Volume 17, Issue 7, Pages 1282-1291 (July 2009) Molecular and Magnetic Resonance Imaging of Human Embryonic Stem Cell–Derived Neural Stem Cell Grafts in Ischemic Rat Brain  Marcel M Daadi, Zongjin Li, Ahmet Arac, Brad A Grueter, Marc Sofilos, Robert C Malenka, Joseph C Wu, Gary K Steinberg  Molecular Therapy  Volume 17, Issue 7, Pages 1282-1291 (July 2009) DOI: 10.1038/mt.2009.104 Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

Figure 1 Generation and characterization of human embryonic stem cell–derived neural stem cells (NSCs) genetically engineered to express the double fusion fLuc and enhanced green fluorescence protein (eGFP) proteins. (a) Schematic representation of the double fusion reporter gene containing fLuc and eGFP driven by human ubiquitin promotor. Neural stem cells were isolated through a multistep process we previously described.10 (b) Flow cytometry analysis demonstrated the purity of the eGFP-expressing hNSCs and the expression of the neural stem cells markers CD133, vimentin and 3CB2. Percent of positive cells is shown in the upper right corner of the flow cytometry graph. Immunocytochemistry confirmed the flow cytometry results and demonstrated the homogenous and uniform expression of the neural stem cell markers (c) vimentin and (d) 3CB2. To determine the electrophysiological property of the neuronal population expressing (e) β-tubulin, hNSCs were induced to differentiate by plating on a glass coverslip in absence of mitogenic factors for 7 days in vitro and prepared for recording. (f) Representative current-clamp trace of action potentials elicited from current injections ranging from −100 to 400 pA from an hNSC-derived neuron. (g) A current-clamp trace representing bursting of action potentials in response to 400 pA current injections. (h) Voltage-activated currents elicited in response to a –50 mV step. (i) Sample trace of spontaneous excitatory postsynaptic currents in a neuron held at −70 mV. Bars = (c,d) 50 µm; (e) 20 µm. Molecular Therapy 2009 17, 1282-1291DOI: (10.1038/mt.2009.104) Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

Figure 2 In vitro and in vivo bioluminescence imaging (BLI) of the hNSCs. (a) In vitro imaging analysis of genetically engineered hNSCs show increasing fLuc activity with cell density and a linear correlation (R2 = 0.98). (b) Data are representative of three independent experiments performed in triplicate. Representative BLI of stroke-lesioned rats transplanted with the hNSCs and monitored for (c) 4 weeks and (d) 8-week post-transplantation survival times. Quantitative analysis of the fLuc activity in these animals shows a stable BLI signal, which suggest the survival of the grafts and the nonproliferative property of the hNSCs. Color scale bar is in photon/s/cm2/sr. Molecular Therapy 2009 17, 1282-1291DOI: (10.1038/mt.2009.104) Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

Figure 3 MRI imaging analysis of the hNSCs grafts in experimental stroke model. (a–c) MRI horizontal and (d–f) frontal scans show dose-dependant size of the superparamagnetic iron oxide (SPIO)-labeled hNSCs grafts as hypointense areas in the striatum (arrow) medially in the penumbral zone of the stroke distinguished as strongly hyperintense areas on T2-weighted images. The cell doses are 50,000 cells (a,d), 200,000 cells (b,e) and 400,000 cells (c,f). (g) Quantitative analysis of graft size, in consecutive coronal MRI scans, 600 µm spaced (Supplementary Figure S1, see Materials and Methods section) in the three animal groups (n = 15) over the post-transplant survival time confirm the BLI data and show a stable graft size demonstrating survival of the graft. Three-dimensional surface rendering reconstruction of grafted rat brain from high resolution T2-MRI illustrate the grafts (green) and stroke (pink, red) in a representative animal from the (h–j) low-dose and (k–m) intermediate-dose group. (n) The MRI measured graft size show a strong correlation (R2 = 0.99) with the cell dose transplanted. (o–q) Histological analysis using Prussian blue staining for SPIO particles demonstrate cytosolic deposition of blue crystals in the grafted hNSCs and migration toward stroke area (asterisk in o,p). Interrupted line in o shows the boundary of stroke zone. Bars = (p) 50 µm; (q) 20 µm. Molecular Therapy 2009 17, 1282-1291DOI: (10.1038/mt.2009.104) Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

Figure 4 Grafts of hNSCs in the stroke-damaged area. (a) Fluorescence confocal micrograph of lectin staining (red) showing the normal distribution of blood vessels in the nonlesioned contralateral side. (b) Photomicrograph taken from the border of stroke-damaged area showing stroke-damaged blood vessels (left to the interrupted white line) some intact vessel (arrow) in the stroke boundary zone of a vehicle treated animal. (c) Confocal photomicrograph showing the robust survival of GFP+ (green) transplanted hNSCs in stroke-damaged zone. Arrow shows some intact part of residual blood vessel with close contact with grafted hNSCs (yellow). Molecular Therapy 2009 17, 1282-1291DOI: (10.1038/mt.2009.104) Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

Figure 5 Transplanted hNSCs into stroke-lesioned brain differentiate into neural lineages. (a) Confocal image showing the co-localization of the human nuclear specific marker hNuc (red, arrow) with the GFP+ (green) hNSCs. Subpopulation of grafted hNSCs (green and red) express nestin (purple, arrow), arrowhead show nestin-negative hNSCs. (c) Confocal photomicrograph showing the non-colocalization of Ki67+ endogenous cells (green, arrowheads) and hNuc+ transplanted hNSCs (red, arrow). Grafted hNuc+ hNSCs (red, arrow) expressed the neuronal lineage markers DCX (d, red). (e) Fluorescence confocal micrographs show the expression of the oligodendrocytes cell surface marker galactocerebroside (GC, red) and GFP+ grafted hNSCs (green, arrow), arrowhead show a GFP+ hNSC− for the GC. (f) Confocal photomicrograph showing hNuc+ hNSC (arrow) expressing the astroglial marker GFAP (red) (g) Example of blood vessel (arrowhead) stained with lectin (red) in stroke-damaged area and fibers extended by a GAFP+ astrocyte (purple) derived from the GFP+ (green) transplanted hNSCs. Bars = (a–d,f) 10 µm; (e,g) 20 µm. Molecular Therapy 2009 17, 1282-1291DOI: (10.1038/mt.2009.104) Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

Figure 6 The hNSCs grafted into ischemic brain show electrophysiological properties of integrated neurons. (a) High magnification confocal image of NeuN+ (red) grafted GFP+ neurons (green). (b) High power confocal photomicrograph shows synaptophysin expression as puncta (red) on grafted GFP+ (green) hNSCs (arrow head). (c) Electron micrograph taken from transplant zone show an axodendritic synapse. The DAB-immunostained GFP is visible in the dendrite. Arrow points at the axon terminal. Bar = 250 nm. (d) Representative current-clamp traces show voltage-dependent responses in a grafted GFP+ hNSC identified under epifluorescence (see Materials and Methods section) elicited in response to a −200–400 pA current injections. (e) A voltage-clamp trace with a small but replicable current in response to a 50 mV step. (f) A voltage clamp trace from cell (d) in which a broad current is elicited in response to a voltage step. (g) Sample trace of spontaneous EPSCs in a grafted cell held at −70 mV. (h) Epifluorescence image of an acute coronal slice containing GFP+ hNSCs (arrow) targeted for the patch clamp recording. Bars = (a,b) 10 µm, (c) 0.2 µm, (h) 20 µm. Molecular Therapy 2009 17, 1282-1291DOI: (10.1038/mt.2009.104) Copyright © 2009 The American Society of Gene Therapy Terms and Conditions