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The striatum and cortex were accurately outlined at low magnification (2.5× objective).
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CellFinder
The optical fractionator probe was selected to perform systematic sampling of the immunoreactive cell population distributed within the serial sections to estimate the population number in the volume of tissue.
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The counting frame of the optical fractionator was defined at 50×50 µm squares and the systematic sampling was performed by translating a grid with 200×200 µm squares onto the sections of interest using the Stereo Investigator software.
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The sample sites were systematically and automatically generated by the computer and examined using a 60× objective of a Nikon Eclipse TE 300 microscope.
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The counting frame displayed inclusion and exclusion lines and only immunoreactive cell bodies falling within the counting frame with no contact with the exclusion lines were counted.
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The cell dispersion was measured by counting the number of cells within 200 µm distance from the graft site.
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The number and distance in µm of cells dispersed beyond 200 µm was also measured.
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An average of 2,000 cells was counted per animal.
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Double labeling was determined using the confocal laser scanning microscope by random sampling of 100 or more cells per marker for each animal, scoring first for hNuc+, followed by DAPI+ nuclei and then the marker of choice.
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The double labeling was always confirmed in x-z and y-z cross-sections produced by the orthogonal projections of z-series.
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Reverse Transcription-Polymerase Chain Reaction (RT-PCR) analysisTotal RNA was extracted from cultured cells using RNAeasy kit (Quiagen).
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Aliquots (1 µg) of total RNA from the cells were reverse transcribed in the presence of 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 0.5 µM dNTPs, and 0.5 µg oligo-dT(12–18) with 200 U Superscript RNase H-Reverse Transcriptase (Invitrogen).
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PCR amplification was performed using standard procedure with Taq Polymerase.
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Aliquots of cDNA equivalent to 50 ng of total RNA were amplified in 25 µl reactions containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2 , 50 pmol of each primer, 400 µM dNTPs, and 0.5 U AmpliTaq DNA polymerase (Perkin-Elmer).
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CellFinder
PCR was performed using the following thermal profile: 4 min at 94°C; 1 min at 94°C, 1 min at 60°C, 1.5 min at 72°C, for 30–40 cycles; 7 min at 72°C, and finally a soak at 4°C overnight.
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The following day, 10 µl aliquots of the amplified products were run on a 2% agarose Tris–acetate gel containing 0.5 mg/ml ethidium bromide.
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The products were visualized through a UV transilluminator, captured in a digital format using Quantify One Gel Analysis software (Bio-Rad Laboratories) on a Macintosh G4 computer.
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The PCR primers specific to each transcript were as follows: GFAP, forward (F), 5′-TCATCGCTCAGGAGGTCCTT–3′ Reverse (R), 5′-CTG TTGCCAGAGATGGAGGTT–3′; MAP2 (F) 5′-GAAGACTCGCATCCGAATGG–3′, (R) 5′-CGCAGGATAGGAGGAAGAGACT–3′; MBP (F) 5′-TTAGCTGAATTC GCGTGTGG–3′, (R) 5′-GAGGAAGTGAATGAGCCGGTTA-3′ were deigned using the Primer Designer software, Version 2.0 (Scientific and Educational Software) [48].
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18S, β-tubulin class III, N-CAM, Nestin, NF-M, Notch-1 primers [51].
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Oct4, Nanog primers [11].
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FOXa2 (HNF3B), Brachyury primers [52].
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Behavioral testsThe cylinder test was used to assess the spontaneous forelimb use during lateral exploration movement [22].
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Rats were placed in a transparent acrylic cylinder (20 cm diameter) for 5 minutes.
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The cylinder encourages use of the forelimbs for vertical exploration.
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A mirror was placed behind the cylinder so that the forelimbs could be viewed at all times.
[ { "end": 61, "label": "Tissue", "start": 52, "text": null } ]
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Testing sessions were videotaped and forelimb use was scored by a blinded operator.
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Movements scored were the independent use of the left or right forelimb or simultaneous use of both the left and right forelimb to contact the wall of the cylinder during a full rear, to initiate a weight-shifting movement, or to regain center of gravity while moving laterally in a vertical posture along the wall.
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Animals were tested for their baselines after stroke and 4 and 8 weeks after cell transplantation.
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Statistical analysisOutcome measurement for each experiment was reported as mean±SEM.
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All data were analyzed using SPSS 11 for Mac OS X (SPSS Inc.).
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Significance of inter-group differences was performed by applying Student's t-test where appropriate.
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The One-Way ANOVA analysis was used to compare group differences for the forelimb use as the dependant variable and groups as the single independent factor variable.
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Differences between the groups were determined using Bonferroni's post hoc test.
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A P-value of less than 0.05 was considered to be statistically significant.
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BackgroundUsing antibodies to specific protein antigens is the method of choice to assign and identify cell lineage through simultaneous analysis of surface molecules and intracellular markers.
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Embryonic stem cell research can be benefited from using antibodies specific to transcriptional factors/markers that contribute to the "stemness" phenotype or critical for cell lineage.
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ResultsIn this report, we have developed and validated antibodies (either monoclonal or polyclonal) specific to human embryonic stem cell antigens and early differentiation transcriptional factors/markers that are critical for cell differentiation into definite lineage.
[ { "end": 137, "label": "CellType", "start": 118, "text": null }, { "end": 137, "label": "CellType", "start": 112, "text": null } ]
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ConclusionThese antibodies enable stem cell biologists to conveniently identify stem cell characteristics and to quantitatively assess differentiation.
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Although the stem cell concept was introduced decades ago, to date, stem cells can only be defined functionally, not morphologically or phenotypically.
[ { "end": 79, "label": "CellType", "start": 69, "text": null } ]
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Two functions define stem cells.
[ { "end": 31, "label": "CellType", "start": 21, "text": null } ]
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Firstly, they are self-renewing, thus able to propagate to generate additional stem cells.
[ { "end": 89, "label": "CellType", "start": 79, "text": null } ]
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Secondly they can differentiate into various progenitor cells, which commit to further maturation along a specific lineage.
[ { "end": 61, "label": "CellType", "start": 45, "text": null } ]
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While stem cells can be best defined functionally, a good number of molecular markers have been used to prospectively identify various stem cell populations.
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Although the functional importance of many of these antigens remains unknown, their unique expression pattern and timing of expression provide a useful tool for scientists to identify as well as isolate stem cells.
[ { "end": 213, "label": "CellType", "start": 203, "text": null } ]
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Embryonic stem cells (ESC), derived from the inner cell mass of pre-implantation embryos, have been recognized as the earliest stem cell population [1,2].
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This pluripotent population can differentiate into all somatic tissue including germ cells.
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In the case of human ESC, they can differentiate into some extra-embryonic derivatives as well.
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Like mouse ESC, human ES cells can be maintained and propagated on mouse fibroblast feeders for extended periods in media containing basic fibroblast growth factor (bFGF) [3].
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Gene expression of undifferentiated human ES cells has been investigated among several ES cell lines by a variety of techniques.
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They include comparison with databases, reverse transcriptase-polymerase chain reaction, focused cDNA microarrays, and immunocytochemistry.
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A list of molecules comprised of known ES-specific or -highly expressed genes and candidates that can serve as markers for human ESCs and may also contribute to the "stemness" phenotype has been established [3-11].
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For example, pluripotent ESC can be characterized by high level expression of Oct3/4 (POU domain, class 5, transcription factor 1, Pou5f1) and Nanog, which are a member of POU domain and homeobox transcription factors respectively.
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A critical amount of Oct3/4 and Nanog expression is required to sustain stem-cell pluripotency and both of these markers are downregulated as cells differentiate in vitro and in vivo [4-9].
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Antibodies to Oct3/4 which cross react with human Oct 3/4 have been widely used to monitor the presence of undifferentiated ESC.
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No single marker however is sufficient or unique for identifying ESCs.
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Oct3/4 for example is expressed by germ cells and may be expressed by specific populations later in development.
[ { "end": 45, "label": "CellType", "start": 35, "text": null } ]
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Likewise, Nanog has been shown to express in other tissues.
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We and other have noted however, that while no single marker is sufficient a constellation of positive and negative markers can in concert unambiguously allow one to define the state of ESC cultures and that surface markers in combination can be used to prospectively sort for ESC.
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Based on published data at the level of gene expression, we have cloned a number of candidate marker genes.
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We have also expressed the recombinant protein and generated a panel of monoclonal or polyclonal antibodies to these proteins.
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Using these antibodies we have confirmed the specificity and selectivity of these antibodies on several ESC lines and established their utility as stem cells markers.
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Our results confirm the expression pattern and timing of these cell markers at the protein level, whereas previous data reported at the level of gene expression.
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Characterization of undifferentiated human ES cells and differentiated EBs by antibodiesAll monoclonal antibodies were initially selected for their abilities to recognize recombinant proteins in direct ELISAs.
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A subset were also tested by Western Blot analysis using recombinant proteins and cell lysate to confirm binding to a single epitope.
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The best clone was later screened for its applications for immunocytochemistry and flow cytometry using various cell lines.
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Human peripheral blood platelets were used for screening mouse anti-human CD9 antibody.
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MCF-7 cells were used for screening mouse anti-human E-Cadherin and PODXL (podocalyxin-like) antibodies.
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MG-63 cells were used for screening mouse anti-human GATA1 (GATA binding protein 1) antibody.
[ { "end": 5, "label": "CellLine", "start": 0, "text": null } ]
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Beta-TC6 cells were used for screening for mouse anti-human/mouse PDX-1 (pancreatic duodenal homeobox-1) antibody.
[ { "end": 8, "label": "CellLine", "start": 0, "text": null } ]
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NTERA-2 cells were used for screening mouse anti-human Oct3/4 and SOX2 (sex-determining region Y-box 2) antibodies.
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All polyclonal antibodies were affinity-purified using recombinant proteins and validated by direct ELISAs and Western.
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Caco-2 cells were used for validation of goat anti-human GATA6 antibody and NTERA-2 cells were used for validation of goat anti-human Nanog and anti-human Oct3/4 antibodies (Summarized in Table 1).Table 1Summary list of antibody verification by western blot.
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AntibodySample used for analysisMol.
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Wt.
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(KD)Gt × hBrachyurymouse ES-derived EB lysate48Ms × hDPPA5N/AN/AGt × hGATA6Caco2 cell lysate65Gt × hNanogNTERA-2 cell lysate33Gt × hOct 3/4NTERA-2 cell lysate39Gt × hPDX1beta-TC 6 cell lysate32Gt × hSOX17mouse ES-derived EB lysate45Ms × hCD9PBMC25Rt × hGATA-1N/AN/AMs × hE-CadherinMCF-7 cell lysate97Ms × hPODXLMCF-7 cell lysate57Ms × hSOX2NTERA-2 cell lysate36N/A: 1.
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DPPA5 is still being subcloned.
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Only Elisa verification is available.2.
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The clone for GATA-1 (MAB1779) does not work for Western blot application but is useful for IHC, The clone picked for Western blot analysis does not work for IHC (MAB17791, see data in ).After antibodies were validated in direct ELISAs, Western blot or cell lines (Fig. 1 and data not shown), they were used to examine the expression of individual molecules in undifferentiated human ES cells and differentiated EBs.
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When examined by immunohistochemistry, high level of expressions of Oct3/4, SOX2, E-Cadherin, PODXL and Nanog were observed in undifferentiated human ES cells (Fig. 2A, 2B and 2C).
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CellFinder
DPPA5 (developmental pluripotency associated 5) expression was also observed in undifferentiated human ES cells (data not shown).
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We noted that a subset of the proteins used were membrane bound proteins.
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To test if any of the antibodies generated could recognize an extracellular epitope and thus be used for live cell sorting, we repeated staining of live cells as previously described.
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The CD9, E-Cadherin and PODXL antibodies recognized an extracellular epitope and their ability to select cells by FACS was confirmed (Fig. 3).
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Minimal or no expressions of Oct3/4, E-Cadherin, PODXL and Nanog were detected in the differentiated EBs (Fig. 2D, 2E and 2F).
[ { "end": 104, "label": "Tissue", "start": 101, "text": null } ]
CellFinder
However, SOX2 expression, which is observed in neural progenitor cells, is persistent in subsets of EBs.
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Figure 1Western blot analysis for Gt × hOct3/4 (A), Gt × hNanog (B) and Ms × hSOX2 (C) in NTERA-2 cell lysate, Ms × hE-Cadherin (D) in MCF-7 cell lysate, Ms × hCD9 (E) in PBMC lysate and Ms × hPDX-1(F) in β-TC-6 cell lysate.
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Numbers indicate the positions of molecular weight markers.
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Figure 2Undifferentiated human ES cells (A, B, and C) and differentiated EBs (D, E and F) were analyzed using antibodies to indicated molecular markers.
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Immunostaining with goat anti-human Oct3/4 (Red in A and D), mouse anti-human SOX2 (Green in A and D), goat anti-human E-Cadherin (Red in B and E), mouse anti-human PODXL (Green in B and E), and goat anti-human Nanog (Red in C and F), are contrasted with DAPI nuclear staining (Blue in C-F).
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CellFinder
Note the dramatic downregulation of ESC specific markers (Oct3/4, E-Cadherin, PODXL, and Nanog) in EBs.
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CellFinder
However, SOX2 expression is persistent in subsets of EB cells.
[ { "end": 61, "label": "CellType", "start": 53, "text": null } ]
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Scale bars = 100 μm.
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Figure 3Human embryonic stem cells stained with anti-CD9 (A), anti-E-Cadherin (B), and anti-PODXL (C) and antigen expression detected by a flow cytometer.
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The specific staining is indicated by green histogram and corresponding isotype control is indicated by black histogram.
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Suspension culture with FGF withdrawal is known to induce differentiation of ES cells to all three germ layer precursors [12].
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The differentiation status of the EB used here was detected to contain all germ cell markers by RT-PCR (Fig. 4).
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In order to examine how more antibodies can be used for characterization of early differentiation events from human ES cells, we examined the expressions of endodermal markers, SOX17, GATA6 and PDX-1, and mesodermal markers, Brachyury and GATA1, in the undifferentiated human ES cells and differentiated EBs.
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CellFinder
Expressions of SOX17, GATA6, PDX-1, Brachyury and GATA1 were not detected in undifferentiated human ES cells (data not shown).
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In contrast to the undifferentiated ES cells, subpopulations of SOX17-, GATA6-, Brachyury- and GATA1-positive cells were observed (Fig 4).
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