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The inner cores of these grafts contained tumorigenic precursor cells (reviewed in [17]).
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These findings suggest that neural cells generated by acute exposure to growth factors and/or morphogens may still be heterogeneous and potentially tumorigenic.
[ { "end": 40, "label": "CellType", "start": 28, "text": null }, { "end": 34, "label": "Tissue", "start": 28, "text": null } ]
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We report an alternative method for the isolation and the perpetuation of a multipotent hNSC line from the hESCs with a primitive mode of self-renewal.
[ { "end": 112, "label": "CellType", "start": 107, "text": null } ]
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We also demonstrate their long-term expansion, non-tumorigenic properties and functional engraftability in an experimental model of stroke.
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1.
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In vitro isolation, growth and differentiation of hESC-derived hNSCsThe hESCs were maintained and expanded on mouse feeder layer in media supplemented with bFGF (Figure 1A).
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After cell dissociation, a portion of the hESCs was cultured in serum free medium containing EGF, bFGF and LIF.
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These factors are known to stimulate the proliferation of human fetal-derived NSCs [18], [19].
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After 3 days in vitro (DIV), there was selective survival and growth of cells that aggregated in clusters or spheres (Figure 1B).
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These primary spheres were harvested and replated in fresh media.
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During the following week, the spheres attached to the flask and a fibroblast-like cell population began to migrate out (Figure 1C).
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Secondary spheres (2° spheres) were generated from these cultures and lifted off by the end of the week leaving a hollow in the middle of the attached cells (Figure 1D).
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The floating 2° spheres were collected and replated in fresh growth medium for 2 weeks.
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The cultures were then passaged by collagenase cell dissociation every 7 DIV for an additional 4 passages (Figure S1).
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At the 5th and 6th passages, spheres were dissociated into a single-cell suspension using trypsin-EDTA.
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At this stage there was a change in the hNSCs' adherent properties, and the cells began to grow as a monolayer with multiple foci of cells throughout the culture (Figure 1F).
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The adherent hNSC culture stained uniformly for nestin (Figure 1K), vimentin (Figure 1L) and with the radial glial marker 3CB2 (Figure 1M) indicating their homogeneity and NSC identity.
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Under these culture conditions, it is noteworthy that we did not observe the formation of rosettes which has been previously reported to occur under certain conditions during neuralization of hESCs [8], [20], [21].
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RT-PCR analysis confirmed that these hNSCs did not express the pluripotency transcripts Oct-4 and Nanog (Figure 1I).
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Furthermore, the hNSCs did not express transcripts for brachyury and foxa2, marker genes for mesoderm and endoderm respectively (negative result, data not shown).10.1371/journal.pone.0001644.g001Figure 1Isolation and purification of hNSCs from the hESCs.
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The hESCs were grown on a mouse feeder layer (A).
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Primary neurospheres (B) were isolated and replated to eliminate other non-neural cells.
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The selectively harvested secondary neurospheres (arrow in C), left behind hollow cores in the surface area (star in D) where they attached earlier.
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They were perpetuated for an additional 5 passages (E).
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These 2° spheres were then passaged using a single cell dissociation protocol (F).
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Arrow in F shows an example of a focus of proliferating cells. (
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G, H) The hNSCs were passaged every 5–7 days, as described in the Methods section.
[ { "end": 15, "label": "CellType", "start": 10, "text": null } ]
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Starting from an initial population of 1 million cells, the cumulative cell number was calculated at each passage as the fold of increase×the total cell number and plotted as logarithm with base 2 in function of time (G).
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The cell perpetuation (G) and population doubling (H) analysis demonstrated the continuous and stable growth of the hNSCs. (
[ { "end": 121, "label": "CellType", "start": 116, "text": null } ]
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I) RT-PCR analysis showing the down regulation of the pluripotency transcripts Oct4 and Nanog in secondary neurospheres and in expanded hNSCs at passage 8 (P8). (
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J) Cytogenetic evaluation of the SD56 hNSCs line at passage 12 by standard G-banding was performed.
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Twenty metaphase cells were analyzed and showed a normal female chromosome complement (46,XX).
[ { "end": 63, "label": "Tissue", "start": 57, "text": null } ]
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Isolated and expanded hNSCs expressed the neural precursor cell markers nestin (K), Vimentin (L) and the radial glial cell marker 3CB2 (M) in virtually all the progeny. (
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N-P) Clonal self-renewal ability of the isolated hNSCs through symmetrical cell division.
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Single (N), two-cell stage (O) and four-cell stage (P) of a hNSC proliferating over a 3-day culture period.
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Note the symmetrical segregation of BrdU and nestin in the progeny.
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Bars: (A, B, C, D) 200 µm; (E, F) 100 µm; (K–M) 20 µm; (N–P) 10 µm.
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To ascertain self-renewal ability under clonal conditions, a single cell suspension was plated at clonal density (1–2 cell/10 µl).
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To determine if the hNSCs divide symmetrically, we pulsed cultures with the thymidine analog, bromodeoxyuridine (BrdU), after plating and looked for the expression of nestin in the progeny.
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Cultures were fixed after 1, 2 or 3 DIV (Figure 1N–P).
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After 2 days, plated single cells first underwent a symmetric cell division and gave rise to daughter cells that were both positive for BrdU and nestin.
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The clone of cells continued to grow over the 3 DIV and all the progeny expressed nestin.
[ { "end": 71, "label": "CellType", "start": 64, "text": null } ]
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BrdU labeling demonstrated that it was rare for only one daughter cell to inherit the BrdU and thus had undergone asymmetric segregation of the chromatids (Figure S2).
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G-band karyotyping of these hNSCs confirmed the normal, non-transformed nature of cells after 12 passages (Figure 1J).
[ { "end": 33, "label": "CellType", "start": 28, "text": null } ]
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We named the derived hNSCs SD56 (intermittently referred to as SD56 hNSCs or hNSCs).Under these defined growth conditions, the hNSCs showed stable growth with a 2.7±0.2 fold increase every 5 to 7 days (Figure 1G).
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The population doubling at each passage averaged at 1.4±0.1 (Figure 1H).
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The viability of hNSCs at each passage was consistent at the approximate value of 98%.
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The projected cumulative cell numbers demonstrated that trillions of cells could be generated over a period of 5 months (Figure 1G).
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We expanded the isolated hNSCs lines for over 20 passages with a stable phenotype.
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An initial cell bank of 75 vials containing 2 to 5 million cells each was generated and cryopreserved.
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Upon removal of the mitogenic factors and plating on a coverslip pre-coated with poly-L-ornithine (PLO) substrate, the hNSCs spontaneously differentiated into neurons, astrocytes and oligodendrocytes, a property that is consistent with normal multipotent hNSCs (Figure 2).
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After 2 DIV, hNSCs expressed transcripts for the neural-specific genes nestin, Notch1 and neural cell adhesion molecule (N-CAM) (Figure 2A) and for the lineage specific markers β-tubulin class III, medium-size neurofilament (NF-M) and microtubule-associated protein 2 (MAP-2) for neurons, GFAP for astrocytes and myelin basic protein (MBP) for oligodendrocytes (Figure 2A).
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Transcripts for the GABAergic cell marker glutamic acid decarboxylase (GAD) were expressed, but transcripts for the tyrosine hydroxylase (TH), a marker for dopaminergic neurons, were undetectable.
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Immunocytochemical analysis (Figure 2B–F) of 10 day-old cultures demonstrated that the proportion of nestin+ cells was 36.6±2.7%, 62.5±2.8% expressed the neuronal marker TuJ1, 1.9±0.3% expressed the astrocytic marker GFAP and 7.1±0.4% were oligodendrocytes and expressed galactocerebrocide (GC) (Figure 2F).
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A subset (9.8±1.6%) of the GFAP+ astrocytes co-expressed nestin.10.1371/journal.pone.0001644.g002Figure 2hESC-derived hNSCs spontaneously differentiated into the 3 principal central nervous system cell types.
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Dissociated hNSCs were washed free of growth factors and plated on poly-L-onithine coated glass coverslips.
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Differentiated cultures were either harvested after 2 DIV for total RNA extraction and RT-PCR analysis or fixed after 10 DIV and processed for indirect immunocytochemistry. (
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A) Differentiated hNSCs expressed the neural-specific transcripts nestin and Notch1 as well as transcripts: for neurons (β-tubulin class III, MAP-2, NCAM and medium-size neurofilament, NF-M), for astrocytes (GFAP) and for oligodendrocytes (MBP).
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The hNSCs expressed transcripts for GAD, but not for TH.
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B, C & D, morphology of NSC-derived progeny differentiated into GFAP+ astrocytes (B), GC-expressing oligodendrocytes (C) and TuJ1+ neurons (D), DAPI (blue) show life cell nuclei. (
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E) Photo showing cultures double-immunostained for TuJ1 (green) and nestin (red) (DAPI, blue). (
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F) Quantitative analysis of immunostained 10 day-old cultures for the 3 neural cell types.
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Results are mean±s.e.m.
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of three independent experiments, each performed in duplicate.
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Bars: (c) 20 µm; (d, e) 10 µm.
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2.
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The isolated hNSCs are normal and do not form tumors in normal nude ratsThe self-renewal and pluripotent abilities of the hESCs are also associated with tumorigenic properties.
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Therefore, the first critical step toward developing therapeutic hNSCs is to verify that they are non-tumorigenic.
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The monolayer culture of SD56 hNSCs clearly demonstrated contact inhibition of growth, a normal karyotype and did not express the pluripotency transcripts Oct-4 and Nanog.
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Removal of mitogenic factors and continued culture on plastic resulted in cell senescence that is characteristic of non-transformed cells.
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To determine whether SD56 hNSCs form tumors in vivo, we transplanted them at high density (see Methods) into the forebrain and subcutaneously into the flank of nude rats.
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The animals were kept for a two-month post-transplant survival period.
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To label mitotically active cells in vivo during S-phase, the rats were injected IP with BrdU (50 mg/kg) every 8 hours during the last 24 hours before euthanasia.
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The transit amplifying endogenous precursors located in the subventricular zone (SVZ) were labeled (Figure S3); however, we were unable to detect grafted SD56 hNSCs co-localizing the human-specific nuclear marker hNuc and BrdU (Figure S3).
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No surviving SD56 hNSCs were detected in the flank of the transplanted animals suggesting that the grafted cells are not tumorigenic.
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3.
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Transplanted cells survived, migrated toward and engrafted into the stroke-damaged host tissueTo investigate the survival and functional engraftment in an injury environment, hNSCs (4×105) were transplanted into the ischemic boundary zone in the rat striatum one week after the middle cerebral artery occlusion (MCAO) was performed.
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Animals were euthanized two months later and the brains processed for histo-pathology and immunocytochemistry.
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Grafted SD56 hNSCs, identified with hNuc, demonstrated a 37.0±15.8% survival rate and a remarkable dispersion toward the stroke-damaged tissue with no sign of overgrowth or tumorigenesis.
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The majority of grafted cells (61.2±4.7%) migrated at least 200 µm away from the injection site and penetrated an average distance of 806.4±49.3 µm into the stroke-damaged tissue (Figure 3A–C).
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Immunostaining with the blood vessel marker, GluT1, revealed dilated vessels in the infarcted striatum and a close association between vessels and the grafted hNSCs (Figure 3B, 3C).
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The grafted cells rarely expressed the proliferation marker Ki67 (Figure 3D), 29.8±4.4% expressed nestin (Figure 3E), 6.5±0.9% expressed doublecortin (DCX) and 60.8±8.1% were TuJ1+ (Figure 3F, G).
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Grafted cells rarely co-expressed the astroglial marker GFAP (Figure 3H) or differentiated into CNPase-expressing oligodendrocytes (Figure 3I).
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Immunostaining for GAD demonstrated that 25.1±2.3% of grafted hNSCs differentiated into GABAergic neurons while less than 2% were positive for glutamate (Figure 3J, K).10.1371/journal.pone.0001644.g003Figure 3Dispersion, engraftment and differentiation of the hNSCs in stroke-lesioned animals.(A) Schematic drawing of a frontal section through the striatum illustrating the dispersion of grafted hNSCs in the focal ischemia-lesioned parenchyma (shaded area). (
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B, C) Photos show frontal sections through the graft in the striatum immunostained with the human specific antibodies: anti-hNuc (green in B & C) and anti-GluT1 (red, B & C) showing blood vessels and dispersed hNSCs in the graft zone.
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C: higher magnification of the inset in B. (D–I) Photos taken from frontal sections through the graft in the striatum double immunoprocessed for cell proliferation and neural lineage markers. (
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D) Note the endogenous Ki67+ cells (red cells, arrow) in the stroke damaged area and the hNuc+ (green)/Ki67- grafted hNSCs (arrowheads). (
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E) Examples of grafted SD56 hNSCs showing co-expression of hNuc (green) and nestin (red). (
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F) Confocal 3D reconstructed orthogonal images of the hNuc+(green)/DCX+(red) NSCs (arrowheads) viewed in the x-z plan on the top and y-z plan on the right. (
[ { "end": 81, "label": "CellType", "start": 77, "text": null } ]
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G) Examples show the majority of grafted NSC progeny co-expressing hNuc (red) and the neuronal marker TuJ1 (green).
[ { "end": 94, "label": "Tissue", "start": 86, "text": null } ]
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Grafted NSCs rarely differentiate into GFAP+ astrocytes (H).
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In I, rare example of grafted NSC progeny becoming an oligodendrocyte identified by the expression of CNPase (green).
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Grafted NSCs expressed the GABAergic marker GAD65/67 (J) and rarely expressed glutamate (K). (
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Abbreviations: Cx: cortex, Str: striatum).
[ { "end": 40, "label": "Tissue", "start": 32, "text": null }, { "end": 25, "label": "Tissue", "start": 19, "text": null } ]
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Bars: (B, C) 100 µm; (D, F) 20 µm; (E, G–K) 10 µm.
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4.
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Transplanted cells improve sensorimotor function of the stroke-disabled forelimbWe asked whether transplanted SD56 hNSCs could enhance the recovery of sensorimotor function that is compromised in the stroke-injured rats.
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We used the cylinder test to measure the sensorimotor asymmetry in forelimb use during spontaneous exploration [22].
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To establish the baseline of the stroke-induced sensorimotor deficit, spontaneous behavior of rats in a transparent cylinder was videotaped one week after stroke (pre-transplant, Figure 4).
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Tests were then conducted 4 and 8 weeks after vehicle and SD56 hNSCs transplantation.
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