Journal: PLoS ONE

Article Title: Pathogen Sensing Pathways in Human Embryonic Stem Cell Derived-Endothelial Cells: Role of NOD1 Receptors

doi: 10.1371/journal.pone.0091119

Figure Lengend Snippet: (A) TLR4 and NOD1 expression in hESC-EC (relative to expression in HUVEC) in vitro . Data are mean ± SEM (n = 3). Statistical significance was determined by one-sample t-test (*p<0.05) for NOD1 vs. TLR4 expression. (B) Representative immunocytochemistry images of hESC-EC (top) and HUVEC (bottom) stained for the NF-κB p65-subunit (red) in response to 1 hour treatment with or without, C12-iE-DAP (NOD1 agonist; 10 µg/ml), LPS (TLR4 agonist; 1 µg/ml) or IL-1β (1 ng/ml). Nuclei were stained with DAPI (blue; 5 µg/ml). Images were acquired using a Cellomics VTi HCS Arrayscanner with a CarlZeiss microscope. (C) LPS (TLR4 agonist; 1 µg/ml) and C12-iE-DAP (NOD1 agonist; 10 µg/ml) induced CXCL8 release after 24 hour stimulation. Data are mean ± SEM. For HUVEC, hESC-EC or BOEC, n = 4–8. For iPSC-EC, n = 2, single isolation. Statistical significance was determined by one-way ANOVA followed by Dunnett's multiple comparison test for each cell type (*p<0.05) and by two-way ANOVA followed by Bonferroni's post-test for between cell types. Analysis was not performed on data from iPSC-EC.

Article Snippet: Experiments were carried out using the H7 hESC line provided under collaboration agreement with the Geron Corporation (Menlo Park, CA, USA) and with permission of the UK Stem Cell Bank.

Techniques: Expressing, In Vitro, Immunocytochemistry, Staining, Microscopy, Isolation, Comparison

Journal: PLoS ONE

Article Title: Pathogen Sensing Pathways in Human Embryonic Stem Cell Derived-Endothelial Cells: Role of NOD1 Receptors

doi: 10.1371/journal.pone.0091119

Figure Lengend Snippet: MSD analysis of cytokine (pg/ml) release from hESC-EC.

Article Snippet: Experiments were carried out using the H7 hESC line provided under collaboration agreement with the Geron Corporation (Menlo Park, CA, USA) and with permission of the UK Stem Cell Bank.

Techniques:

Journal: PLoS ONE

Article Title: Pathogen Sensing Pathways in Human Embryonic Stem Cell Derived-Endothelial Cells: Role of NOD1 Receptors

doi: 10.1371/journal.pone.0091119

Figure Lengend Snippet: TLR4 and NOD1 expression in (A) hESC-EC and (B) HUVEC before (pre-implant; open bars) and 21 days after (post-implant; filled bars) implantation in vivo (‘conditioning’). Data are mean ± SEM and are normalized at unity (1) to gene levels in pre-implant cells. HUVEC; NOD1 pre-implant n = 8, post implant n = 4: HUVEC; TLR4 pre-implant n = 10, post implant n = 3. hESC-ECs; NOD1 pre-implant n = 6, post implant n = 5: hESC-ECs; TLR4 pre-implant n = 10, post implant n = 6. Data was obtained from 2 independent experiments (using up to 12 rats per group). Statistical significance was determined by one-sample t-test where results were compared to a theoretical control of 1 (*p<0.05). ND = none detectable.

Article Snippet: Experiments were carried out using the H7 hESC line provided under collaboration agreement with the Geron Corporation (Menlo Park, CA, USA) and with permission of the UK Stem Cell Bank.

Techniques: Expressing, In Vivo, Control

Journal: PLoS ONE

Article Title: Pathogen Sensing Pathways in Human Embryonic Stem Cell Derived-Endothelial Cells: Role of NOD1 Receptors

doi: 10.1371/journal.pone.0091119

Figure Lengend Snippet: (A) Effect of LPS (1 µg/ml) or C12-iE-DAP (10 µg/ml) on CXCL8 release from hESC-EC and HUVEC after 24 hours. (B) Effect of Haemophilus influenzae (HIN) (10 5 –10 8 CFU/ml) on CXCL8 release from hESC-EC (solid line) or HUVEC (dashed line) after 24 hours. Data are mean ± SEM; n = 3 representative of 6 hESC-EC isolations. Statistical significance for responses to drugs or bacteria was determined by one-way ANOVA followed by Dunnett's multiple comparison test (p<0.05).

Article Snippet: Experiments were carried out using the H7 hESC line provided under collaboration agreement with the Geron Corporation (Menlo Park, CA, USA) and with permission of the UK Stem Cell Bank.

Techniques: Bacteria, Comparison

Journal: PLoS ONE

Article Title: Pathogen Sensing Pathways in Human Embryonic Stem Cell Derived-Endothelial Cells: Role of NOD1 Receptors

doi: 10.1371/journal.pone.0091119

Figure Lengend Snippet: (A) Relative expression (vs. GAPDH) of NOD1 following 48 hour incubation with NOD1 siRNA normalized to non-targeting siRNA; n = 6. (B) CXCL8 release from hESC-EC following 48 hour pre-incubation with non-targeting siRNA (open bars) or NOD1-siRNA (filled bars) and 24 hour treatment +/− C12-iE-DAP (10 µg/ml) or Haemophilus influenzae (HIN) (10 7 –10 8 CFU/ml); n = 7–8. (C) Effect of GSK'214 (300 nM; RIP2 inhibitor) or GSK'217 (300 nM; NOD1 inhibitor), given 30 minutes before a 24 hour treatment with HIN (10 7 CFU/ml) or C12-iE-DAP (10 µg/ml) on CXCL8 release; n = 4. It should be noted that GSK drugs increased CXCL8 release under basal conditions; for each experiment this was subtracted from treatment groups. For panel A, statistical significance was determined by one-sample t-test. For panel B statistical significance within siRNA groups was determined by one-way ANOVA followed by Dunnett's multiple comparison test (*p<0.05), and between groups by two-way ANOVA followed by Bonferroni's post-test (+p<0.05). For panel C statistical significance for the effects of inhibitor of C12-iE-DAP or HIN induced CXCL8 was determined by one-way ANOVA followed by Dunnett's multiple comparison test (*p<0.05).

Article Snippet: Experiments were carried out using the H7 hESC line provided under collaboration agreement with the Geron Corporation (Menlo Park, CA, USA) and with permission of the UK Stem Cell Bank.

Techniques: Expressing, Incubation, Comparison

Journal: Stem cells (Dayton, Ohio)

Article Title: Human Embryonic Stem Cell-Derived Oligodendrocyte Progenitor Cell Transplants Improve Recovery after Cervical Spinal Cord Injury

doi: 10.1002/stem.245

Figure Lengend Snippet: hESC-derived OPC expression profile. (a): More than 90% of cells labeled with the chondroitin sulfate proteoglycan NG2 (red), associated with oligodendrocyte precursors. (b): A total of 72 ± 2% of cells expressed the cell surface tyrosine kinase receptor for platelet-derived growth factor, PDGFRα (red), associated with oligodendrocyte progenitors. No cells were detected that expressed the embryonic stem cell markers Oct-4 and SSEA4 or the neural development transcription factor Pax6 (black bars) (c). Fewer than 5% of cells expressed the less mature OPC marker, A2B5, or the more mature oligodendroglial markers, O4 and RIP (red bars) (c). Some cells (<5% each) were positive for the non-oligodendroglial markers GFAP and Tuj1 (green bars) (c). (d): Detection of selected mRNA expression by RT-PCR. Scale bar: a, 60μm; b 45 μm.

Article Snippet: Derivation of OPCs from hESCs The WA07 (H7) hESC line at passage 32 was obtained from Geron (Menlo Park, CA).

Techniques: Derivative Assay, Expressing, Labeling, Marker, Reverse Transcription Polymerase Chain Reaction

Journal: Stem cells (Dayton, Ohio)

Article Title: Human Embryonic Stem Cell-Derived Oligodendrocyte Progenitor Cell Transplants Improve Recovery after Cervical Spinal Cord Injury

doi: 10.1002/stem.245

Figure Lengend Snippet: Transplantation of hESC-derived OPCs into acute SCI resulted in cell survival, limited migration from the site of implantation, and differentiation to mature oligodendrocytes. (a): The distribution of hESC-derived OPCs was detected using anti-human-positive nuclei staining (brown). No human cells were detected in control animals. (b): Close image of transplant area reveals non-human cells (blue-green) among the anti-human nuclei-positive, transplanted cells (brown). Within the transplant area, human nuclei positive cells were homogeneously distributed and were not located in discrete clusters or rosette-like formations in any of the transplanted animals as might suggest neuroblastoma/glioma. (c): Distribution of total numbers of anti-human nuclei-positive cells within spinal cord transverse sections along the cranial-caudal axis. Cells were significantly localized to the injury epicenter (* p < 0.001, error bars represent ±SEM). Inset panels illustrate limited distribution of transplanted OPCs (black punctuation) in dorsal column. (d): Clusters of anti-human nuclei-positive cells (green) double labeled with Olig1 (red) within a region of high concentration of human nuclei-positive cells. (e): 2–3 mm from the injury epicenter, human-specific CXCR4 (red) cells localized to sites of rat/mouse-specific CXCL12 (green), suggesting that the transplanted cells responded to host cell-migration cues. (f): Anti-human nuclei-positive cells (HuNu, green) double-labeled (arrows) with the mature oligodendrocyte marker APC/CC1 (red) detected within a region of white matter. Endogenous APC/CC1-positive oligodendrocytes were present within the white matter of transplants and controls. (g): Double-labeling of anti-human nuclei (HuNu, green) and APC/CC1 (red) was confirmed by 3-D reconstruction of confocal scanned thin-plane images (large arrow in f and g). No human cells were found that colabeled with the astrocytes marker, GFAP, the neuronal marker, NeuN, or the pluripotent marker, Oct-4 (data not shown). Scale bar: a, 1.25 mm; b, d, 45 μm; e, 200 μm; f, 90 μm; g, 8 μm.

Article Snippet: Derivation of OPCs from hESCs The WA07 (H7) hESC line at passage 32 was obtained from Geron (Menlo Park, CA).

Techniques: Transplantation Assay, Derivative Assay, Migration, Staining, Control, Labeling, Concentration Assay, Marker

Journal: Stem cells (Dayton, Ohio)

Article Title: Human Embryonic Stem Cell-Derived Oligodendrocyte Progenitor Cell Transplants Improve Recovery after Cervical Spinal Cord Injury

doi: 10.1002/stem.245

Figure Lengend Snippet: hESC-derived OPC-transplants to cervical SCI significantly improved forelimb motor function. (a): Recovery of forelimb mean stride length significantly improved in the transplanted group relative to the non-transplanted group. The improvement was significant from at least 4 weeks after transplantation through the duration of the testing (* p < 0.01). The mean stride length of uninjured animals that were trained to the same apparatus for 9 weeks was 17.5 cm (dashed line). (b): The range of proximal forelimb motion in degrees was significantly increased in the transplant group compared to the non-transplanted group (* p < 0.01). (c): The increased range of proximal forelimb motion was resolved to the lift-off (rear-most) position of the step as opposed to the placement (fore-most) by measurement of the inside angle between the forelimb and the floor at lift-off and placement (* p < 0.01). (d): The occurrence of steps that pass the perpendicular plane is significantly increased from frequent (>50%) in the transplant group versus occasional (≤50%) in the control group (** p < 0.001). Error bars represent ±SEM.

Article Snippet: Derivation of OPCs from hESCs The WA07 (H7) hESC line at passage 32 was obtained from Geron (Menlo Park, CA).

Techniques: Derivative Assay, Transplantation Assay, Control

Journal: Stem cells (Dayton, Ohio)

Article Title: Human Embryonic Stem Cell-Derived Oligodendrocyte Progenitor Cell Transplants Improve Recovery after Cervical Spinal Cord Injury

doi: 10.1002/stem.245

Figure Lengend Snippet: Histopathogenesis in transplanted and non-transplanted animals. (a): Microthin sections from non-transplanted animals demonstrate myelin pathology concentrated around the dorsal and medial-ventral white matter and cavitation that eliminates a large portion of gray matter. (b): Transplanted animals lack cavitation and myelin pathology is less extensive compared to non-transplanted animals. (c): The dorsal column along the cavitation (cav) contains mostly Schwann cell (SC)-remyelinated axons (sc) with an edge that is consistent with scar formation. (d): In contrast to non-transplanted animals (c), the dorsal column of transplanted animals contains mostly normally myelinated axons (n) and no delineation between the transplanted (trans) and host cells. (e): Myelin pathology in non-transplanted animals shows thin oligodendrocyte remyelination (o), partitioned SC-remyelination (sc), and few small diameter axons. Inset illustrates the thin myelin sheath characteristic of oligodendrocyte remyelination. (f): Transplanted animal spinal cords contained hypercellularity and extra-axonal swelling, spared myelination (n), as well as more small diameter axons compared to controls. (g): Quantification of myelin pathology revealed a significant (*** p < 0.001) decrease in normally myelinated axons and an increase in SC-remyelinated (*** p < 0.001) and demyelinated (* p < 0.05) axons in the non-transplanted group relative to the transplanted group. These data indicate that normally myelinated and oligodendrocyte-remyelinated axons constitute about 95% of the total axon population within the area of dorsal column pathology in transplanted animals. (h): hESC-derived OPC transplants had significantly (*** p < 0.001) more oligodendrocyte-remyelinated axons relative to SC-remyelinated and demyelinated axons than controls. The transplants increased oligodendrocyte-remyelination efficiency to approximately 6.8-fold. Error bars represent ±SEM. Scale bar: a, b, 1.25 mm; c, d, 60 μm; e, 30 μm; f, 40 μm.

Article Snippet: Derivation of OPCs from hESCs The WA07 (H7) hESC line at passage 32 was obtained from Geron (Menlo Park, CA).

Techniques: Derivative Assay