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1.
Figure 5

Figure 5. Reporter gene imaging of hES cell and hESC-EC fate after transplantation. From: Comparison of Reporter Gene and Iron Particle Labeling for Tracking Fate of Human Embryonic Stem Cells and Differentiated Endothelial Cells in Living Subjects.

(A) A representative animal injected with 1×106 hESC-ECs (right hind limb) shows significant bioluminescence activity at day 2, which decreases progressively over the following 4 weeks. In contrast, undifferentiated hES cells (left hind limb) show the lowest bioluminescence signals at day 7, which increases dramatically during week 2 and week 4. (B) Detailed quantitative analysis of signals from all animals transplanted with hES cells versus hESC-ECs. Signal activity is expressed as photons/sec/cm2/sr. Note the Y-axis is shown as log 10 scale.

Zongjin Li, et al. Stem Cells. 2008 April;26(4):864-873.
2.
Figure 3

Figure 3. Iron particle labeling of hES cells and hESC-ECs. From: Comparison of Reporter Gene and Iron Particle Labeling for Tracking Fate of Human Embryonic Stem Cells and Differentiated Endothelial Cells in Living Subjects.

(A) Prussian blue staining for iron shows cytosolic deposition of blue crystals. Upper panel is 100X and lower panel is 1000X magnification. Scale bar=100μm (upper) and 10μm (lower). (B) Representative in vitro cellular MR images. Iron-labeled hESC-ECs demonstrated larger area of signal dephasing. The cell suspensions in 96-well plates each contain 1×104, 5×104, 1×105 iron-labeled cells (from left to right). (C) Trypan blue cell viability assay show no significant difference between control unlabeled cells and iron labeled cells for both cell populations (hESC and hESC-EC).

Zongjin Li, et al. Stem Cells. 2008 April;26(4):864-873.
3.

Figure 4. Serial in vivo MR imaging of iron-labeled cells from day 2 to week 4. From: Comparison of Reporter Gene and Iron Particle Labeling for Tracking Fate of Human Embryonic Stem Cells and Differentiated Endothelial Cells in Living Subjects.

(A) Representative in vivo gradient-recalled echo (GRE) imaging. No hypointense signal is found in the MR image of control mouse injected with unlabeled cells. MR signals showed no significant difference from day 2 to day 28. MR image by GRE at day 28 shows bulking expansion of the left hind limb injected with hES cells due to teratoma formation (arrow head). (B) Detailed quantitative analysis of GRE signals from all animals transplanted with hES cells and hESC-ECs (signal activity is expressed as authority unit (AU)). (C) Representative in vivo Off-Resonance (OR) imaging. (D) The analysis of quantitative OR signal from all animals transplanted with iron-labeled cells. No significant differences in the OR signal analysis was observed from day 2 to day 28. Abbreviations: GRE, gradient-recalled echo; OR, off-resonance.

Zongjin Li, et al. Stem Cells. 2008 April;26(4):864-873.
4.
Figure 6

Figure 6. Histologic analysis of double labeled hES cells and hESC-ECs. From: Comparison of Reporter Gene and Iron Particle Labeling for Tracking Fate of Human Embryonic Stem Cells and Differentiated Endothelial Cells in Living Subjects.

(A) Staining for macrophages and iron 4 weeks after transplantation of hESC-ECs. Immunostaining of Mac-3 for macrophages (I, III) and Prussian blue for iron (II, IV) were counterstained with hematoxylin and nuclear fast red, respectively. Note macrophages loaded with iron particles can be found in between muscle bundles. Scale bar=100μm (I, III) and 20μm (II, IV). (B) Immunofluorescence staining of GFP for transplanted double fusion hESC-ECs, CD31 for microvasculature of hindlimb, and Mac-3 for macrophages at 4 weeks after transplantation. There were no transplanted GFP+ hESC-ECs found nearby macrophages. Nuclei were stained with DAPI (blue). Scale bar=20μm. (C) Staining for macrophages and iron 4 weeks after implantation of undifferentiated hES cells. Prussian blue positive cells are distributed between normal skeletal muscles (*) and teratoma (#). Scale bar=100μm (I, III) and 20μm (II, IV). (D) Immunofluorescence staining for GFP, CD31 and macrophages 4 weeks after transplantation of hESCs. GFP staining showed teratoma formation (#) and clear edge (dashed line) separating from the normal muscle fibers (*). Nuclei were stained with DAPI (blue). Scale bar= 20μm.

Zongjin Li, et al. Stem Cells. 2008 April;26(4):864-873.
5.

Figure 2. Stable lentiviral transduction of hES cells with the double fusion reporter genes. From: Comparison of Reporter Gene and Iron Particle Labeling for Tracking Fate of Human Embryonic Stem Cells and Differentiated Endothelial Cells in Living Subjects.

(A) Schema of the double fusion reporter gene containing fusion of Fluc-eGFP. The double fusion reporter gene was cloned into a self-inactivating lentiviral vector downstream from the constitutive ubiquitin promoter. (B) Control nontransduced hES cells and transduced hES cells showed similar expression pattern of Oct-4 under fluorescence microscopy. DAPI staining is used as a nuclear marker. Scale bar=10μm. (C) Ex vivo imaging analysis of stably transduced hES cells show increasing bioluminescence signals with cell numbers of hES cells (r2=0.99) and with hESC-ECs (r2=0.99). Compared to hES cells, hESC-ECs expressed higher bioluminescence activity. Data are representative of three independent experiments. (D) Flow cytometric analysis of double fusion hESC-ECs. Percent positive cells are shown in upper right hand corner. Double fusion hESC-ECs were isolated from day-12 hEB by FACSan and subcultured. Normal hESC-ECs were used as control. Isotype-matched antibodies were used in flow cytometry for background fluorescence. (E) Uptake of DiI-ac-LDL (red) by double fusion hESC-ECs. Nuclei were stained with DAPI (blue). Data are representative of three independent experiments. Scale bar=50μm. Abbreviations: DAPI, 4′, 6-diamidino-2-phenylindole; EC, endothelial cell; LDL, low density lipoprotein.

Zongjin Li, et al. Stem Cells. 2008 April;26(4):864-873.
6.

Figure 1. In vitro endothelial differentiation of hES cells. From: Comparison of Reporter Gene and Iron Particle Labeling for Tracking Fate of Human Embryonic Stem Cells and Differentiated Endothelial Cells in Living Subjects.

(A) Schematic outline of the differentiation procedures. Undifferentiated hES cells were grown to 60%–70% confluence on mouse embryonic fibroblasts (MEFs) or on Matrigel in MEF conditioned medium, dated as day 0. At day 12, hEBs were collected and digested by Liberase Blendzyme IV and CD31+ cells were isolated by FACS and sub-cultured in EGM-2 medium to expand and induce endothelial maturation. Scale bar=20μm (upper and lower) and 100μm (middle). (B) Whole-mount immunochemistry of day-12 hEB. Areas of CD31 cells (red) within hEBs are organized in elongated clusters (I) and channels (II, arrowhead). Cell nuclei stained with DAPI (blue). Scale bar=50μm (I) and 10μm (II). (C) Flow cytometric analysis of endothelial cell markers (CD31, VE-cadherin, and KDR). Percent positive cells are shown. hESC-ECs were isolated from day-12 hEB by FACSan and subcultured. HUVECs were used as positive control. Isotype-matched antibodies were used in flow cytometry for background fluorescence. (D) Comparison of mRNA expression levels of hESC, hEB and hESC-EC between HUVEC. The quantification was performed by real-time RT-PCR. Experiments were performed in triplicates. #P>0.05, *P<0.05 compared to HUVEC. (E) Endothelial tube formation by hESC-ECs and HUVECs after 12 hours of plating on Matrigel in 24-well plates. Scale bar=20μm. Abbreviations: hEB, human embryoid body; hESC, human ES cell; FACS, fluorescence activated cell sorting; HUVEC, human umbilical vein endothelial cell.

Zongjin Li, et al. Stem Cells. 2008 April;26(4):864-873.

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