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Stable asymmetry in forelimb use was observed 7 days post-stroke (pre-transplant, Figure 4).
|
[] |
CellFinder
|
Ischemic rats used their impaired forelimbs (contralateral to lesion) during lateral exploration less than they did before stroke.
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CellFinder
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Transplantation of SD56 hNSCs significantly enhanced the independent use of the impaired contralateral forelimb 4 weeks post transplantation (P<0.05 vs pre-transplant).
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CellFinder
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Eight weeks after transplantation the improvement in the use of the impaired forelimb was stable and significant when compared to the pre-transplant group and significantly improved in comparison to vehicle treated group at 8 weeks (Figure 4).
|
[] |
CellFinder
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In the vehicle treated group, the independent use of the contralateral forelimb remained impaired 4 and 8 weeks post-injection.
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CellFinder
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In an independent study and using the same MCAO rat animal model, we found that transplantation of dermal fibroblasts did not improve the stroke-induced motor deficits (unpublished data).10.1371/journal.pone.0001644.g004Figure 4Transplantation of NSCs improves sensorimotor function of the stroke-disabled forelimb.
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CellFinder
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Forelimb use during spontaneous lateral exploration was measured in the cylinder test (see Method and Results sections for details).
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CellFinder
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Groups of vehicle injected (n = 7) and hNSCs (n = 10) transplanted are represented.
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CellFinder
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The animals were tested as described in Method section.
|
[] |
CellFinder
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Note the significant increase in the independent use of the impaired contralateral forelimb at 4 and 8 weeks post transplantation (P<0.05 vs pre-transplant group).
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[
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CellFinder
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The contralateral forelimb remained impaired in the vehicle treated group at 4 and 8 weeks post-injection.
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CellFinder
|
Bars represent percentages±s.e.m.
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[] |
CellFinder
|
of steps taken by the ipsilateral, contralateral and both forelimbs simultaneously. *
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CellFinder
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P<0.05 vs pre-transplant group; #P<0.05 vs vehicle groups.
|
[] |
CellFinder
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Our results indicate that a self-renewable and homogenous population of hNSCs, SD56, was derived from hESCs using defined media supplemented with a specific combination of growth factors.
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CellFinder
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The SD56 hNSCs grew as an adherent monolayer culture, uniformly expressed molecular features of hNSCs including nestin, vimentin and the radial glial marker 3CB2, and did not express the pluripotency markers Oct4 or Nanog.
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CellFinder
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The self-renewal property of the hNSCs was characterized by a predominant symmetrical mode of cell division.
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CellFinder
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They exhibited no chromosomal abnormalities and demonstrated non-tumorigenic properties after implantation into ischemic brains and into naïve nude rat brains and flanks.
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CellFinder
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Furthermore, the transplanted SD56 hNSCs migrated toward the stroke-damaged adult brain parenchyma, engrafted and improved the independent use of the stroke-impaired forelimb.
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[
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CellFinder
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Maintenance of stem cells requires symmetrical and asymmetrical cell divisions to both expand and to give rise to specialized progeny of a specific tissue (reviewed in [23]).
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[
{
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CellFinder
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In vivo, a complex cellular micro-environment or niche ensures the self-maintenance property of NSCs [24], [25], [26], [27].
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[
{
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CellFinder
|
In vitro, defined growth factors and extracellular matrices support stem cell self-renewal [28], [29].
|
[] |
CellFinder
|
The embryonic stem cells can propagate in a predominantly proliferative symmetrical mode, leading to homogeneous cell cultures growing relatively quickly with minimal cell differentiation [30], [31], [32], [33], [34].
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{
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CellFinder
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Tissue specific stem cells, however, self-renew in a predominant asymmetric mode to maintain them selves and compensate for the loss of differentiated cells due to disease or injury.
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[
{
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CellFinder
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Thus, NSCs isolated from developing or adult brain grow as a mixture of undifferentiated and differentiated cells due the predominant asymmetrical mode of cell division [35], [36], [37], [38], [39], [40], [41].
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[
{
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CellFinder
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A recent study has reported that a murine ESC-derived NSC line (LC1) is propagated as an adherent homogenous culture with a dominant mode of symmetrical self-renewal [21].
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CellFinder
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A combination of EGF and FGF2 was sufficient to propagate these NSCs as an adherent monolayer.
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[
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CellFinder
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However, the SD56 hNSC line described here required the combination of EGF, bFGF and LIF for self-maintenance.
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CellFinder
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Although there are morphological and molecular similarities between our hNSCs and the NSCs previously described [21], the methods of isolation and growth are different.
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CellFinder
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In addition to the different combination of growth factors used, the hNSC line we have isolated did not go through the rosette-structure stage.
|
[] |
CellFinder
|
The in vitro analysis of BrdU incorporation and nestin expression indicated that our hNSCs divide predominantly symmetrically.
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[
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CellFinder
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This type of growth pattern is characteristic of primitive normal stem cells undergoing mostly symmetric cell division to increase the stem cell pool at the early stage of development or during tissue regeneration after injury [23].
|
[] |
CellFinder
|
RT-PCR and immunocytochemistry analysis demonstrated that these undifferentiated SD56 cells did not express any pluripotency, endodermal or mesodermal markers.
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CellFinder
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Furthermore, the SD56 hNSCs described here exhibited the multipotential characteristic to differentiate into neurons, astrocytes and oligodendrocytes both in vitro and upon transplantation.
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CellFinder
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Together these findings suggest that the hNSC line we isolated are appropriately programmed and share similar characteristics with the definitive NSCs of the developing brain.
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[
{
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CellFinder
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The SD56 hNSCs demonstrated a remarkable ability to migrate toward the stroke-damaged parenchyma of the adult rat brain.
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CellFinder
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This directed migration by the majority of the grafted cells could be due to their uniform cellular composition, which results in an equal response to the host microenvironment.
|
[] |
CellFinder
|
In previous studies, subpopulations of transplanted hESCs that were enriched in neural cells migrated in host microenvironments conducive to cell migration, such as the developing brain or in structures such as the rostral migratory stream [13], [20].
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CellFinder
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In the adult lesioned brain, the grafted hESC-derived neural cells proliferated and formed rosettes [14], teratomas [12], [15] or a cellular mass that induced a gliotic host response whereby local astrocytes demarcated the grafts [16].
|
[
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CellFinder
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Enriched neural cultures derived from mouse [42] and monkey ESCs [43] have produced behavioral improvements when transplanted into animal models of stroke and brain injury.
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CellFinder
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However, in these cases, the transplanted non-human ESCs also formed a mass with signs of overgrowth in the core, as well as deformations [44], [45], [46].
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CellFinder
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ESCs plated at low density acquire neural identity within few hours after plating [47].
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CellFinder
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Interestingly, nearly all viable cells expressed nestin, the early neural fate marker Sox1, and the pluripotency marker Oct4.
|
[] |
CellFinder
|
Together, these studies are seminal and suggest that complete neuralization may not be achieved through certain enrichment processes, consequently the neural cells could revert to a pluripotent stage [17].
|
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CellFinder
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The dispersion of the grafted hNSCs within host parenchyma may allow for more graft-host interactions that could stabilize differentiation, inhibit growth and prevent gliotic host response.
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[
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CellFinder
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In the present study, SD56 hNSC-transplanted animals demonstrated a stable improvement in the sensorimotor function when evaluated for spontaneous exploratory activity in the cylinder test that detects long-lasting deficits in forelimb use in the experimental models of stroke [22].
|
[
{
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CellFinder
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The transplantation of hNSCs significantly enhanced the independent use of the impaired contralateral forelimb 8 weeks post transplantation.
|
[
{
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"label": "CellType",
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}
] |
CellFinder
|
This is the first report demonstrating that the transplantation of hNSCs derived from hESCs can improve neurologic behavior after experimental stroke.
|
[
{
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{
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CellFinder
|
Together, these findings are encouraging and suggest that these cells are promising for future development.
|
[] |
CellFinder
|
In addition to their therapeutic application, the hNSCs isolated under the reported conditions offer a means to interrogate host environments and unravel mechanistic features of self-renewal, non-tumorigenicity and functional engraftability in animal models of neurological disorders.
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[
{
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CellFinder
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hESC and NSC CulturesThe hESC line H9 (WiCell Research Institute) was propagated every 5 to 7 days on irradiated mouse embryonic fibroblasts.
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{
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CellFinder
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The cell culture media consisted of a 1∶1 mixture of Dulbecco's modified Eagle's medium (DMEM) and F12 nutrient, 20% serum replacement (Invitrogen), 0.1 mM β-mercaptoethanol, 2 µg/ml heparin and 4 ng/ml bFGF (R&D Systems).
|
[] |
CellFinder
|
To generate the NSCs, dissociated hESCs were cultured in a chemically defined medium composed of DMEM/F12 (1∶1) including glucose (0.6%), glutamine (2 mM), sodium bicarbonate (3 mM), and HEPES buffer (5 mM) [all from Sigma except glutamine (Invitrogen)].
|
[
{
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{
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CellFinder
|
A defined hormone mix and salt mixture (Sigma), including insulin (25 mg/ml), transferrin (100 mg/ml), progesterone (20 nM), putrescine (60 mM), and selenium chloride (30 nM) was used in place of serum.
|
[] |
CellFinder
|
The medium was supplemented with EGF (20 ng/ml), bFGF (10 ng/ml) and LIF (10 ng/ml).
|
[] |
CellFinder
|
Dissociated hNSCs were plated at a density of 100,000 cell/ml in Corning T75 (Invitrogen) culture flasks in the defined media together with the growth factors.
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[
{
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CellFinder
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After 5–7 DIV, the adherent culture was incubated in 0.025%trypsin/0.01% EDTA (w/v) for 1 min followed by the addition of trypsin inhibitor (Invitrogen) then gently triturated to achieve single cell suspension.
|
[] |
CellFinder
|
The cells were then washed twice with fresh medium and reseeded in fresh growth factor-containing media at 100,000 cells/ml.
|
[] |
CellFinder
|
This procedure was performed for 21 passages and the fold of increase and population doubling were calculated at each passage.
|
[] |
CellFinder
|
For clonal analysis, single spheres or confluent hNSC cultures were single cell dissociated and serially diluted to yield 1–2 cell/10 µl.
|
[] |
CellFinder
|
A 10-µl-cell suspension was then added to each of 96 or 24 well plates containing 200–300 µl of growth media.
|
[] |
CellFinder
|
Wells containing one viable cell were marked the next day and re-scored 5 to 7 days later for cell proliferation.
|
[] |
CellFinder
|
The differentiation of the hNSCs was performed as previously described [48].
|
[
{
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CellFinder
|
Dissociated hNSCs were plated at a density of 106 cells/ml in control (media/hormone mix) medium devoid of any growth factors and supplemented with 1% fetal bovine serum (FBS) on poly-L-ornithine-coated (15 mg/ml; Sigma) glass coverslips in 24-well Nunclon culture dishes with 0.5 ml/well.
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{
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CellFinder
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After 2, 7–15 DIV cultures were fixed and processed for immunocytochemistry or used for RT-PCR analysis.
|
[] |
CellFinder
|
Karyotype analysisLong-term cultures of hNSCs were incubated at 37°C and harvested for metaphase chromosomes when the cultures were 75% confluent.
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[
{
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}
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CellFinder
|
Metaphase chromosomes were obtained by standard chromosome harvest methods by exposure to Colcemid at 0.1 µg/ml for 1 hour at 37°C, a 2-minute exposure to trypsin/EDTA, hypotonized with 0.057 M KCl and fixed with 3∶1 methanol:acetic acid.
|
[] |
CellFinder
|
Slide preparations were made by dropping the fixed cell pellet onto cold, wet slides and air-dried.
|
[] |
CellFinder
|
After incubating the slides at 90°C for 30 minutes, chromosomes were trypsin banded and then Wright/Giemsa stained for G-banding analysis.
|
[] |
CellFinder
|
Twenty metaphase cells were completely analyzed and a normal female chromosome complement was found (46,XX).
|
[
{
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CellFinder
|
Tumorigenicity in nude ratsAll animal experiments were conducted according to the National Institute of Health (NIH) guidelines and approved by the IACUC.
|
[] |
CellFinder
|
Normal adult NIH-Nude rats (n = 5, 8 week-old, Taconic, Germantown, New York, United States) were used to test the tumorigenic potential of the SD56 hNSCs.
|
[
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CellFinder
|
Undifferentiated hNSCs from passage 9 were single cell dissociated using trypsin-EDTA and suspended at the concentration of 125,000 cell/µl in preparation for cell transplantation.
|
[
{
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] |
CellFinder
|
Two µl of the cell suspension were stereotaxically transplanted into 4 sites within the striatum at the following coordinates: AP: +1.0 mm, ML: +3.2 mm, DV: −5.0; AP: +0.5 mm, ML: +3.0 mm, DV: −5.0; AP: −0.5 mm, ML: +3.0 mm, DV: −5.0; AP: −1.0 mm, ML: +3.5 mm, DV: −5.0 mm with the incisor bar set at 3.4 mm.
|
[] |
CellFinder
|
The injection rate was 1 µl/min, and the cannula was left in place for an additional 5 min before retraction.
|
[] |
CellFinder
|
For the flank tumor assay, 2×106 cells (125,000 cell/µl) were injected subcutaneously to the side of the adult nude rats.
|
[
{
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}
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CellFinder
|
To label mitotically active cells in vivo during S-phase, the rats were injected IP with the BrdU (50 mg/kg, Sigma) every 8 hours during the last 24 hours before euthanasia.
|
[] |
CellFinder
|
After 2-month survival time, rats were euthanized and a postmortem examination for tumor formation was performed.
|
[] |
CellFinder
|
Induction of Focal Ischemia and Cell TransplantationAll animal experimentations were conducted according to the National Institute of Health (NIH) guidelines and approved by the IACUC.
|
[] |
CellFinder
|
Sprague Dawley adult male rats (n = 17, 275g–310g, Charles River Laboratories, Wilmington, Massachusetts, United States) were subjected to one and a half hour suture occlusion of the middle cerebral artery (MCAO), as previously described [49] and immunosuppressed 2 days before cell transplantation and daily thereafter for one week with i.p.
|
[
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CellFinder
|
injections of cyclosporine A (20 mg/ml, Sandimmune, Novartis Pharmaceuticals).
|
[] |
CellFinder
|
Thereafter oral cyclosporine was used at 210 µg/ml in drinking water until euthanasia.
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[] |
CellFinder
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Undifferentiated SD56 hNSCs from passages between P9 and P13 were single cell dissociated using trypsin-EDTA in preparation for cell transplantation.
|
[
{
"end": 27,
"label": "CellType",
"start": 22,
"text": null
},
{
"end": 21,
"label": "CellLine",
"start": 17,
"text": null
}
] |
CellFinder
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One week after the stroke lesion, 2 µl of the hNSCs, at a concentration of 50,000 cell/µl, were stereotaxically transplanted into 4 sites within the lesioned striatum (n = 10) at the following coordinates: AP: +1.0 mm, ML: +3.2 mm, DV: −5.0; AP: +0.5 mm, ML: +3.0 mm, DV: −5.0; AP: −0.5 mm, ML: +3.0 mm, DV: −5.0; AP: −1.0 mm, ML: +3.5 mm, DV: −5.0 mm with the incisor bar set at 3.4 mm.
|
[
{
"end": 51,
"label": "CellType",
"start": 46,
"text": null
}
] |
CellFinder
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The injection rate was 1 µl/min, and the cannula was left in place for an additional 5 min before retraction.
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[] |
CellFinder
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As a control group, we used rats subjected to ischemia and injected with the vehicle (n = 7).
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[] |
CellFinder
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All animals underwent baseline motor behavioral assessment before and after the ischemic lesion, and 4 & 8 weeks after cell transplantation.
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The animals were killed after 2-month survival time by transcardial perfusion with phosphate buffered saline (PBS) followed by 4% paraformaldehyde.
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CellFinder
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The brains were cryoprotected in an increasing gradient of 10, 20 and 30% sucrose solution and cryostat sectioned at 40 µm and processed for immunocytochemistry.
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[] |
CellFinder
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ImmunocytochemistryCultures were fixed with 4% paraformaldehyde for 15 min.
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CellFinder
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Both cells and brain sections were rinsed in PBS for 3×5 min then incubated for 2 hrs (cultures) or overnight (brain sections) with the appropriate primary antibodies for multiple labeling.
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CellFinder
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Secondary antibodies raised in the appropriate hosts and conjugated to FITC, RITC, AMCA, CY3 or CY5 chromogenes (Jackson ImmunoResearch) were used.
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CellFinder
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Cells and sections were counterstained with the nuclear marker 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI).
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[] |
CellFinder
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Positive and negative controls were included in each run.
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[] |
CellFinder
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Immunostained sections were coverslipped using fluorsave (Calbiochem) as the mounting medium.
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[] |
CellFinder
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The following antibodies were used: Anti-human Nuclei (hNuc, monoclonal 1∶100, Chemicon), Anti-TuJ1 (monoclonal 1∶100, Covance; Polyclonal 1∶200, Aves Labs); anti-GAD65/67 (polyclonal 1∶1000, Chemicon); Anti-glial fibrillary acidic protein (GFAP, monoclonal 1∶1000, Chemicon; polyclonal 1∶200, Aves Labs); Anti-galactocerebrocide (GC, monoclonal 1∶250, Chemicon); Anti-CNPase (polyclonal 1∶200, Aves Labs); Anti-Glucose Transporter type 1 (Glut-1 polyclonal, 1∶500, Chemicon); Anti-Nestin (polyclonal 1∶1000, Chemicon); Anti-vimentin (monoclonal 1∶500, Calbiochem); Anti-3CB2 (monoclonal 1∶500, Developmental Studies Hybridoma Bank); Anti-doublecortin (DCX, polyclonal 1∶250, SantaCruz Biotechnology); Anti-Ki67 (polyclonal 1∶250, SantaCruz Biotechnology).
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[] |
CellFinder
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Fluorescence was detected, analyzed and photographed with a Zeiss LSM550 laser scanning confocal photomicroscope.
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[] |
CellFinder
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For each animal, quantitative estimates of the total number of grafted cells were stereologically determined using the optical fractionator procedure [50].
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[] |
CellFinder
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A computer-assisted image analysis system was performed using Stereo Investigator software (MicroBrightField, Inc.).
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[] |
CellFinder
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The rostral and caudal limits of the reference volume were determined by first and last frontal sections containing grafted cells.
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[
{
"end": 11,
"label": "Tissue",
"start": 4,
"text": null
},
{
"end": 22,
"label": "Tissue",
"start": 16,
"text": null
}
] |
CellFinder
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