell lines switch from a proliferating to a nonproliferating
phenotype after RA exposure and treatment with mitotic
inhibitors. Both hNT2.17 [43]andhNT2.19[47] cell lines
downregulate their expression of the tumor-proteins TGF-
α and FGF-4 with RA exposure and di
erentiation in vitro.
Bromodeoxyuridine (BrdU) immunostaining has also has
been used as a marker for proliferating cells in v itro [68]
and in vivo [69], since dividing cells incorporate BrdU-
labeled uridine into newly made deoxyribonucleic acid
(DNA). Figure 2 illustrates the loss of the BrdU signal with
dierentiation in culture in each cell line. The hNT2.17
and hNT2.19 cells were exposed to 1 μMBrdUinvitro
during either proliferation or dierentiation before anti-
BrdU immunostaining. Following 3 days of proliferation in
the presence of BrdU, the BrdU signal was intense and found
in all the dividing cells hNT2.17 (Figure 2(a)) and hNT2.19
(Figure 2(d)) cells. After 1 week of BrdU exposure dur ing
the first week of dierentiation, hNT2.17 (Figure 2(b))and
hNT2.19 (Figure 2(e)) cells remained viable, as evidenced
by DAPI staining. The same field of dierentiated hNT2.17
(Figure 2(c))andhNT2.19(Figure 2(f)) cells showed no
Page 7
8 Pain Research and Treatment
(a) (b)
(c) (d)
Figure 1: Human neuronal hNT2.17 GABA and hNT2.19 5HT cell lines in vitro. The GABA hNT2.17 (a, c) and 5HT hNT2.19 (b, d) cell
lines were subcloned by serial dilution and treated for 2 wk with retinoic acid and mitotic inhibitors. They were further dierentiated for
2 wk before either phase microscopy (a, b) or stained with anti-GABA (c) or -5HT (d) antibodies, respectively. Magnification: bar
= 50 nm,
(a–c); 25 nm (d).
BrdU signal, demonstrating the inability to incorporate
BrdU during dierentiation in these two cell lines.
3.3. The hN2.17 and hNT2.19 Cell Lines Synthesize and
Secrete the Neurotransmitters GABA and 5HT, Respectively,
In Vitro. Since the neurotransmitters GABA and 5HT each
play a major role in antinociception with nervous system
injury, stable human neuronal cell lines with these specific
neurotransmitter phenotypes, and able to secrete the GABA
or 5HT directly into the cellular environment, near the
spinal cord, are a good choice for transplant in models of
neuropathic pain. The hNT2.17 GABA and hNT2.19 5HT
cell lines were dierentiated for 2 weeks in vitro before
HPLC analysis of GABA in hNT2.17 (Figure 3(a))or5HTin
hNT2.19 (Figure 3(b)) content, basal secretion in the pres-
ence of basal KCl (2.95 mmol/L), and stimulated release in
the presence of high KCl (100 mmol/L) in the medium. The
hNT2.17 cell line was able to synthesize significant amounts
of the GABA neurotransmitter, matching the immunohis-
tochemical staining patterns seen above. GABA content
(Figure 3(a)) was 1567.88 (mean) pmoles per 10 million
cells. The hNT2.17 cell line also demonstrated significant
GABA release under basal or potassium-stimulated condi-
tions at the time point during dierentiation when these cells
were transplanted in the QUIS SCI pain model [43]. GABA
release under basal (mean of 281.95 pmoles per 10 million
cells) or stimulated KCl conditions (mean of 471.16 pmoles
per 10 million cells) during a period of 15 minutes was able
to account for about 18% and more than 30%, respectively,
of the total GABA content in the cell cultures. Although
not shown here, glycine content, secretion, or release was
approximately 10 times higher than that for GABA in the
hNT2.17 [43]. Very dierent results are seen for the hNT2.19
cell line, which does not contain GABA, but instead syn-
thesizes, secretes, and releases 5HT [47]. The hNT2.19 cell
line synthesized significant amounts of the 5HT neurotrans-
mitter, a gain matching the immunohistochemical staining
patterns seen above. 5HT content (Figure 3(b)) was 485.13
(mean) pmoles per 10 million cells. The hNT2.19 cell line
also demonstrated significant 5HT release under basal or
potassium-stimulated conditions at the time point during
dierentiation when these cells were transplanted in the
severe contusive SCI pain model [47]. 5HT release under
basal (mean of 73.38 pmoles per 10 million cells) or stim-
ulated KCl conditions (mean of 85.64 pmoles per 10 million
cells) during a period of 15 minutes was able to account for
about 15% and more than 17%, respectively, of the total 5HT
content in the cell cultures.
3.4. Recovery of Sensory Function in Peripheral Models of Neu-
ropathic Pain with Transplant of GABA hNT2.17 Cells. Two
common models [12] of pain-like sensations of peripheral
Page 8
Pain Research and Treatment 9
(a) (b) (c)
(d) (e) (f)
Figure 2: hNT2.17 and hNT2.19 cells are nontumorigenic: BrdU stain. hNT2.17 (a–c) and hNT2.19 (d–f) cells were exposed to 1 μM
bromodeoxyuridine (BrdU) (a, d) during 3 days of proliferation or (b, c, e, f) for 1 week during dierentiation in vitro. Proliferating cells (a,
d) incorporate abundant BrdU during proliferation. Viable dierentiated cells (b, e) were labeled with DAPI (4
6-diamidino-2-phenylindal-
2HCl) stain, while the same field (c, f) of dierentiated cells did not incorporate any BrdU during dierentiation. After 2 weeks of treatment
with retinoic acid (RA) and mitotic inhibitors, hNT2.17 and hNT2.19 cells cease dividing and dierentiation proceeds without further cell
division. Magnification bar
= 20 nm (d); 30 nm (a, b, c, e, f).
origin and neuropathy have been used to examine the eects
on behavioral hypersensitivity and transplants of hNT2-
derived cell lines, especially the GABA hNT2.17 cell line: uni-
lateral chronic constriction injury to the sciatic nerve (CCI)
[30, 70] and diabetic peripheral neuropathy (DPN) pain
with injection of streptozotocin (STZ) [71, 72]. Use of both
models has revealed some interesting data concerning the
inhibitory neurotransmitter GABA cell line hNT2.17, since
a cell therapy approach to influence the GABA inhibitory
system [11, 13, 30, 32, 73] has a long history, a nd similar
pharmacologic intervention [7477] is the most commonly
used approach in these models.
3.4.1. Chronic Constriction Injury (CCI) and Transplant of
hNT2.17 Cells. Both the hNT2.17 and hNT2.19 cell lines
were subcloned from the parental NT2 cell line [35], but
the parental hNT2 cell line has never been examined for it’s
potential to aect neuropathic pain in the CCI model. A
comparison of the sensory eects in the CCI model by sub-
arachnoid lumbar grafts of either the parental hNT2 (Figures
4(a) and 4(b)) or the GABA hNT2.17 cells (Figures 4(c) and
4(d))isillustratedinFigure 4.WhenviablehNT2graftsare
placed two weeks after the CCI, and tactile allodynia (TA) (a)
is examined, the maximum recover y-of-function is seen at
six weeks, but never achieves more than about 40% of normal
tactile responses by the end of the experiment (42 days). Both
CCI alone and grafts of nonviable hNT2 cells had no eect
on sensory recovery, and viable hNT2 cell grafts induced an
improvement in TA and TH over injury alone or nonviable
graft placement. When, the experiment is repeated with the
same number of grafted cells placed again two weeks after
CCI, where the grafted cells are either the viable or nonviable
subcloned GABA hNT2.17 cells (c), recovery of sensory func-
tion is also seen within one week after cell transplant, but
achieves about 60% of normal tactile responses at 21 days
and 100% recovery by seven weeks. Again, grafts of nonviable
hNT2.17 cells or CCI only had no eect on the development
of tactile allodynia. The hNT2.17 grafts were far more
eective than similar grafts of the parental hNT2 cells.
Similar results are seen in results of an examination of TH
responses with the gr afts of either the parental hNT2 cells (b)
or the subcloned GABA hNT2.17 cells (d), but the maximum
eect of GABA hNT2.17 is immediate (100% recovery of
normal TH responses), seen at 21 days. In comparison, graft
of parental hNT2 cells does not achieve 100% recovery-
of-thermal function until 8 weeks, with only about 65%
recovery at 21 days. Also, like TA responses, there is no eect
on TH recovery by either CCI or grafts of nonviable hNT2
or hNT2.17 cells. Overall, the viable hN2.17 grafts were
far more eective on recovery of normal sensory function,
compared to transplant of viable parental hNT2 cells.
3.4.2. Diabetic Peripheral Neuropathy (DPN) Pain and Trans-
plant of hNT2.17 Cells. DPN pain as an animal model of pain
of peripheral origin takes advantage of the toxic, destructive
eect of STZ, delivered either i.p. or i.v. into the tail vein,
to the pancreas [71]. In our hands, an i.v. route of admin-
istration of the drug is the most dependable, and induces
vigorous TA and TH within 3 days after injection, at a dose
of 50 mg/Kg. When viable hNT2.17 cells are transplanted in
Page 9
10 Pain Research and Treatment
GABA (pmol/10
7
cells)
Conditions
Content
Basal K+
High K+
0
500
1000
1500
2000
(a)
0
250
500
750
Conditions
5HT (pmol/10
7
cells)
Content
Basal K+
High K+
(b)
Figure 3: HPLC of GABA and serotonin in hNT2.17 and hNT2.19 cells. The hNT2.17 (a) and hNT2.19 (b) cell lines were dierentiated,
after RA and mitotic inhibitor treatment, for two weeks in 6-well substrate-coated plates before cell lysis and examination of cell content
for GABA (a) or 5HT (b) by HPLC methods. For GABA or 5HT secretion (basal) and release (stimulated), sister cultures of the hNT2.17
or hNT2.19 cells were dierentiated for two weeks before cells were exposed to basal (2.95 mM) or high (100 mM) concentrations of KCl
for potassium (K+)-stimulated s ecretion/release for GABA or 5HT. Data represent the mean + SEM from 3-4 samples from >4 independent
experiments for each neurotransmitter.
the lumbar subarachnoid space at 5 days after STZ injection
(Figure 5(a)), and the transplant site examined at 42 days
with either anti-NUMA (a) or anti-GABA (b) immuno-
histochemistry, many viable, GABAergic hN2.17 cells can
been seen in the subarachnoid space (arrows), with a 14-day
course of CsA immunosuppression, peritransplant time. STZ
injection induces permanent, increasing, and vigorous TA (c)
and TH (d) behaviors within 3 days of drug administration,
where only grafts of viable GABA hNT2.17 cells, placed
5 days after STZ, are able to recover permanent normal
sensory responses (100% by 35 days in TA; 100% by 21 days
in TH behaviors), compared to STZ alone or STZ/nonviable
grafts. However, this sensory recovery-of-function did not
aect the great increase in blood glucose le vels, seen in the
presence or absence of grafts, with STZ injection (e).
3.5. Recovery-of-Function in Central (SCI) Models of Neu-
ropathic Pain and Motor Dysfunction with Transplant of
hNT2.17 and hNT2.19 Cells. Two well-described models of
SCI, with accompanying loss of sensory and motor function
have been used to examine the eects of transplants of hNT2-
derived cell lines, namely the quisqualic (QUIS) chemical
lesion, induced by the unilateral spinal injection of quisqualic
acid [78] and the se vere contusive SCI model [56], induced
by weight-drop spinal contusion (NYU impactor). Even with
significant dierences in these injuries and outcomes [12],
both induce permanent TA and TH behaviors, with the QUIS
model also inducing permanent > excessive
= grooming
behaviors in the ipsilateral hindpaw. Permanent motor dys-
function (bilateral) is induced by severe contusive SCI. Per-
haps the QUIS model may be characterized as a pure sensory
model of central pain, since no motor paralysis is induced,
while the severe contusive SCI induces both motor and
sensory loss-of-function. Both oer certain advantages and
disadvantages, not the least of which might be the greatly
increased animal survival in the QUIS model, compared
to severe contusive SCI, with no loss of bladder function,
no paralysis, as long as excessive grooming behaviors are
not too severe. And only a severe contusion (>25 mm weight-
drop) SCI can be used for an examination of sensory
dysfunction, since <25 mm weight-drop (e.g., 12.5 or 6 mm)
does not provide consistent TA and TH [79], since injury
severity and sensory/motor outcomes depend on weight-
drop distance with the NYU impactor device [80]. Less
severe contusion SCI models are more often used in a
variety of cell ther a py approaches to recovery -of-function,
for example, Schwann cell grafts for sensory/motor recover
and regeneration [81].
3.5.1. Graft Sites after SCI and Transplant of hNT2.17 or
hNT2.19 Cells. Graft sites after the transplant of human cells
can easily be located with anti-human cell immunohisto-
chemistry for either the markers NuMA or TuJ1, both used to
find the graft of parental hNT2 cells in previous studies [51].
Both markers have also been used previously to locate g rafted
hNT2.17 [43]andhNT2.19[47] cells in various models of
Page 10
Pain Research and Treatment 11
Weeks after chronic constriction injury
112345678
3
1
1
3
5
7
9
11
13
15
(ligated minus nonligated)
Mechanical threshold (grams)
Naive
CCI
CCI/nonviable hNT2 cells
CCI/viable hNT2 cells
Transplant
Tactile allodynia
(a)
Withdrawal latency (seconds)
4
3
2
1
0
1
2
3
4
112345678
Transplant
Weeks after chronic constriction injury
(ligated minus nonligated)
Naive
CCI
CCI/nonviable hNT2 cells
CCI/viable hNT2 cells
Thermal hyperalgesia
(b)
6
4
2
0
2
5
9
7
4
13
11
15
Mechanical threshold (grams)
123456781
Naive
CCI/nonviable hNT2.17 cells
CCI/viable hNT2.17 cells
Transplant
Weeks after chronic constriction injury
(ligated minus nonligated)
CCI only
Tactile allodynia
(c)
12345678
1
Naive
CCI/nonviable hNT2.17 cells
CCI/viable hNT2.17 cells
4
3
2
1
0
1
2
3
4
Withdrawal latency (seconds)
Transplant
Weeks after chronic constriction injury
(ligated minus nonligated)
CCI only
Thermal hyperalgesia
(d)
Figure 4: Sensory behaviors after initiation of peripheral CCI pain and intrathecal transplant of parental hNT2 and hNT2.17 cells. For tactile
allodynia after CCI and transplant of parental (a) hNT2 cells, adult female rats were either left unoperated, underwent CCI, or transplanted
with nonviable hNT2 or viable hNT2 cells two weeks following CCI, one day following behavioral testing. Nonviable cells were prepared by
suspension of the cells in water, centrifugation, and resuspension in buer before transplant. All rats received 10 mg/Kg i.p. CsA at the time
points corresponding to one day before and 13 days after cell transplant (daily injections). Animals were tested for hindpaw withdr awal to
a graded series of von Frey hairs once every week for one week before and eight weeks following CCI and before and after transplants. Only
animals that demonstrated tactile allodynia two weeks after CCI were transplanted. The data reported are the mean
± SEM of the dierence
scores for ligated paw minus the sham-operated paw of 14 animals in each group. The results with viable hNT2 cell transplants diered
significantly from the CCI or nonviable graft conditions at each time point. P<0.001. For thermal hyperalgesia after CCI and transplant of
parental (b) hNT2 cells, animals were tested for hindpaw withdrawal once every week for one week before and eight weeks following CCI and
before and after transplants. Only animals that demonstrated thermal hyperalgesia 2 weeks after CCI were transplanted. The data reported
are the mean
± SEM of the dierence values for ligated paw minus the sham-operated paw of 14 animals in each group. The viable hNT2
transplants diered significantly from the CCI and nonviable graft condition at each time point. P<0.001. For tactile allodynia after CCI
and transplant of GABA (c) hNT2.17 cells, adult female rats were either left unoperated, underwent CCI, or transplanted with nonviable
hNT2.17 or viable hNT2.17 cells two weeks following CCI, one day following behavioral testing. Animals were tested for hindpaw withdrawal
once every week for one week before and eight weeks following CCI and b efore and after transplants. Only animals that demonstrated tactile
allodynia two weeks after CCI were transplanted. The data reported are the mean
± SEM of the dierence scores for ligated paw minus the
sham-operated paw of 14 animals in each group. The viable hNT2.17 cell transplants diered significantly from the CCI or nonviable graft
conditions at each time point. P<0.001. For thermal hyperalgesia after CCI and transplant of (d) hNT2.17 cells, animals were tested for
hindpaw withdrawal once every week for one week before and eight weeks following CCI and before and after transplants. Only animals that
demonstrated thermal hyperalgesia 2 weeks after CCI were transplanted. The data reported are the mean
± SEM of the dierence values for
ligated paw minus the sham-operated paw of 14 animals in each group. The viable hNT2.17 transplants diered significantly from the CCI
and nonviable graft condition at each time point. P<0.001.
Page 11
12 Pain Research and Treatment
(a) (b)
40
35
30
25
20
15
7 0 7 1421283542
Days after STZ
STZ/nonviable hNT2.17
STZ/viable hNT2.17
Saline injection
STZ injection
Transplant
(grams)
Cutaneous allodynia: STZ-diabetes
(c)
STZ/nonviable hNT2.17
STZ/viable hNT2.17
Saline injection
STZ injection
70 7 1421283542
Days after STZ
16
12
8
4
0
(seconds)
Transplant
Thermal hyperalgesia: STZ-diabetes
(d)
Figure 5: Continued.
Page 12
Pain Research and Treatment 13
650
450
250
50
0 7 14 21 28 35 42 49 56
Saline injection
Blood glucose (mg/dL)
50 mg/kg STZ injection
STZ/nonviable hNT2.17 graft
STZ/viable hNT2.17 graft
Days after STZ
Graft
10 mg/kg CsA ip
(e)
Figure 5: Sensory behaviors after diabetic peripheral neuropathy (DPN) and intrathecal transplant of hNT2.17 cells. The hNT2.17 cells
were transplanted into the lumbar subarachnoid space 5 days after a STZ injection (50 mg/Kg, i.v.) and cords fixed at 42 days after STZ and
transplants. (a) Cords were examined for graft survival with the human NuMA marker (arrows) and adjacent sections (b) stained with the
antibody marker for GABA (arrows). Many surviving hNT2.17 cells, expressing both NuMA and GABA could be found on the pial surface
(arrows), especially over the dorsal lumbar spinal cord. For cell transplant and sensory behavior evaluation, nonviable cells were prepared by
suspension of the cells in water, centrifugation, and resuspension in buer before transplant. All rats received 10 mg/Kg i.p. CsA at the time
points corresponding to one day before and 13 days after cell transplant (daily injections). Tactile allodynia behaviors (c) were examined
for both a baseline period before STZ injection and for 42 days following STZ. Two groups of STZ-injected rats (n
= 6) were transplanted
with either 1
× 10
6
hNT2.17 viable or nonviable hNT2.17 cells at 5 days following STZ, a time when the behavioral hypersensitivity to
nonnoxious tactile stimulation was already apparent in the rats. In these three groups (nontransplanted and transplanted), both hindpaws
develop (pooled data) increased hypersensitiv ity, but only the rats with viable hNT2.17 cell grafts recover permanent tactile responses. Saline
injected rats never develop tactile allodynia and serve as positive controls. The data (mean + SEM) is from six rats in each group. For thermal
hyperalgesia (d), the same rats were examined on alternate days for responses to noxious thermal stimulation and like tactile allodynia,
thermal hyperalgesia was apparent immediately before the transplant time, 5 days after STZ. In these three groups (non-tr a nsplanted and
transplanted), both hindpaws develop (pooled data) increased hypersensitivity, but only the rats with viable hNT2.17 cell grafts recover
permanent normal thermal responses. Saline injected rats never develop thermal hyperalgesia and serve as positive controls. The data (mean
+ SEM) is from six rats in each group. (e) All rats were examined for blood glucose levels before and after STZ and transplant of hNT2.17
cells. Data are the mean
± SEM from >six rats in each group. No treatment had an eect on the vigorous increase in blood glucose levels
induced by 50 mg/Kg STZ injection; only the saline injected animals showed no increase in blood glucose.
pain. Here we illustrate anti-NuMA (Figures 6(a) and 6(b))
and -TuJ1 (Figures 6(c) and 6(d)) stained hNT2.17 (a, b)
and hNT2.19 (c, d) grafts in the QUIS and contusive SCI
models, respectively. Both cell line grafts survive well and
maintain their neurotransmitter phenotypes at least 6 weeks
after transplant in these two models of SCI pain, excitotoxic
(QUIS), and severe contusive SCI.
3.5.2. Direct Comparison of Behavioral Sensitivity Following
Grafts of Either hNT2.17 Or hNT2.19 Cells in the Same Model
of QUIS SCI. The hNT2.17 and hNT2.19 cell lines were
directly compared for their ability to attenuate TA and TH
behaviors in the same central model of neuropathic pain, the
excitotoxic SCI model, il lust rated (Figures 7(a) and 7(b)).
In these experiments, a laminectomy alone injury was com-
pared to QUIS SCI alone, with naive animals providing base-
line data. There was no significant dierence with TA (a) or
TH (b) in the data from the naive or laminectomy alone ani-
mals throughout the course of the experiment (63 days after
QUIS). QUIS alone SCI induced a vigorous TA (a) and TH
(b) behavioral response as soon as 14 days after the injury,
that persisted and worsened over the course of the experi-
ment and was dierent at every time point from the naive
or laminectomy data. For TA behaviors (a), lumbar sub-
arachnoidtransplantat14daysafterQUISofeitherhNT2.17
or hNT2.19 cells (10
6
cells/injection) immediately attenuated
the TA behaviors (to near normal) at 21 days. The atten-
uation persisted, with little dierence between the cell line
grafts, until 42 days, when the attenuation briefly dropped
until 56 days, when attenuation began to recover to near
normal levels by the end of the experiment, where data was
indistinguishable from naive animals. For TH behaviors (b),
transplant of either cell line had an immediate eect on TH,
attenuating the behaviors to near normal levels at 21 days,
an eect which persisted throughout the 63-day experiment.
Overall, there was little dierence on attenuation of TA and
TH behaviors between transplants of the GABA hNT2.17
or 5HT hNT2.19 cell lines in the same QUIS model of
Page 13
14 Pain Research and Treatment
(a) (b)
(c) (d)
Figure 6: Comparison of graft sites of hNT2.17 and hNT2.19 in the QUIS and severe contusive-SCI models, respectively, at 6 weeks after
cell transplant. (a) Sagittal section of anti-GABA-immunostained QUIS + hNT2.17 transplant lumbar spinal cord 6 weeks after grafting.
Easily detectible hNT2.17 cells stain for GABA (arrows) on the pial membranes. (b) Sagittal section of anti-NuMA-immunostained QUIS
+ hNT2.17 transplant lumbar spinal cord 6 weeks after grafting. Easily detectible hNT2.17 cells stain for NuMA (arrows) on the pial
membranes in adjacent sections. The hNT2.19 were alternately injected into the subarachnoid space two weeks after severe contusive SCI.
Cell graft sites were co-localized with 5HT (c) and the human-specific marker TuJ1(d) (neuron-specific class III β-tubulin). There are many
surviving hNT2.19 (d) grafted cells visible on the pial surface, which stain for TuJ1 (arrows) at the end of the experiment, 56 days after SCI
and about 6 weeks after cell transplant. Adjacent sections w ith the same grafted hNT2.19 (c) are labeled for 5HT (arrows). Magnification
bar
= 20 μm.
neuropathic pain; both achie ving near normal recover y-of-
sensory function immediately after transplant, an eect that
persisted through 63 days.
3.5.3. Percent Comparison of Sensory Recovery in the QUIS
and Severe Contusive SCI Models with Transplant of hNT2.17
and hNT2.19 Cells. Although the two central models of SCI
pain used here cannot easily be directly compared, each cell
line was examined for its sensory behavioral eects, hNT2.17
cells in the QUIS model and hNT2.19 cells in the contusive
SCI model, and the data normalized to percent of laminec-
tomy control data results for each experiment (Figures 8(a)
and 8(b)). For TA behaviors (a), QUIS SCI alone induced
a 44.4% increase in TA (55.6% of laminectomy) by 14 days
after injection. In comparison, contusive SCI induced a
51.2% increase in TA (48.8% of laminectomy) by 14 days fol-
lowing the contusion. Both lumbar subarachnoid grafts were
placed immediately following measure of this data, at 14 days
after SCI: hNT2.17 (10
6
cells/injection) in QUIS-injured ani-
mals and hNT2.19 (10
6
cells/injection) in contusive SCI ani-
mals that demonstrated the same increases in TA behaviors.
By 21 days (7 days after transplant in both models), when
QUIS SCI alone had induced a 48.6% increase in TA (51.4%
of laminectomy), the addition of hNT2.17 grafts to the
QUIS SCI had attenuated the TA to 84.3% of laminectomy.
Similarly, at 21 days, the addition of hNT2.19 grafts to the
contusive SCI had attenuated the TA behaviors to 78.8% of
laminectomy, compared to a 54% increase (46% of laminec-
tomy) with the contusive SCI alone. These comparisons
continue throughout the 56-day experiment: the QUIS SCI
increases in TA, with the highest allodynia reached at 56 days,
with 59.4% (40.6% of laminectomy), when the addition of
hNT2.17 grafts had decreased the TA to 89.6% of laminec-
tomy control. Similarly, the contusive SCI alone had the
worst TA at 56 days, 47.6% of laminectomy, while the addi-
tion of hNT2.19 grafts to the SCI showed near normal recov-
ery, 92.2% of laminectomy. For TH behaviors (b), at 14 days
the QUIS SCI induced a 25.6% increase (74.4% of laminec-
tomy), compared to a 44.9% increase (55.1% of laminec-
tomy) for contusive SCI. By 21 days, the addition of hNT2.17
grafts to the QUIS SCI had improved the TH to 94.4%
of laminectomy, compared to only 67.4% of laminectomy
for QUIS SCI. By the end of the experiment (56 days), the
addition of hNT2.17 grafts had induced a 100% recovery,
Page 14
Pain Research and Treatment 15
15
20
10
25
30
35
40
(days)
14 4 7 18 28 49 60 7039
Force (grams)
QUIS
QUIS
Naive
QUIS/NT2.17 cells
QUIS/NT2.19 cells
Laminectomy
Transplant
Tactile allodynia/QUIS/NT2 cell lines
(a)
QUIS
Naive
QUIS/NT2.17
QUIS/NT2.19
Laminectomy
(days)
14 4 7 18 28 49 60 7039
QUIS
8
10
6
12
14
16
18
Withdrawal latency (seconds)
Transplant
Thermal hyperalgesia/QUIS/NT2 cell lines
(b)
Figure 7: Comparison of sensory recovery after transplant of hNT2.17 or hNT2.19 cell lines in the same QUIS model of SCI. Rats were
spinally injected with QUIS in a rat model of SCI and chronic pain. All animals in the study received CsA (10 mg/Kg) 1 day before and
for 13 days after the two week time-point when some animals were injected with either the hNT2.17 or hNT2.19 cells. Animals were either
left untreated (na
¨
ıve), injected with QUIS alone or laminectomy only or QUIS plus hNT2.17 or hNT2.19 cells (10
6
cells/injection) into the
lumbar subara chnoid space at two weeks after QUIS. Animals were tested before the SCI (baseline) and once a week following QUIS and
treatments for hypersensitivity to tactile (a) or thermal (b) stimuli in hindpaws below the SCI. QUIS injury negatively aected hindpaw
responses bilaterally, but the ipsilateral hindpaw is most aected by the injection of quisqualic acid (shown here). Neither hindpaw recovers
normal tactile or thermal responses after QUIS alone by 60 days after the injection. Both ipsilateral and contralateral hindpaws recovered
near-normal sensory responses to tactile and thermal stimuli after grafting the GABAergic hNT2.17 or serotonergic hNT2.19, compared to
the QUIS injury alone. Data represent the mean value
± SEM (n = 4–6 animals in each group) at each time point before and 63 days after
QUIS.
compared to only 74% for QUIS SCI alone. Similar results
were found for the contusive SCI model. At 14 days, the
contusive SCI had increased the TH behavior 54.1% (45.9%
of laminectomy. By 21 days, this TH had not improved much
(54.7% of laminectomy), but the addition of hNT2.19 grafts
had improved TH recovery to 81.8% of laminectomy alone.
At 56 days, the SCI alone was only 50.7% of laminectomy,
compared to 89.4% fo9r the addition of hNT2.19 grafts
the contusive SCI paradigm. Even though the cell lines are
dierent and the SCI models used are categorically dierent,
the eect on TA and TH behaviors with the hNT2.17 and
hNT2.19 cell grafts are quite similar.
3.5.4. Percent Compar ison of Open-Field Motor Behaviors after
Severe Contusive SCI and Following Either Intraspinal Or
Intrathecal Transplant of hNT2.19 Cells. The grafts of the
serotonergic hNT2.19 cells are able to modestly promote the
recovery of motor function after severe contusive SCI [46],
but it’s informative to compare these motor eects when the
hNT2.19 grafts are placed at two dierent locations in the
same contusive SCI model. Both the open-field motor behav-
iors (Figure 9(a)) and fine-motor behaviors (Figure 9(b))are
compared at each week after SCI, and reported as a percent
of laminectomy control, when the same dose of hNT2.19
(10
6
cells/injection) is provided, at either a lumbar subarach-
noid or intraspinal site (immediately above/below the lesion
site). The open-field BBB score (a) was used to study the
functional recovery stages following the injury, by categoriz-
ing the rat hindlimb movements, trunk position and stability,
coordination, stepping, and paw placement and tail position.
The fine-motor BBB subscore (b) described frequency of
clearance of toes while moving the hindlimbs and is a mea-
sure of improvement which accompanies sucient improve-
ment in BBB scores. At seven days after the SCI, the SCI
had induced 18.3% of laminectomy BBB scores (a) in the
intraspinal model. By 14 days, the SCI group had only 27.8%
of the laminectomy BBB scores in the intraspinal model. At
56 days, the SCI group had recovered only 39.7% of the
laminectomy BBB scores in the intraspinal model. When
the hNT2.19 cells are placed int raspinally, recovery reached
46.1% of laminectomy BBB scores (7 days after transplant).
By 21 days, recovery improved to 46.8%; by 56 days, BBB
scores had improved to 50.81% of laminectomy BBB scores,
in the presence of intraspinal hNT2.19 grafts. Results were
very dierent in the intrathecal model for BBB scores (a).
At 7 days after SCI, BBB scores were 21.7% of laminectomy
for the SCI group; at 21 days, the BBB scores were 36%
of laminectomy scores for the SCI group and 35.5% for
Page 15
16 Pain Research and Treatment
110
100
90
80
70
0 14212835424956
(days)
QUIS SCI only
QUIS/hNT2.17 cells
Contus SCI only
SCI/hNT2.19 cells
60
50
30
40
20
10
0
Laminectomy only (%)
(a)
110
100
90
80
70
0 14212835424956
(days)
QUIS SCI only
QUIS/hNT2.17 cells
Contus SCI only
SCI/hNT2.19 cells
60
50
30
40
20
10
0
Laminectomy only (%)
(b)
Figure 8: Percent comparison of sensory behaviors after QUIS SCI or severe contusive SCI and with transplant of hNT2.17 or hNT2.19 cells
in vivo—type of hNT2-derived cell graft. Rats were either injured with QUIS injection SCI or with a weight-drop device (NYU impactor,
25 mm; severe contusive SCI) in rat models of SCI and chronic behavioral hypersensitivity. All animals in the study received CsA (10 mg/Kg)
1 day before and for 13 days after the two week time-point (14 days) when some animals were injected with hNT2.17 (QUIS model) or
hNT2.19 cells (severe contusion SCI). Animals either received one of the SCIs alone, laminectomy alone, or one of the SCIs plus hNT2.17
or hNT2.19 cells (10
6
cells/injection) into the subarachnoid space at two weeks after SCI. Animals were tested before the SCI (baseline), and
once a week following SCI and treatments for hypersensitivity to tactile (a) or thermal (b) stimuli in hindpaws below the SCI. Data are the
mean of percent of laminectomy control data in each model, where SCI injury alone negatively aected hindpaw responses and laminectomy
alone had no eect. Data represent the mean value (n
= 4–9 animals in each group) at each time point before and 56 days after SCI. In these
dierent models, the hNT2.17 and hNT2.19 and cell grafts p otently and comparably attenuated tactile allodynia (a) and thermal hyperalgesia
(b) induced by either SCI. Recovery of normal behaviors was near-complete by experiment’s end with graft of either hNT2.17 or hNT2.19
cells.
the SCI/hNT2.19 graft group (7 days after transplant). At 56
days, BBB scores were 46.1% of laminectomy scores for the
SCI group and 37.3% for the SCI/hNT2.17 intr a thecal trans-
plant group. There was no recovery in BBB scores when the
hNT2.19 grafts were placed intrathecally, compared to the
modest recovery seen when grafts are placed intraspinally.
Similar results are seen with an examination of the data from
the BBB subscores (b), and the results normalized in the
intraspinal and intrathecal m odels to percent of laminectomy
control. At 7 days after SCI, the SCI had induced 7.4% of
the laminectomy BBB subscores in the intraspinal model.
By 14 days, the SCI group had induced only 14.7% of
the laminectomy BBB subscores, while the addition of
intraspinal hNT2.19 grafts had improved the BBB subscores
to 22.8% (7 days after graft). By 56 days, when SCI had
recovered only 19.7% of the laminectomy BBB subscores, the
addition of intraspinal hNT2.19 gr afts had improved the BBB
subscores to 32.9% of laminectomy controls. When hNT2.19
are placed intrathecally, they never improve BBB subscores
above that seen for SCI alone (even lower the subscore,
compared to SCI). Overall, only intraspinal hNT2.19 gr afts
are able to significantly, if modestly, improve BBB and BBB
subscores, compared to intrathecal placement of hNT2.19
grafts in the same severe contusive SCI model.
4. Discussion
4.1. NT2 Cells for Transplant. The teratocarcinoma human
NT2 (NT2/D1; hNT2) parental cell line was the source of
the hNT2.17 and hNT2.19 cell lines and is derived from the
embryonic carcinoma (EC) cell type, after dierentiation in
response to retinoic acid (RA). A derivative of the original
polyclonal TERA-2 EC cell line, hNT2 are cells with phe-
notypic properties of neurons after dierentiation and this
resultant, exclusively neuronal, phenotype with RA treat-
ment has remained a hallmark of this human cell line. The
RA-dierentiated neurons, often called NT2-N cells, are
similar to developing human spinal cord or CNS neurons,
reminiscent of terminally dierentiated postmitotic neurons.
Such neurons express typical neuronal markers [82], w ith
a stable polarized phenotype [83]ofcentral,notperipheral
nervous system neurons. Spinal implantation of dierenti-
ated hNT2 cells in rats results in grafted neurons with 1-2%
survival (with brief immunosuppression) at three months
after transplantation. These surviving neurons display a
robust axo-dendritic sprouting and expression of markers
typical of mature neurons and grafted neurons with a mostly
GABA phenotype [64]. More recently, the same hNT2 cell
grafts uniformly showed robust cell survival and progressive
Page 16
Pain Research and Treatment 17
1 7 14 21 28 35 42 49 56
Days after SCI
SCI intraspinal model
SCI/hNT2.19 intraspinal
SCI intrathecal model
SCI/hNT2.19 intrathecal
70
60
50
30
40
20
10
0
Laminectomy only (%)
(a)
1 7 14 21 28 35 42 49 56
Days after SCI
SCI intraspinal model
SCI/hNT2.19 intraspinal
SCI intrathecal model
SCI/hNT2.19 intrathecal
60
50
30
40
20
10
0
Laminectomy only (%)
(b)
Figure 9: Percent comparison of open-field motor behaviors after severe contusive SCI and following either intraspinal or intrathecal
transplant of hNT2.19 cells in vivo: location of graft of hNT2.19. Rats were injured with a weight-drop device (NYU impactor, 25 mm;
severe contusive SCI) in a rat model of SCI and chronic motor dysfunction. Some rats received only a laminectomy, and data are a percent
of laminectomy control data (for each independent transplant location experiment) for open-field BBB scores (a) and BBB subscores (b)
after contusive SCI only and SCI + hNT2.19 cell grafts, where gr afts are placed either intraspinally or in the lumbar subarachnoid space
(intrathecally). Gross open-field motor behavioral results (a) ( BBB) showing gradual recovery of motor scores beginning at 1 week after SCI,
with additional partial and persistent recovery with the addition of intraspinal hNT2.19 grafts. There is no improvement over SCI alone
when the grafts are placed intr a thecally. Data represent the mean value (n
= 6 animals in each group) at each time point before and for
56 days after SCI. The BBB subscore (b) demonstrated a significant improvement in the subscore, beginning at 2 weeks after SCI, with the
addition of the intraspinal, but not the intrathecally-placed hNT2.19 cells. Data represent the mean value (n
= 6 animals in each group) at
each time point before and for 56 days after SCI. Only intraspinally placed hNT2.19 cells improved BBB and BBB subscores.
neuronal maturation, with most transplants demonstrating a
GABA phenotype, after injection into the lumbar spinal cord
segments of naive immunosuppressed minipigs [84]. Both
the hNT2.17 and hNT2.19 cells, derived from this hNT2 lin-
eage, have been suciently characterized in vitro and in vivo
to determine their exclusive neuronal and neurotransmitter
phenotype [43, 47, 61].
4.1.1. Phenotype of hNT2-Derived Cell Lines. The fact that
the parental hNT2 cell line contains multiple, distinct cell
types, and hence exists more as a cell population rather than
as a pure cell line, serves as an advantage when these cells are
intended to be subcloned into multiple and distinct cell lines.
A few characterization studies [85, 86]describeavariety
of phenotypes possible, including cholinergic, GABAergic,
catecholaminergic, serotonergic, and peptidergic, expressed
by hNT2N neurons after 2B4 weeks of dierentiation in
vitro. The most common phenotype observed in the parental
hNT2 neurons is the GABAergic type (about 62% in [85];
about 15% in [86]). The other common phenotypic markers
seen in parental hNT2 neurons were glutamatergic or do-
paminergic, but in our hands these neurotransmitters were
not seen in either the subcloned hNT2.17 or hNT2.19 cells.
Another common hNT2 phenotype, depending on the cell
preparation method, was for 5HT-expressing hNT2 cells,
ranging from about 2% [86] to 30% [85]. Further increasing
the proportion of 5HT producing neurons seems to require
particular dierentiation protocols involving the timed
application of various growth factors [87], methods not used
in the current subcloning of the hNT2.19 cell line. Our dif-
ferentiation method is similar to that of the Guillemain study
[85], which provides about 30% 5HTcontaining neurons.
This explains the relative ease of finding both a GABA or
5HT-subclone, such as the hNT2.17 and hNT2.19 cell lines,
since our methods for subcloning depended on examining
single cells which formed colonies (and established the sub-
sequent cell lines). Using the rapid aggregation method for
dierentiation [82], and determination of good cell survival
and doubling times in culture, as well as anti-neurotransmit-
ter antibody markers for the choice of candidates for useful
cell lines, meant final ly screening about 100–150 cell lines,
to derive a few hNT2-lines, and finally the hNT2.17 and
hNT2.19 cell lines. A similar approach could only be hurried
with the use of automated mechanical cell-picking devices
for subcloning, rather than the laborious art of using cloning
rings and individual cell line analysis, as was done here.
There is much interest in directing the eventual and
terminal phenotype of stem cells [88], and, interestingly,
the parental hNT2 cell line (see: patent application US
2008/0160614A1, Stable Dierentiation of Adult Stem Cells,
Page 17
18 Pain Research and Treatment
Saporta et al.) without the use of growth factors or retinoic
acid, since the growth factors and/or retinoic acid can be dif-
ficult to completely remove during commercial production
for clinical use. These eorts are based on the observation
that particular phenotypes expressed in dierentiated hNT2
cells (usually neurons) do not appear to be stable (beyond
30+ days in culture). In our hands, the GABA phenotype (of
hNT2.17) and 5HT phenotype (of hNT2.19) remain 100%
after 30+ days in vitro and >6 weeks after gr afting [43] into
the lumbar subarachnoid space, but with further studies,
that phenotypic instability observed by others may indeed
prove true. Alternative methods for generating a specific,
and useful, phenotype, at least in immortalized cell lines
of murine origin, often involve both immortalization with
an oncogene [89], such the temperature-sensitive mutant of
SV40 large T-antigen (ts-Tag) [90], as well as the possible
addition of a rate-limiting neurotransmitter enzyme gene,
such as GAD or TH, or addition of (or in vitro exposure to)
aneurotrophingenesuchasBDNF,todriveterminaldier-
entiation. Such studies and methods have occupied our in-
terests for years [16, 2830, 9198], in fact leading to the con-
clusions that GABA and 5HT were the most eective agents
for anti-nociception in cell therapy (with studies of bio-
engineered rat cell lines), but neither cell-transfection tech-
nology nor non-human cells will likely ever be useful (or
meet FDA approval; see: [99]) for safe human cell therapy.
Perhaps only the simplest manipulations, stem cell sources
or otherwise, for production of cell sources for cell therapy
will meet current approval. The parental hNT2 cell line has
of course been approved for clinical trials [40], and it might
be expected that these hNT2-derived and subcloned cell lines
could also meet FDA approval. But bio-engineering cells,
tailoring cells to meet specific phenotype requirements, with
current gene transfection vectors, await thorough vetting for
safety, similar to that seen in the development of new gene
therapy approaches [100].
Additionally, the colocalization (and release) of other
neurotransmitters which might be relevant for anti-nocicep-
tion, that is, glycine in hNT2.17 cells [43, 61 ], or a specific
synaptic amino-acid transporter, such as vesicular inhibitory
amino acid transporter (VIAAT), also found in hNT2.17 cells
[43], suggests that these hNT2-derived cell lines are mature,
functional, human neurons, and likely remain so after graft-
ing in a murine or human host, able to secrete or release neu-
roactive agents into the neural environment, and they also
will likely integrate into the neural matrix, when conditions
are appropriate, as has been seen for the hNT2 parental cells
[65, 101]. Our single study of intraspinal transplant of the
dierentiated serotonergic hNT2.19 cells [46] demonstrated
amodesteec t on recovery-of-motor function after SCI with
grafts, without easily revealing surviving gr afted cells (in the
nude rat). Further studies will be needed to understand how
well these two cell lines integrate into white- and grey-matter
in the PNS or CNS, but they clearly maintain their specific
neurotransmitter phenotype when placed in a subarachnoid
location, near the lumbar cord.
4.1.2. Tumorgenic ity Considerations. Part of the concern for
safety in these cell lines for clinical use [37] is the issue of
expression of a tumorigenic phenotype in the human host.
Postmortem examination of a single patient following par-
ental hNT2 graft for stroke showed that no tumor was iden-
tified anywhere in the brain, and a monoclonal antibody to
Ki-67, a protein expressed in cycling cells, immunolabeled
1% of cells, consistent with the absence of a neoplasm [65].
These hNT2 cells which require dierentiation before graft-
ing to eliminate the possibility of tumor formation, have
been well-studied in animal models [38, 39]. Other studies
provide some understanding of the central role of retinoic
acid regulation of dierentiation [35, 102] and suppression
of tumor genes, such as FGF-4 [103]andTGF-
α [103, 104],
in RA-sensitive cell lines, such as the hNT2. Downregulation
of FGF-4 and TGF-α, as well as expression of cytoskeletal
proteins [83], serves as markers of dierentiation with lack
of tumorogenicity, and FGF-4 and TGF-α are expressed in
both the proliferating, but not dierentiated, parental hNT2
[105] and our hNT2-derived [43, 47] cell lines. With RA-
exposure these cell lines terminally dierentiate, express a
number of markers of mature neurons [43, 47, 61, 83], with
FGF-4 and TGF-α eectively inhibited in dierentiated cells.
In addition, immunohistochemistry for the BrDU marker in
proliferating versus dierentiated cells, shows similar results,
for example, the hNT2.17 and hNT2.19 cells used for grafting
do not take up BrDU, and the overall conclusion is that
these cell lines are not tumorigenic when used after RA-
exposure and dierentiation, when placed within or outside
of the spinal cord. Over more than 10 years of using these
two cell lines as graft sources in na
¨
ıve and immunosup-
pressed rats, with and without chemical- or mechanically-
induced injuries, no rats have ever developed tumors or
further mechanical or sensory impairment, even in animals
followed out to 3 months after cell placement. The hNT2-
derived cells were always well-dierentiated (for at least 2
weeks following 2 weeks of retinoic-acid treatment), exclu-
sively neuronal in phenotype, with no reversion to a tumori-
genic (genetic) phenotype. Histopathological examination
[10] of the subarachnoid and spinal cord transplant site in
a quisqualic (QUIS) lesion SCI paradigm concluded that
hNT2.17 (grafted) cells were identified on H+E staining as
small round basophilic cells without significant dendritic or
axonal processes. The H+E staining showed no dierence
between the cytoarchitecture of QUIS and QUIS plus trans-
plant and showed no cord damage in the naive plus trans-
plant cord group, compared to naive. Myelin staining showed
significant demyelination in all animals undergoing a QUIS
injury that did not improve with hNT2.17 tr a nsplants. No
demyelination was seen in conjunction with cell grafts,
implying their safety as a graft source Such long-term data
will likely be required in mouse tumor studies under GLP
conditions for eventual clinical use.
4.2. Survival of Grafted Cells. Graft survival and surv iving
cell numbers have been examined in a series of studies of
the hNT2.17 cell line grafts in the excitotoxic QUIS model
of SCI pain [43, 61]. With their identity as a model of human
neurons, it is not surprising that the hNT2 cell line has
been used to examine a variety of genetic elements that can
influence cell survival, for example, RA-induced activation
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Pain Research and Treatment 19
of the p53 gene [106], or expression of the neurotrophin
BDNF [107] in hNT2 cells, suggesting that these factors likely
drive early neuron survival in normal brain development.
Using the transplantation of hNT2 cells into the CNS of
immunodeficient mice as an in vivo model system for
studies of the formation and remodeling of the developing
central nervous system [108], survival, and integration
of undierentiated grafted cells are seen to depend on
graft site, suggesting that areas of the brain, such as the
caudoputamen, contain environmental cues that lead to the
progressive dierentiation of large numbers of NT2 cells into
postmitotic, immature, neuron-like cells, whereas transplant
of undierentiated cells into the subarachnoid space induces
both tumors and massive cell death. When hNT2 cells
are predierentiated with RA, the bcl-2 gene expression is
upregulated and after transplant into the rat striatum, 85%
of implanted neurons expressed bcl-2, with 12% of the hNT2
neurons surviving the transplantation at 1 month. Collec-
tively, these data suggest that only predierentiated hNT2
cells can be used for transplant and RA induction is required
for dierentiation before grafting. In our experience, both
pre-dierentiation and an optimal course of CsA immuno-
suppression around the time of transplant are required for
good graft survival in the subarachnoid space [43]. Since
transplant into a nonhuman host such as the rat, these
xenografts, require at least 1-2 weeks of immunosuppression.
The most interesting data comes from examination of
graft sites from either intraspinal transplant of hNT2.19
cells in contusive SCI [46], or cervical subdural grafts of
hNT2.17 cells in QUIS SCI [61], where motor and sensory
recovery does not require long-term hNT2.17 graft survival,
since transplanted cells could not be identified at the 6–
8 week experiment’s end. But even optimal survival of these
xenografts in a subarachnoid location was about 1% after
8weekswithSCI[43], suggesting that at least some course
of immunosuppression might be required in humans, or
repeated, additional i.t. injection of antinociceptive hNT2-
derived cells would be necessary if eectiveness is reduced
over time in humans, as has been observed w ith i.t. injection
of anti-nociceptive chroman cells for terminal cancer pain
in Phase II trials [109].
4.3. Requirement for Immunosuppression. Requirements for
immunosuppression of i.t. cell grafts suggest complex issues
[110], even with toxicity, such as nephrotoxicity, hyperten-
sion, hypertrichosis, infection, hyperkalemia, and neuropa-
thy, associated with CsA use and cell transplant [111]. There
is a contention of the CNS being a privileged transplant
site, but the issue is controversial, even with the use of allo-
genic stem cells transplants into the nervous system [112].
But some trends are seen with the hNT2.17 grafts in the
QUIS model of pain: a minimal or optimal course of immu-
nosuppression with CsA, about 1 to 2 weeks after transplants,
is required; this minimal CsA course ensures optimal ecacy
in reversal of the behavioral hypersensitivity associated with
SCI-pain; less than minimal immunosuppression (1 day)
only provides minimal ecacy; longer than the optimal
timecourseofCsAdoesnotimproveecacy significantly.
The dierences in recovery of TA versus TH when less
than 2 weeks of immunosuppression are provided is also
interesting to note. A mixed CsA eect occurs after 1 week of
treatment and seems to preserve the antiallodynic eects of
the transplants but not the antihyperalgesic eects. Certainly
the grafts do not survive as well as when 2 weeks of CsA is
provided for immunosuppression. We examined immunos-
tained sections at the end of these experiments (data not
shown), and although reliable quantification of grafts is
almost impossible, there were clearly fewer surviv ing grafts
with less than 2 weeks of CsA. A critical” number of func-
tioning grafted cells could influence or permanently aect TA
versus TH. Also, TA is likely modulated dierently than TH;
these dierences are also seen in the timing studies. We have
seen similar di
erences in previous studies of the eects of
timing with rat cell therapy and the CCI peripheral model of
neuropathic pain [113].
4.4. Timing of Graft Placement. The issue of altered ecacy
with changes in the time of placement of cell grafts was first
examined with the use of GABA-secreting cell grafts in the
CCI-model of peripheral nerve injury pain and the GAD67-
rat cell line [113], where lumbar subarachnoid transplant
of these cells only permanently and completely attenuated
TA and TH behaviors induced by the unilateral nerve injury
when an early time point (2 weeks after CCI) was used for
cell injection, even though many surviving grafted cell were
found, no matter when cells were grafted. A similar result is
seen with the use of hNT2.17 lumbar grafts in the QUIS
model of SCI pain [61]. In both studies, even with the great
dierences in the rat pain models and the characteristics of
these GABA-secreting cell lines (rat and human derivations;
bioengineered versus “natural” GABA phenotypes), the pro-
file of eects is remarkably similar. Early transplant times (2
weeks after nerve or spinal cord injury) provide complete and
permanent attenuation of behavioral hypersensitivity. Later
transplant times (6 weeks after nerve or spinal cord injury),
when both TA and TH behaviors are well established has, at
best, a temporary and/or partial eect. Although a percent-
age of patients report neuropathic pain immediately with
injury, onset time is often 3–6 months later with both periph-
eral and CNS injuries. Another common observation is that
while acute neuropathic pain more often responds to treat-
ment, chronic neuropathic pain can be resistant to a variety
of treatments [114]. Where pharmacologic pain treatment is
eective, onset of relief can be immediate [115], but these
same medications, where eective, are best in chronic pain
and must be individualized to specific pain scenarios, with
the appearance of side-eects at higher doses [77]. Our data
suggest that the earlier the initial GABA hNT2.17 grafting
the better the outcome, although even a graft provided 6
weeks after SCI in the rat provided >70% behavioral ecacy.
Understanding the mechanisms in both the initiation and
maintenance of pain behaviors in any type of pain, as well
as for it
=s relief, are the keys to understanding any eventual
use of cell therapy in humans.
4.5. Graft Location in Recovery-of-Sensory Function. For dec-
ades, development of the idea of cell therapy for pain has
envisioned and tested the lumbar subarachnoid location for
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20 Pain Research and Treatment
anti-nociceptive transplants [11, 116]. Whether primar y cell
sources, human [117]orxenografts[118], stem cells, immor-
talized cell lines [30, 119, 120], or encapsulated cell or tissues
for immunoprotection [121], cell transplants have usually
been examined for their eects on lumbar dorsal horn sen-
sory systems in models of pain. But several epidemiologi cal
studies suggest that most or many SCIs are at a cervical
spinal level [122], although chronic pain is a common report
following thoracic SCIs [123]. Most studies report pain in
distal limbs, even following clinically complete injury. But
neuropathic pain, associated with SCI [124] or peripheral
injuries [125] in upper limbs is also a common complaint,
such that testing graft location, cervical as well as lumbar,
in cell therapy studies in a SCI pain model is a reasonable
preclinical approach. Here a cervical graft of GABA hNT2.17
cells completely reverses behavioral sensitivity measured in
the hindlimbs equally as well as a lumbar graft. Although
these diering graft sites were examined some 6 weeks after
transplant with no surviving cervical grafts visible, such
cervical grafts were equally ecacious for the behavioral
hypersensitivity from a low thoracic S CI. Although the lum-
bar i.t. space is larger than the cer vical space, it has relatively
stagnant CSF flow to compensate for intraspinal/intracranial
pressure changes. CSF flow is greatest in the cervical space,
perhaps a result of the “bottleneck” at the entrance to the
brain. However long the g rafts might survive on the cervical
pia (to produce identical behavioral eects as lumbar place-
ment), by the end of the experiment, they will have likely
eroded into the CSF flow. Similar results are seen on both
forelimb and hindlimb behavioral hypersensitivity, when cell